WorldWideScience

Sample records for fiv gag virus

  1. Diagnosing feline immunodeficiency virus (FIV) infection in FIV-vaccinated and FIV-unvaccinated cats using saliva.

    Science.gov (United States)

    Westman, Mark E; Malik, Richard; Hall, Evelyn; Norris, Jacqueline M

    2016-06-01

    We recently showed that two immunochromatography point-of-care FIV antibody test kits (Witness FeLV/FIV and Anigen Rapid FIV/FeLV) were able to correctly assign FIV infection status, irrespective of FIV vaccination history, using whole blood as the diagnostic specimen. A third FIV antibody test kit, SNAP FIV/FeLV Combo (an enzyme-linked immunosorbent assay [ELISA]), was unable to differentiate antibodies produced in response to FIV vaccination from those incited by FIV infection. The aim of this study was to determine if saliva is a suitable diagnostic specimen using the same well characterized feline cohort. FIV infection status of these cats had been determined previously using a combination of serology, polymerase chain reaction (PCR) testing and virus isolation. This final assignment was then compared to results obtained using saliva as the diagnostic specimen utilizing the same three point-of-care FIV antibody test kits and commercially available PCR assay (FIV RealPCR). In a population of cats where one third (117/356; 33%) were FIV-vaccinated, both immunochromatography test kits accurately diagnosed FIV infection using saliva via a centrifugation method, irrespective of FIV vaccination history. For FIV diagnosis using saliva, the specificity of Anigen Rapid FIV/FeLV and Witness FeLV/FIV was 100%, while the sensitivity of these kits was 96% and 92% respectively. SNAP FIV/FeLV Combo respectively. SNAP FIV/FeLV Combo had a specificity of 98% and sensitivity of 44%, while FIV RealPCR testing had a specificity of 100% and sensitivity of 72% using saliva. A revised direct method of saliva testing was trialed on a subset of FIV-infected cats (n=14), resulting in 14, 7 and 0 FIV positive results using Anigen Rapid FIV/FeLV, Witness FeLV/FIV and SNAP FIV/FeLV Combo, respectively. These results demonstrate that saliva can be used to diagnose FIV infection, irrespective of FIV vaccination history, using either a centrifugation method (Anigen Rapid FIV/FeLV and Witness

  2. Determining the feline immunodeficiency virus (FIV) status of FIV-vaccinated cats using point-of-care antibody kits.

    Science.gov (United States)

    Westman, Mark E; Malik, Richard; Hall, Evelyn; Sheehy, Paul A; Norris, Jacqueline M

    2015-10-01

    This study challenges the commonly held view that the feline immunodeficiency virus (FIV) infection status of FIV-vaccinated cats cannot be determined using point-of-care antibody test kits due to indistinguishable antibody production in FIV-vaccinated and naturally FIV-infected cats. The performance of three commercially available point-of-care antibody test kits was compared in a mixed population of FIV-vaccinated (n=119) and FIV-unvaccinated (n=239) cats in Australia. FIV infection status was assigned by considering the results of all antibody kits in concert with results from a commercially available PCR assay (FIV RealPCR™). Two lateral flow immunochromatography test kits (Witness FeLV/FIV; Anigen Rapid FIV/FeLV) had excellent overall sensitivity (100%; 100%) and specificity (98%; 100%) and could discern the true FIV infection status of cats, irrespective of FIV vaccination history. The lateral flow ELISA test kit (SNAP FIV/FeLV Combo) could not determine if antibodies detected were due to previous FIV vaccination, natural FIV infection, or both. The sensitivity and specificity of FIV RealPCR™ for detection of viral and proviral nucleic acid was 92% and 99%, respectively. These results will potentially change the way veterinary practitioners screen for FIV in jurisdictions where FIV vaccination is practiced, especially in shelter scenarios where the feasibility of mass screening is impacted by the cost of testing.

  3. Pharmacological Inhibition of Feline Immunodeficiency Virus (FIV

    Directory of Open Access Journals (Sweden)

    Dorothee Bienzle

    2012-04-01

    Full Text Available Feline immunodeficiency virus (FIV is a member of the retroviridae family of viruses and causes an acquired immunodeficiency syndrome (AIDS in domestic and non-domestic cats worldwide. Genome organization of FIV and clinical characteristics of the disease caused by the virus are similar to those of human immunodeficiency virus (HIV. Both viruses infect T lymphocytes, monocytes and macrophages, and their replication cycle in infected cells is analogous. Due to marked similarity in genomic organization, virus structure, virus replication and disease pathogenesis of FIV and HIV, infection of cats with FIV is a useful tool to study and develop novel drugs and vaccines for HIV. Anti-retroviral drugs studied extensively in HIV infection have targeted different steps of the virus replication cycle: (1 inhibition of virus entry into susceptible cells at the level of attachment to host cell surface receptors and co-receptors; (2 inhibition of fusion of the virus membrane with the cell membrane; (3 blockade of reverse transcription of viral genomic RNA; (4 interruption of nuclear translocation and viral DNA integration into host genomes; (5 prevention of viral transcript processing and nuclear export; and (6 inhibition of virion assembly and maturation. Despite much success of anti-retroviral therapy slowing disease progression in people, similar therapy has not been thoroughly investigated in cats. In this article we review current pharmacological approaches and novel targets for anti-lentiviral therapy, and critically assess potentially suitable applications against FIV infection in cats.

  4. Construction and Testing of orfA +/- FIV Reporter Viruses

    Directory of Open Access Journals (Sweden)

    Eric M. Poeschla

    2012-01-01

    Full Text Available Single cycle reporter viruses that preserve the majority of the HIV-1 genome, long terminal repeat-promoted transcription and Rev-dependent structural protein expression are useful for investigating the viral life cycle. Reporter viruses that encode the viral proteins in cis in this way have been lacking for feline immunodeficiency virus (FIV, where the field has used genetically minimized transfer vectors with viral proteins supplied in trans. Here we report construction and use of a panel of single cycle FIV reporter viruses that express fluorescent protein markers. The viruses can be produced to high titer using human cell transfection and can transduce diverse target cells. To illustrate utility, we tested versions that are (+ and (- for OrfA, an FIV accessory protein required for replication in primary lymphocytes and previously implicated in down-regulation of the primary FIV entry receptor CD134. We observed CD134 down-regulation after infection with or without OrfA, and equivalent virion production as well. These results suggest a role for FIV proteins besides Env or OrfA in CD134 down-regulation.

  5. Eight-year observation and comparative study of specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) subtypes A and B: terminal acquired immunodeficiency syndrome in a cat infected with FIV petaluma strain.

    Science.gov (United States)

    Kohmoto, M; Uetsuka, K; Ikeda, Y; Inoshima, Y; Shimojima, M; Sato, E; Inada, G; Toyosaki, T; Miyazawa, T; Doi, K; Mikami, T

    1998-03-01

    Three specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) strains Petaluma, TM1 and TM2, respectively were observed for over 8 years. Without showing any significant clinical signs of immunodeficiency syndrome (AIDS) for 8 years and 4 months of asymptomatic phase, the Petaluma-infected cat exhibited severe stomatitis/gingivitis, anorexia, emaciation, hematological and immunological disorders such as severe anemia, lymphopenia, thrombocytopenia, and decrease of CD4/CD8 ratio to 0.075, and finally died with hemoperitoneum at 8 years and 8 months post-infection. Histopathological studies revealed that the cat had systemic lymphoid atrophy and bone marrow disorders indicating acute myelocytic leukemia (aleukemic type). Plasma viral titer of the cat at AIDS phase was considerably high and anti-FIV antibody titer was slightly low as compared with the other FIV-infected cats. In addition, immunoblotting analysis using serially collected serum/plasma samples of these cats revealed that antibodies against FIV proteins were induced in all the infected cats, however in the Petaluma-infected cat anti-Gag antibodies disappeared during the asymptomatic period. These results suggested that plasma viral load and anti-FIV Gag antibody response correlated with disease progression, and supported FIV-infected cats as a suitable animal model of human AIDS.

  6. Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein.

    NARCIS (Netherlands)

    E.J. Tijhaar (Edwin); C.H.J. Siebelink (Kees); J.A. Karlas (Jos); M.C. Burger; F.R. Mooi (Frits); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractSalmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens

  7. Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein.

    NARCIS (Netherlands)

    E.J. Tijhaar (Edwin); C.H.J. Siebelink (Kees); J.A. Karlas (Jos); M.C. Burger; F.R. Mooi (Frits); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractSalmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens

  8. Immunomodulator expression in trophoblasts from the feline immunodeficiency virus (FIV-infected cat

    Directory of Open Access Journals (Sweden)

    Donaldson Janet R

    2011-07-01

    Full Text Available Abstract Background FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF and anti-inflammatory cytokines (IL-4, IL-5, and IL-10; CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor; the chemokine receptor CXCR4 (FIV co-receptor; SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy. Methods We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression. Results We detected IL-4, IL-5, IL-6, IL-1β, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens. Conclusion Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in

  9. Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein.

    Directory of Open Access Journals (Sweden)

    María J Esteva

    Full Text Available To gain insight into the functional relationship between the capsid (CA domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively, we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex. Furthermore, we show here that the carboxyl-terminus domain (CTD of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD. Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

  10. Contrasting clinical outcomes in two cohorts of cats naturally infected with feline immunodeficiency virus (FIV)

    Science.gov (United States)

    Bęczkowski, Paweł M.; Litster, Annette; Lin, Tsang Long; Mellor, Dominic J.; Willett, Brian J.; Hosie, Margaret J.

    2015-01-01

    Despite over 25 years of feline immunodeficiency virus (FIV) research, relatively little is known about the longitudinal course of FIV infection following natural infection. In contrast to published reports of experimental infections using lethal strains of the virus, clinical signs of naturally acquired FIV infection can be mild or inapparent, rather than life-threatening. In this prospective, longitudinal controlled study, based in Chicago, IL (n = 17) and Memphis, TN (n = 27), we investigated two cohorts of privately owned, naturally infected cats kept under different housing conditions. Cats in the Chicago cohort (Group 1) were kept in households of ≤2 cats, while the Memphis cohort (Group 2) comprised part of a large multi-cat household of over 60 cats kept indoors only, with unrestricted access to one another. The majority of cats from Group 1 did not display clinical signs consistent with immunodeficiency during the 22-month observation period. In contrast, the outcome of infection in Group 2 was dramatically different; 17/27 (63%) of cats lost a median of 51.3% of their bodyweight (P cats classified as ‘healthy’ and ‘not healthy’ at either cohort. FIV load at enrolment was significantly lower in Group 1 than in Group 2 (P cats at either group. In conclusion, the results of this study suggest that management and housing conditions impact on disease progression and survival times of FIV-positive cats. PMID:25595267

  11. The Foamy Virus Gag Proteins: What Makes Them Different?

    OpenAIRE

    Erik Müllers

    2013-01-01

    Gag proteins play an important role in many stages of the retroviral replication cycle. They orchestrate viral assembly, interact with numerous host cell proteins, engage in regulation of viral gene expression, and provide the main driving force for virus intracellular trafficking and budding. Foamy Viruses (FV), also known as spumaviruses, display a number of unique features among retroviruses. Many of these features can be attributed to their Gag proteins. FV Gag proteins lack characteristi...

  12. Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Siddhartha A.K.; Zuo, Xiaobing; Clark, Patrick K.; Campbell, Stephen J.; Wang, Yun-Xing; Rein, Alan (SAIC); (NCI)

    2012-05-09

    Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of {approx}7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.

  13. Prevalence of feline calicivirus, feline leukaemia virus and antibodies to FIV in cats with chronic stomatitis.

    Science.gov (United States)

    Knowles, J O; Gaskell, R M; Gaskell, C J; Harvey, C E; Lutz, H

    1989-04-01

    The prevalence of feline calicivirus (FCV), feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) antibodies were assessed in 78 British and 18 North American household cats with chronic stomatitis and in appropriate controls. In British cats, FCV was significantly (P less than 0.005) more prevalent in both hospital (92 per cent) and general practice (79 per cent) cases compared to their controls (19 per cent in both cases). A similar difference in prevalence of FCV was noted in North American cats where 50 per cent of cases were positive compared to 0 per cent of controls (P less than 0.01). FeLV prevalence was low in all chronic stomatitis populations. A significantly higher prevalence of antibody to FIV was found in British hospital cases (81 per cent) compared with time-matched controls (16 per cent) (P less than 0.001): a similar rate was found in the general practice cases (75 per cent) for which no controls were available. In the North American sample, FIV antibody status was similar in cases (54 per cent positive) and their age, sex and breed matched controls (50 per cent). The possible role of FCV and FIV in the pathogenesis of feline chronic stomatitis is discussed.

  14. The Foamy Virus Gag Proteins: What Makes Them Different?

    Directory of Open Access Journals (Sweden)

    Erik Müllers

    2013-03-01

    Full Text Available Gag proteins play an important role in many stages of the retroviral replication cycle. They orchestrate viral assembly, interact with numerous host cell proteins, engage in regulation of viral gene expression, and provide the main driving force for virus intracellular trafficking and budding. Foamy Viruses (FV, also known as spumaviruses, display a number of unique features among retroviruses. Many of these features can be attributed to their Gag proteins. FV Gag proteins lack characteristic orthoretroviral domains like membrane-binding domains (M domains, the major homology region (MHR, and the hallmark Cys-His motifs. In contrast, they contain several distinct domains such as the essential Gag-Env interaction domain and the glycine and arginine rich boxes (GR boxes. Furthermore, FV Gag only undergoes limited maturation and follows an unusual pathway for nuclear translocation. This review summarizes the known FV Gag domains and motifs and their functions. In particular, it provides an overview of the unique structural and functional properties that distinguish FV Gag proteins from orthoretroviral Gag proteins.

  15. Sequences within both the 5' UTR and Gag are required for optimal in vivo packaging and propagation of mouse mammary tumor virus (MMTV genomic RNA.

    Directory of Open Access Journals (Sweden)

    Farah Mustafa

    Full Text Available BACKGROUND: This study mapped regions of genomic RNA (gRNA important for packaging and propagation of mouse mammary tumor virus (MMTV. MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV. Studies of FIV and Mason-Pfizer monkey virus (MPMV, a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5' untranslated region (5' UTR and 5' end of gag constitute important packaging determinants for gRNA. METHODOLOGY: Three series of MMTV transfer vectors containing incremental amounts of gag or 5' UTR sequences, or incremental amounts of 5' UTR in the presence of 400 nucleotides (nt of gag were constructed to delineate the extent of 5' sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env, and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA. PRINCIPAL FINDINGS: MMTV requires the entire 5' UTR and a minimum of ~120 nucleotide (nt at the 5' end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5' UTR were defective for both efficient packaging and propagation into target cells. CONCLUSIONS/SIGNIFICANCE: These results reveal that the 5' end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.

  16. Evaluation of Clinical Findings, Some Hematological and Biochemical Findings, and Age and Sex Status in Feline Immunodeficiency Virus (FIV) Seropositive Cats With Clinical Symptoms and Without Clinical Symptoms

    OpenAIRE

    DENİZ, Abdülkerim

    2001-01-01

    This study was performed to investigate the clinical findings, some hematological and biochemical findings, and sex and age status in feline immunodeficiency virus (FIV) seropositive cats. In 37 FIV-seropositive cats, different clinical symptoms were observed (group 1). However, seven of the FIV-seropositive cats showed no clinical symptoms (group 2). The control group was made up of 116 healthy cats. FIV infection was seen more in male cats (78.4%) than in female cats. 62% of the FIV-seropos...

  17. Evaluation of Clinical Findings, Some Hematological and Biochemical Findings, and Age and Sex Status in Feline Immunodeficiency Virus (FIV) Seropositive Cats With Clinical Symptoms and Without Clinical Symptoms

    OpenAIRE

    DENİZ, Abdülkerim

    2014-01-01

    This study was performed to investigate the clinical findings, some hematological and biochemical findings, and sex and age status in feline immunodeficiency virus (FIV) seropositive cats. In 37 FIV-seropositive cats, different clinical symptoms were observed (group 1). However, seven of the FIV-seropositive cats showed no clinical symptoms (group 2). The control group was made up of 116 healthy cats. FIV infection was seen more in male cats (78.4%) than in female cats. 62% of the FIV-seropos...

  18. Immunomodulator expression in trophoblasts from the feline immunodeficiency virus FIV infected cat

    Science.gov (United States)

    FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection m...

  19. Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein.

    Directory of Open Access Journals (Sweden)

    Guangzhi Zhang

    Full Text Available The Gag protein of the mouse mammary tumor virus (MMTV is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV, assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.

  20. Renal Alterations in Feline Immunodeficiency Virus (FIV-Infected Cats: A Natural Model of Lentivirus-Induced Renal Disease Changes

    Directory of Open Access Journals (Sweden)

    Mauro Pistello

    2012-08-01

    Full Text Available Human immunodeficiency virus (HIV is associated with several renal syndromes including acute and chronic renal failures, but the underlying pathogenic mechanisms are unclear. HIV and feline immunodeficiency virus (FIV share numerous biological and pathological features, including renal alterations. We investigated and compared the morphological changes of renal tissue of 51 experimentally and 21 naturally infected cats. Compared to the latter, the experimentally infected cats exhibited some mesangial widening and glomerulonephritis, milder proteinuria, and lower tubular and interstitial alterations. The numbers of giant protein tubular casts and tubular microcysts were also lower. In contrast, diffuse interstitial infiltrates and glomerular and interstitial amyloidosis were detected only in naturally infected cats. Similar alterations are found in HIV infected patients, thus supporting the idea of a causative role of FIV infection in renal disease, and underlining the relevance of the FIV and its natural host as an animal model for investigating lentivirus-associated nephropathy.

  1. Kunjin replicon-based simian immunodeficiency virus gag vaccines

    NARCIS (Netherlands)

    Anruka, I.; Mokhonov, V.; Rattanasena, P.; Mokhonova, E.; Leung, J.Y.; Pijlman, G.P.; Cara, A.; Schroder, W.A.; Khromykh, A.A.; Suhrbier, A.

    2008-01-01

    An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines

  2. Kunjin replicon-based simian immunodeficiency virus gag vaccines

    NARCIS (Netherlands)

    Anruka, I.; Mokhonov, V.; Rattanasena, P.; Mokhonova, E.; Leung, J.Y.; Pijlman, G.P.; Cara, A.; Schroder, W.A.; Khromykh, A.A.; Suhrbier, A.

    2008-01-01

    An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines enc

  3. An initial examination of the potential role of T-cell immunity in protection against feline immunodeficiency virus (FIV) infection.

    Science.gov (United States)

    Aranyos, Alek M; Roff, Shannon R; Pu, Ruiyu; Owen, Jennifer L; Coleman, James K; Yamamoto, Janet K

    2016-03-14

    The importance of vaccine-induced T-cell immunity in conferring protection with prototype and commercial FIV vaccines is still unclear. Current studies performed adoptive transfer of T cells from prototype FIV-vaccinated cats to partial-to-complete feline leukocyte antigen (FLA)-matched cats a day before either homologous FIVPet or heterologous-subtype pathogenic FIVFC1 challenge. Adoptive-transfer (A-T) conferred a protection rate of 87% (13 of 15, p 13 × 10(6) cells were required for A-T protection against FIVFC1 strain, reported to be a highly pathogenic virus resistant to vaccine-induced neutralizing-antibodies. The addition of FLA-matched B cells alone was not protective. The poor quality of the anti-FIV T-cell immunity induced by the vaccine likely contributed to the lack of protection in an FLA-matched recipient against FIVFC1. The quality of the immune response was determined by the presence of high mRNA levels of cytolysin (perforin) and cytotoxins (granzymes A, B, and H) and T helper-1 cytokines (interferon-γ [IFNγ] and IL2). Increased cytokine, cytolysin and cytotoxin production was detected in the donors which conferred protection in A-T studies. In addition, the CD4(+) and CD8(+) T-cell proliferation and/or IFNγ responses to FIV p24 and reverse transcriptase increased with each year in cats receiving 1X-3X vaccine boosts over 4 years. These studies demonstrate that anti-FIV T-cell immunity induced by vaccination with a dual-subtype FIV vaccine is essential for prophylactic protection against AIDS lentiviruses such as FIV and potentially HIV-1.

  4. Phosphoinositides direct equine infectious anemia virus gag trafficking and release.

    Science.gov (United States)

    Fernandes, Fiona; Chen, Kang; Ehrlich, Lorna S; Jin, Jing; Chen, Min H; Medina, Gisselle N; Symons, Marc; Montelaro, Ronald; Donaldson, Julie; Tjandra, Nico; Carter, Carol A

    2011-04-01

    Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release. © 2011 John Wiley & Sons A/S.

  5. Serological survey of Toxoplasma gondii, Dirofilaria immitis, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infections in pet cats in Bangkok and vicinities, Thailand

    Science.gov (United States)

    The seroprevalence of Toxoplasma gondii, Dirofilaria immitis (heartworm), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was examined using serum or plasma samples from 746 pet cats collected between May and July 2009 from clinics and hospitals located in and around ...

  6. Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

    Directory of Open Access Journals (Sweden)

    Koester Mario

    2006-09-01

    Full Text Available Abstract Background Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. Results The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV at the plasma membrane (PM and the formation of virus like particles in multivesicular bodies (MVBs. In our study we show that caveolin-1 (Cav-1, a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. Conclusion This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly.

  7. Oral Immunization with Recombinant Listeria monocytogenes Controls Virus Load after Vaginal Challenge with Feline Immunodeficiency Virus

    OpenAIRE

    Stevens, Rosemary; Howard, Kristina E.; Nordone, Sushila; Burkhard, MaryJo; Dean, Gregg A

    2004-01-01

    Recombinant Listeria monocytogenes has many attractive characteristics as a vaccine vector against human immunodeficiency virus (HIV). Wild-type and attenuated Listeria strains expressing HIV Gag have been shown to induce long-lived mucosal and systemic T-cell responses in mice. Using the feline immunodeficiency virus (FIV) model of HIV we evaluated recombinant L. monocytogenes in a challenge system. Five cats were immunized with recombinant L. monocytogenes that expresses the FIV Gag and del...

  8. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

    Energy Technology Data Exchange (ETDEWEB)

    Dick, Robert A.; Datta, Siddhartha A.K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M. (NCI); (Cornell); (CM); (NIST)

    2016-05-06

    Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

    Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our

  9. Serological survey of Toxoplasma gondii, Dirofilaria immitis, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infections in pet cats in Bangkok and vicinities, Thailand.

    Science.gov (United States)

    Sukhumavasi, Woraporn; Bellosa, Mary L; Lucio-Forster, Araceli; Liotta, Janice L; Lee, Alice C Y; Pornmingmas, Pitcha; Chungpivat, Sudchit; Mohammed, Hussni O; Lorentzen, Leif; Dubey, J P; Bowman, Dwight D

    2012-08-13

    The seroprevalence of Toxoplasma gondii, Dirofilaria immitis (heartworm), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was examined using serum or plasma samples from 746 pet cats collected between May and July 2009 from clinics and hospitals located in and around Bangkok, Thailand. The samples were tested for heartworm, FIV, and FeLV using a commercial ELISA. Of the 746 samples, 4.6% (34/746) were positive for heartworm antigen, 24.5% (183/746) had circulating FeLV antigen, and 20.1% (150/746) had antibodies against FIV. In addition, the first 348 submitted samples were tested for T. gondii antibodies using a modified agglutination test (MAT, cut off 1:25); 10.1% (35/348) were seropositive. Of the 348 cats sampled for all four pathogens, 11, 10, and 1 were positive for T. gondii antibodies and FIV antibodies, FeLV antigen, or D. immitis antigen, respectively. Of the 35 T. gondii-seropositive cats, 42.9% (15/35) were co-infected with at least one of the other three pathogens. The presence of antibodies to FIV was significantly associated with both age and gender, while FeLV antigen presence was only associated with age. In the case of FIV, males were twice as likely to be infected as females, and cats over 10 years of age were 13.5 times more likely to be infected than cats less than 1 year of age. FeLV antigen was more common in younger cats, with cats over 10 years of age being 10 times less likely to be FeLV positive than cats under 1 year of age. This is the first survey for these four pathogens affecting feline health in Thailand.

  10. Coinfection of Leishmania chagasi with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in cats from an endemic area of zoonotic visceral leishmaniasis.

    Science.gov (United States)

    Sobrinho, Ludmila Silva Vicente; Rossi, Cláudio Nazaretian; Vides, Juliana Peloi; Braga, Eveline Tozzi; Gomes, Ana Amélia Domingues; de Lima, Valéria Marçal Félix; Perri, Sílvia Helena Venturoli; Generoso, Diego; Langoni, Hélio; Leutenegger, Christian; Biondo, Alexander Welker; Laurenti, Márcia Dalastra; Marcondes, Mary

    2012-06-08

    The aim of the present study was to determine the coinfection of Leishmania sp. with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in a population of cats from an endemic area for zoonotic visceral leishmaniasis. An overall 66/302 (21.85%) cats were found positive for Leishmania sp., with infection determined by direct parasitological examination in 30/302 (9.93%), by serology in 46/302 (15.23%) and by both in 10/302 (3.31%) cats. Real time PCR followed by amplicon sequencing successfully confirmed Leishmania infantum (syn Leishmania chagasi) infection. Out of the Leishmania infected cats, coinfection with FIV was observed in 12/66 (18.18%), with T. gondii in 17/66 (25.75%) and with both agents in 5/66 (7.58%) cats. FeLV was found only in a single adult cat with no Leishmania infection. A positive association was observed in coinfection of Leishmania and FIV (p0.05). In conclusion, cats living in endemic areas of visceral leishmaniasis are significantly more likely to be coinfected with FIV, which may present confounding clinical signs and therefore cats in such areas should be always carefully screened for coinfections. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Expression of regulatory T cell (Treg) activation markers in endometrial tissues from early and late pregnancy in the feline immunodeficiency virus (FIV)-infected cat.

    Science.gov (United States)

    Lockett, N N; Scott, V L; Boudreaux, C E; Clay, B T; Pruett, S B; Ryan, P L; Coats, K S

    2010-09-01

    Regulatory T cells (Tregs) support pregnancy maintenance by suppressing placental inflammation, while diminished Treg function may accompany reproductive failure. Experimental FIV infection frequently results in vertical transmission and increased pregnancy failure in the cat. The mechanism of reproductive compromise is unknown. We hypothesized that FIV infection alters endometrial Treg population dynamics and function, potentiating vertical transmission and reproductive failure. RNA collected from early and late gestation reproductive tissue and fetuses from FIV infected and control cats was probed for expression of FIV gag and Treg markers CD25, FOXP3, and CTLA4, using real time reverse-transcriptase (RT)-PCR. Frequent placental and fetal infection and reproductive failure were detected at early and late pregnancy. Expression of FOXP3 and CTLA4 was higher in early gestation tissues from control cats. FIV infection significantly reduced expression of FOXP3 and CTLA4 at early, but not late pregnancy. At late pregnancy, CTLA4 was expressed to higher levels in infected tissues. The number of tissues with decreased co-expression of FOXP3 and CTLA4 was significant in infected cats at early pregnancy. No significant changes in CD25 expression occurred between FIV-infected and control animals at early or late pregnancy. Differences in Treg marker expression were not significant between viable and non-viable pregnancies in infected cats. The detection of Treg markers in these feline tissues provides the first evidence of feline endometrial Tregs and suggests that such cells diminish as pregnancy progresses. These cells may be depleted or rendered less functional by viral infection, but understanding their role in pregnancy requires further study.

  12. Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

    Institute of Scientific and Technical Information of China (English)

    Xia FENG; Shuang-qing YU; Tsugumine Shu; Tetsuro Matano; Mamoru Hasegawa; Xiao-li WANG; Hong-tao MA; Hong-xia LI; Yi ZENG

    2008-01-01

    Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendal virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

  13. Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tomo, Naoki; Goto, Toshiyuki; Morikawa, Yuko

    2013-03-26

    Yeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system. The nearly full-length HIV-1 genome containing the entire 5' long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells

  14. Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication

    Directory of Open Access Journals (Sweden)

    Reh Juliane

    2011-08-01

    Full Text Available Abstract Background Foamy viruses (FVs unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol protein interactions, ensures Pol particle encapsidation. Results Several Prototype FV (PFV Gag-Pol fusion protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85PR-RT and p40IN Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71Gag

  15. Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging.

    Science.gov (United States)

    Maldonado, José O; Angert, Isaac; Cao, Sheng; Berk, Serkan; Zhang, Wei; Mueller, Joachim D; Mansky, Louis M

    2017-07-19

    Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500-2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag-Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag-Gag interactions that occur during

  16. FIV establishes a latent infection in feline peripheral blood CD4+ T lymphocytes in vivo during the asymptomatic phase of infection

    Directory of Open Access Journals (Sweden)

    Murphy Brian

    2012-02-01

    Full Text Available Abstract Background Feline immunodeficiency virus (FIV is a lentivirus of cats that establishes a lifelong persistent infection with immunologic impairment. Results In an approximately 2 year-long experimental infection study, cats infected with a biological isolate of FIV clade C demonstrated undetectable plasma viral loads from 10 months post-infection onward. Viral DNA was detected in CD4+CD25+ and CD4+CD25- T cells isolated from infected cats whereas viral RNA was not detected at multiple time points during the early chronic phase of infection. Viral transcription could be reactivated in latently infected CD4+ T cells ex vivo as demonstrated by detectable FIV gag RNA and 2-long terminal repeat (LTR circle junctions. Viral LTR and gag sequences amplified from peripheral blood mononuclear cells during early and chronic stages of infection demonstrated minimal to no viral sequence variation. Conclusions Collectively, these findings are consistent with FIV latency in peripheral blood CD4+ T cells isolated from chronically infected cats. The ability to isolate latently FIV-infected CD4+ T lymphocytes from FIV-infected cats provides a platform for the study of in vivo mechanisms of lentiviral latency.

  17. Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages.

    Science.gov (United States)

    Inlora, Jingga; Chukkapalli, Vineela; Bedi, Sukhmani; Ono, Akira

    2016-10-01

    The subcellular sites of HIV-1 assembly, determined by the localization of the structural protein Gag, vary in a cell-type-dependent manner. In T cells and transformed cell lines used as model systems, HIV-1 assembles at the plasma membrane (PM). The binding and localization of HIV-1 Gag to the PM are mediated by the interaction between the matrix (MA) domain, specifically the highly basic region, and a PM-specific acidic phospholipid, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. In primary macrophages, prominent accumulation of assembling or assembled particles is found in the virus-containing compartments (VCCs), which largely consist of convoluted invaginations of the PM. To elucidate the molecular mechanism of HIV-1 Gag targeting to the VCCs, we examined the impact of overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P2, in primary macrophages. We found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression, suggesting an important role for the MA-PI(4,5)P2 interaction in HIV-1 assembly in primary macrophages. However, our analysis of HIV-1 Gag derivatives with MA changes showed that this interaction contributes to Gag membrane binding but is dispensable for specific targeting of Gag to the VCCs per se We further determined that deletion of the NC domain abolishes VCC-specific localization of HIV-1 Gag. Notably, HIV-1 Gag localized efficiently to the VCCs when the NC domain was replaced with a leucine zipper dimerization motif that promotes Gag multimerization. Altogether, our data revealed that targeting of HIV-1 Gag to the VCCs requires NC-dependent multimerization. In T cells and model cell lines, HIV-1 Gag localizes to the PM in a manner dependent on the MA-PI(4,5)P2 interaction. On the other hand, in primary macrophages, HIV-1 Gag localizes to convoluted intracellular membrane structures termed virus-containing compartments (VCCs). Although these

  18. Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag.

    Science.gov (United States)

    Grewe, Bastian; Hoffmann, Bianca; Ohs, Inga; Blissenbach, Maik; Brandt, Sabine; Tippler, Bettina; Grunwald, Thomas; Uberla, Klaus

    2012-03-01

    In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

  19. Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV following in vivo escape from neutralising antibody

    Directory of Open Access Journals (Sweden)

    Samman Ayman

    2010-04-01

    Full Text Available Abstract Background In the acute phase of infection with feline immunodeficiency virus (FIV, the virus targets activated CD4+ T cells by utilising CD134 (OX40 as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. Results Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. Conclusions The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

  20. Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production

    Directory of Open Access Journals (Sweden)

    De Camilli Pietro

    2003-12-01

    Full Text Available Abstract Background The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. Results A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. Conclusions This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production.

  1. Covalent conjugation of the equine infectious anemia virus Gag with SUMO.

    Science.gov (United States)

    Wang, Jinzhong; Wen, Shuping; Zhao, Rui; Qi, Jing; Liu, Zhao; Li, Weiwei; An, Jing; Wood, Charles; Wang, Ying

    2017-05-06

    The conjugation of small ubiquitin-like modifier (SUMO) to the target protein, namely, SUMOylation, is involved in the regulation of many important biological events including host-pathogen interaction. Some viruses have evolved to exploit the host SUMOylation machinery to modify their own protein. Retroviral Gag protein plays critical roles in the viral life cycle. The HIV-1 p6 and the Moloney murine leukemia virus CA have been reported to be conjugated with SUMO. In this study, we report for the first time, to our knowledge, the covalent conjugation of equine infectious anemia virus (EIAV) Gag with SUMO. The C-terminal p9 domain of Gag is a main target for SUMOylation and SUMO is attached to multiple sites of p9, including K30 whose mutation abolished p9 SUMOylation completely. The SUMOylation of p9, but not the p9-K30 mutant, was also detected in equine fibroblastic cells ATCC(®) CCL-57™. Ubc9 and its C93 residue are indispensable for the SUMOylation of p9. Using confocal microscopy, it is found that EIAV Gag localizes primarily, if not exclusively, in the cytoplasm of the cell and the co-localization of EIAV Gag with Ubc9 was observed. Our findings that EIAV Gag is SUMOylated at p9-K30, together with previous findings on the defects of p9-K30 mutant in viral DNA translocation from cytoplasm to the nucleus, suggests that SUMOylation of Gag may be involved in such functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates.

    Science.gov (United States)

    Boldbaatar, Bazartseren; Bazartseren, Tsevel; Koba, Ryota; Murakami, Hironobu; Oguma, Keisuke; Murakami, Kenji; Sentsui, Hiroshi

    2013-04-01

    In the current study, primers described previously and modified versions of these primers were evaluated for amplification of full-length gag genes from different equine infectious anemia virus (EIAV) strains from several countries, including the USA, Germany and Japan. Each strain was inoculated into a primary horse leukocyte culture, and the full-length gag gene was amplified by reverse transcription polymerase chain reaction. Each amplified gag gene was cloned into a plasmid vector for sequencing, and the detectable copy numbers of target DNA were determined. Use of a mixture of two forward primers and one reverse primer in the polymerase chain reaction enabled the amplification of all EIAV strains used in this study. However, further study is required to confirm these primers as universal for all EIAV strains. The nucleotide sequence of gag is considered highly conserved, as evidenced by the use of gag-encoded capsid proteins as a common antigen for the detection of EIAV in serological tests. However, significant sequence variation in the gag genes of different EIAV strains was found in the current study.

  3. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O. [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States); Tumpey, Terrence M. [Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Atlanta, GA (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States)

    2016-01-15

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

  4. Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections in stray and pet cats (Felis catus) in northwest China: co-infections and risk factors.

    Science.gov (United States)

    Cong, Wei; Meng, Qing-Feng; Blaga, Radu; Villena, Isabelle; Zhu, Xing-Quan; Qian, Ai-Dong

    2016-01-01

    This study was conducted to estimate the prevalence of Toxoplasma gondii, Dirofilaria immitis, feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) infections among stray and pet cats in Lanzhou, northwest China, and to identify the influence of age, gender, and regions on seropositivity. T. gondii antibodies were examined in cat sera by the modified agglutination test (MAT). The circulating antigens of D. immitis and FeLV and specific antibodies to FIV were examined using kits commercially available. The overall prevalence of T. gondii, FIV, FeLV, and D. immitis was 19.34, 9.12, 11.33, and 3.04 %, respectively. For the genetic characterization of T. gondii genotypes in cats, genomic DNA was extracted from the seropositive cats and the T. gondii B1 gene was amplified using a semi-nested PCR. DNA samples giving positive B1 amplification were then genotyped using multilocus PCR-RFLP. Two T. gondii genotypes (ToxoDB#9 and ToxoDB#1) were identified. Results of the multivariate logistic regression analysis showed that older cats are more likely to be seropositive than juveniles for T. gondii, FIV, FeLV, and D. immitis. This is the first report of T. gondii genotypes in cats in northwest China. Moreover, the present study is the first study of retrovirus and D. immitis seroprevalence in cats in China. The results revealed that T. gondii, FIV, and FeLV infections are common in stray and pet cats in northwest China.

  5. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

    Science.gov (United States)

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-04-25

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

  6. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

    Directory of Open Access Journals (Sweden)

    Melanie Friedrich

    2016-04-01

    Full Text Available The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l- domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

  7. Maternal hematological and virological characteristics during early feline immunodeficiency virus (FIV) infection of cats as predictors of fetal infection and reproductive outcome at early gestation.

    Science.gov (United States)

    Boudreaux, Crystal E; Lockett, Nikki N; Chemerys, Daniellé N; Clay, Brittany T; Scott, Veronica L; Willeford, Bridget; Brown, Timothy; Coats, Karen S

    2009-10-15

    The FIV-infected cat is a small animal model for HIV mother-to-child transmission (MTCT) because the two lentiviruses are biologically related and produce similar clinical syndromes. Both viruses are vertically transmissible and may negatively impact reproductive outcome. Maternal hematological and virological parameters are predictors of MTCT in HIV-infected women. Our purpose was to determine whether similar maternal characteristics during early pregnancy in FIV-infected cats influence pregnancy outcome. We inoculated 10 cats with FIV-B-2542; 10 cats were uninoculated. We quantified longitudinal CD4:CD8 T cell ratios, proviral load, and plasma viremia, monitored longitudinal serostatus, and documented clinical and reproductive outcome during early pregnancy. Inoculated queens were seropositive and provirus positive by week 4 post-infection (p.i.). CD4:CD8 ratios were depressed in the infected group by month 3.5 p.i. Proviral load was variable in the animals throughout the course of infection; plasma viremia was below the level of detection in all animals. Reduced litter sizes and increased fetal demise occurred in infected queens. Viral RNA, but not proviral DNA, was detected in representative placentas (14 of 14; 100%) and fetuses (12 of 14; 86%) collected from infected queens. However, maternal virological and hematological characteristics did not correlate either positively or negatively with reproductive outcome.

  8. Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Vliet, A.L.W. van; Egberink, H.F.; Hesselink, W.; Ronde, A. de

    1993-01-01

    The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence

  9. Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Vliet, A.L.W. van; Egberink, H.F.; Hesselink, W.; Ronde, A. de

    1993-01-01

    The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence cleav

  10. Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Vliet, A.L.W. van; Egberink, H.F.; Hesselink, W.; Ronde, A. de

    1993-01-01

    The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence cleav

  11. Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

    Institute of Scientific and Technical Information of China (English)

    DAI Chunming; ZHANG Xiaoyan; WANG Shuhui; LIU Ying; DUAN Danli; SHEN Rongxian; SHAO Yiming

    2004-01-01

    In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA+rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.

  12. Design, fabrication, and characterization of polymeric bioMEMS for the detection of feline immunodeficiency virus (FIV)

    Science.gov (United States)

    Cohen, Brian; Gadre, Anand; Kaloyeros, Alain E.

    2007-02-01

    This project comprises the development of a novel polymeric BioMEMS device capable of rapidly detecting FIV in a minimally invasive manner. FIV severely inhibits the infected feline from mounting an immune response, and causes susceptibility to other types of diseases. Vaccines against FIV do exist, but have some strong limitations to their effectiveness; so early detection is the best method to combat the spread of the disease. Current testing methods look for antibodies to the FIV protein p24 in feline blood using established Enzyme Linked ImmunoSorbent Assay (ELISA) protocols. The focus of this research is to design and construct a device that can detect antibodies to p24 in a salivary sample by non-intrusive electrochemical means. The device is constructed upon a silicon substrate with gold microelectrodes coated with polypyrrole (PPy), an electrically conducting and biocompatible polymer. In the current phase of the research, the PPy deposition process has been optimized with regards to film thickness, uniformity and conductivity. Microfluidic channels have been fabricated using SU-8, an epoxy based polymer that enables the test sample and other solutions to pass freely through the device. The PPy will be coated with anti-FIV p24 antibodies that can capture FIV p24 antigens present in a salivary sample. Future research will involve the analysis of PPy/antibody interaction and its effect on functionality. The capture of such antigens will interfere with a reduction-oxidation (redox) reaction in a subsequently added ionic solution. This interference will change the characteristic resistance of the solution yielding a qualitative test for the presence of the viral antigens in the sample and hence determining the occurrence of infection.

  13. Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ying-Chuan; Perryman, Alexander L.; Olson, Arthur J.; Torbett, Bruce E.; Elder, John H.; Stout, C. David, E-mail: dave@scripps.edu [The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)

    2011-06-01

    Crystal structures of the 6s-98S FIV protease chimera with darunavir and lopinavir bound have been determined at 1.7 and 1.8 Å resolution, respectively. A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC{sub 50} values in the nanomolar range.

  14. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    OpenAIRE

    Maxwell, S.; Arlinghaus, R B

    1981-01-01

    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution co...

  15. Construction of Recombinant Fowlpox Virus (rFpv) Expressing HIV-1 Gag-pol Protein

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The induction of human immunodeficiency virus(HIV)-specific T-cell response is generally considered as critical to the development of effective immunity to HIV type 1 ( HIV-1 ). Recombinant Avipoxvirus vectors are used widely for vaccination against HIV-1, where the induction of a cytotoxic CD8 + T-cell(CTL) response seems to be an important component of protective immunity. A recombinant fowlpox virus(rFPV/Gag-pol) expressing the Gag-pol protein of HIV was constructed and characterized. The specific expression protein in CEF cells infected by recombinant fowlpox and the specific antibody in the sera of mice immunized with rFPV were analyzed via Westeru-blot.

  16. FIV diversity: FIV Ple subtype composition may influence disease outcome in African lions.

    Science.gov (United States)

    Troyer, Jennifer L; Roelke, Melody E; Jespersen, Jillian M; Baggett, Natalie; Buckley-Beason, Valerie; MacNulty, Dan; Craft, Meggan; Packer, Craig; Pecon-Slattery, Jill; O'Brien, Stephen J

    2011-10-15

    Feline immunodeficiency virus (FIV) infects domestic cats and at least 20 additional species of non-domestic felids throughout the world. Strains specific to domestic cat (FIV(Fca)) produce AIDS-like disease progression, sequelae and pathology providing an informative model for HIV infection in humans. Less is known about the immunological and pathological influence of FIV in other felid species although multiple distinct strains of FIV circulate in natural populations. As in HIV-1 and HIV-2, multiple diverse cross-species infections may have occurred. In the Serengeti National Park, Tanzania, three divergent subtypes of lion FIV (FIV(Ple)) are endemic, whereby 100% of adult lions are infected with one or more of these strains. Herein, the relative distribution of these subtypes in the population are surveyed and, combined with observed differences in lion mortality due to secondary infections based on FIV(Ple) subtypes, the data suggest that FIV(Ple) subtypes may have different patterns of pathogenicity and transmissibility among wild lion populations. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    Science.gov (United States)

    Maxwell, S; Arlinghaus, R B

    1981-09-01

    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).

  18. Gag genetic heterogeneity of equine infectious anemia virus (EIAV) in naturally infected horses in Canada.

    Science.gov (United States)

    Nagarajan, Malliga M; Simard, Carole

    2007-11-01

    Gag genetic heterogeneity of equine infectious anemia virus (EIAV) variants in naturally infected horses in Canada was studied since very limited information is available on the variability of EIAV Gag sequences in public database. A phylogenetic analysis based on 414nts of Gag gene sequences amplified by a nested polymerase chain reaction (PCR) revealed the distinct divergence of these variants compared to other published strains in a corresponding region. Significant predicted amino acid sequence variations were also identified in an immunorelevant region within this fragment which corresponded to a previously characterized cytotoxic T lymphocytes (CTL) epitope cluster (EC2, aa 77-119). Furthermore, alignment of the predicted full-length Gag protein gene sequences of some of these variants associated with clinical cases of EIA in Canada with the published sequences of EIAV originating from other countries revealed conserved and variant sequences in regions corresponding to other characterized CTL epitope clusters, EC1, EC3 and EC4. Conserved sequences identified among different variant strains might have an important implication for their screening and selection of putative peptide epitopes to mediate relevant immune response and cross protection against divergent field strains of EIAV.

  19. N-Terminally Myristoylated Feline Foamy Virus Gag Allows Env-Independent Budding of Sub-Viral Particles

    Directory of Open Access Journals (Sweden)

    Yong-Boum Kim

    2011-11-01

    Full Text Available Foamy viruses (FVs are distinct retroviruses classified as Spumaretrovirinae in contrast to the other retroviruses, the Orthoretrovirinae. As a unique feature of FVs, Gag is not sufficient for sub-viral particle (SVP release. In primate and feline FVs (PFV and FFV, particle budding completely depends on the cognate FV Env glycoproteins. It was recently shown that an artificially added N-terminal Gag myristoylation signal (myr-signal overcomes this restriction in PFV inducing an Orthoretrovirus-like budding phenotype. Here we show that engineered, heterologous N-terminal myr-signals also induce budding of the distantly related FFV Gag. The budding efficiency depends on the myr-signal and its location relative to the N-terminus of Gag. When the first nine amino acid residues of FFV Gag were replaced by known myr-signals, the budding efficiency as determined by the detection of extracellular SVPs was low. In contrast, adding myr-signals to the intact N‑terminus of FFV Gag resulted in a more efficient SVP release. Importantly, budding of myr-Gag proteins was sensitive towards inhibition of cellular N-myristoyltransferases. As expected, the addition or insertion of myr-signals that allowed Env-independent budding of FFV SVPs also retargeted Gag to plasma membrane-proximal sites and other intracellular membrane compartments. The data confirm that membrane-targeted FV Gag has the capacity of SVP formation.

  20. Gag-Pol Transframe Domain p6* Is Essential for HIV-1 Protease-Mediated Virus Maturation.

    Directory of Open Access Journals (Sweden)

    Fu-Hsien Yu

    Full Text Available HIV-1 protease (PR is encoded by pol, which is initially translated as a Pr160gag-pol polyprotein by a ribosomal frameshift event. Within Gag-Pol, truncated p6gag is replaced by a transframe domain (referred to as p6* or p6pol located directly upstream of PR. p6* has been proposed as playing a role in modulating PR activation. Overlapping reading frames between p6* and p6gag present a challenge to researchers using genetic approaches to studying p6* biological functions. To determine the role of p6* in PR activation without affecting the gag reading frame, we constructed a series of Gag/Gag-Pol expression vectors by duplicating PR with or without p6* between PR pairs, and observed that PR duplication eliminated virus production due to significant Gag cleavage enhancement. This effect was mitigated when p6* was placed between the two PRs. Further, Gag cleavage enhancement was markedly reduced when either one of the two PRs was mutationally inactivated. Additional reduction in Gag cleavage efficiency was noted following the removal of p6* from between the two PRs. The insertion of a NC domain (wild-type or mutant directly upstream of PR or p6*PR did not significantly improve Gag processing efficiency. With the exception of those containing p6* directly upstream of an active PR, all constructs were either noninfectious or weakly infectious. Our results suggest that (a p6* is essential for triggering PR activation, (b p6* has a role in preventing premature virus processing, and

  1. Feline immunodeficiency virus (FIV, feline leukaemia virus (FeLV and Leishmania sp. in domestic cats in the Midwest of Brazil

    Directory of Open Access Journals (Sweden)

    Daniella Poffo

    Full Text Available ABSTRACT: This search aimed to investigate FIV and FeLV infections in domestic cats, analysing the epidemiological profile of the disease as well as additional infection with Leishmania sp. We evaluated 88 domestic cats for the presence of FIV, FeLV and Leishmania sp. infection. Eleven (12.5% cats were positive for FIV infection, four (4.5% were positive for FeLV, and two were co-infected. However, none was infected with Leishmania sp. The prevalence for FIV infection was higher than FeLV, and those observed in other regions, but no factor was associated with the infection by FIV and FeLV in this study.

  2. Structure and Immunogenicity of Alternative Forms of the Simian Immunodeficiency Virus Gag Protein Expressed Using Venezuelan Equine Encephalitis Virus Replicon Particles

    OpenAIRE

    Cecil, Chad; West, Ande; Collier, Martha; Jurgens, Christy; Madden, Victoria; Whitmore, Alan; Johnston, Robert; Moore, Dominic T.; Swanstrom, Ronald; Davis, Nancy L.

    2007-01-01

    Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-), or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infect...

  3. Role of the HIV-1 Matrix Protein in Gag Intracellular Trafficking and Targeting to the Plasma Membrane for Virus Assembly

    Directory of Open Access Journals (Sweden)

    Ruba H Ghanam

    2012-02-01

    Full Text Available Human immunodeficiency virus type-1 (HIV-1 encodes a polypeptide called Gag that is able to form virus-like particles (VLPs in vitro in the absence of any cellular or viral constituents. During the late phase of the HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM for assembly. In the past two decades, in vivo, in vitro and structural studies have shown that Gag trafficking and targeting to the PM are orchestrated events that are dependent on multiple factors including cellular proteins and specific membrane lipids. The matrix (MA domain of Gag has been the focus of these studies as it appears to be engaged in multiple intracellular interactions that are suggested to be critical for virus assembly and replication. The interaction between Gag and the PM is perhaps the most understood. It is now established that the ultimate localization of Gag on punctate sites on the PM is mediated by specific interactions between the MA domain of Gag and phosphatidylinositol-4,5-bisphosphate (PI(4,5P2, a minor lipid localized on the inner leaflet of the PM. Structure-based studies revealed that binding of PI(4,5P2 to MA induces minor conformational changes, leading to exposure of the myristyl (myr group. Exposure of the myr group is also triggered by binding of calmodulin, enhanced by factors that promote protein self-association like the capsid domain of Gag, and is modulated by pH. Despite the steady progress in defining both the viral and cellular determinants of retroviral assembly and release, Gag’s intracellular interactions and trafficking to its assembly sites in the infected cell are poorly understood. In this review, we summarize the current understanding of the structural and functional role of MA in HIV replication.

  4. NMR structure of the myristylated feline immunodeficiency virus matrix protein.

    Science.gov (United States)

    Brown, Lola A; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G; Kuo, Lillian; Freed, Eric O; Summers, Michael F

    2015-04-30

    Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag's N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  5. Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection.

    NARCIS (Netherlands)

    J.A. Karlas (Jos); C.H.J. Siebelink (Kees); M.A. van Peer (Maartje); W. Huisman (Willem); A.M. Cuisinier; G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Albert)

    1999-01-01

    textabstractCats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore, virus-neutralizin

  6. Japanese encephalitis virus-based replicon RNAs/particles as an expression system for HIV-1 Pr55 Gag that is capable of producing virus-like particles.

    Science.gov (United States)

    Yun, Sang-Im; Song, Byung-Hak; Koo, Yongbum; Jeon, Iksoo; Byun, Sung June; Park, Jong-Hyeon; Joo, Yi-Seok; Kim, Seok-Yong; Lee, Young-Min

    2009-09-01

    Ectopic expression of the structural protein Pr55(Gag) of HIV-1 has been limited by the presence of inhibitory sequences in the gag coding region that must normally be counteracted by HIV-1 Rev and RRE. Here, we describe a cytoplasmic RNA replicon based on the RNA genome of Japanese encephalitis virus (JEV) that is capable of expressing HIV-1 gag without requiring Rev/RRE. This replicon system was constructed by deleting all three JEV structural protein-coding regions (C, prM, and E) from the 5'-proximal region of the genome and simultaneously inserting an HIV-1 gag expression cassette driven by the internal ribosome entry site of encephalomyocarditis virus into the 3'-proximal noncoding region of the genome. Transfection of this JEV replicon RNA led to expression of Pr55(Gag) in the absence of Rev/RRE in the cytoplasm of hamster BHK-21, human HeLa, and mouse NIH/3T3 cells. Production of the Pr55(Gag) derived from this JEV replicon RNA appeared to be increased by approximately 3-fold when compared to that based on an alphavirus replicon RNA. Biochemical and morphological analyses demonstrated that the Pr55(Gag) proteins were released into the culture medium in the form of virus-like particles. We also observed that the JEV replicon RNAs expressing the Pr55(Gag) could be encapsidated into single-round infectious JEV replicon particles when transfected into a stable packaging cell line that provided the three JEV structural proteins in trans. This ectopic expression of the HIV-1 Pr55(Gag) by JEV-based replicon RNAs/particles in diverse cell types may represent a useful molecular platform for various biological applications in medicine and industry.

  7. Evaluation of Different Antiretroviral Drug Protocols on Naturally Infected Feline Immunodeficiency Virus (FIV Cats in the late Phase of the Asymptomatic Stage of Infection

    Directory of Open Access Journals (Sweden)

    Paola B. Pisano

    2012-05-01

    Full Text Available The aim of this study was to evaluate the efficacy of the antiretrovirals: Zidovudine (ZDV alone; ZDV + Recombinant Human Interferon-α (rHuIFN-α; ZDV + Lamivudine (3TC and ZDV + valproic acid (Valp on naturally feline immunodeficiency virus (FIV-infected cats, in the late phase of the asymptomatic stage of infection. The follow-up was performed over one year, through clinical evaluation and the determination of viral loads and CD4+/CD8+ ratios. Neurological signs were studied by visual and auditory evoked potentials (VEP, AEP and the responses were abnormal in 80% of the FIV-infected cats. After one year, an improvement in VEP and AEP was observed in the ZDV + Valp group and a worsening in the group receiving ZDV + rHuIFN-α. The CD4+/CD8+ ratio showed a significant increase (both intra and inter-groups only in ZDV and ZDV + 3TC, between their pre-treatment and one year values, as well as among the other groups. Viral load only showed a significant decrease in ZDV and ZDV + 3TC groups, when comparing the values at one year of treatment vs. pre-treatment values and when the different groups were compared. In addition, the viral load decrease was significantly more pronounced in the ZDV + 3TC vs. ZDV group. We conclude that ZDV and ZDV + 3TC produce significant reductions in viral load and stimulate a recovery of the CD4+/CD8+ ratio, compared with the other protocols. It is clear that the addition of 3TC resulted in a greater reduction in viral load than use of ZDV as a single drug. Therefore, the combination ZDV + 3TC could be more effective than the sole use of ZDV.

  8. Structural characterization of membrane-bound human immunodeficiency virus-1 Gag matrix with neutron reflectometry

    Science.gov (United States)

    Eells, Rebecca; Barros, Marilia; Scott, Kerry M.; Karageorgos, Ioannis; Heinrich, Frank; Lösche, Mathias

    2017-01-01

    The structural characterization of peripheral membrane proteins represents a tremendous challenge in structural biology due to their transient interaction with the membrane and the potential multitude of protein conformations during this interaction. Neutron reflectometry is uniquely suited to address this problem because of its ability to structurally characterize biological model systems nondestructively and under biomimetic conditions that retain full protein functionality. Being sensitive to only the membrane-bound fraction of a water-soluble peripheral protein, neutron reflectometry obtains a low-resolution average structure of the protein-membrane complex that is further refined using integrative modeling strategies. Here, the authors review the current technological state of biological neutron reflectometry exemplified by a detailed report on the structure determination of the myristoylated human immunodeficiency virus-1 (HIV-1) Gag matrix associated with phosphoserine-containing model membranes. The authors found that the HIV-1 Gag matrix is able to adopt different configurations at the membrane in a pH-dependent manner and that the myristate group orients the protein in a way that is conducive to PIP2-binding. PMID:28511544

  9. Control of simian immunodeficiency virus replication by vaccine-induced Gag- and Vif-specific CD8+ T cells.

    Science.gov (United States)

    Iwamoto, Nami; Takahashi, Naofumi; Seki, Sayuri; Nomura, Takushi; Yamamoto, Hiroyuki; Inoue, Makoto; Shu, Tsugumine; Naruse, Taeko K; Kimura, Akinori; Matano, Tetsuro

    2014-01-01

    For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8(+) T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8(+) T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8(+) T-cell responses. In the first group vaccinated with Gag-expressing vectors (n = 5 animals), three animals that showed efficient Gag-specific CD8(+) T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors (n = 6 animals), three animals that elicited Vif-specific CD8(+) T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8(+) T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8(+) T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8(+) T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8(+) T-cell responses by prophylactic AIDS vaccines.

  10. Mechanistic Studies and Modeling Reveal the Origin of Differential Inhibition of Gag Polymorphic Viruses by HIV-1 Maturation Inhibitors.

    Science.gov (United States)

    Lin, Zeyu; Cantone, Joseph; Lu, Hao; Nowicka-Sans, Beata; Protack, Tricia; Yuan, Tian; Yang, Hong; Liu, Zheng; Drexler, Dieter; Regueiro-Ren, Alicia; Meanwell, Nicholas A; Cockett, Mark; Krystal, Mark; Lataillade, Max; Dicker, Ira B

    2016-11-01

    HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC50 values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral

  11. Simian sarcoma virus-encoded gag-related protein: in vitro cleavage by Friend leukemia virus-associated proteolytic activity.

    Science.gov (United States)

    Hafenrichter, R; Thiel, H J

    1985-05-01

    The simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) which is not processed in SSV nonproducer cells (SSV-NP cells) (H.-J. Thiel, T. J. Matthews, E. M. Broughton, K. J. Weinhold, D. P. Bolognesi, T. Graf, and H. Beug (1981a), Virology 114, 124-131). In order to cleave SSV p65, retroviral particles containing this antigen were incubated with extracts from the heterologous helper virus Friend leukemia virus (FLV). Superinfection of SSV-NP cells by FLV has been previously shown to result in processing of SSV p65 in vivo (H.-J. Thiel, F. Weiland, R. Hafenrichter, T. J. Matthews, and K. J. Weinhold (1982), Virology 123, 229-234). In vitro cleavage was most efficient in the presence of a nonionic detergent (greater than 0.1% Nonidet-P40) and a reducing agent (greater than 5 mM dithiothreitol) at a pH of 7.0. The products, termed SSV p55 (p15, p12, p30), SSV p30, SSV p25 (p15, p12), and SSV p10, were characterized by (1) molecular weight, (2) kinetics experiments, (3) incorporation of different radiolabeled amino acids, and (4) comparison with SSAV structural proteins. Kinetics experiments with two amino acids ([3H]leucine, [35S]cysteine) revealed that initial processing of SSV p65 produced SSV p55 and SSV p10, with subsequent processing of SSV p55 occurring thereafter. In contrast to the Moloney system, the major intermediate p40 (p30, p10) could not be clearly demonstrated. A direct comparison of SSAV p10 and the cleavage product SSV p10 by SDS-PAGE suggests that SSAV pr65gag and SSV p65 differ slightly by molecular weight.

  12. NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

    Directory of Open Access Journals (Sweden)

    Lola A. Brown

    2015-04-01

    Full Text Available Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2 is mediated by Gag’s N-terminally myristylated matrix (MA domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV, a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S. These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  13. Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection

    NARCIS (Netherlands)

    J.A. Karlas (Jos); C.H.J. Siebelink (Kees); M.A. Peer; W. Huisman (Willem); A.M. Cuisinier; G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Ab)

    1999-01-01

    textabstractCats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore,

  14. Contribution of Gag and protease to variation in susceptibility to protease inhibitors between different strains of subtype B human immunodeficiency virus type 1.

    Science.gov (United States)

    Sutherland, Katherine A; Mbisa, Jean L; Cane, Patricia A; Pillay, Deenan; Parry, Chris M

    2014-01-01

    Recent reports have shown that human immunodeficiency virus type 1 (HIV-1) Gag can directly affect susceptibility to protease inhibitors (PIs) in the absence of known resistance mutations in protease. Inclusion of co-evolved Gag alongside protease in phenotypic drug susceptibility assays can alter PI susceptibility in comparison with protease with a WT Gag. Using a single-replication-cycle assay encompassing full-length Gag together with protease we demonstrated significant variation in PI susceptibility between a number of PI-naïve subtype B viruses. Six publicly available subtype B molecular clones, namely HXB2, NL4-3, SF2, YU2, JRFL and 89.6, displayed up to nine-fold reduced PI susceptibility in comparison with the assay reference strain. For two molecular clones, YU2 and JRFL, Gag contributed solely to the observed reduction in susceptibility, with the N-terminal region of Gag contributing significantly. Gag and protease from treatment-naïve, patient-derived viruses also demonstrated significant variation in susceptibility, with up to a 17-fold reduction to atazanavir in comparison with the assay reference strain. In contrast to the molecular clones, protease was the main determinant of the reduced susceptibility. Common polymorphisms in protease, including I13V, L63P and A71T, were shown to contribute to this reduction in PI susceptibility, in the absence of major resistance mutations. This study demonstrated significant variation in PI susceptibility of treatment-naïve patient viruses, and provided further evidence of the independent role of Gag, the protease substrate and in particular the N-terminus of Gag in PI susceptibility. It also highlighted the importance of considering co-evolved Gag and protease when assessing PI susceptibility.

  15. First Molecular Characterization of Feline Immunodeficiency Virus in Domestic Cats from Mainland China

    Science.gov (United States)

    Zhang, Jilei; Wang, Liang; Li, Jing; Kelly, Patrick; Price, Stuart; Wang, Chengming

    2017-01-01

    The feline immunodeficiency virus (FIV) is a retrovirus of the Lentivirus genus that was initially isolated from a colony of domestic cats in California in 1986 and has now been recognized as a common feline pathogen worldwide. To date, there is only one recent serology-based report on FIV in mainland China which was published in 2016. We designed this study to investigate the molecular prevalence and diversity of feline immunodeficiency virus (FIV) in domestic cats from mainland China. We studied the prevalence of FIV in whole blood samples of 615 domestic cats in five cities (Beijing, Guangzhou, Nanjing, Shanghai and Yangzhou) of mainland China and examined them using FRET-PCR (Fluorescence Resonance Energy Transfer-Polymerase Chain Reaction) and regular PCRs for the gag and env genes. Overall, 1.3% (8/615) of the cats were positive for provirus DNA with nucleotide analysis using PCRs for the gag and env sequences showing the cats were infected with FIV subtype A. This is the first molecular characterization of FIV in mainland China and the first description of subtype A in continental Asia. PMID:28107367

  16. Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production.

    Science.gov (United States)

    Zheng, Wenwen; Ling, Limian; Li, Zhaolong; Wang, Hong; Rui, Yajuan; Gao, Wenying; Wang, Shaohua; Su, Xing; Wei, Wei; Yu, Xiao-Fang

    2017-04-01

    The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55(Gag)) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55(Gag) compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55(Gag) interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55(Gag), which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4(+) T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55(Gag) interaction provides a potential target for the future development of antiviral strategies.IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55(Gag)) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55(Gag) although HIV-1 Vif proteins

  17. Human Immunodeficiency Virus Type 2 (HIV-2 Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner.

    Directory of Open Access Journals (Sweden)

    Justine E Alford

    Full Text Available Although human immunodeficiency virus (HIV types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release.

  18. Natural transmission of feline immunodeficiency virus from infected queen to kitten

    Directory of Open Access Journals (Sweden)

    Medeiros Sheila de

    2012-05-01

    Full Text Available Abstract Background Feline immunodeficiency virus (FIV is a naturally occurring lentivirus that infects cats. The primary mode of transmission occurs through bite wounds, and other routes are difficult to observe in nature. Findings The purpose of this study was to evaluate FIV transmission from queen to kitten in a colony of naturally infected stray cats. With this aim, a queen was monitored over a period of three years. A blood sample was taken to amplify and sequence gag, pol and env regions of the virus from the queen, two kittens and other cats from the colony. Conclusion Phylogenetic analysis showed evidence of queen to kitten transmission.

  19. Optimization of vesicular stomatitis virus-G pseudotyped feline immunodeficiency virus vector for minimized cytotoxicity with efficient gene transfer.

    Science.gov (United States)

    Song, Jae Jin; Lee, Boyoung; Chang, Jin Woo; Kim, Joo-Hang; Kwon, Yunhee Kim; Lee, Heuiran

    2003-05-01

    FIV-based lentiviral vector has shown a unique opportunity as an efficient gene delivery vehicle, especially to nondividing human cells. Here, we genetically reconstructed the FIV-based vector by serially deleting residual virus genes of gag and vif, leading to minimized cytotoxicity together with efficient virus production and gene transfer. The modified FIV- based vector was generated by transiently transfecting 293T cells with three plasmids of the gene transfer vector with minimal gag region, the packaging plasmid without vif and the VSV-G-expressing plasmid. The vector was routinely generated as many as 1 x 10(7) transducing particles per ml and easily concentrated by simple centrifugation. The cytotoxic effect significantly decreased in sensitive cells to FIV infection even at high multiplicity of infection (MOI), such as 500. Moreover, the transduction efficiency was consistently retained after cell cycle was arrested in a variety of human cells. Taken together, our results suggest that the modified VSV-G pseudotyped FIV-based vector efficiently transduce dividing and nondividing human cells with minimal cytotoxicity.

  20. HLA-associated alterations in replication capacity of chimeric NL4-3 viruses carrying gag-protease from elite controllers of human immunodeficiency virus type 1.

    Science.gov (United States)

    Miura, Toshiyuki; Brockman, Mark A; Brumme, Zabrina L; Brumme, Chanson J; Pereyra, Florencia; Trocha, Alicja; Block, Brian L; Schneidewind, Arne; Allen, Todd M; Heckerman, David; Walker, Bruce D

    2009-01-01

    Human immunodeficiency virus type 1 (HIV-1)-infected persons who maintain plasma viral loads of viruses (VRC) obtained from EC (n = 54) compared to those from chronic progressors (CP; n = 41) by constructing chimeric viruses using patient-derived gag-protease sequences amplified from plasma HIV RNA and inserted into an NL4-3 backbone. The chimeric viruses generated from EC displayed lower VRC than did viruses from CP (P viruses from B*57(+) EC (n = 18) demonstrated lower VRC than did viruses from B*57(+) CP (n = 8, P = 0.0245). Differences in VRC between EC and CP were also observed for viruses obtained from individuals expressing no described "protective" alleles (P = 0.0065). Intriguingly, two common HLA alleles, A*02 and B*07, were associated with higher VRC (P = 0.0140 and 0.0097, respectively), and there was no difference in VRC between EC and CP sharing these common HLA alleles. These findings indicate that cytotoxic T-lymphocyte (CTL) selection pressure on gag-protease alters VRC, and HIV-specific CTLs inducing escape mutations with fitness costs in this region may be important for strict viremia control in EC of HIV.

  1. Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

    Science.gov (United States)

    Steppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Kramberger, Petra; Tover, Andres; Berger, Eva; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Jungbauer, Alois

    2017-02-01

    The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 10(12) /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 10(11) particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 10(9) particles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

    Science.gov (United States)

    Wilkes, Rebecca Penrose; Kania, Stephen A; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chang, Hsiu-Hui; Ma, Li-Juan; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2015-07-01

    Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats.

  3. Structure and immunogenicity of alternative forms of the simian immunodeficiency virus gag protein expressed using Venezuelan equine encephalitis virus replicon particles.

    Science.gov (United States)

    Cecil, Chad; West, Ande; Collier, Martha; Jurgens, Christy; Madden, Victoria; Whitmore, Alan; Johnston, Robert; Moore, Dominic T; Swanstrom, Ronald; Davis, Nancy L

    2007-06-05

    Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-) or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gagmyr+) and distinctive cytoplasmic aggregates (VRP-Gagmyr-) with each cell type. VRP were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-gamma ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-gamma-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gagmyr+-VRP. Antibody responses to MA/CA-VRP and Gagmyr+-VRP were not significantly different.

  4. Gag Protein Epitopes Recognized by ELA-A-Restricted Cytotoxic T Lymphocytes from Horses with Long-Term Equine Infectious Anemia Virus Infection

    Science.gov (United States)

    Zhang, Wei; Lonning, Scott M.; McGuire, Travis C.

    1998-01-01

    Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV. PMID:9811694

  5. Using Dynamic Stochastic Modelling to Estimate Population Risk Factors in Infectious Disease: The Example of FIV in 15 Cat Populations

    OpenAIRE

    David Fouchet; Guillaume Leblanc; Frank Sauvage; Micheline Guiserix; Hervé Poulet; Dominique Pontier

    2009-01-01

    BACKGROUND: In natural cat populations, Feline Immunodeficiency Virus (FIV) is transmitted through bites between individuals. Factors such as the density of cats within the population or the sex-ratio can have potentially strong effects on the frequency of fight between individuals and hence appear as important population risk factors for FIV. METHODOLOGY/PRINCIPAL FINDINGS: To study such population risk factors, we present data on FIV prevalence in 15 cat populations in northeastern France. ...

  6. Gag Protein Epitopes Recognized by CD4+ T-Helper Lymphocytes from Equine Infectious Anemia Virus-Infected Carrier Horses

    Science.gov (United States)

    Lonning, S. M.; Zhang, W.; McGuire, T. C.

    1999-01-01

    Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV. PMID:10196322

  7. Localization of human T-cell lymphotropic virus-1 gag proviral sequences in dermato-immunological disorders with eosinophilia.

    Science.gov (United States)

    Nagy, K; Marschalkó, Márta; Kemény, B; Horváth, A

    2005-01-01

    The mechanisms leading to the development of eosinophilia were investigated in 65 patients with immunodermatological disorders, including the role of eosinophilotactic cytokines and the possible involvement of human T-cell leukemia virus, HTLV. HTLV-1 gag proviral sequences were revealed in two cases of lymphoproliferative disorders such as angiolymphoid hyperplasia with eosinophilia (ALHE) and CD4+ cutaneous lymphoma, respectively. Increased level of GM-CSF was detected in 33% of disorders studied. Elevated level of IL-5 and eotaxin was detected in 27% and 30%, respectively, of patients with bullous diseases. Elevated level of GM-CSF and eotaxin was found in 33% and 46%, respectively, of patients with inflammatory diseases. Neither of the four cytokines, however proved to be responsible alone or together for the induction of eosinophilia. The possible indirect role of human retroviruses through induction of eosinophilic chemotactic cytokines is hypothesized.

  8. The human foamy virus pol gene is expressed as a Pro-Pol polyprotein and not as a Gag-Pol fusion protein.

    OpenAIRE

    Löchelt, M; Flügel, R M

    1996-01-01

    It has been reported recently that the human foamy virus (HFV) Pol polyprotein of 120 kDa is synthesized in the absence of the active HFV aspartic protease. To gain more information on how the 120-kDa Pro-Pol protein is synthesized, mutant HFV genomes were constructed and the resulting proviruses were analyzed with respect to HFV pol expression and infectivity. HFV proviruses that contain termination codons in the nucleocapsid domain of gag and thus lack a gag-pol overlap region assumed to be...

  9. Virus de la inmunodeficiencia felina (VIF: evaluación de las globulinas en pacientes infectados espontáneamente Feline immunodeficiency virus (FIV: study of globulins in patients with natural infections

    Directory of Open Access Journals (Sweden)

    N Gómez

    2006-12-01

    Full Text Available Fueron estudiadas las posibles correlaciones de parámetros tales como la Alfa glicoproteína ácida (AGP, proteína de fase aguda, fracciones electroforéticas de las proteínas séricas y títulos de Toxoplasma gondii en gatos infectados por el Virus de Inmunodeficiencia Felina (VIF. Los títulos de Toxoplasma gondii obtenidos por Inmunofluorescencia Indirecta (IFI no correlacionaron con los valores de Proteínas Totales ni con los de las globulinas. Sí se halló múltiple correlación entre todas las proteínas estudiadas (r: 0,98, pStatistical correlation between parameters such as globulins, Alpha- Glycoprotein AGP, serum proteins fractions by electrophoresis and Toxoplasma gondii titles in cats infected with Feline Immunodeficiency Virus (FIV were studied. Indirect Immunofluorecence titles to Toxoplasma gondii did not showed correlation with Total proteins and globulins. It was observed correlation between all types of proteins studied (r: 0,98, p<0,04. Total proteins versus globulins showed positive correlation (r:0,93, p <0,0001. Total protein versus alpha-globulin evidenced negative correlation (r:-0,75, p<0,01. AGP and alpha-globulins did not showed correlation and it was detected negative correlation with gamma-globulins (r:-0, 94, p<0,0001 and with globulins (r:-0,67, p<0,03. The patients evaluated showed a high level of Total proteins because of the increase of globulins. Gamma-Globulins were detected increased but there was not correlation with Toxoplasma gondii titles. It was not observed correlation between AGP and Alfa-globulins.

  10. A Mason-Pfizer Monkey virus Gag-GFP fusion vector allows visualization of capsid transport in live cells and demonstrates a role for microtubules.

    Directory of Open Access Journals (Sweden)

    Jasmine Clark

    Full Text Available Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV, are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.

  11. Oral immunization with recombinant listeria monocytogenes controls virus load after vaginal challenge with feline immunodeficiency virus.

    Science.gov (United States)

    Stevens, Rosemary; Howard, Kristina E; Nordone, Sushila; Burkhard, MaryJo; Dean, Gregg A

    2004-08-01

    Recombinant Listeria monocytogenes has many attractive characteristics as a vaccine vector against human immunodeficiency virus (HIV). Wild-type and attenuated Listeria strains expressing HIV Gag have been shown to induce long-lived mucosal and systemic T-cell responses in mice. Using the feline immunodeficiency virus (FIV) model of HIV we evaluated recombinant L. monocytogenes in a challenge system. Five cats were immunized with recombinant L. monocytogenes that expresses the FIV Gag and delivers an FIV Env-expressing DNA vaccine (LMgag/pND14-Lc-env). Control cats were either sham immunized or immunized with wild-type L. monocytogenes (LM-wt). At 1 year after vaginal challenge, provirus could not be detected in any of the nine tissues evaluated from cats immunized with the recombinant bacteria but was detected in at least one tissue in 8 of 10 control animals. Virus was isolated from bone marrow of four of five LMgag/pND14-Lc-env-immunized cats by use of a stringent coculture system but required CD8(+) T-cell depletion, indicating CD8(+) T-cell suppression of virus replication. Control animals had an inverted CD4:CD8 ratio in mesenteric lymph node and were depleted of both CD4(+) and CD8(+) intestinal epithelial T cells, while LMgag/pND14-Lc-env-immunized animals showed no such abnormalities. Vaginal FIV-specific immunoglobulin A was present at high titer in three LMgag/pND14-Lc-env-immunized cats before challenge and in all five at 1 year postchallenge. This study demonstrates that recombinant L. monocytogenes conferred some control of viral load after vaginal challenge with FIV.

  12. Feline immunodeficiency virus can be experimentally transmitted via milk during acute maternal infection.

    OpenAIRE

    Sellon, R K; Jordan, H L; Kennedy-Stoskopf, S; Tompkins, M B; Tompkins, W A

    1994-01-01

    Postnatal transmission of feline immunodeficiency virus (FIV) in neonates nursed by acutely infected mothers and infection resulting from oral inoculation of kittens with FIV were evaluated. Ten of 16 kittens nursed by four queens with FIV infection established immediately postpartum developed FIV infection. Five of 11 neonates orally administered cell-free FIV culture supernatant developed FIV infection. Kittens that developed FIV infection had greater proportions of CD4+ and Pan-T+ lymphocy...

  13. Advances in FIV vaccine technology

    OpenAIRE

    Uhl, Elizabeth W.; Martin, Marcus; Coleman, James K.; Yamamoto, Janet K.

    2008-01-01

    Advances in vaccine technology are occurring in the molecular techniques used to develop vaccines and in the assessment of vaccine efficacy, allowing more complete characterization of vaccine-induced immunity correlating to protection. FIV vaccine development has closely mirrored and occasionally surpassed the development of HIV-1 vaccine, leading to first licensed technology. This review will discuss technological advances in vaccine designs, challenge infection assessment, and characterizat...

  14. First isolation and nucleotide comparison of the gag gene of the caprine arthritis encephalitis virus circulating in naturally infected goats from Argentina

    Directory of Open Access Journals (Sweden)

    Carlos Javier Panei

    2017-02-01

    Full Text Available Caprine arthritis encephalitis virus (CAEV has been reported in different countries worldwide, based on serological and molecular detection. In Argentina, the prevalence of CAEV infections is increasing, with goats showing symptoms associated mostly with cachexia and arthritis. Although in Argentina the virus has been detected by serology, it has never been isolated or characterized. Thus, the objectives of this work were to isolate and analyze the nucleotide sequences of the gag gene of Argentine CAEV strains and compare them with those of other SRLVs previously reported. Nucleotide sequence comparison showed homology with CAEV-Co, the CAEV prototype. Phylogenetic analyses showed that the Argentine strains clustered with genotype B, subtype B1. Because the molecular characterization of the gag region is suitable for phylogenetic studies and may be applied to monitor the control of SRLV, molecularly characterizing the Argentine CAEV strains may help develop a proper plan of eradication of CAEV infections.

  15. Impairment of alternative splice sites defining a novel gammaretroviral exon within gag modifies the oncogenic properties of Akv murine leukemia virus

    DEFF Research Database (Denmark)

    Sørensen, Annette Balle; Lund, Anders H; Kunder, Sandra

    2007-01-01

    to be associated with specific tumor diagnoses or individual viral mutants. CONCLUSION: We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential......BACKGROUND: Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine...... leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus. RESULTS...

  16. Placental immunopathology in the FIV-infected cat: a role for inflammation in compromised pregnancy?

    Science.gov (United States)

    Coats, Karen S; Boudreaux, Crystal E; Clay, Brittany T; Lockett, Nikki N; Scott, Veronica L

    2010-03-15

    In utero transmission of feline immunodeficiency virus (FIV) occurs frequently in queens experimentally infected with FIV-B-2542 and other FIV isolates. Fetal infection has been detected as early as 3-4 weeks gestation, and the incidence of fetal infection increases with progressing gestation. Reproductive failure occurs commonly, including fetal resorptions and developmentally-arrested fetuses, demonstrating that fetal demise occurs early in gestation. Precise, temporal immunomodulation within the placenta is essential for successful pregnancy. Placental Th1 and Th2 cytokines must be appropriately balanced, typically favoring Th2 cytokines at the maternal-fetal interface. Abnormal inflammatory cytokine expression often accompanies miscarriage. Regulatory T cells (Tregs) play an essential role in maternal tolerance of the semi-allogeneic fetus by suppressing inflammation. We are using the FIV-infected cat to examine the relationship between lentivirus-induced placental immunopathology and reproductive outcome. Using TaqMan real time reverse transcriptase (RT)-PCR, we measured relative expression of key immunomodulators in the placentas of FIV-B-2542-infected and control cats, including placentas from both viable and nonviable pregnancies. Our data associate significantly-increased expression of inflammatory cytokines with failed pregnancies, identify Treg markers in the placentas, and provide preliminary evidence that Tregs or other cells bearing similar activation markers may be involved in pregnancy maintenance. Our data suggest that placental inflammation in the FIV-infected cat may compromise pregnancy.

  17. Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

    Science.gov (United States)

    Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

    2013-06-24

    Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins

  18. Membrane interaction of retroviral Gag proteins

    Directory of Open Access Journals (Sweden)

    Robert Alfred Dick

    2014-04-01

    Full Text Available Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses-- MA, CA, and NC-- provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding.

  19. HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles

    Energy Technology Data Exchange (ETDEWEB)

    Haffar, O.K.; Smithgall, M.D.; Moran, P.A.; Travis, B.M.; Zarling, J.M.; Hu, S.L. (Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute-Seattle, WA (USA))

    1991-08-01

    Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultraviolet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, the authors results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS.

  20. Expression of CD134 and CXCR4 mRNA in term placentas from FIV-infected and control cats.

    Science.gov (United States)

    Scott, Veronica L; Burgess, Shane C; Shack, Leslie A; Lockett, Nikki N; Coats, Karen S

    2008-05-15

    Feline immunodeficiency virus (FIV) causes a natural infection of domestic cats that resembles HIV-1 in pathogenesis and disease progression. Feline AIDS is characterized by depression of the CD4+ T cell population and fatal opportunistic infections. Maternal-fetal transmission of FIV readily occurs under experimental conditions, resulting in infected viable kittens and resorbed or arrested fetal tissues. Although both FIV and HIV use the chemokine receptor CXCR4 as a co-receptor, FIV does not utilize CD4 as the primary receptor. Rather, CD134 (OX40), a T cell activation antigen and co-stimulatory molecule, is the primary receptor for FIV. We hypothesized that placental expression of CD134 and CXCR4 may render the placenta vulnerable to FIV infection, possibly facilitating efficient vertical transmission of FIV, and impact pregnancy outcome. The purpose of this project was to quantify the relative expression of CD134 and CXCR4 mRNA from the term placentas of three groups of cats: uninfected queens producing viable offspring, experimentally-infected queens producing only viable offspring, and experimentally-infected queens producing viable offspring among mostly non-viable fetuses. Total RNA was extracted from term placental tissues from all groups of cats. Real-time one-step reverse transcriptase-PCR was used to measure gene expression. The FIV receptors CD134 and CXCR4 were expressed in all late term feline placental tissues. Placentas from FIV-infected queens producing litters of only viable offspring expressed more CD134 and CXCR4 mRNA than those from uninfected queens, suggesting that infection may cause upregulation of the receptors. On the other hand, placentas from FIV-infected cats with non-successful pregnancies expressed similar levels of CD134 mRNA and slightly less CXCR4 mRNA than those from uninfected queens. Thus, it appears that cells expressing these receptors may play a role in pregnancy maintenance.

  1. Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env, and receptor via distortion of the endoplasmic reticulum.

    Science.gov (United States)

    Tang, Yan-Dong; Na, Lei; Zhu, Chun-Hui; Shen, Nan; Yang, Fei; Fu, Xian-Qiu; Wang, Yu-Hong; Fu, Li-Hua; Wang, Jia-Yi; Lin, Yue-Zhi; Wang, Xue-Feng; Wang, Xiaojun; Zhou, Jian-Hua; Li, Cheng-Yao

    2014-11-01

    Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) upregulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximately 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the α-helix domain and radical S-adenosylmethionine (SAM) domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin α-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the α-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level. In previous studies, viperin was indicated as restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms, including the reduction

  2. Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

    Directory of Open Access Journals (Sweden)

    José L. Affranchino

    2014-01-01

    Full Text Available The lentiviral envelope glycoproteins (Env mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles.

  3. Complexity of the inoculum determines the rate of reversion of SIV Gag CD8 T cell mutant virus and outcome of infection.

    Directory of Open Access Journals (Sweden)

    Liyen Loh

    2009-04-01

    Full Text Available Escape mutant (EM virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively "fit" compared to wild-type (WT virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load in vivo in HIV-1 infection. The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing > or =10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIV(mn229 viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIV(mac239, demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until > or =8 days after inoculation and took > or =8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection.

  4. HIV-1 Gag-virus-like particles inhibit HIV-1 replication in dendritic cells and T cells through IFN-α-dependent upregulation of APOBEC3G and 3F.

    Science.gov (United States)

    Chang, Myint Oo; Suzuki, Tomoyuki; Yamamoto, Norio; Watanabe, Megumi; Takaku, Hiroshi

    2012-01-01

    Human immunodeficiency virus-1 (HIV-1) infection and the acquired immune deficiency syndrome (AIDS) pandemic remain global threats in the absence of a protective or a therapeutic vaccine. HIV-1 replication is reportedly inhibited by some cellular factors, including APOBEC3G (A3G) and APOBEC3F (A3F), which are well known inhibitors of HIV-1. Recently, HIV-1 Gag-virus-like particles (Gag-VLPs) have been shown to be safe and potent HIV-1 vaccine candidates that can elicit strong cellular and humoral immunity without need of any adjuvant. In this report, we stimulated human monocyte-derived dendritic cells (DCs) with Gag-VLPs and we demonstrated that Gag-VLP-treated DCs (VLP-DCs) produced interferon alpha (IFN-α), along with an increase in mRNA and protein expression of A3G and A3F. Gag-VLPs inhibited HIV-1 replication not only in DCs themselves, but also in cocultured T cells in an IFN-α-dependent manner. In addition, A3G/3F content in HIV virions released from VLP-DCs increased. Both the increase in A3G/3F expression and the inhibition of HIV-1 replication were reversed by anti-IFN-α or anti-IFNAR antibodies. Our findings in this study provide insight into the mechanism of Gag-VLP-induced inhibition of HIV-1 replication in DCs and T cells.

  5. Neutralizing antibodies in cats infected with feline immunodeficiency virus.

    NARCIS (Netherlands)

    F. Tozzini; D. Matteucci; P. Bandecchi; F. Baldinotti; C.H.J. Siebelink (Kees); A.D.M.E. Osterhaus (Albert); M. Bendinelli

    1993-01-01

    textabstractSera from cats experimentally infected with five isolates of feline immunodeficiency virus (FIV) from various geographical regions and from FIV enzyme-linked immunosorbent assay-seropositive field cats from four European countries neutralized the Petaluma strain of FIV (FIV-P), originall

  6. Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

    Directory of Open Access Journals (Sweden)

    Ristic Natalia

    2010-09-01

    Full Text Available Abstract Background The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1 sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. Results HIV-1 lacking SL1 (NLΔSL1 did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K on day 18 postinfection and C1907T in the SP1 domain (P10L on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1

  7. First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus-Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens.

    Science.gov (United States)

    Nyombayire, Julien; Anzala, Omu; Gazzard, Brian; Karita, Etienne; Bergin, Philip; Hayes, Peter; Kopycinski, Jakub; Omosa-Manyonyi, Gloria; Jackson, Akil; Bizimana, Jean; Farah, Bashir; Sayeed, Eddy; Parks, Christopher L; Inoue, Makoto; Hironaka, Takashi; Hara, Hiroto; Shu, Tsugumine; Matano, Tetsuro; Dally, Len; Barin, Burc; Park, Harriet; Gilmour, Jill; Lombardo, Angela; Excler, Jean-Louis; Fast, Patricia; Laufer, Dagna S; Cox, Josephine H

    2017-01-01

     We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)-vectored, human immunodeficiency virus type 1 (HIV-1) vaccine.  Sixty-five HIV-1-uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 prime-boost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35-vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH).  All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot-determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeV-Gag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8(+) T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic.  SeV-Gag primed functional, durable HIV-specific T-cell responses and boosted antibody responses. The prime

  8. Recombination in feline immunodeficiency virus from feral and companion domestic cats

    Directory of Open Access Journals (Sweden)

    Rodrigo Allen G

    2008-06-01

    Full Text Available Abstract Background Recombination is a relatively common phenomenon in retroviruses. We investigated recombination in Feline Immunodeficiency Virus from naturally-infected New Zealand domestic cats (Felis catus by sequencing regions of the gag, pol and env genes. Results The occurrence of intragenic recombination was highest in env, with evidence of recombination in 6.4% (n = 156 of all cats. A further recombinant was identified in each of the gag (n = 48 and pol (n = 91 genes. Comparisons of phylogenetic trees across genes identified cases of incongruence, indicating intergenic recombination. Three (7.7%, n = 39 of these incongruencies were found to be significantly different using the Shimodaira-Hasegawa test. Surprisingly, our phylogenies from the gag and pol genes showed that no New Zealand sequences group with reference subtype C sequences within intrasubtype pairwise distances. Indeed, we find one and two distinct unknown subtype groups in gag and pol, respectively. These observations cause us to speculate that these New Zealand FIV strains have undergone several recombination events between subtype A parent strains and undefined unknown subtype strains, similar to the evolutionary history hypothesised for HIV-1 "subtype E". Endpoint dilution sequencing was used to confirm the consensus sequences of the putative recombinants and unknown subtype groups, providing evidence for the authenticity of these sequences. Endpoint dilution sequencing also resulted in the identification of a dual infection event in the env gene. In addition, an intrahost recombination event between variants of the same subtype in the pol gene was established. This is the first known example of naturally-occurring recombination in a cat with infection of the parent strains. Conclusion Evidence of intragenic recombination in the gag, pol and env regions, and complex intergenic recombination, of FIV from naturally-infected domestic cats in New Zealand was found. Strains

  9. Recombination in feline immunodeficiency virus from feral and companion domestic cats.

    Science.gov (United States)

    Hayward, Jessica J; Rodrigo, Allen G

    2008-06-17

    Recombination is a relatively common phenomenon in retroviruses. We investigated recombination in Feline Immunodeficiency Virus from naturally-infected New Zealand domestic cats (Felis catus) by sequencing regions of the gag, pol and env genes. The occurrence of intragenic recombination was highest in env, with evidence of recombination in 6.4% (n = 156) of all cats. A further recombinant was identified in each of the gag (n = 48) and pol (n = 91) genes. Comparisons of phylogenetic trees across genes identified cases of incongruence, indicating intergenic recombination. Three (7.7%, n = 39) of these incongruencies were found to be significantly different using the Shimodaira-Hasegawa test.Surprisingly, our phylogenies from the gag and pol genes showed that no New Zealand sequences group with reference subtype C sequences within intrasubtype pairwise distances. Indeed, we find one and two distinct unknown subtype groups in gag and pol, respectively. These observations cause us to speculate that these New Zealand FIV strains have undergone several recombination events between subtype A parent strains and undefined unknown subtype strains, similar to the evolutionary history hypothesised for HIV-1 "subtype E".Endpoint dilution sequencing was used to confirm the consensus sequences of the putative recombinants and unknown subtype groups, providing evidence for the authenticity of these sequences. Endpoint dilution sequencing also resulted in the identification of a dual infection event in the env gene. In addition, an intrahost recombination event between variants of the same subtype in the pol gene was established. This is the first known example of naturally-occurring recombination in a cat with infection of the parent strains. Evidence of intragenic recombination in the gag, pol and env regions, and complex intergenic recombination, of FIV from naturally-infected domestic cats in New Zealand was found. Strains of unknown subtype were identified in all three gene

  10. Bovine Leukemia ProVirus: Evidence of Presence of Part of Gag Gene in Seminal Plasma of Naturally Infected Bulls

    Directory of Open Access Journals (Sweden)

    Razi Jafari

    2010-06-01

    Full Text Available It is of critical importance to understand the modalities of BLV presence in semen, especially with regard to artificial insemination (AI. Presence of bovine leukemia provirus was demonstrated in fresh and frozen semen samples by researchers. In this study paired blood and semen samples from 45 bulls were assessed for the presence of part of gag gene and antibodies to BLV in blood, semen and cell-free fraction of the semen (seminal plasma. Proviral DNA was detected in 5 out of 45 seminal plasma samples. PCR products were sequenced and submitted to gene bank. This data strongly suggested that seminal plasma of seropositive bulls can be positive in PCR.

  11. Genomic organization, sequence divergence, and recombination of feline immunodeficiency virus from lions in the wild

    Directory of Open Access Journals (Sweden)

    Sondgeroth Kerry

    2008-02-01

    Full Text Available Abstract Background Feline immunodeficiency virus (FIV naturally infects multiple species of cat and is related to human immunodeficiency virus in humans. FIV infection causes AIDS-like disease and mortality in the domestic cat (Felis catus and serves as a natural model for HIV infection in humans. In African lions (Panthera leo and other exotic felid species, disease etiology introduced by FIV infection are less clear, but recent studies indicate that FIV causes moderate to severe CD4 depletion. Results In this study, comparative genomic methods are used to evaluate the full proviral genome of two geographically distinct FIV subtypes isolated from free-ranging lions. Genome organization of FIVPle subtype B (9891 bp from lions in the Serengeti National Park in Tanzania and FIVPle subtype E (9899 bp isolated from lions in the Okavango Delta in Botswana, both resemble FIV genome sequence from puma, Pallas cat and domestic cat across 5' LTR, gag, pol, vif, orfA, env, rev and 3'LTR regions. Comparative analyses of available full-length FIV consisting of subtypes A, B and C from FIVFca, Pallas cat FIVOma and two puma FIVPco subtypes A and B recapitulate the species-specific monophyly of FIV marked by high levels of genetic diversity both within and between species. Across all FIVPle gene regions except env, lion subtypes B and E are monophyletic, and marginally more similar to Pallas cat FIVOma than to other FIV. Sequence analyses indicate the SU and TM regions of env vary substantially between subtypes, with FIVPle subtype E more related to domestic cat FIVFca than to FIVPle subtype B and FIVOma likely reflecting recombination between strains in the wild. Conclusion This study demonstrates the necessity of whole-genome analysis to complement population/gene-based studies, which are of limited utility in uncovering complex events such as recombination that may lead to functional differences in virulence and pathogenicity. These full-length lion

  12. Effects of an oral superoxide dismutase enzyme supplementation on indices of oxidative stress, proviral load, and CD4:CD8 ratios in asymptomatic FIV-infected cats.

    Science.gov (United States)

    Webb, Craig B; Lehman, Tracy L; McCord, Kelly W

    2008-10-01

    This study was designed to test the effect of antioxidant supplementation on feline immunodeficiency virus (FIV)-infected felines. Six acutely FIV-infected cats (> or =16 weeks post-inoculation) were given a propriety oral superoxide dismutase (SOD) supplement (Oxstrin; Nutramax Laboratories) for 30 days. Following supplementation, the erythrocyte SOD enzyme concentration was significantly greater in the supplemented FIV-infected group than the uninfected control group or the unsupplemented FIV-infected group. The CD4+ to CD8+ ratio increased significantly (0.66-0.88) in the SOD supplemented FIV-infected cats but not in the unsupplemented FIV-infected cats. Proviral load and reduced glutathione (GSH) levels in leukocyte cell types did not change significantly following supplementation. Antioxidant supplementation resulted in an increase in SOD levels, confirming the oral bioavailability of the compound in FIV-infected cats. This result warrants further investigation with trials of antioxidant therapy in FIV-infected cats that are showing clinical manifestations of their disease, as well as in other feline patients where oxidative stress likely contributes to disease pathogenesis, such as diabetes mellitus and chronic renal failure.

  13. Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection.

    Science.gov (United States)

    Tasker, Séverine; Caney, Sarah M A; Day, Michael J; Dean, Rachel S; Helps, Chris R; Knowles, Toby G; Lait, Philippa J P; Pinches, Mark D G; Gruffydd-Jones, Timothy J

    2006-10-31

    The purpose of this study was to investigate the effect of chronic feline immunodeficiency virus (FIV) infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection. Six cats chronically infected with FIV-Glasgow8 (Group X) and six FIV-free cats (Group Y) were infected with M. haemofelis on Day 0 by intravenous blood inoculation. From Day 0 until Day 86 post-infection (pi), blood samples were collected for M. haemofelis and FIV provirus quantitative real-time PCR and haematology. Three of the six cats in each of Groups X and Y were randomly selected to receive marbofloxacin treatment (2 mg/kg PO q24 h) from Day 16 to 43 pi, with the remaining cats being untreated controls with no antibiotic treatment. The M. haemofelis copy numbers and haematological data were compared between Groups X and Y, and between marbofloxacin-treated and control cats using a Mann-Whitney U-test. M. haemofelis infection was associated with development of macrocytic hypochromic anaemia. In some cats, marked variation in M. haemofelis copy number over time (>100,000-fold difference within 48 h in some cats) and/or cycling of copy number was seen. No correlation was found between FIV provirus copy number and M. haemofelis copy number or haematological variables. No significant effect of chronic FIV infection on M. haemofelis copy number kinetics or haematological changes due to M. haemofelis infection was found, other than MCHC (P=0.03). Marbofloxacin treatment was associated with a significant decrease in M. haemofelis copy number (P=0.002), although consistent clearance of infection was not demonstrated. This study reveals the presence of marked fluctuations in M. haemofelis copy number kinetics in vivo and a significant response to marbofloxacin antibiotic treatment.

  14. Oral Recombinant Feline Interferon-Omega as an alternative immune modulation therapy in FIV positive cats: clinical and laboratory evaluation.

    Science.gov (United States)

    Gil, S; Leal, R O; McGahie, D; Sepúlveda, N; Duarte, A; Niza, M M R E; Tavares, L

    2014-02-01

    Recombinant-Feline Interferon-Omega (rFeIFN-ω) is an immune-modulator licensed for use subcutaneously in Feline Immunodeficiency virus (FIV) therapy. Despite oral protocols have been suggested, little is known about such use in FIV-infected cats. This study aimed to evaluate the clinical improvement, laboratory findings, concurrent viral excretion and acute phase proteins (APPs) in naturally FIV-infected cats under oral rFeIFN-ω therapy (0.1 MU/cat rFeIFN-ω PO, SID, 90 days). 11 FIV-positive cats were treated with oral rFeIFN-ω (PO Group). Results were compared to previous data from 7 FIV-positive cats treated with the subcutaneous licensed protocol (SC Group). Initial clinical scores were similar in both groups. Independently of the protocol, rFeIFN-ω induced a significant clinical improvement of treated cats. Concurrent viral excretion and APP's variation were not significant in the PO Group. Oral rFeIFN-ω can be an effective alternative therapy for FIV-infected cats, being also an option for treatment follow-up in cats submitted to the licensed protocol.

  15. A Single Amino Acid Difference within the α-2 Domain of Two Naturally Occurring Equine MHC Class I Molecules Alters the Recognition of Gag and Rev Epitopes by Equine Infectious Anemia Virus-Specific CTL1

    Science.gov (United States)

    Mealey, Robert H.; Lee, Jae-Hyung; Leib, Steven R.; Littke, Matt H.; McGuire, Travis C.

    2012-01-01

    Although CTL are critical for control of lentiviruses, including equine infectious anemia virus, relatively little is known regarding the MHC class I molecules that present important epitopes to equine infectious anemia virus-specific CTL. The equine class I molecule 7-6 is associated with the equine leukocyte Ag (ELA)-A1 haplotype and presents the Env-RW12 and Gag-GW12 CTL epitopes. Some ELA-A1 target cells present both epitopes, whereas others are not recognized by Gag-GW12-specific CTL, suggesting that the ELA-A1 haplotype comprises functionally distinct alleles. The Rev-QW11 CTL epitope is also ELA-A1-restricted, but the molecule that presents Rev-QW11 is unknown. To determine whether functionally distinct class I molecules present ELA-A1-restricted CTL epitopes, we sequenced and expressed MHC class I genes from three ELA-A1 horses. Two horses had the 7-6 allele, which when expressed, presented Env-RW12, Gag-GW12, and Rev-QW11 to CTL. The other horse had a distinct allele, designated 141, encoding a molecule that differed from 7-6 by a single amino acid within the α-2 domain. This substitution did not affect recognition of Env-RW12, but resulted in more efficient recognition of Rev-QW11. Significantly, CTL recognition of Gag-GW12 was abrogated, despite Gag-GW12 binding to 141. Molecular modeling suggested that conformational changes in the 141/Gag-GW12 complex led to a loss of TCR recognition. These results confirmed that the ELA-A1 haplotype is comprised of functionally distinct alleles, and demonstrated for the first time that naturally occurring MHC class I molecules that vary by only a single amino acid can result in significantly different patterns of epitope recognition by lentivirus-specific CTL. PMID:17082657

  16. Positive selection pressure introduces secondary mutations at Gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations

    DEFF Research Database (Denmark)

    Banke, S.; Lillemark, M.R.; Gerstoft, J.;

    2009-01-01

    mutations). Additional sequences from 13 patients were included for longitudinal analysis. We assessed positive selection pressure on the gag/protease region using a test for the overall influence of positive selection and a total of five tests to identify positively selected single codons. We found...... that positive selection pressure was the driving evolutionary force for the gag region in all three patient groups. An increase in positive selection was observed in gag cleavage site regions p7/p1/p6 only after the acquisition of major PI mutations, suggesting that amino acids in gag cleavage sites under...

  17. Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity.

    Science.gov (United States)

    Monde, Kazuaki; Terasawa, Hiromi; Nakano, Yusuke; Soheilian, Ferri; Nagashima, Kunio; Maeda, Yosuke; Ono, Akira

    2017-04-26

    Human endogenous retroviruses (HERVs), the remnants of ancient retroviral infections, constitute approximately 8% of human genomic DNA. Since HERV-K Gag expression is induced by HIV-1 Tat in T cells, induced HERV-K proteins could affect HIV-1 replication. Indeed, previously we showed that HERV-K Gag and HIV-1 Gag coassemble and that this appears to correlate with the effect of HERV-K Gag expression on HIV-1 particle release and its infectivity. We further showed that coassembly requires both MA and NC domains, which presumably serve as scaffolding for Gag via their abilities to bind membrane and RNA, respectively. Notably, however, despite possessing these abilities, MLV Gag failed to coassemble with HIV-1 Gag and did not affect assembly and infectivity of HIV-1 particles. It is unclear how the specificity of coassembly is determined. Here, we showed that coexpression of HERV-K Gag with HIV-1 Gag changed size and morphology of progeny HIV-1 particles and severely diminished infectivity of such progeny viruses. We further compared HERV-K-MLV chimeric constructs to identify molecular determinants for coassembly specificity and for inhibition of HIV-1 release efficiency and infectivity. We found that the CA N-terminal domain (NTD) of HERV-K Gag is important for the reduction of the HIV-1 release efficiency, whereas both CA-NTD and major homology region of HERV-K Gag contribute to colocalization with HIV-1 Gag. Interestingly, these regions of HERV-K Gag were not required for reduction of progeny HIV-1 infectivity. Our results showed that HERV-K Gag CA is important for reduction of HIV-1 release and infectivity but the different regions within CA are involved in the effects on the HIV-1 release and infectivity. Altogether, these findings revealed that HERV-K Gag interferes the HIV-1 replication by two distinct molecular mechanisms.

  18. Terminal-repeat retrotransposons with GAG domain in plant genomes: a new testimony on the complex world of transposable elements.

    Science.gov (United States)

    Chaparro, Cristian; Gayraud, Thomas; de Souza, Rogerio Fernandes; Domingues, Douglas Silva; Akaffou, Sélastique; Laforga Vanzela, Andre Luis; Kochko, Alexandre de; Rigoreau, Michel; Crouzillat, Dominique; Hamon, Serge; Hamon, Perla; Guyot, Romain

    2015-01-07

    A novel structure of nonautonomous long terminal repeat (LTR) retrotransposons called terminal repeat with GAG domain (TR-GAG) has been described in plants, both in monocotyledonous, dicotyledonous and basal angiosperm genomes. TR-GAGs are relatively short elements in length (element into the virus-like particle. GAG precursors show similarities with both Copia and Gypsy GAG proteins, suggesting evolutionary relationships of TR-GAG elements with both families. Despite the lack of the enzymatic machinery required for their mobility, strong evidences suggest that TR-GAGs are still active. TR-GAGs represent ubiquitous nonautonomous structures that could be involved in the molecular diversities of plant genomes.

  19. Safety and Immunogenicity of the Mycobacterium tuberculosis {Delta}lysA {Delta}panCD Vaccine in Domestic Cats Infected with Feline Immunodeficiency Virus

    Science.gov (United States)

    Feline immunodeficiency virus (FIV)+ and FIV- cats (n = 4/group) received 2 x 10**6 cfu Mycobacterium tuberculosis Delta-lysA Delta-panCD intramuscularly. Vaccination elicited antibody responses; albeit, at lower levels in FIV+ cats as compared to FIV- cats. Delayed-type hypersensitivity responses ...

  20. Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

    Science.gov (United States)

    Hammer, Diana; Wild, Jens; Ludwig, Christine; Asbach, Benedikt; Notka, Frank; Wagner, Ralf

    2008-06-01

    Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

  1. Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes.

    Science.gov (United States)

    Mealey, Robert H; Sharif, Amin; Ellis, Shirley A; Littke, Matt H; Leib, Steven R; McGuire, Travis C

    2005-08-15

    Cytotoxic T lymphocytes (CTL) are critical for control of lentiviruses, including equine infectious anemia virus (EIAV). Measurement of equine CTL responses has relied on chromium-release assays, which do not allow accurate quantitation. Recently, the equine MHC class I molecule 7-6, associated with the ELA-A1 haplotype, was shown to present both the Gag-GW12 and Env-RW12 EIAV CTL epitopes. In this study, 7-6/Gag-GW12 and 7-6/Env-RW12 MHC class I/peptide tetrameric complexes were constructed and used to analyze Gag-GW12- and Env-RW12-specific CTL responses in two EIAV-infected horses (A2164 and A2171). Gag-GW12 and Env-RW12 tetramer-positive CD8+ cells were identified in nonstimulated peripheral blood mononuclear cells as early as 14 days post-EIAV inoculation, and frequencies of tetramer-positive cells ranged from 0.4% to 6.7% of nonstimulated peripheral blood CD8+ cells during the 127-day study period. Although both horses terminated the initial viremic peak, only horse A2171 effectively controlled viral load. Neutralizing antibody was present during the initial control of viral load in both horses, but the ability to maintain control correlated with Gag-GW12-specific CD8+ cells in A2171. Despite Env-RW12 dominance, Env-RW12 escape viral variants were identified in both horses and there was no correlation between Env-RW12-specific CD8+ cells and control of viral load. Although Gag-GW12 CTL escape did not occur, a Gag-GW12 epitope variant arose in A2164 that was recognized less efficiently than the original epitope. These data indicate that tetramers are useful for identification and quantitation of CTL responses in horses, and suggest that the observed control of EIAV replication and clinical disease was associated with sustained CTL recognition of Gag-specific epitopes.

  2. DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV.

    Science.gov (United States)

    Hu, Xintao; Valentin, Antonio; Dayton, Frances; Kulkarni, Viraj; Alicea, Candido; Rosati, Margherita; Chowdhury, Bhabadeb; Gautam, Rajeev; Broderick, Kate E; Sardesai, Niranjan Y; Martin, Malcolm A; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2016-11-15

    HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24(gag) plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27(Gag) Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B(+) CD107a(+)) targeting subdominant CE epitopes, compared with the responses elicited by the p57(gag) pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth. Copyright © 2016 by The American Association of Immunologists, Inc.

  3. Immunogenicity of seven new recombinant yellow fever viruses 17D expressing fragments of SIVmac239 Gag, Nef, and Vif in Indian rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Mauricio A Martins

    Full Text Available An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV. Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (rYF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIVmac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5 vectors resulted in robust expansion of SIV-specific CD8(+ T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4(+ cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D - a clinically relevant vaccine vector - can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D

  4. Generation of a recombinant Gag virus-like-particle panel for the evaluation of p24 antigen detection by diagnostic HIV tests.

    Directory of Open Access Journals (Sweden)

    Beatrice N Vetter

    Full Text Available Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens.We generated a panel of 43 recombinantly expressed virus-like particles (VLPs, containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection.Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection.The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

  5. Human immunodeficiency virus type 2 (HIV-2) Gag is trafficked in an AP-3 and AP-5 dependent manner

    OpenAIRE

    Alford, Justine E.; Marongiu, Michela; Gemma L Watkins; Anderson, Emma C.

    2016-01-01

    Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HI...

  6. HIV Pol inhibits HIV budding and mediates the severe budding defect of Gag-Pol.

    Directory of Open Access Journals (Sweden)

    Xin Gan

    Full Text Available The prevailing hypothesis of HIV budding posits that the viral Gag protein drives budding, and that the Gag p6 peptide plays an essential role by recruiting host-cell budding factors to sites of HIV assembly. HIV also expresses a second Gag protein, p160 Gag-Pol, which lacks p6 and fails to bud from cells, consistent with the prevailing hypothesis of HIV budding. However, we show here that the severe budding defect of Gag-Pol is not caused by the absence of p6, but rather, by the presence of Pol. Specifically, we show that (i the budding defect of Gag-Pol is unaffected by loss of HIV protease activity and is therefore an intrinsic property of the Gag-Pol polyprotein, (ii the N-terminal 433 amino acids of Gag and Gag-Pol are sufficient to drive virus budding even though they lack p6, (iii the severe budding defect of Gag-Pol is caused by a dominant, cis-acting inhibitor of budding in the HIV Pol domain, and (iv Gag-Pol inhibits Gag and virus budding in trans, even at normal levels of Gag and Gag-Pol expression. These and other data support an alternative hypothesis of HIV budding as a process that is mediated by the normal, non-viral pathway of exosome/microvesicle biogenesis.

  7. Characterization of cytoplasmic Gag-gag interactions by dual-color z-scan fluorescence fluctuation spectroscopy.

    Science.gov (United States)

    Fogarty, Keir H; Chen, Yan; Grigsby, Iwen F; Macdonald, Patrick J; Smith, Elizabeth M; Johnson, Jolene L; Rawson, Jonathan M; Mansky, Louis M; Mueller, Joachim D

    2011-03-16

    Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addition of a distinctly colored reference protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus.

    Science.gov (United States)

    Theimer, C A; Giedroc, D P

    2000-03-01

    The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G x U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CdeltaA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (+/-0.2) kcal mol(-1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (deltaG(0)37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability deltadeltaH = 6.6 (+/-2.1) kcal mol(-1) with deltadeltaG(0)37 comparable to that predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G x U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)6(3+) ions stabilize the A14 and deltaA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus deltaA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617).

  9. Recommendations for the measurement of FIV(1) values in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Visser, Frank J; Ramlal, Sunil; Dekhuijzen, P N Richard; Heijdra, Yvonne F

    2008-01-01

    In contrast to static inspiratory parameters such as vital capacity and inspiratory capacity, information on forced inspiratory volume in 1 s (FIV(1)) in patients with chronic obstructive pulmonary disease (COPD) is limited. It was the aim of this study to investigate the influence of the preceding expiratory manoeuvre and the optimal number of manoeuvres on FIV(1) values. In 169 patients with COPD, FIV(1) manoeuvres were performed after a forced (FIV(1)-Fe) and a slow (FIV(1)-Se) expiration. To investigate the optimal number of the FIV(1)-Se manoeuvres, 8 attempts were performed. The variability of FIV(1)-Fe was greater than that of FIV(1)-Se. The mean difference between FIV(1)-Se and FIV(1)-Fe was 0.21 litres (p Obstructive Lung Disease (GOLD) stage. The higher the GOLD stage, the greater the difference between the 2 techniques. The correlation coefficient between FIV(1)-Se and FIV(1)-Fe was high (r = 0.89, p = 0.01), but there was a poor agreement between these parameters (limits of agreement -0.52 to 0.94 litres). Five manoeuvres were needed to obtain an optimal FIV(1)-Se. There was no association with the GOLD stage. In COPD patients, FIV(1)-Se are less variable than FIV(1)-Fe, the agreement between the 2 manoeuvres is poor, and at least 5 FIV(1)-Se manoeuvres are needed to get an acceptable FIV(1). This holds for all GOLD stages. 2007 S. Karger AG, Basel.

  10. Virus de la inmunodeficiencia felina (VIF): evaluación de las globulinas en pacientes infectados espontáneamente Feline immunodeficiency virus (FIV): study of globulins in patients with natural infections

    OpenAIRE

    N Gómez; M. A. Gisbert; L Ramayo; Bratanich, A; Castillo, V; A Suraniti

    2006-01-01

    Fueron estudiadas las posibles correlaciones de parámetros tales como la Alfa glicoproteína ácida (AGP, proteína de fase aguda), fracciones electroforéticas de las proteínas séricas y títulos de Toxoplasma gondii en gatos infectados por el Virus de Inmunodeficiencia Felina (VIF). Los títulos de Toxoplasma gondii obtenidos por Inmunofluorescencia Indirecta (IFI) no correlacionaron con los valores de Proteínas Totales ni con los de las globulinas. Sí se halló múltiple correlación entre todas la...

  11. Accessory Genes Confer a High Replication Rate to Virulent Feline Immunodeficiency Virus

    OpenAIRE

    Troyer, Ryan M.; Thompson, Jesse; Elder, John H.; VandeWoude, Sue

    2013-01-01

    Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between tw...

  12. Seroprevalence and genomic divergence of circulating strains of feline immunodeficiency virus among Felidae and Hyaenidae species.

    Science.gov (United States)

    Troyer, Jennifer L; Pecon-Slattery, Jill; Roelke, Melody E; Johnson, Warren; VandeWoude, Sue; Vazquez-Salat, Nuria; Brown, Meredith; Frank, Laurence; Woodroffe, Rosie; Winterbach, Christiaan; Winterbach, Hanlie; Hemson, Graham; Bush, Mitch; Alexander, Kathleen A; Revilla, Eloy; O'Brien, Stephen J

    2005-07-01

    Feline immunodeficiency virus (FIV) infects numerous wild and domestic feline species and is closely related to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Species-specific strains of FIV have been described for domestic cat (Felis catus), puma (Puma concolor), lion (Panthera leo), leopard (Panthera pardus), and Pallas' cat (Otocolobus manul). Here, we employ a three-antigen Western blot screening (domestic cat, puma, and lion FIV antigens) and PCR analysis to survey worldwide prevalence, distribution, and genomic differentiation of FIV based on 3,055 specimens from 35 Felidae and 3 Hyaenidae species. Although FIV infects a wide variety of host species, it is confirmed to be endemic in free-ranging populations of nine Felidae and one Hyaenidae species. These include the large African carnivores (lion, leopard, cheetah, and spotted hyena), where FIV is widely distributed in multiple populations; most of the South American felids (puma, jaguar, ocelot, margay, Geoffroy's cat, and tigrina), which maintain a lower FIV-positive level throughout their range; and two Asian species, the Pallas' cat, which has a species-specific strain of FIV, and the leopard cat, which has a domestic cat FIV strain in one population. Phylogenetic analysis of FIV proviral sequence demonstrates that most species for which FIV is endemic harbor monophyletic, genetically distinct species-specific FIV strains, suggesting that FIV transfer between cat species has occurred in the past but is quite infrequent today.

  13. Oxidative stress during acute FIV infection in cats.

    Science.gov (United States)

    Webb, Craig; Lehman, Tracy; McCord, Kelly; Avery, Paul; Dow, Steven

    2008-03-15

    Oxidative stress is thought to contribute to the pathogenesis of HIV infection in humans. For example, CD4(+) T cells are particularly affected in HIV patients and oxidative stress may also contribute to impairment of neutrophil function in HIV/AIDS patients. Since cats infected with FIV develop many of the same immunological abnormalites as HIV-infected humans, we investigated effects of acute FIV infection on oxidative stress in cats. Cats were infected with a pathogenic strain of FIV and viral load, changes in neutrophil number, total blood glutathione, malondiadehye, antioxidant enzyme concentrations, and reduced glutathione (GSH) concentration in leukocytes were measured sequentially during the first 16 weeks of infection. We found that superoxide dismutase and glutathione peroxidase concentrations in whole blood increased significantly during acute FIV infection. In addition, neutrophil numbers increased significantly during this time period, though their intracellular GSH concentrations did not change. In contrast, the numbers of CD4(+) T cells decreased significantly and their intracellular GSH concentration increased significantly, while intracellular GSH concentrations were unchanged in CD8(+) T cells. However, by 16 weeks of infection, many of the abnormalities in oxidative balance had stabilized or returned to pre-inoculation values. These results suggest that acute infection with FIV causes oxidative stress in cats and that CD4(+) T cells appear to be preferentially affected. Further studies are required to determine whether early treatment with anti-oxidants may help ameliorate the decline in CD4(+) T cell number and function associated with acute FIV infection in cats.

  14. Productive infection of human peripheral blood mononuclear cells by feline immunodeficiency virus: implications for vector development.

    Science.gov (United States)

    Johnston, J; Power, C

    1999-03-01

    Feline immunodeficiency virus (FIV) is a lentivirus causing immune suppression and neurological disease in cats. Like primate lentiviruses, FIV utilizes the chemokine receptor CXCR4 for infection. In addition, FIV gene expression has been demonstrated in immortalized human cell lines. To investigate the extent and mechanism by which FIV infected primary and immortalized human cell lines, we compared the infectivity of two FIV strains, V1CSF and Petaluma, after cell-free infection. FIV genome was detected in infected human peripheral blood mononuclear cells (PBMC) and macrophages at 21 and 14 days postinfection, respectively. Flow cytometry analysis of FIV-infected human PBMC indicated that antibodies to FIV p24 recognized 12% of the cells. Antibodies binding the CCR3 chemokine receptor maximally inhibited infection of human PBMC by both FIV strains compared to antibodies to CXCR4 or CCR5. Reverse transcriptase levels increased in FIV-infected human PBMC, with detection of viral titers of 10(1.3) to 10(2.1) 50% tissue culture infective doses/10(6) cells depending on the FIV strain examined. Cell death in human PBMC infected with either FIV strain was significantly elevated relative to uninfected control cultures. These findings indicate that FIV can productively infect primary human cell lines and that viral strain specificity should be considered in the development of an FIV vector for gene therapy.

  15. A determinant of feline immunodeficiency virus involved in Crandell feline kidney cell tropism.

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees); J.A. Karlas (Jos); G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Albert); M.L. Bosch (Marnix)

    1995-01-01

    textabstractViral progeny of the molecular clone 19k1 of feline immunodeficiency virus (FIV) can infect feline T-cells but not Crandell feline kidney (CrFK) cells. In contrast, the biological isolate FIV-AM6c, which was CrFK adapted by co-cultivation of FIV-AM6 infected thymocytes with CrFK cells, c

  16. HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

    Directory of Open Access Journals (Sweden)

    Jing Jin

    Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

  17. Terminal-Repeat Retrotransposons with GAG Domain in Plant Genomes: A New Testimony on the Complex World of Transposable Elements

    Science.gov (United States)

    Chaparro, Cristian; Gayraud, Thomas; de Souza, Rogerio Fernandes; Domingues, Douglas Silva; Akaffou, Sélastique; Laforga Vanzela, Andre Luis; de Kochko, Alexandre; Rigoreau, Michel; Crouzillat, Dominique; Hamon, Serge; Hamon, Perla; Guyot, Romain

    2015-01-01

    A novel structure of nonautonomous long terminal repeat (LTR) retrotransposons called terminal repeat with GAG domain (TR-GAG) has been described in plants, both in monocotyledonous, dicotyledonous and basal angiosperm genomes. TR-GAGs are relatively short elements in length (<4 kb) showing the typical features of LTR-retrotransposons. However, they carry only one open reading frame coding for the GAG precursor protein involved for instance in transposition, the assembly, and the packaging of the element into the virus-like particle. GAG precursors show similarities with both Copia and Gypsy GAG proteins, suggesting evolutionary relationships of TR-GAG elements with both families. Despite the lack of the enzymatic machinery required for their mobility, strong evidences suggest that TR-GAGs are still active. TR-GAGs represent ubiquitous nonautonomous structures that could be involved in the molecular diversities of plant genomes. PMID:25573958

  18. Imbalance of placental regulatory T cell and Th17 cell population dynamics in the FIV-infected pregnant cat

    Directory of Open Access Journals (Sweden)

    Boudreaux Crystal E

    2012-05-01

    Full Text Available Abstract Background An appropriate balance in placental regulatory T cells (Tregs, an immunosuppressive cell population, and Th17 cells, a pro-inflammatory cell population, is essential in allowing tolerance of the semi-allogeneic fetus. TGF-β and IL-6 are cytokines that promote differentiation of Tregs and Th17 cells from a common progenitor; aberrant expression of the cytokines may perturb the balance in the two cell populations. We previously reported a pro-inflammatory placental environment with decreased levels of FoxP3, a Treg marker, and increased levels of IL-6 in the placentas of FIV-infected cats at early pregnancy. Thus, we hypothesized that FIV infection in the pregnant cat causes altered placental Treg and Th17 cell populations, possibly resulting in placental inflammation. Methods We examined the effect of FIV infection on Treg and Th17 populations in placentas at early pregnancy using quantitative confocal microscopy to measure FoxP3 or RORγ, a Th17 marker, and qPCR to quantify expression of the key cytokines TGF-β and IL-6. Results FoxP3 and RORγ were positively correlated in FIV-infected placentas at early pregnancy, but not placentas from normal cats, indicating virus-induced alteration in the balance of these cell populations. In control cats the expression of IL-6 and RORγ was positively correlated as predicted, but this relationship was disrupted in infected animals. TGF-β was reduced in infected queens, an occurrence that could dysregulate both Treg and Th17 cell populations. Co-expression analyses revealed a highly significant positive correlation between IL-6 and TGF-β expression in control animals that did not occur in infected animals. Conclusion Collectively, these data point toward potential disruption in the balance of Treg and Th17 cell populations that may contribute to FIV-induced inflammation in the feline placenta.

  19. Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

    Science.gov (United States)

    Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

    2016-06-01

    MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses.

  20. Determinants of Glycosaminoglycan (GAG Structure

    Directory of Open Access Journals (Sweden)

    Kristian Prydz

    2015-08-01

    Full Text Available Proteoglycans (PGs are glycosylated proteins of biological importance at cell surfaces, in the extracellular matrix, and in the circulation. PGs are produced and modified by glycosaminoglycan (GAG chains in the secretory pathway of animal cells. The most common GAG attachment site is a serine residue followed by a glycine (-ser-gly-, from which a linker tetrasaccharide extends and may continue as a heparan sulfate, a heparin, a chondroitin sulfate, or a dermatan sulfate GAG chain. Which type of GAG chain becomes attached to the linker tetrasaccharide is influenced by the structure of the protein core, modifications occurring to the linker tetrasaccharide itself, and the biochemical environment of the Golgi apparatus, where GAG polymerization and modification by sulfation and epimerization take place. The same cell type may produce different GAG chains that vary, depending on the extent of epimerization and sulfation. However, it is not known to what extent these differences are caused by compartmental segregation of protein cores en route through the secretory pathway or by differential recruitment of modifying enzymes during synthesis of different PGs. The topic of this review is how different aspects of protein structure, cellular biochemistry, and compartmentalization may influence GAG synthesis.

  1. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    Energy Technology Data Exchange (ETDEWEB)

    López, Claudia S., E-mail: lopezcl@ohsu.edu [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Sloan, Rachel; Cylinder, Isabel [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Kozak, Susan L.; Kabat, David [Biochemistry and Molecular Biology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Barklis, Eric, E-mail: barklis@ohsu.edu [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States)

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

  2. Class II transactivator (CIITA enhances cytoplasmic processing of HIV-1 Pr55Gag.

    Directory of Open Access Journals (Sweden)

    Kristen A Porter

    Full Text Available BACKGROUND: The Pr55(gag (Gag polyprotein of HIV serves as a scaffold for virion assembly and is thus essential for progeny virion budding and maturation. Gag localizes to the plasma membrane (PM and membranes of late endosomes, allowing for release of infectious virus directly from the cell membrane and/or upon exocytosis. The host factors involved in Gag trafficking to these sites are largely unknown. Upon activation, CD4+ T cells, the primary target of HIV infection, express the class II transcriptional activator (CIITA and therefore the MHC class II isotype, HLA-DR. Similar to Gag, HLA-DR localizes to the PM and at the membranes of endosomes and specialized vesicular MHC class II compartments (MIICs. In HIV producer cells, transient HLA-DR expression induces intracellular Gag accumulation and impairs virus release. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that both stable and transient expression of CIITA in HIV producer cells does not induce HLA-DR-associated intracellular retention of Gag, but does increase the infectivity of virions. However, neither of these phenomena is due to recapitulation of the class II antigen presentation pathway or CIITA-mediated transcriptional activation of virus genes. Interestingly, we demonstrate that CIITA, apart from its transcriptional effects, acts cytoplasmically to enhance Pr160(gag-pol (Gag-Pol levels and thereby the viral protease and Gag processing, accounting for the increased infectivity of virions from CIITA-expressing cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that CIITA enhances HIV Gag processing, and provides the first evidence of a novel, post-transcriptional, cytoplasmic function for a well-known transactivator.

  3. Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

    Directory of Open Access Journals (Sweden)

    Shephard Enid G

    2011-10-01

    Full Text Available Abstract Background HIV-1 Gag virus like particles (VLPs used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates

  4. Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch.

    Science.gov (United States)

    Datta, Siddhartha A K; Zhao, Zhuojun; Clark, Patrick K; Tarasov, Sergey; Alexandratos, Jerry N; Campbell, Stephen J; Kvaratskhelia, Mamuka; Lebowitz, Jacob; Rein, Alan

    2007-01-19

    Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

  5. Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag

    Directory of Open Access Journals (Sweden)

    Tiffiny Rye-McCurdy

    2016-09-01

    Full Text Available Retroviruses specifically package full-length, dimeric genomic RNA (gRNA even in the presence of a vast excess of cellular RNA. The “psi” (Ψ element within the 5′-untranslated region (5′UTR of gRNA is critical for packaging through interaction with the nucleocapsid (NC domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1 Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity.

  6. Evolution of feline immunodeficiency virus in Felidae: implications for human health and wildlife ecology.

    Science.gov (United States)

    Pecon-Slattery, Jill; Troyer, Jennifer L; Johnson, Warren E; O'Brien, Stephen J

    2008-05-15

    Genetic analyses of feline immunodeficiency viruses provide significant insights on the worldwide distribution and evolutionary history of this emerging pathogen. Large-scale screening of over 3000 samples from all species of Felidae indicates that at least some individuals from most species possess antibodies that cross react to FIV. Phylogenetic analyses of genetic variation in the pol-RT gene demonstrate that FIV lineages are species-specific and suggest that there has been a prolonged period of viral-host co-evolution. The clinical effects of FIV specific to species other than domestic cat are controversial. Comparative genomic analyses of all full-length FIV genomes confirmed that FIV is host specific. Recently sequenced lion subtype E is marginally more similar to Pallas cat FIV though env is more similar to that of domestic cat FIV, indicating a possible recombination between two divergent strains in the wild. Here we review global patterns of FIV seroprevalence and endemnicity, assess genetic differences within and between species-specific FIV strains, and interpret these with patterns of felid speciation to propose an ancestral origin of FIV in Africa followed by interspecies transmission and global dissemination to Eurasia and the Americas. Continued comparative genomic analyses of full-length FIV from all seropositive animals, along with whole genome sequence of host species, will greatly advance our understanding of the role of recombination, selection and adaptation in retroviral emergence.

  7. Roles played by acidic lipids in HIV-1 Gag membrane binding.

    Science.gov (United States)

    Olety, Balaji; Ono, Akira

    2014-11-26

    The MA domain mediates plasma membrane (PM) targeting of HIV-1 Gag, leading to particle assembly at the PM. The interaction between MA and acidic phospholipids, in addition to N-terminal myristoyl moiety, promotes Gag binding to lipid membranes. Among acidic phospholipids, PI(4,5)P2, a PM-specific phosphoinositide, is essential for proper HIV-1 Gag localization to the PM and efficient virus particle production. Recent studies further revealed that MA-bound RNA negatively regulates HIV-1 Gag membrane binding and that PI(4,5)P2 is necessary to overcome this RNA-imposed block. In this review, we will summarize the current understanding of Gag-membrane interactions and discuss potential roles played by acidic phospholipids.

  8. Disseminated Mycobacterium genavense infection in a FIV-positive cat.

    Science.gov (United States)

    Hughes, M S; Ball, N W; Love, D N; Canfield, P J; Wigney, D I; Dawson, D; Davis, P E; Malik, R

    1999-03-01

    An 8-year-old FIV-positive Australian cat was presented with coughing, periocular alopecia, pyrexia and inappetence. Skin scrapings demonstrated Demodex cati mites. Antibiotics were administered and it was treated successfully for periocular demodectic mange, but the cat continued to exhibit respiratory signs and lose weight. Further investigation revealed an ascarid infection and active chronic inflammation of undetected cause affecting the lower airways. Repetitive treatment with pyrantel failed to eradicate the ascarid infection. The cat became cachectic and developed moist ulcerative dermatitis of the neck, severe non-regenerative anaemia, leucopenia and thrombocytopenia. Necropsy and histopathology revealed mycobacteriosis affecting skin, lungs, spleen, lymph nodes, liver and kidney. Attempted culture of frozen tissues at a mycobacteria reference laboratory was unsuccessful. Paraffin-embedded, formalin-fixed tissue was retrieved and examined using PCR to amplify part of the 16S rRNA gene. A diagnosis of disseminated Mycobacterium genavense infection was made based on the presence of acid fast bacteria in many tissues and partial sequence of the 16S rRNA gene. Although M genavense has been identified previously as a cause of disseminated disease in AIDS patients, this is the first report of infection in a cat. It was suspected that the demodecosis, recurrent ascarid infections and disseminated M genavense infection resulted from an immune deficiency syndrome consequent to longstanding FIV infection.

  9. Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3

    Directory of Open Access Journals (Sweden)

    Elder John H

    2007-01-01

    Full Text Available Abstract We have obtained the 1.7 Å crystal structure of FIV protease (PR in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR. The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants 1234. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with Ki values of 1.5 nM, 10 nM, and 41 nM, respectively 234. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed theformation of additinal van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.

  10. Evaluation of subunit vaccines against feline immunodeficiency virus infection

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Willemse, M.J.; Stam, J.G.; Vliet, A.L.W. van; Pouwels, H.; Chalmers, S.K.; Sondermeijer, P.J.; Hesselink, W.; Ronde, A. de

    1996-01-01

    Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase

  11. The oral and conjunctival microbiotas in cats with and without feline immunodeficiency virus infection

    OpenAIRE

    Weese, Scott J; Nichols, Jamieson; Jalali, Mohammad; Litster, Annette

    2015-01-01

    International audience; AbstractThe oral and conjunctival microbiotas likely play important roles in protection from opportunistic infections, while also being the source of potential pathogens. Yet, there has been limited investigation in cats, and the impact of comorbidities such as feline immunodeficiency virus (FIV) infection has not been reported. Oral and conjunctival swabs were collected from cats with FIV infection and FIV-uninfected controls, and subjected to 16S rRNA gene (V4) PCR a...

  12. Spatial analysis of feline immunodeficiency virus infection in cougars.

    Science.gov (United States)

    Wheeler, David C; Waller, Lance A; Biek, Roman

    2010-07-01

    The cougar (Puma concolor) is a large predatory feline found widely in the Americas that is susceptible to feline immunodeficiency virus (FIV), a fast-evolving lentivirus found in wild feline species that is analogous to simian immunodeficiency viruses in wild primates and belongs to the same family of viruses as human immunodeficiency virus. FIV infection in cougars can lead to a weakened immune system that creates opportunities for other infecting agents. FIV prevalence and lineages have been studied previously in several areas in the western United States, but typically without spatially explicit statistical techniques. To describe the distribution of FIV in a sample of cougars located in the northern Rocky Mountain region of North America, we first used kernel density ratio estimation to map the log relative risk of FIV. The risk surface showed a significant cluster of FIV in northwestern Montana. We also used Bayesian cluster models for genetic data to investigate the spatial structure of the feline immunodeficiency virus with virus genetic sequence data. A result of the models was two spatially distinct FIV lineages that aligned considerably with an interstate highway in Montana. Our results suggest that the use of spatial information and models adds novel insight when investigating an infectious animal disease. The results also suggest that the influence of landscape features likely plays an important role in the spatiotemporal spread of an infectious disease within wildlife populations.

  13. GAGs and GAGs diseases: when pathophysiology supports the clinic.

    Science.gov (United States)

    Costantini, Elisabetta; Lazzeri, Massimo; Porena, Massimo

    2013-01-01

    The urinary epithelium has been the subject of considerable interest and much research in recent years. What has radically changed in the last decade is the concept of what the bladder epithelium really is. It is currently no longer considered just a simple barrier and a non-specific defence against infections, and it has been recognized as a specialized tissue regulating complex bladder functions and playing a fundamental and active role in the pathogenesis of cystitis. Researchers have been focussing on the receptors and mediators that are active in the sub-epithelial layer, with the hope that understanding the role of the urothelium defect will offer the opportunity for new therapeutic strategies. On the surface of the urothelial umbrella-cells there is a thick layer of glycoproteins and proteoglycans, which together are called Glycosaminoglycans (GAGs). They constitute a hydrophilic mucosal coating and act as a barrier against solutes found in urine. In recent years they have received special attention because injury to Gags, due to different noxae, has been identified as the first step in the genesis of chronic inflammatory bladder diseases, such as recurrent urinary tract infections, chemical or radiation cystitis, interstitial cystitis and/or Bladder Pain Syndrome. Aim of this study is to define the importance of the urothelium starting from the anatomy and physiology of the bladder wall. Furthermore, we will underline the role of glycosaminoglycans, focusing both on their pathophysiological role in the principal bladder diseases and on the therapeutic aspects from the clinical point of view.

  14. Toxicity associated with high dosage 9-[(2R,5R-2,5-dihydro-5-phosphonomethoxy)-2-furanyl]adenine therapy off attempts to abort early FIV infection.

    Science.gov (United States)

    Hartmann, K; Ferk, G; North, T W; Pedersen, N C

    1997-09-01

    9-[(2R,5R-2,5-dihydro-5-phosphonomethoxy)-2-furanyl]adenine, or D4API, was tested in the feline immunodeficiency virus (FIV) infection model and found to be significantly more inhibitory in vitro than its parent compound 9-phosphonylmethoxethyl adenine (PMEA). Cytotoxicity was less than for PMEA or azidothymidine (AZT) for culture periods of 7 days, but more toxic after 10 days. D4API was rapidly absorbed by cats following subcutaneous inoculation, with a plasma half-life of less than 1 h after intravenous inoculation and between 2 and 3 h after subcutaneous injection. Peripheral blood mononuclear cells collected from cats given a single dose of D4API were refractory, however, to FIV infection in vitro for up to 24 h. Given its prolonged intracellular phase and high selectivity index, high dose D4API therapy was tested for its ability to abort an acute (i.e. 2 week) FIV infection. A divided daily dose of D4API, which was one-fourth the toxic dose and 125 times the concentration that would totally inhibit virus replication in vitro, completely abrogated the anticipated viremia and antibody responses. Unfortunately, a majority of treated/uninfected and treated/infected test cats died acutely of drug toxicity after 47 days of treatment. Toxicity in vivo mirrored what was observed in vitro, being precipitous and cumulative in nature. Toxic signs included widespread hepatic and lymphoid necrosis. A surviving treated/FIV infected cat remained healthy to day 175 when the study was terminated; antibodies appeared 2 months later than in untreated/infected cats and virus was only detectable at low levels on day 175. In contrast, untreated/infected cats were viremic and antibody positive from 3 to 4 weeks post-infection onwards. Therefore, it was possible to alter, but not abort, an early FIV infection with prolonged, high-dose D4API treatment.

  15. A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins.

    Directory of Open Access Journals (Sweden)

    Julia Gunzenhäuser

    Full Text Available The assembly process of the human immunodeficiency virus 1 (HIV-1 is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.

  16. Gagging Problem Assessment : a re-evaluation

    NARCIS (Netherlands)

    van Linden van den Heuvell, G. F. E. C.; de Boer, B.; ter Pelkwijk, B. J.; Bildt, M. M.; Stegenga, B.

    The Gagging Problem Assessment (GPA) is an instrument to evaluate dental gagging. Although the GPA seemed to be reliable and valid in a pilot study, a replication study with more subjects was needed. Based on the pilot study, the number of items was reduced, resulting in the revised version of the

  17. Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy▿†

    Science.gov (United States)

    Weber, Jan; Vazquez, Ana C.; Winner, Dane; Rose, Justine D.; Wylie, Doug; Rhea, Ariel M.; Henry, Kenneth; Pappas, Jennifer; Wright, Alison; Mohamed, Nizar; Gibson, Richard; Rodriguez, Benigno; Soriano, Vicente; King, Kevin; Arts, Eric J.; Olivo, Paul D.; Quiñones-Mateu, Miguel E.

    2011-01-01

    Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens. PMID:21628544

  18. Early Pathogenesis of Transmucosal Feline Immunodeficiency Virus Infection

    OpenAIRE

    Obert, Leslie A.; Hoover, Edward A.

    2002-01-01

    To identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-...

  19. Feline immunodeficiency virus: Studies on pathogenesis and vaccine development

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees)

    1995-01-01

    textabstractFeline immunodeficiency virus (FIV) is classified as a member of the genus Lentivirus (subfamily Lentivirinae) of the Retroviridae family on basis of its morphology, biochemical characteristics, genomic organization, Mg'+ dependent reverse transcriptase, and nucleotide sequence homology

  20. Feline immunodeficiency virus: Studies on pathogenesis and vaccine development

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees)

    1995-01-01

    textabstractFeline immunodeficiency virus (FIV) is classified as a member of the genus Lentivirus (subfamily Lentivirinae) of the Retroviridae family on basis of its morphology, biochemical characteristics, genomic organization, Mg'+ dependent reverse transcriptase, and nucleotide sequence homology

  1. Prevalence of feline immunodeficiency virus infection in domesticated and feral cats in eastern Australia.

    Science.gov (United States)

    Norris, Jacqueline M; Bell, Erin T; Hales, Louise; Toribio, Jenny-Ann L M L; White, Joanna D; Wigney, Denise I; Baral, Randolph M; Malik, Richard

    2007-08-01

    Serum samples from 340 pet cats presented to three inner city clinics in Sydney Australia, 68 feral cats from two separate colonies in Sydney, and 329 cattery-confined pedigree and domestic cats in eastern Australia, were collected over a 2-year period and tested for antibodies directed against feline immunodeficiency virus (FIV) using immunomigration (Agen FIV Rapid Immunomigration test) and enzyme-linked immunosorbent assay methods (Snap Combo feline leukaemia virus antigen/FIV antibody test kit, IDEXX Laboratories). Western blot analysis was performed on samples in which there was discrepancy between the results. Information regarding breed, age, gender, housing arrangement and health status were recorded for all pet and cattery-confined cats, while the estimated age and current physical condition were recorded for feral cats. The FIV prevalence in the two feral cat populations was 21% and 25%. The majority of FIV-positive cats were male (60-80%). The FIV prevalence in cattery-confined cats was nil. The prevalence of FIV in the pet cat sample population was 8% (27/340) with almost equal prevalence in 'healthy' (13/170) and 'systemically unwell' (14/170) cats. The age of FIV-positive pet cats ranged from 3 to 19 years; all FIV-positive cats were domestic shorthairs with outside access. The median age of FIV-positive pet cats (11 years) was significantly greater than the median age of FIV-negative pet cats (7.5 years: Pcats (21/172; 12%) was three times that in female pet cats (6/168; 4%; Pcat population given outside access and continued FIV infection present in the feral population, this study highlights the need to develop rapid, accurate and cost-effective diagnostic methods that are not subject to false positives created by concurrent vaccination against FIV. This is especially important in re-homing stray cats within animal shelters and monitoring the efficacy of the new vaccine, which has not been challenged against Australian strains. The absence of FIV

  2. Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stochiometry

    Directory of Open Access Journals (Sweden)

    Fogarty Keir H

    2010-09-01

    Full Text Available Abstract Background Human T-lymphotropic virus type 1 (HTLV-1 is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells. Results The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM. Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS. The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions. Conclusions In summary, our studies represent the first quantitative biophysical

  3. Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters

    Science.gov (United States)

    Yandrapalli, Naresh; Lubart, Quentin; Tanwar, Hanumant S.; Picart, Catherine; Mak, Johnson; Muriaux, Delphine; Favard, Cyril

    2016-01-01

    The self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P2 lipid (PIP2). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP2 lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP2/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly. PMID:28008947

  4. Characterization of a highly pathogenic molecular clone of feline immunodeficiency virus clade C.

    Science.gov (United States)

    de Rozières, Sohela; Mathiason, Candace K; Rolston, Matthew R; Chatterji, Udayan; Hoover, Edward A; Elder, John H

    2004-09-01

    We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number AF474246) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4(+)-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV.

  5. Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

    Institute of Scientific and Technical Information of China (English)

    Yong-Nan Xu; Joon-Ho Yoon; Dae-Hwan Ko; Teoan Kim; Nam-Hyung Kim; Sang-Jun Uhm; Bon-Chul Koo; Mo-Sun Kwon; Ji-Yeol Roh; Jung-Seok Yang; Hyun-Yong Choi; Young-Tae Heo; Xiang-Shun Cui

    2013-01-01

    The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins.Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer,but these approaches have been largely ineffective; however,a third approach,lentivirus-mediated transgenesis,has successfully produced transgenic livestock.In this study,we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors,which expressed enhanced green fluorescent protein (EGFP) systemically.Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV)carrying EGFP were injected into the perivitelline space of MII oocytes.EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5% ± 2.2% v.s.22.9% ± 2.9%).Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers.Ten heifers were successfully impregnated and delivered 10 healthy calves.All of these calves expressed EGFP as detected by in vivo imaging,PCR and Southern blotting.In addition,we established an EGFP-expressing cell line from TG calves,which was followed by nuclear transfer (NT).Recloned 8-cell embryos also expressed EGFP,and there were no differences in the rates of fusion,cleavage and development between cells derived from TG and non-TG calves,which were subsequently used for NT.These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle.Moreover,our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.

  6. Tsg101 Regulates PI(4,5P2/Ca2+ Signaling for HIV-1 Gag Assembly

    Directory of Open Access Journals (Sweden)

    Lorna S Ehrlich

    2014-05-01

    Full Text Available Our previous studies identified the 1,4,5-inositol triphosphate receptor (IP3R, a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3, a product of phospholipase C-mediated PI(4,5P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5P2. Raising cytosolic Ca2+ promotes viral particle production and our studies indicate that IP3R and the ER Ca2+ store are the physiological providers of Ca2+ for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca2+ level originating from the ER store than control cells, suggesting that Gag induced release of store Ca2+. This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca2+ elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence and immunoelectron microscopy or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an ability to

  7. Tsg101 regulates PI(4,5)P2/Ca(2+) signaling for HIV-1 Gag assembly.

    Science.gov (United States)

    Ehrlich, Lorna S; Medina, Gisselle N; Photiadis, Sara; Whittredge, Paul B; Watanabe, Susan; Taraska, Justin W; Carter, Carol A

    2014-01-01

    Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca(2+) from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca(2+) promotes viral particle production and our studies indicate that IP3R and the ER Ca(2+) store are the physiological providers of Ca(2+) for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca(2+) level originating from the ER store than control cells, suggesting that Gag induced release of store Ca(2+). This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca(2+) elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence (TIRF) and immunoelectron microscopy (IEM) or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag

  8. HIV-1 Gag-specific exosome-targeted T cell-based vaccine stimulates effector CTL responses leading to therapeutic and long-term immunity against Gag/HLA-A2-expressing B16 melanoma in transgenic HLA-A2 mice

    Directory of Open Access Journals (Sweden)

    Rong Wang

    2014-01-01

    Full Text Available Human immunodeficiency virus type-1 (HIV-1-specific dendritic cell (DC vaccines have been applied to clinical trials that show only induction of some degree of immune responses, warranting the search of other more efficient vaccine strategies. Since HIV-1-specific CD8+ cytotoxic T lymphocytes (CTLs have been found to recognize some HIV-1 structural protein Gag conserved and cross-strain epitopes, Gag has become one of the most attractive target candidates for HIV-1 vaccine development. In this study, we generated HIV-1 Gag-specific Gag-Texo vaccine by using ConA-stimulated polyclonal CD8+ T-cells with uptake of Gag-expressing adenoviral vector AdVGag-transfected DC (DCGag-released exosomes (EXOs, and assessed its stimulation of Gag-specific CD8+ CTL responses and antitumor immunity. We demonstrate that Gag-Texo and DCGag vaccines comparably stimulate Gag-specific effector CD8+ CTL responses. Gag-Texo-stimulated CTL responses result in protective immunity against Gag-expressing BL6-10Gag melanoma in 8/8 wild-type C57BL/6 mice. In addition, we show that Gag-Texo vaccine also induces CTL responses leading to protective and long-term immunity against Gag/HLA-A2-expressing BL6-10Gag/A2 melanoma in 8/8 and 2/8 transgenic HLA-A2 mice, respectively. The average number of lung tumor colonies in mice with 30-days post-immunization is only 23, which is significantly less than that (>300 in control ConA-T-immunized HLA-A2 mice. Furthermore, Gag-Texo vaccine also induces some degree of therapeutic immunity. The average number (50 and size (0.23 mm in diameter of lung tumor colonies in Gag-Texo-immunized HLA-A2 mice bearing 6-day-established lung BL6-10Gag/A2 melanoma metastasis are significantly less than the average number (>300 and size (1.02 mm in diameter in control ConA-T-immunized HLA-A2 mice. Taken together, HIV-1 Gag-Texo vaccine capable of stimulating Gag-specific CTL responses and therapeutic immunity may be useful as a new immunotherapeutic

  9. Seroprevalence of Toxoplasma gondii and concurrent Bartonella spp., feline immunodeficiency virus, feline leukemia virus, and Dirofilaria immitis infections in Egyptian cats

    Science.gov (United States)

    Toxoplasma gondii and Bartonella spp. are zoonotic pathogens of cats. Feline Immunodeficiency Virus (FIV), and Feline Leukemia Virus (FeLv) are related to Human Immunodeficiency Virus, and Human Leukemia Virus, respectively, and these viruses are immunosuppressive. In the present study, the prevalen...

  10. Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, A.; Oroszlan, S.

    1984-03-01

    Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of (/sup 3/H)myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chromatography to be (/sup 3/H)myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid. It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins. The possible role of myristylation of transforming proteins is discussed.

  11. Could FIV zoonosis responsible of the breakdown of the pathocenosis which has reduced the European CCR5-Delta32 allele frequencies?

    Directory of Open Access Journals (Sweden)

    Faure Eric

    2008-10-01

    Full Text Available Abstract Background In Europe, the north-south downhill cline frequency of the chemokine receptor CCR5 allele with a 32-bp deletion (CCR5-Δ32 raises interesting questions for evolutionary biologists. We had suggested first that, in the past, the European colonizers, principally Romans, might have been instrumental of a progressively decrease of the frequencies southwards. Indeed, statistical analyses suggested strong negative correlations between the allele frequency and historical parameters including the colonization dates by Mediterranean civilisations. The gene flows from colonizers to native populations were extremely low but colonizers are responsible of the spread of several diseases suggesting that the dissemination of parasites in naive populations could have induced a breakdown rupture of the fragile pathocenosis changing the balance among diseases. The new equilibrium state has been reached through a negative selection of the null allele. Results Most of the human diseases are zoonoses and cat might have been instrumental in the decrease of the allele frequency, because its diffusion through Europe was a gradual process, due principally to Romans; and that several cat zoonoses could be transmitted to man. The possible implication of a feline lentivirus (FIV which does not use CCR5 as co-receptor is discussed. This virus can infect primate cells in vitro and induces clinical signs in macaque. Moreover, most of the historical regions with null or low frequency of CCR5-Δ32 allele coincide with historical range of the wild felid species which harbor species-specific FIVs. Conclusion We proposed the hypothesis that the actual European CCR5 allelic frequencies are the result of a negative selection due to a disease spreading. A cat zoonosis, could be the most plausible hypothesis. Future studies could provide if CCR5 can play an antimicrobial role in FIV pathogenesis. Moreover, studies of ancient DNA could provide more evidences regarding

  12. Could FIV zoonosis responsible of the breakdown of the pathocenosis which has reduced the European CCR5-Delta32 allele frequencies?

    Science.gov (United States)

    Faure, Eric

    2008-01-01

    Background In Europe, the north-south downhill cline frequency of the chemokine receptor CCR5 allele with a 32-bp deletion (CCR5-Δ32) raises interesting questions for evolutionary biologists. We had suggested first that, in the past, the European colonizers, principally Romans, might have been instrumental of a progressively decrease of the frequencies southwards. Indeed, statistical analyses suggested strong negative correlations between the allele frequency and historical parameters including the colonization dates by Mediterranean civilisations. The gene flows from colonizers to native populations were extremely low but colonizers are responsible of the spread of several diseases suggesting that the dissemination of parasites in naive populations could have induced a breakdown rupture of the fragile pathocenosis changing the balance among diseases. The new equilibrium state has been reached through a negative selection of the null allele. Results Most of the human diseases are zoonoses and cat might have been instrumental in the decrease of the allele frequency, because its diffusion through Europe was a gradual process, due principally to Romans; and that several cat zoonoses could be transmitted to man. The possible implication of a feline lentivirus (FIV) which does not use CCR5 as co-receptor is discussed. This virus can infect primate cells in vitro and induces clinical signs in macaque. Moreover, most of the historical regions with null or low frequency of CCR5-Δ32 allele coincide with historical range of the wild felid species which harbor species-specific FIVs. Conclusion We proposed the hypothesis that the actual European CCR5 allelic frequencies are the result of a negative selection due to a disease spreading. A cat zoonosis, could be the most plausible hypothesis. Future studies could provide if CCR5 can play an antimicrobial role in FIV pathogenesis. Moreover, studies of ancient DNA could provide more evidences regarding the implications of

  13. Expression from second-generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells.

    Science.gov (United States)

    Price, Mary A; Case, Scott S; Carbonaro, Denise A; Yu, Xiao-Jin; Petersen, Denise; Sabo, Kathleen M; Curran, Michael A; Engel, Barbara C; Margarian, Hovanes; Abkowitz, Janis L; Nolan, Garry P; Kohn, Donald B; Crooks, Gay M

    2002-11-01

    Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

  14. Infección con el virus de la inmunodeficiencia felina: desarrollo y aplicación de una técnica de doble reacción en cadena de la polimerasa (PRC) para el diagnóstico

    OpenAIRE

    Oliva, Graciela A.; Vila Roza, M. V.; Teodoroff, T.; Castellano, María Cecilia; Pecoraro, Marcelo R.; Arias, Daniel Osvaldo; González, E. T.; Etcheverrigaray, María Elisa; Galosi, Cecilia Mónica

    2000-01-01

    A Nested PCR technique for direct identification of Feline Immunodeficiency Virus (FIV) was developed. Two methods for proviral DNA extraction and two pairs of primers were evaluated. The method for amplification was standardized and its specificity was determined by the restriction endonucleases digestion of PCR products. Antibodies specific to FIV were determined using a commercial kit. Thirty heparinized blood samples obtained from cats with and without clinical signs consistent with FIV i...

  15. Selection of HLA-B57-associated Gag A146P mutant by HLA-B∗48:01-restricted Gag140-147-specific CTLs in chronically HIV-1-infected Japanese.

    Science.gov (United States)

    Naruto, Takuya; Murakoshi, Hayato; Chikata, Takayuki; Koyanagi, Madoka; Kawashima, Yuka; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

    2011-08-01

    We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B∗57-restricted CTLs, was selected by HLA-B∗48:01-restricted Gag138-147(LI10)-specific CTLs in a Japanese cohort in which HLA-B∗57 individuals were not detected. We herein demonstrated Gag140-147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B∗48:01(+) individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs.

  16. Vif of feline immunodeficiency virus from domestic cats protects against APOBEC3 restriction factors from many felids.

    Science.gov (United States)

    Zielonka, Jörg; Marino, Daniela; Hofmann, Henning; Yuhki, Naoya; Löchelt, Martin; Münk, Carsten

    2010-07-01

    To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Deltavif FIV, felid A3Z2s did not show any antiviral activity against Deltavif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether Vif(FIV) can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.Vif(FIV) was constructed. This HIV-1.Vif(FIV) was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor.

  17. Role of Gag and lipids during HIV-1 assembly in CD4 T cells and Macrophages

    Directory of Open Access Journals (Sweden)

    Charlotte eMariani

    2014-06-01

    Full Text Available HIV-1 is an RNA enveloped virus that preferentiallyinfects CD4+ T lymphocytes andalso macrophages. In CD4+ T cells, HIV-1mainly buds from the host cell plasma membrane.The viral Gag polyprotein targets theplasma membrane and is the orchestrator ofthe HIV assembly as its expression is sufficientto promote the formation of virus-likeparticles particles carrying a lipidic envelopederiving from the host cell membrane. Certainlipids are enriched in the viral membraneand are thought to play a key role in theassembly process and the envelop composition.A large body of work performed oninfected CD4+ T cells has provided importantknowledge about the assembly process andthe membrane virus lipid composition. WhileHIV assembly and budding in macrophages isthought to follow the same general Gag-drivenmechanism as in T-lymphocytes, the HIV cyclein macrophage exhibits specific features.In these cells, new virions bud from the limitingmembrane of seemingly intracellular compartments,where they accumulate while remaininginfectious. These structures are now oftenreferred to as Virus Containing Compartments(VCCs. Recent studies suggest that VCCsrepresent intracellularly sequestered regionsof the plasma membrane, but their precisenature remains elusive. The proteomic andlipidomic characterization of virions producedby T cells or macrophages has highlightedthe similarity between their composition andthat of the plasma membrane of producercells, as well as their enrichment in acidiclipids, some components of raft lipids andin tetraspanin-enriched microdomains. Greatchances are that Gag promotes the coalescenceof these components into an assemblyplatform from which viral budding takesplace. How Gag exactly interacts with membranelipids and what are the mechanisms involvedin the interaction between the differentmembrane nanodomains within the assemblyplatform remains unclear. Here we review recentliterature regarding the role of Gag andlipids

  18. Early pathogenesis of transmucosal feline immunodeficiency virus infection.

    Science.gov (United States)

    Obert, Leslie A; Hoover, Edward A

    2002-06-01

    To identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-ISH and in retropharyngeal, tracheobronchial, or external iliac lymph nodes and sometimes in spleen or blood mononuclear cells by day 2, indicating that regional and distant spread of virus-infected cells occurred rapidly after mucosal exposure. By day 8, viral RNA, DNA, and culturable virus were uniformly detected in regional and distant tissues, connoting systemic infection. TSA-ISH proved more sensitive than DNA PCR in detecting early FIV-infected cells. In mucosal tissues, the earliest demonstrable FIV-bearing cells were either within or subjacent to the mucosal epithelium or were in germinal centers of regional lymph nodes. The FIV(+) cells were of either of two morphological types, large stellate or small round. Those FIV RNA(+) cells which could be colabeled for a phenotype marker, were labeled for either dendritic-cell-associated protein p55 or T-lymphocyte receptor antigen CD3. These studies indicate that FIV crosses mucous membranes within hours after exposure and rapidly traffics via dendritic and T cells to systemic lymphoid tissues, a pathway similar to that thought to occur in the initial phase of infection by the human and simian immunodeficiency viruses.

  19. Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

    Directory of Open Access Journals (Sweden)

    Woods Matthew W

    2011-11-01

    Full Text Available Abstract Background The identification and characterization of several interferon (IFN-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5 that blocks a unique late stage of the HIV-1 life cycle. Results HERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus. Conclusions HERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.

  20. Recommendations for the measurement of FIV(1) values in chronic obstructive pulmonary disease.

    NARCIS (Netherlands)

    Visser, F.J.; Ramlal, S.; Dekhuijzen, P.N.R.; Heijdra, Y.F.

    2008-01-01

    BACKGROUND: In contrast to static inspiratory parameters such as vital capacity and inspiratory capacity, information on forced inspiratory volume in 1 s (FIV(1)) in patients with chronic obstructive pulmonary disease (COPD) is limited. OBJECTIVES: It was the aim of this study to investigate the inf

  1. Recommendations for the measurement of FIV(1) values in chronic obstructive pulmonary disease.

    NARCIS (Netherlands)

    Visser, F.J.; Ramlal, S.; Dekhuijzen, P.N.R.; Heijdra, Y.F.

    2008-01-01

    BACKGROUND: In contrast to static inspiratory parameters such as vital capacity and inspiratory capacity, information on forced inspiratory volume in 1 s (FIV(1)) in patients with chronic obstructive pulmonary disease (COPD) is limited. OBJECTIVES: It was the aim of this study to investigate the

  2. Prevalence and risk factors of feline leukaemia virus and feline immunodeficiency virus in peninsular Malaysia

    Directory of Open Access Journals (Sweden)

    Bande Faruku

    2012-03-01

    Full Text Available Abstract Background Feline leukaemia virus (FeLV and feline immunodeficiency virus (FIV are major causes of morbidity and mortality in domestic and wild felids. Despite the clinical importance of feline retroviruses and the growing interest in cats as pets, information about FeLV and FIV in Malaysia is presently insufficient to properly advise veterinarians and pet owners. A cross-sectional study was carried out from January 2010 to December 2010 to determine the prevalence and risk factors associated with FeLV and FIV among domestic cats in peninsular Malaysia. Plasma samples were harvested from the blood of 368 domestic cats and screened for evidence of FeLV p27 antigen and FIV antibodies, using an immunochromatographic kit. Additionally, data on cat demographics and health were collected using a structured questionnaire, and were evaluated as potential risk factors for FeLV or FIV status. Results Of the 368 cats that were evaluated in this study, 12.2% (45/368; 95% CI = 8.88 - 15.58 were positive for FeLV p27 antigen, 31.3%, (115/368; 95% CI = 26.51 - 35.99 were seropositive to FIV antibodies, and 4.3% (16/368; 95% CI = 2.27 - 6.43 had evidence of both viruses. Factors found to significantly increase the risk for FeLV seropositivity include sex, age, behaviour, sickness, and living in a multi-cat household. Seropositive response to FIV was significantly associated with sex, neuter status, age, behaviour, and health status. Conclusions The present study indicates that FeLV and FIV are common among domestic cats in peninsular Malaysia, and that factors related to cat demographics and health such as age, sex, behaviour, health status and type of household are important predictors for seropositive status to FeLV or FIV in peninsular Malaysia. High prevalence of FeLV or FIV observed in our study is of concern, in view of the immunosuppressive potentials of the two pathogens. Specific measures for control and prevention such as screening and

  3. Prevalence and risk factors of feline leukaemia virus and feline immunodeficiency virus in peninsular Malaysia.

    Science.gov (United States)

    Bande, Faruku; Arshad, Siti Suri; Hassan, Latiffah; Zakaria, Zunita; Sapian, Nurul Asyikin; Rahman, Noor Alimah; Alazawy, Amer

    2012-03-22

    Feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) are major causes of morbidity and mortality in domestic and wild felids. Despite the clinical importance of feline retroviruses and the growing interest in cats as pets, information about FeLV and FIV in Malaysia is presently insufficient to properly advise veterinarians and pet owners. A cross-sectional study was carried out from January 2010 to December 2010 to determine the prevalence and risk factors associated with FeLV and FIV among domestic cats in peninsular Malaysia. Plasma samples were harvested from the blood of 368 domestic cats and screened for evidence of FeLV p27 antigen and FIV antibodies, using an immunochromatographic kit. Additionally, data on cat demographics and health were collected using a structured questionnaire, and were evaluated as potential risk factors for FeLV or FIV status. Of the 368 cats that were evaluated in this study, 12.2% (45/368; 95% CI = 8.88 - 15.58) were positive for FeLV p27 antigen, 31.3%, (115/368; 95% CI = 26.51 - 35.99) were seropositive to FIV antibodies, and 4.3% (16/368; 95% CI = 2.27 - 6.43) had evidence of both viruses. Factors found to significantly increase the risk for FeLV seropositivity include sex, age, behaviour, sickness, and living in a multi-cat household. Seropositive response to FIV was significantly associated with sex, neuter status, age, behaviour, and health status. The present study indicates that FeLV and FIV are common among domestic cats in peninsular Malaysia, and that factors related to cat demographics and health such as age, sex, behaviour, health status and type of household are important predictors for seropositive status to FeLV or FIV in peninsular Malaysia. High prevalence of FeLV or FIV observed in our study is of concern, in view of the immunosuppressive potentials of the two pathogens. Specific measures for control and prevention such as screening and routine vaccination are needed to ensure that FeLV and FIV

  4. Fragmentation of SIV-gag vaccine induces broader T cell responses.

    Directory of Open Access Journals (Sweden)

    Adel Benlahrech

    Full Text Available BACKGROUND: High mutation rates of human immunodeficiency virus (HIV allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition. METHODOLOGY/PRINCIPAL FINDINGS: three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an in vitro human priming system. The breadth of the CD8 T cell response, defined by the number of distinct epitopes, was assessed by IFN-γ-ELISPOT and memory phenotype and cytokine production evaluated by flow cytometry. We observed an increase of two- to six-fold in the number of epitopes recognised in the ubiquitin-fused fragments compared to the ubiquitin-fused full-length gag. In contrast, although proteasomal targeting was achieved, there was a marked reduction in the number of epitopes recognised in the ubiquitin-fused full-length gag compared to the full-length unmodified gene, but there were no differences in the number of epitope responses induced by non-ubiquitinated full-length gag and the ubiquitin-fused mini genes. Fragmentation and ubiquitination did not affect T cell memory differentiation and polyfunctionality, though most responses were directed against the Ad5 vector. CONCLUSION/SIGNIFICANCE: Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response

  5. Induction of Multifunctional Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T Cells Capable of Proliferation in Healthy Subjects by Using a Prime-Boost Regimen of DNA- and Modified Vaccinia Virus Ankara-Vectored Vaccines Expressing HIV-1 Gag Coupled to CD8+ T-Cell Epitopes†

    Science.gov (United States)

    Goonetilleke, Nilu; Moore, Stephen; Dally, Len; Winstone, Nicola; Cebere, Inese; Mahmoud, Abdul; Pinheiro, Susana; Gillespie, Geraldine; Brown, Denise; Loach, Vanessa; Roberts, Joanna; Guimaraes-Walker, Ana; Hayes, Peter; Loughran, Kelley; Smith, Carole; De Bont, Jan; Verlinde, Carl; Vooijs, Danii; Schmidt, Claudia; Boaz, Mark; Gilmour, Jill; Fast, Pat; Dorrell, Lucy; Hanke, Tomas; McMichael, Andrew J.

    2006-01-01

    A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8+ T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2× MVA.HIVA) (n = 8) or two doses of placebo (2× placebo) (n = 4). The second group received 2× pTHr.HIVA followed by one dose of MVA.HIVA (n = 8) or 3× placebo (n = 4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-γ) ELISPOT (group mean, 210 spot-forming cells/106 cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2× MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-γ ELISPOT assay, positive responses mainly mediated by CD4+ T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2× MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4+ T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8+ T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1β. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees. PMID:16641265

  6. Increased T cell breadth and antibody response elicited in prime-boost regimen by viral vector encoded homologous SIV Gag/Env in outbred CD1 mice.

    Science.gov (United States)

    Andersson, Anne-Marie Carola; Holst, Peter Johannes

    2016-12-20

    A major obstacle for the development of HIV vaccines is the virus' worldwide sequence diversity. Nevertheless, the presence of T cell epitopes within conserved regions of the virus' structural Gag protein and conserved structures in the envelope (env) sequence raises the possibility that cross-reactive responses may be induced by vaccination. In this study, the aim was to investigate the importance of antigenic match on immunodominance and breadth of obtainable T cell responses. Outbred CD1 mice were immunized with either heterologous (SIVmac239 and HIV-1 clade B consensus) or homologous (SIVmac239) gag sequences using adenovirus (Ad5) and MVA vectors. Env (SIVmac239) was co-encoded in the vectors to study the induction of antibodies, which is a primary target of current HIV vaccine designs. All three vaccines were designed as virus-encoded virus-like particle vaccines. Antibody responses were analysed by ELISA, avidity ELISA, and neutralization assay. T cell responses were determined by intracellular cytokine staining of splenocytes. The homologous Env/Gag prime-boost regimen induced higher Env binding antibodies, and induced stronger and broader Gag specific CD8+ T cell responses than the homologous Env/heterologous Gag prime-boost regimen. Homologous Env/heterologous Gag immunization resulted in selective boosting of Env specific CD8+ T cell responses and consequently a paradoxical decreased recognition of variant sequences including conserved elements of p24 Gag. These results contrast with related studies using Env or Gag as the sole antigen and suggest that prime-boost immunizations based on homologous SIVmac239 Gag inserts is an efficient component of genetic VLP vaccines-both for induction of potent antibody responses and cross-reactive CD8+ T cell responses.

  7. Shared usage of the chemokine receptor CXCR4 by primary and laboratory-adapted strains of feline immunodeficiency virus.

    Science.gov (United States)

    Richardson, J; Pancino, G; Merat, R; Leste-Lasserre, T; Moraillon, A; Schneider-Mergener, J; Alizon, M; Sonigo, P; Heveker, N

    1999-05-01

    Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1alpha (SDF-1alpha). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1alpha, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.

  8. Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.

    Science.gov (United States)

    Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B

    2005-10-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

  9. Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis

    Science.gov (United States)

    Rivero, Matias; Cohen, Éric A.; Lopez-Lastra, Marcelo; Mouland, Andrew J.

    2013-01-01

    The precursor group-specific antigen (pr55Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55Gag expression, the synthesis of other HIV-1 proteins, including that of pr160Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55Gag for virus assembly and production. PMID:23861855

  10. Dual mechanisms of translation initiation of the full-length HIV-1 mRNA contribute to gag synthesis.

    Directory of Open Access Journals (Sweden)

    Anne Monette

    Full Text Available The precursor group-specific antigen (pr55(Gag is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES-mediated mechanisms. In support of this notion, pr55(Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55(Gag expression, the synthesis of other HIV-1 proteins, including that of pr160(Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55(Gag for virus assembly and production.

  11. HIV-1 Subtype C Mosaic Gag Expressed by BCG and MVA Elicits Persistent Effector T Cell Responses in a Prime-Boost Regimen in Mice.

    Directory of Open Access Journals (Sweden)

    Tsungai Ivai Jongwe

    Full Text Available Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG ΔpanCD auxotroph and modified vaccinia Ankara (MVA vaccines expressing HIV-1C mosaic Gag (GagM were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-GagM vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-GagM and boosting with MVA-GagM elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-GagM only and MVA-GagM only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4+ and CD8+ T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (104 pfu can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C.

  12. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

    Directory of Open Access Journals (Sweden)

    Nguyen Albert T

    2011-12-01

    Full Text Available Abstract Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1 maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1 is cleaved to p24 (CA and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR. Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

  13. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

    Science.gov (United States)

    2011-01-01

    Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors. PMID:22151792

  14. Parameters of disease progression in long-term experimental feline retrovirus (feline immunodeficiency virus and feline leukemia virus) infections: hematology, clinical chemistry, and lymphocyte subsets.

    Science.gov (United States)

    Hofmann-Lehmann, R; Holznagel, E; Ossent, P; Lutz, H

    1997-01-01

    After several years of latency, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) cause fatal disease in the cat. The aim of this study was to determine laboratory parameters characteristic of disease progression which would allow a better description of the asymptomatic phase and a better understanding of the pathogenesis of the two infections. Therefore, experimentally infected cats (FIV and/or FeLV positive) and control animals were observed over a period of 6.5 years under identical conditions. Blood samples were analyzed for the following: complete hematology, clinical chemistry, serum protein electrophoresis, and determination of CD4+ and CD8+ lymphocyte subsets. The following hematological and clinical chemistry parameters were markedly changed in the FIV-infected animals from month 9 onwards: glucose, serum protein, gamma globulins, sodium, urea, phosphorus, lipase, cholesterol, and triglyceride. In FeLV infection, the markedly changed parameters were mean corpuscular volume, mean corpuscular hemoglobin, aspartate aminotransferase, and urea. In contrast to reports of field studies, neither FIV-positive nor FeLV-positive animals developed persistent leukopenia, lymphopenia, or neutropenia. A significant decrease was found in the CD4+/CD8+ ratio in FIV-positive and FIV-FeLV-positive animals mainly due to loss of CD4+ lymphocytes. In FeLV-positive cats, both CD4+ and, to a lesser degree, CD8+ lymphocytes were decreased in long-term infection. The changes in FIV infection may reflect subclinical kidney dysfunction, changes in energy and lipid metabolism, and transient activation of the humoral immune response as described for human immunodeficiency virus (HIV) infections. The changes in FeLV infection may also reflect subclinical kidney dysfunction and, in addition, changes in erythrocyte and immune function of the animals. No severe clinical signs were observed in the FIV-positive cats, while FeLV had a severe influence on the life

  15. Seroprevalence of Toxoplasma gondii and concurrent bartonella spp., feline immunodeficiency virus, and feline leukemia infections in cats from Grenada, West Indies

    Science.gov (United States)

    Toxoplasma gondii and Bartonella spp. are zoonotic pathogens of cats. Feline Immunodeficiency Virus (FIV), and Feline Leukemia Virus (FeLv) are related to Human Iimmunodeficiency Virus, and Human Leukemia Virus, respectively, and these viruses are immunosuppressive. In the present study, the prevale...

  16. Feline immunodeficiency virus testing in stray, feral, and client-owned cats of Ottawa.

    Science.gov (United States)

    Little, Susan E

    2005-10-01

    Feline immunodeficiency virus (FIV) seroprevalence is evaluated in 3 groups of cats. Seventy-four unowned urban strays were tested, as well as 20 cats from a small feral cat colony, and 152 client-owned cats. Of the 246 cats tested, 161 (65%) were male and 85 (35%) were female. Seroprevalence for FIV was 23% in the urban strays, 5% in the feral cat colony, and 5.9% in the client-owned cats. Ten cats (4%) were also positive for Feline leukemia virus (FeLV) antigen, including 2 cats coinfected with FeLV and FIV. Seroprevalence for FIV in cats from Ottawa is similar to that found in other nonrandom studies of cats in North America.

  17. Increased t cell breadth and antibody response elicited in prime-boost regimen by viral vector encoded homologous siv gag/env in outbred cd1 mice

    DEFF Research Database (Denmark)

    Andersson, Anne Marie Carola; Holst, Peter Johannes

    2016-01-01

    Background: A major obstacle for the development of HIV vaccines is the virus' worldwide sequence diversity. Nevertheless, the presence of T cell epitopes within conserved regions of the virus' structural Gag protein and conserved structures in the envelope (env) sequence raises the possibility...

  18. Molecular characterization of the HIV-1 gag nucleocapsid gene associated with vertical transmission

    Directory of Open Access Journals (Sweden)

    Ramakrishnan Rajesh

    2006-04-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 nucleocapsid (NC plays a pivotal role in the viral lifecycle: including encapsulating the viral genome, aiding in strand transfer during reverse transcription, and packaging two copies of the viral genome into progeny virions. Another gag gene product, p6, plays an integral role in successful viral budding from the plasma membrane and inclusion of the accessory protein Vpr within newly budding virions. In this study, we have characterized the gag NC and p6 genes from six mother-infant pairs following vertical transmission by performing phylogenetic analysis and by analyzing the degree of genetic diversity, evolutionary dynamics, and conservation of functional domains. Results Phylogenetic analysis of 168 gag NC and p6 genes sequences revealed six separate subtrees that corresponded to each mother-infant pair, suggesting that epidemiologically linked individuals were closer to each other than epidemiologically unlinked individuals. A high frequency (92.8% of intact open reading frames of NC and p6 with patient and pair specific sequence motifs were conserved in mother-infant pairs' sequences. Nucleotide and amino acid distances showed a lower degree of viral heterogeneity, and a low degree of estimates of genetic diversity was also found in NC and p6 sequences. The NC and p6 sequences from both mothers and infants were found to be under positive selection pressure. The two important functional motifs within NC, the zinc-finger motifs, were highly conserved in most of the sequences, as were the gag p6 Vpr binding, AIP1 and late binding domains. Several CTL recognition epitopes identified within the NC and p6 genes were found to be mostly conserved in 6 mother-infant pairs' sequences. Conclusion These data suggest that the gag NC and p6 open reading frames and functional domains were conserved in mother-infant pairs' sequences following vertical transmission, which confirms the

  19. Prevalence of feline leukaemia virus and antibodies to feline immunodeficiency virus in cats in the United Kingdom.

    Science.gov (United States)

    Hosie, M J; Robertson, C; Jarrett, O

    1989-09-09

    A representative sample of the pet cat population of the United Kingdom was surveyed. Blood samples from 1204 sick and 1007 healthy cats of known breed, age and sex were tested for antibodies to feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). The prevalence of FIV was 19 per cent in sick cats and 6 per cent in healthy cats, and the prevalence of FeLV was 18 per cent in sick cats and 5 per cent in healthy cats; both infections were more common in domestic cats than in pedigree cats. Feline immunodeficiency virus was more prevalent in older cats but FeLV was more prevalent in younger cats. There was no difference between the prevalence of FeLV in male and female cats but male cats were more likely to be infected with FIV than female cats. No interaction was demonstrated between FIV and FeLV infections. Of the cats which were in contact with FIV in households with more than one cat, 21 per cent had seroconverted. The prevalence of FeLV viraemia in cats in contact with FeLV was 14 per cent. The clinical signs associated with FIV were pyrexia, gingivitis/stomatitis and respiratory signs, and with FeLV, pyrexia and anaemia. It was concluded that both viruses were significant causes of disease, and that the cats most likely to be infected with FIV were older, free-roaming male cats and for FeLV, younger, free-roaming cats.

  20. Modulation of HIV-1 infectivity and cyclophilin A-dependence by Gag sequence and target cell type

    Directory of Open Access Journals (Sweden)

    Dam Elisabeth

    2009-03-01

    Full Text Available Abstract Background HIV-1 Gag proteins are essential for virion assembly and viral replication in newly infected cells. Gag proteins are also strong determinants of viral infectivity; immune escape mutations in the Gag capsid (CA protein can markedly reduce viral fitness, and interactions of CA with host proteins such as cyclophilin A (CypA and TRIM5α can have important effects on viral infectivity. Little information, however, is available concerning the extent that different primary Gag proteins affect HIV-1 replication in different cell types, or the impact on viral replication of differences in the expression by target cells of proteins that interact with CA. To address these questions, we compared the infectivity of recombinant HIV-1 viruses expressing Gag-protease sequences from primary isolates in different target cells in the presence or absence of agents that disrupt cyclophilin A – CA interactions and correlated these results with the viral genotype and the expression of cyclophilin A and TRIM5α by the target cells. Results Viral infectivity was governed by the nature of the Gag proteins in a target cell-specific fashion. The treatment of target cells with agents that disrupt CypA-CA interactions often produced biphasic dose-response curves in which viral infectivity first increased and subsequently decreased as a function of the dose used. The extent that treatment of target cells with high-dose CypA inhibitors impaired viral infectivity was dependent on several factors, including the viral genotype, the nature of the target cell, and the extent that treatment with low-dose CypA inhibitors increased viral infectivity. Neither the presence of polymorphisms in the CA CypA-binding loop, the level of expression of CypA, or the level of TRIM5α expression could, alone, explain the differences in the shape of the dose-response curves observed or the extent that high-dose CypA inhibitors reduced viral infectivity. Conclusion Multiple

  1. HIV-1 Gag Blocks Selenite-Induced Stress Granule Assembly by Altering the mRNA Cap-Binding Complex

    Directory of Open Access Journals (Sweden)

    Alessandro Cinti

    2016-03-01

    Full Text Available Stress granules (SGs are dynamic accumulations of stalled preinitiation complexes and translational machinery that assemble under stressful conditions. Sodium selenite (Se induces the assembly of noncanonical type II SGs that differ in morphology, composition, and mechanism of assembly from canonical SGs. Se inhibits translation initiation by altering the cap-binding activity of eukaryotic translation initiation factor 4E (eIF4E-binding protein 1 (4EBP1. In this work, we show that human immunodeficiency virus type 1 (HIV-1 Gag is able to block the assembly of type II noncanonical SGs to facilitate continued Gag protein synthesis. We demonstrate that expression of Gag reduces the amount of hypophosphorylated 4EBP1 associated with the 5′ cap potentially through an interaction with its target, eIF4E. These results suggest that the assembly of SGs is an important host antiviral defense that HIV-1 has evolved for inhibition through several distinct mechanisms.

  2. Relationship of lymphoid lesions to disease course in mucosal feline immunodeficiency virus type C infection.

    Science.gov (United States)

    Obert, L A; Hoover, E A

    2000-09-01

    Feline immunodeficiency virus (FIV) infection typically has a prolonged and variable disease course in cats, which can limit its usefulness as a model for human immunodeficiency virus infection. A clade C FIV isolate (FIV-C) has been associated with high viral burdens and rapidly progressive disease in cats. FIV-C was transmissible via oral-nasal, vaginal, or rectal mucosal exposure, and infection resulted in one of three disease courses: rapid, conventional/slow, or regressive. The severity of the pathologic changes paralleled the disease course. Thymic depletion was an early lesion and was correlated with detection of FIV RNA in thymocytes by in situ hybridization. The major changes in thymic cell populations were depletion of p55+/S100+ dendritic cells, CD3- cells, CD4+/CD8- cells, and CD4+/CD8+ cells and increases in apoptosis, CD45R+ B cells, and lymphoid follicles. In contrast to thymic depletion, peripheral lymphoid tissues often were hyperplastic. Mucosally transmitted FIV-C is thymotropic and induces a spectrum of lymphoid lesions and disease mirroring that seen with the human and simian immunodeficiency virus infections.

  3. Interactions between nattokinase and heparin/GAGs.

    Science.gov (United States)

    Zhang, Fuming; Zhang, Jianhua; Linhardt, Robert J

    2015-12-01

    Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK's potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6-O-sulfo as well as the N-sulfo groups but not the 2-O-sulfo groups within heparin, are essential for heparin's interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin's interaction with antithrombin and fibroblast growth factors.

  4. Mechanisms of human immunodeficiency virus type 2 RNA packaging

    DEFF Research Database (Denmark)

    Ni, Na; Nikolaitchik, Olga A; Dilley, Kari A;

    2011-01-01

    Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2...... proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results...... do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins...

  5. Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s.

    Science.gov (United States)

    Gu, Qinyong; Zhang, Zeli; Cano Ortiz, Lucía; Franco, Ana Cláudia; Häussinger, Dieter; Münk, Carsten

    2016-12-01

    Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain.

  6. Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2007-10-01

    Full Text Available Abstract Background Treatment of feline immunodeficiency virus (FIV infection has been hampered by the absence of a specific combination antiretroviral treatment (ART. Integrase strand transfer inhibitors (INSTIs are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD. Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound

  7. Use of training dentures in management of gagging

    Directory of Open Access Journals (Sweden)

    Shweta Yadav

    2011-01-01

    Full Text Available Gagging is a frequent impediment to the performance of dental procedures. This stimulation of the gagging reflex, or more accurately, the vomiting reflex, is a special problem in prosthodontic service. A hypersensitive gagging reflex often prevents the dentist from carrying out critical procedures or causes them to performat a less than satisfactory level. In addition, once having suffered an unpleasant gagging experience in a dentist′s office, the patients develop a fear of further visits to dentists. The purpose of this paper is to describe methods of managing the gagging patient that has a sound rationale based on modified treatment approaches starting from impression making to design of the prosthesis aided by training dentures to help the patient to tolerate prosthesis in mouth before fabrication of definite prosthesis.

  8. Heparin (GAG-hed inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    Directory of Open Access Journals (Sweden)

    López-Bayghen Esther

    2006-08-01

    Full Text Available Abstract Background High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR, plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs, such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Methods Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. Results We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Conclusion Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell

  9. Development of clinical disease in cats experimentally infected with feline immunodeficiency virus.

    Science.gov (United States)

    English, R V; Nelson, P; Johnson, C M; Nasisse, M; Tompkins, W A; Tompkins, M B

    1994-09-01

    Cats naturally infected with feline immunodeficiency virus (FIV) develop an AIDS-like syndrome whereas experimentally infected cats do not. To investigate the role of cofactors in the development of this disease in cats, 7 specific pathogen-free (SPF) and 12 random-source (RS) cats were infected with FIV. Over 4 years, infected cats developed similar phenotypic and functional immune abnormalities characterized by early and chronic inversion of CD4+:CD8+ cell ratios and significantly decreased mitogen responses compared with controls. Beginning 18-24 months after infection, 10 RS cats developed chronic clinical disease typical of feline AIDS, including stomatitis and recurrent upper respiratory disease; 4 SPF cats also developed chronic clinical disease, 2 with neurologic disease and 2 with B cell lymphomas. Thus, immunologic background is important in the type of disease that develops in cats infected with FIV, and FIV represents a promising animal model for studying the immunopathogenesis of AIDS in humans.

  10. Domain- and nucleotide-specific Rev response element regulation of feline immunodeficiency virus production.

    Science.gov (United States)

    Na, Hong; Huisman, Willem; Ellestad, Kristofor K; Phillips, Tom R; Power, Christopher

    2010-09-01

    Computational analysis of feline immunodeficiency virus (FIV) RNA sequences indicated that common FIV strains contain a rev response element (RRE) defined by a long unbranched hairpin with 6 stem-loop sub-domains, termed stem-loop A (SLA). To examine the role of the RNA secondary structure of the RRE, mutational analyses were performed in both an infectious FIV molecular clone and a FIV CAT-RRE reporter system. These studies disclosed that the stems within SLA (SA1, 2, 3, 4, and 5) of the RRE were critical but SA6 was not essential for FIV replication and CAT expression. These studies also revealed that the secondary structure rather than an antisense protein (ASP) mediates virus expression and replication in vitro. In addition, a single synonymous mutation within the FIV-RRE, SA3/45, reduced viral reverse transcriptase activity and p24 expression after transfection but in addition also showed a marked reduction in viral expression and production following infection.

  11. Intracellular characterization of Gag VLP production by transient transfection of HEK 293 cells.

    Science.gov (United States)

    Cervera, Laura; González-Domínguez, Irene; Segura, María Mercedes; Gòdia, Francesc

    2017-11-01

    Transient transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 min of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 10(10) VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 h post-transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet

  12. The Effect of Glycosaminoglycans (GAGs on Amyloid Aggregation and Toxicity

    Directory of Open Access Journals (Sweden)

    Clara Iannuzzi

    2015-02-01

    Full Text Available Amyloidosis is a protein folding disorder in which normally soluble proteins are deposited extracellularly as insoluble fibrils, impairing tissue structure and function. Charged polyelectrolytes such as glycosaminoglycans (GAGs are frequently found associated with the proteinaceous deposits in tissues of patients affected by amyloid diseases. Experimental evidence indicate that they can play an active role in favoring amyloid fibril formation and stabilization. Binding of GAGs to amyloid fibrils occurs mainly through electrostatic interactions involving the negative polyelectrolyte charges and positively charged side chains residues of aggregating protein. Similarly to catalyst for reactions, GAGs favor aggregation, nucleation and amyloid fibril formation functioning as a structural templates for the self-assembly of highly cytotoxic oligomeric precursors, rich in β-sheets, into harmless amyloid fibrils. Moreover, the GAGs amyloid promoting activity can be facilitated through specific interactions via consensus binding sites between amyloid polypeptide and GAGs molecules. We review the effect of GAGs on amyloid deposition as well as proteins not strictly related to diseases. In addition, we consider the potential of the GAGs therapy in amyloidosis.

  13. Association of VH4-59 Antibody Variable Gene Usage with Recognition of an Immunodominant Epitope on the HIV-1 Gag Protein.

    Directory of Open Access Journals (Sweden)

    Valentine U Chukwuma

    Full Text Available The human antibody response against HIV-1 infection recognizes diverse antigenic subunits of the virion, and includes a high level of antibodies to the Gag protein. We report here the isolation and characterization of a subset of Gag-specific human monoclonal antibodies (mAbs that were prevalent in the antibody repertoire of an HIV-infected individual. Several lineages of Gag-specifc mAbs were encoded by a single antibody heavy chain variable region, VH4-59, and a representative antibody from this group designated mAb 3E4 recognized a linear epitope on the globular head of the p17 subunit of Gag. We found no evidence that mAb 3E4 exhibited any function in laboratory studies aimed at elucidating the immunologic activity, including assays for neutralization, Ab-dependent cell-mediated virus inhibition, or enhanced T cell reactivity caused by Gag-3E4 complexes. The findings suggest this immunodominant epitope in Gag protein, which is associated with VH4-59 germline gene usage, may induce a high level of B cells that encode binding but non-functional antibodies that occupy significant repertoire space following HIV infection. The studies define an additional specific molecular mechanism in the immune distraction activity of the HIV virion.

  14. Evaluation of vaccination strategies against infection with feline immunodeficiency virus (FIV) based on recombinant viral vectors expressing FIV Rev and OrfA

    NARCIS (Netherlands)

    Huisman, W.; Schrauwen, E.J.A.; Tijhaar, E.; Süzer, Y.; Pas, S.D.; Amerongen, van G.; Sutter, G.; Rimmelzwaan, G.F.; Osterhaus, A.D.M.E.

    2008-01-01

    In recent years it has become clear that cell-mediated immunity is playing a role in the control of lentivirus infections. In particular, cytotoxic T lymphocyte responses have been associated with improved outcome of infection, especially those directed against the regulatory proteins like Rev and T

  15. Evidence of feline immunodeficiency virus, feline leukemia virus, and Toxoplasma gondii in feral cats on Mauna Kea, Hawaii

    Science.gov (United States)

    Danner, R.M.; Goltz, Dan M.; Hess, S.C.; Banko, P.C.

    2007-01-01

    We determined prevalence to feline immunodeficiency virus (FIV) antibodies, feline leukemia virus (FeLV) antigen, and Toxoplasma gondii antibodies in feral cats (Felis catus) on Mauna Kea Hawaii from April 2002 to May 2004. Six of 68 (8.8%) and 11 of 68 (16.2%) cats were antibody positive to FIV and antigen positive for FeLV, respectively; 25 of 67 (37.3%) cats were seropositive to T. gondii. Antibodies to FeLV and T. gondii occurred in all age and sex classes, but FIV occurred only in adult males. Evidence of current or previous infections with two of these infectious agents was detected in eight of 64 cats (12.5%). Despite exposure to these infectious agents, feral cats remain abundant throughout the Hawaiian Islands. ?? Wildlife Disease Association 2007.

  16. Evidence of feline immunodeficiency virus, feline leukemia virus, and Toxoplasma gondii in feral cats on Mauna Kea, Hawaii.

    Science.gov (United States)

    Danner, Raymond M; Goltz, Daniel M; Hess, Steven C; Banko, Paul C

    2007-04-01

    We determined prevalence to feline immunodeficiency virus (FIV) antibodies, feline leukemia virus (FeLV) antigen, and Toxoplasma gondii antibodies in feral cats (Felis catus) on Mauna Kea Hawaii from April 2002 to May 2004. Six of 68 (8.8%) and 11 of 68 (16.2%) cats were antibody positive to FIV and antigen positive for FeLV, respectively; 25 of 67 (37.3%) cats were seropositive to T. gondii. Antibodies to FeLV and T. gondii occurred in all age and sex classes, but FIV occurred only in adult males. Evidence of current or previous infections with two of these infectious agents was detected in eight of 64 cats (12.5%). Despite exposure to these infectious agents, feral cats remain abundant throughout the Hawaiian Islands.

  17. Rescue of HIV-1 release by targeting widely divergent NEDD4-type ubiquitin ligases and isolated catalytic HECT domains to Gag.

    Directory of Open Access Journals (Sweden)

    Eric R Weiss

    Full Text Available Retroviruses engage the ESCRT pathway through late assembly (L domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA. The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release.

  18. Adaptation and Study of AIDS Viruses in Animal and Cell Culture Systems

    Science.gov (United States)

    1989-01-30

    category one, e.g , Friend Murine -6- Leukemia Virus (FMuLV), Feline Leukemia Virus (FeLV), and the Macaque Type D SAIDS retrovirus (SRV) have been...10). One other animal lentivirus, Feline Immunodeficiency Virus (FIV), has had some utility in the study of protective immunity and in screening...et al. (58) transplanted RNA mumps virus infected human HeLa cells, or RNA vesicular stomatitis virus-infected hamster BHK cells into nude mice

  19. Epidemiology of Feline Foamy Virus and Feline Immunodeficiency Virus Infections in Domestic and Feral Cats: a Seroepidemiological Study

    Science.gov (United States)

    Winkler, I. G.; Löchelt, M.; Flower, R. L. P.

    1999-01-01

    Although foamy viruses (Spumaviruses) have repeatedly been isolated from both healthy and diseased cats, cattle, and primates, the primary mode of transmission of those common viruses remains undefined. A database of the feline foamy virus (FeFV) and feline immunodeficiency virus (FIV) antibody status, age, and sex of 389 domestic cats presented to veterinarians was assembled. A similar database for 66 feral (wild) cats was also assembled. That FeFV antibody status reflects infection was validated by PCR. Both FeFV and FIV infection rates were found to gradually increase with age, and over 70% of cats older than 9 years were seropositive for FeFV. In domestic cats, the prevalence of FeFV infection was similar in both sexes. In feral cats, FeFV infection was more prevalent in female cats than in male cats. Although both FeFV and FIV have been reported to be transmitted by biting, the patterns of infection observed are more consistent with an interpretation that transmission of these two retroviruses is not the same. The prevalence of FIV infection is highest in nondesexed male cats, the animals most likely to display aggressive behavior. The gradual increase in the proportion of FeFV-infected animals is consistent with transmission of foamy viruses by intimate social contact between animals and less commonly by aggressive behavior. PMID:10449463

  20. Bicyclams, selective antagonists of the human chemokine receptor CXCR4, potently inhibit feline immunodeficiency virus replication

    NARCIS (Netherlands)

    Horzinek, M.C.; Egberink, H.F.; Clercq, E. de; Vliet, A.L.W. van; Balzarini, J.; Bridger, G.J.; Henson, G.; Schols, D.

    1999-01-01

    Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evalua

  1. The oral and conjunctival microbiotas in cats with and without feline immunodeficiency virus infection.

    Science.gov (United States)

    Weese, Scott J; Nichols, Jamieson; Jalali, Mohammad; Litster, Annette

    2015-03-03

    The oral and conjunctival microbiotas likely play important roles in protection from opportunistic infections, while also being the source of potential pathogens. Yet, there has been limited investigation in cats, and the impact of comorbidities such as feline immunodeficiency virus (FIV) infection has not been reported. Oral and conjunctival swabs were collected from cats with FIV infection and FIV-uninfected controls, and subjected to 16S rRNA gene (V4) PCR and next generation sequencing. 9,249 OTUs were identified from conjunctival swabs, yet the most common 20 (0.22%) OTUs accounted for 76% of sequences. The two most abundant OTUs both belonged to Staphylococcus, and accounted for 37% of sequences. Cats with FIV infection had significantly lower relative abundances of Verrucomicrobia, Fibrobacteres, Spirochaetes, Bacteroidetes and Tenericutes, and a higher relative abundance of Deinococcus-Thermus. There were significant differences in both community membership (P = 0.006) and community structure (P = 0.02) between FIV-infected and FIV-uninfected cats. FIV-infected cats had significantly higher relative abundances of Fusobacteria and Actinobacteria in the oral cavity, and significantly higher relative abundances of several bacterial classes including Fusobacteria (0.022 vs 0.007, P = 0.006), Actinobacteria (0.017 vs 0.003, P = 0.003), Sphingobacteria (0.00015 vs 0.00003, P = 0.0013) and Flavobacteria (0.0073 vs 0.0034, P = 0.030). The feline conjunctival and oral microbiotas are complex polymicrobial communities but dominated by a limited number of genera. There is an apparent impact of FIV infection on various components of the microbiota, and assessment of the clinical relevance of these alterations in required.

  2. Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

    OpenAIRE

    Ahi, Yadvinder S.; Shu Zhang; Yashna Thappeta; Audrey Denman; Amin Feizpour; Suryaram Gummuluru; Bjoern Reinhard; Delphine Muriaux; Fivash, Matthew J.; Alan Rein

    2016-01-01

    ABSTRACT Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, th...

  3. Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

    Science.gov (United States)

    Nowicka-Sans, Beata; Protack, Tricia; Lin, Zeyu; Li, Zhufang; Zhang, Sharon; Sun, Yongnian; Samanta, Himadri; Terry, Brian; Liu, Zheng; Chen, Yan; Sin, Ny; Sit, Sing-Yuen; Swidorski, Jacob J; Chen, Jie; Venables, Brian L; Healy, Matthew; Meanwell, Nicholas A; Cockett, Mark; Hanumegowda, Umesh; Regueiro-Ren, Alicia; Krystal, Mark; Dicker, Ira B

    2016-07-01

    BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1

  4. Vaccination against feline immunodeficiency virus using fixed infected cells

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Vliet, A.L.W. van; Egberink, H.F.; Hesselink, W.; Alphen, W.E. van; Joosten, I.; Boog, C.J.P.; Ronde, A. de

    1995-01-01

    Crandell feline kidney cells and feline thymocytes, either feline immunodeficiency virus (FIV) infected or uninfected, were fixed with paraformaldehyde and used to vaccinate cats. The cells were mixed with a 30:70 water/mineral oil emulsion containing 250 mu g ml−1 N-acetyl-d-glucosaminyl-beta-(1 4)

  5. Feline immunodeficiency virus testing in stray, feral, and client-owned cats of Ottawa

    OpenAIRE

    Susan E. Little

    2005-01-01

    Feline immunodeficiency virus (FIV) seroprevalence is evaluated in 3 groups of cats. Seventy-four unowned urban strays were tested, as well as 20 cats from a small feral cat colony, and 152 client-owned cats. Of the 246 cats tested, 161 (65%) were male and 85 (35%) were female. Seroprevalence for FIV was 23% in the urban strays, 5% in the feral cat colony, and 5.9% in the client-owned cats. Ten cats (4%) were also positive for Feline leukemia virus (FeLV) antigen, including 2 cats coinfected ...

  6. [The role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and the inhibitors].

    Science.gov (United States)

    Jiang, Yan; Liu, Xin-yong

    2010-02-01

    The late stages of the HIV-1 replication cycle are important to the overall replication cycle. During the late stages, HIV-1 replication undergoes the processes of assembly, release, and maturation, resulting in the production of a mature virus particle capable of infecting a new target cell. The structural protein Gag and its related gene (protein) play a central role in these pathways. The different regions of Gag worked in concert to drive production of a mature infectious particle through protein-protein, protein-RNA and protein-lipid interactions. The designed drug aimed directly at these stages can efficiently block the maturation and infectivity of HIV-1. In this article, the role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and related inhibitors is reviewed.

  7. Feline Immunodeficiency Virus in South America

    Directory of Open Access Journals (Sweden)

    Bruno M. Teixeira

    2012-03-01

    Full Text Available The rapid emergence of AIDS in humans during the period between 1980 and 2000 has led to extensive efforts to understand more fully similar etiologic agents of chronic and progressive acquired immunodeficiency disease in several mammalian species. Lentiviruses that have gene sequence homology with human immunodeficiency virus (HIV have been found in different species (including sheep, goats, horses, cattle, cats, and several Old World monkey species. Lentiviruses, comprising a genus of the Retroviridae family, cause persistent infection that can lead to varying degrees of morbidity and mortality depending on the virus and the host species involved. Feline immunodeficiency virus (FIV causes an immune system disease in domestic cats (Felis catus involving depletion of the CD4+ population of T lymphocytes, increased susceptibility to opportunistic infections, and sometimes death. Viruses related to domestic cat FIV occur also in a variety of nondomestic felids. This is a brief overview of the current state of knowledge of this large and ancient group of viruses (FIVs in South America.

  8. A modified edentulous maxillary custom tray to help prevent gagging.

    Science.gov (United States)

    Callison, G M

    1989-07-01

    The gag reflex can be a normal, healthy defense mechanism to prevent foreign objects from entering the trachea. During certain dental procedures, however, gagging can greatly complicate the final result, especially during the maxillary complete denture final impression. A modification can be made to the maxillary custom acrylic resin tray to aid in securing a clinically acceptable elastomeric final impression. This modification involves forming a vacuum chamber at the posterior extent of the custom tray to which a saliva ejector tip is embedded. When the saliva ejector is connected to the low-volume evacuation hose, the chamber will trap any excess impression material that might extrude from the posterior border of the loaded tray. This results in a reduced chance of eliciting the patient's gag reflex.

  9. Ocorrência do vírus da imunodeficiência felina e do vírus da leucemia felina em gatos domésticos mantidos em abrigos no município de Belo Horizonte Occurrence of feline immunodeficiency virus and feline leukemia virus in Sheltered domestic cats of Belo Horizonte

    Directory of Open Access Journals (Sweden)

    B.M. Teixeira

    2007-08-01

    Full Text Available Investigou-se a ocorrência da infecção pelo vírus da imunodeficiência felina (FIV e pelo vírus da leucemia felina (FeLV em gatos domésticos, provenientes de dois abrigos, no município de Belo Horizonte. Amostras de sangue de 145 animais foram coletadas e testadas para detecção do FIV pela reação em cadeia da polimerase (PCR. Destas amostras, 40 foram testadas para o antígeno p26 de FeLV por meio de ELISA. Observaram-se duas fêmeas (1,4% e quatro machos (2,8% positivos para FIV e nove fêmeas (22,5% e quatro machos (10,0% positivos para FeLV.The occurrence of the feline immunodeficiency virus (FIV and feline leukemia virus (FeLV was investigated in domestic cats from two shelters of Belo Horizonte. Samples from 145 cats were collected and tested for FIV by the polymerase chain reaction (PCR. Forty out of 145 samples were tested for FeLV p27 antigen by a commercial ELISA kit. Two females (1.4% and four males (2.8% were positive for FIV. For FeLV tests, 13 cats (32.5% were positive, being nine females (22.5% and four males (10.0%.

  10. Gag Proteins of Drosophila Telomeric Retrotransposons: Collaborative Targeting to Chromosome Ends

    Science.gov (United States)

    Fuller, Adelaide M.; Cook, Elizabeth G.; Kelley, Kerry J.; Pardue, Mary-Lou

    2010-01-01

    TAHRE, the least abundant of the three retrotransposons forming telomeres in Drosophila melanogaster, has high sequence similarity to the gag gene and untranslated regions of HeT-A, the most abundant telomere-specific retrotransposon. Despite TAHRE's apparent evolutionary relationship to HeT-A, we find TAHRE Gag cannot locate to telomere-associated “Het dots” unless collaborating with HeT-A Gag. TAHRE Gag is carried into nuclei by HeT-A or TART Gag, but both TART and TAHRE Gags need HeT-A Gag to localize to Het dots. When coexpressed with the appropriate fragment of HeT-A and/or TART Gags, TAHRE Gag multimerizes with either protein. HeT-A and TART Gags form homo- and heteromultimers using a region containing major homology region (MHR) and zinc knuckle (CCHC) motifs, separated by a pre_C2HC motif (motifs common to other retroelements). This region's sequence is strongly conserved among the three telomeric Gags, with precise spacing of conserved residues. Nontelomeric Gags neither interact with the telomeric Gags nor have this conserved spacing. TAHRE Gag is much less able to enter the nucleus by itself than HeT-A or TART Gags. The overall telomeric localization efficiency for each of the three telomeric Gag proteins correlates with the relative abundance of that element in telomere arrays, suggesting an explanation for the relative rarity of TAHRE elements in telomere arrays and supporting the hypothesis that Gag targeting to telomeres is important for the telomere-specific transposition of these elements. PMID:20026680

  11. Characterization of glycosaminoglycan (GAG) sulfatases from the human gut symbiont Bacteroides thetaiotaomicron reveals the first GAG-specific bacterial endosulfatase.

    Science.gov (United States)

    Ulmer, Jonathan E; Vilén, Eric Morssing; Namburi, Ramesh Babu; Benjdia, Alhosna; Beneteau, Julie; Malleron, Annie; Bonnaffé, David; Driguez, Pierre-Alexandre; Descroix, Karine; Lassalle, Gilbert; Le Narvor, Christine; Sandström, Corine; Spillmann, Dorothe; Berteau, Olivier

    2014-08-29

    Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973-25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Preferential CTL targeting of Gag is associated with relative viral control in long-term surviving HIV-1 infected former plasma donors from China

    Institute of Scientific and Technical Information of China (English)

    Mingming Jia; Quanbi Zhao; Dan Li; Hong Peng; Marcus Altfeld; Bruce D Walker; Xu G Yu; Yiming Shao; Kunxue Hong; Jianping Chen; Yuhua Ruan; Zhe Wang; Bing Su; Guoliang Ren; Xiaoqing Zhang; Zhen Liu

    2012-01-01

    It is generally believed that CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection,and this effect may partly depend on which HIV product is preferentially targeted.To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs),143 antiretroviral therapy naive FPDs infected with HIV-1 clade B' strains were assessed for HIV-1-specific CTL responses with an IFN-γElispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome.By using a Spearman's rank correlation analysis,we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts,as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts,In addition,Vpr-specifc CTL responses showed a similar protective effect with Gag responses,but with a much lower frequency of recognition.Significantly,we also observed an association between HLA-A*30/B*13/Cw*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses.Thus,our data demonstrate the prominent role of Gag-specific CTL responses in disease control.The advantage of HLA-A*30/B*13/Cw*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.

  13. Anxiety, fainting and gagging in dentistry: Separate or overlapping constructs?

    NARCIS (Netherlands)

    Houtem, C.M.H.H. van

    2016-01-01

    This thesis aimed to increase the knowledge about severe forms of anxiety, gagging and fainting in dentistry and to investigate whether these phenomena are overlapping or separate constructs. In Chapter 2 a literature review of twin studies showed that the estimated heritability of specific phobias

  14. Development of the gagging problem assessment : A pilot study

    NARCIS (Netherlands)

    van den Heuvell, G. F. E. C. van Linden; Ter Pelkwijk, B. J.; Stegenga, B.

    An accepted management strategy for gagging problems appears not to exist. A reliable and valid instrument is needed to develop an evidence based treatment for this clinical problem. The aim of this pilot study was to evaluate the reliability and validity of a newly developed measurement tool for

  15. Development of the gagging problem assessment : A pilot study

    NARCIS (Netherlands)

    van den Heuvell, G. F. E. C. van Linden; Ter Pelkwijk, B. J.; Stegenga, B.

    2008-01-01

    An accepted management strategy for gagging problems appears not to exist. A reliable and valid instrument is needed to develop an evidence based treatment for this clinical problem. The aim of this pilot study was to evaluate the reliability and validity of a newly developed measurement tool for de

  16. Polypeptide composition and gag gene-coded products of type-D oncovirus from HEp-2 cells.

    Science.gov (United States)

    Morozov, V A

    1982-01-01

    The protein composition of type-D oncovirus HEp-2, isolated from cell-free medium of continuous human HEp-2 cell line, has been investigated using electrophoresis on gradient polyacrylamide gels with sodium dodecyl sulfate (SDS). Labeling with 14C-amino acids revealed five viral polypeptides with molecular weights of 70 000 (gp70), 27 000 (p27), 19 000 (p19), 15 000 (p15), 12 000-10 000 (p12-10). The 70 000 dalton protein was shown to be the only glycoprotein by incorporation of radioactive glucosamine. A polypeptide with molecular weight of 78 000 has been specifically precipitated from pulse-labeled type-D oncovirus producing HEp-2 cells with goat anti Mason-Pfizer p27 serum. This protein was shown to be gag gene-coded polyprotein precursor (Pr78gag) of the major virus polypeptide p27. Pulse-labeled HEp-2 and Mason-Pfizer infected Tu 197 cells were rinsed, lysed, clarified and precipitated with goat anti Mason-Pfizer p27 serum. In both cases Pr78gag was detected.

  17. Gag mutations strongly contribute to HIV-1 resistance to protease inhibitors in highly drug-experienced patients besides compensating for fitness loss.

    Directory of Open Access Journals (Sweden)

    Elisabeth Dam

    2009-03-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 resistance to protease inhibitors (PI results from mutations in the viral protease (PR that reduce PI binding but also decrease viral replicative capacity (RC. Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6 with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

  18. Broadening of the T-cell repertoire to HIV-1 Gag p24 by vaccination of HLA-A2/DR transgenic mice with overlapping peptides in the CAF05 adjuvant

    DEFF Research Database (Denmark)

    Korsholm, Karen S; Karlsson, Ingrid; Tang, Sheila T

    2013-01-01

    Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus......, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T....../DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines....

  19. Tsg101 regulates PI(4,5)P2/Ca2+ signaling for HIV-1 Gag assembly

    OpenAIRE

    Ehrlich, Lorna S.; Sara ePhotiadis; Medina, Gisselle N.; Whittredge, Paul B.; Taraska, Justin W; Susan eWatanabe; Carol A Carter

    2014-01-01

    Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,...

  20. Ocelots on Barro Colorado Island are infected with feline immunodeficiency virus but not other common feline and canine viruses.

    Science.gov (United States)

    Franklin, Samuel P; Kays, Roland W; Moreno, Ricardo; TerWee, Julie A; Troyer, Jennifer L; VandeWoude, Sue

    2008-07-01

    Transmission of pathogens from domestic animals to wildlife populations (spill-over) has precipitated local wildlife extinctions in multiple geographic locations. Identifying such events before they cause population declines requires differentiating spillover from endemic disease, a challenge complicated by a lack of baseline data from wildlife populations that are isolated from domestic animals. We tested sera collected from 12 ocelots (Leopardus pardalis) native to Barro Colorado Island, Panama, which is free of domestic animals, for antibodies to feline herpes virus, feline calicivirus, feline corona virus, feline panleukopenia virus, canine distemper virus, and feline immunodeficiency virus (FIV), typically a species-specific infection. Samples also were tested for feline leukemia virus antigens. Positive tests results were only observed for FIV; 50% of the ocelots were positive. We hypothesize that isolation of this population has prevented introduction of pathogens typically attributed to contact with domestic animals. The high density of ocelots on Barro Colorado Island may contribute to a high prevalence of FIV infection, as would be expected with increased contact rates among conspecifics in a geographically restricted population.

  1. Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium.

    Science.gov (United States)

    Cervera, Laura; Gutiérrez-Granados, Sonia; Martínez, Marta; Blanco, Julià; Gòdia, Francesc; Segura, María Mercedes

    2013-07-20

    Virus-like particles (VLPs) offer great promise as candidates for new vaccine strategies. Large-scale approaches for the manufacturing of HIV-1 Gag VLPs have mainly focused on the use of the baculovirus expression system. In this work, the development and optimization of an HIV-1 Gag VLP production protocol by transient gene expression in mammalian cell suspension cultures is reported. To facilitate process optimization, a Gag-GFP fusion construct enabling the generation of fluorescent VLPs was used. The great majority of Gag-GFP present in cell culture supernatants was shown to be correctly assembled into virus-like particles of the expected size and morphology consistent with immature HIV-1 particles. Medium optimization was performed using design of experiments (DoE). Culture medium supplementation with non-animal derived components including recombinant proteins and lipids of synthetic or non-animal-derived origin resulted in improved HEK 293 cell growth and VLP production. The maximum cell density attained using the optimized Freestyle culture medium was 5.4×10(6)cells/mL in batch mode, almost double of that observed using the unsupplemented medium (2.9×10(6)cells/mL). Best production performance was attained when cells were transfected at mid-log phase (2-3×10(6)cells/mL) with medium exchange at the time of transfection using standard amounts of plasmid DNA and polyethylenimine. By using an optimized production protocol, VLP titers were increased 2.4-fold obtaining 2.8μg of Gag-GFP/mL or 2.7×10(9)VLPs/mL according to ELISA and nanoparticle tracking quantification analyses, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. The rectal microbiota of cats infected with feline immunodeficiency virus infection and uninfected controls.

    Science.gov (United States)

    Weese, J S; Nichols, J; Jalali, M; Litster, A

    2015-10-22

    Rectal swabs were collected from 31 cats, 16 with FIV infection and 15 uninfected controls, to evaluate and compare the rectal bacterial microbiota in cats with feline immunodeficiency virus (FIV) infection and uninfected controls. The rectal microbiota was characterized via next generation sequencing of 16S rRNA gene (V4 region) polymerase chain reaction products. Eighteen different phyla were identified. Firmicutes dominated in both groups, followed by Proteobacteria and Actinobacteria, but there were no significant differences between groups. When predominant orders are compared, FIV-infected cats had significant higher median relative abundances of Bifidobacteriales (P=0.022), Lactobacillales (P=0.022) and Aeromonadales (P=0.043). No differences were identified in the 50 most common genera when adjusted for false discovery rate. There were significant differences in community membership (Jaccard index, unifrac P=0.008, AMOVA Pmicrobiota differed between cats with FIV infection and uninfected controls. Some of the changes that were noted have been associated with 'dysbiosis' and proinflammatory states in other species, so it is possible that subclinical alteration in the intestinal microbiota could influence the health of FIV-infected cats. Evaluation of the reasons for microbiota alteration and the potential impact on cat health is required.

  3. Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors.

    Science.gov (United States)

    Lotery, Andrew J; Derksen, Todd A; Russell, Stephen R; Mullins, Robert F; Sauter, Sybille; Affatigato, Louisa M; Stone, Edwin M; Davidson, Beverly L

    2002-04-10

    We hypothesize that recombinant feline immunodeficiency viral (rFIV) vectors may be useful for gene transfer to the nonhuman primate retina. We performed vitrectomies and subretinal injections in the right eyes of 11 cynomolgus monkeys. Vesicular stomatitis virus glycoprotein-pseudotyped rFIV that expressed the Escherichia coli beta-galactosidase gene was injected into eight eyes. Sham vehicle or lactose buffer injections were also performed in two of these eight study eyes. rFIV pseudotyped with an amphotropic envelope was used in two eyes, and in one animal injections of lactose buffer only were given. After surgery the animals were clinically evaluated by retinal photography and electroretinography. beta-Galactosidase expression was evaluated, at a final end point, in histological sections. We found photoreceptor and Müller cells to have the greatest transgene expression. Focal inflammatory responses localized to the injection site were seen histologically in all eyes. No difference in transduction efficiency was seen between injections near the macula and more peripheral injections. Visual function as assessed by electroretinography was not significantly affected by vector or vehicle injections. We conclude that rFIV vectors administered beneath the retina can transduce a variety of retinal cells in the nonhuman primate retina. rFIV vectors have therapeutic potential and could be exploited to develop gene therapy for the human eye.

  4. Feline immunodeficiency virus and feline leukemia virus infection in free-ranging guignas (Leopardus guigna) and sympatric domestic cats in human perturbed landscapes on Chiloé Island, Chile.

    Science.gov (United States)

    Mora, Mónica; Napolitano, Constanza; Ortega, René; Poulin, Elie; Pizarro-Lucero, José

    2015-01-01

    Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two of the most common viruses affecting domestic cats (Felis catus). During the last two decades, reports show that both viruses also infect or affect other species of the family Felidae. Human landscape perturbation is one of the main causes of emerging diseases in wild animals, facilitating contact and transmission of pathogens between domestic and wild animals. We investigated FIV and FeLV infection in free-ranging guignas (Leopardus guigna) and sympatric domestic cats in human perturbed landscapes on Chiloé Island, Chile. Samples from 78 domestic cats and 15 guignas were collected from 2008 to 2010 and analyzed by PCR amplification and sequencing. Two guignas and two domestic cats were positive for FIV; three guignas and 26 domestic cats were positive for FeLV. The high percentage of nucleotide identity of FIV and FeLV sequences from both species suggests possible interspecies transmission of viruses, facilitated by increased contact probability through human invasion into natural habitats, fragmentation of guigna habitat, and poultry attacks by guignas. This study enhances our knowledge on the transmission of pathogens from domestic to wild animals in the global scenario of human landscape perturbation and emerging diseases.

  5. The Foreseeable Harms of Trump's Global Gag Rule.

    Science.gov (United States)

    Bingenheimer, Jeffrey B; Skuster, Patty

    2017-09-01

    As one of his first acts as President of the United States, Donald Trump signed an executive order reinstating a version of the global gag rule. Under this rule, US grantees are barred from receiving global health funding if they engage in abortion-related work: not only abortion services, but also abortion referrals and counseling or advocacy for the liberalization of abortion laws. Critics of the Trump global gag rule generally raise three classes of objections: (1) that the rule fails to accomplish its presumed objective of reducing the number of abortions; (2) that it negatively affects the health and well-being of individuals and populations in affected countries; and (3) that it interferes with governments' ability to meet their international obligations. In this commentary, we examine the scientific and policy bases for these criticisms. © 2017 The Population Council, Inc.

  6. Chronic oral infections of cats and their relationship to persistent oral carriage of feline calici-, immunodeficiency, or leukemia viruses.

    Science.gov (United States)

    Tenorio, A P; Franti, C E; Madewell, B R; Pedersen, N C

    1991-08-01

    Two hundred and twenty-six cats from the Veterinary Medical Teaching Hospital (VMTH), a cat shelter, and a purebred cattery were tested for chronic feline calicivirus (FCV), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) infections. Chronic oral carriage of FCV was present in about one-fifth of the cats in each of the groups. FIV infection was not present in the purebred cattery, was moderately prevalent (8%) in the pet population of cats examined at the VMTH for various complaints and was rampant in the cat shelter (21%). Unexpectedly high FeLV infection rates were found in the hospital cat population (28%) and in the purebred cattery (36%), but not in the cat shelter (1.4%). FCV and FeLV infections tended to occur early in life, whereas FIV infections tended to occur in older animals. From 43 to 100% of the cats in these environments had oral cavity disease ranging from mild gingivitis (23-46%), proliferative gingivitis (18-20%), periodontitis (3-32%) and periodontitis with involvement of extra-gingival tissues (7-27%). Cats infected solely with FCV did not have a greater likelihood of oral lesions, or more severe oral disease, than cats that were totally virus free. This was also true for cats infected solely with FeLV, or for cats dually infected with FeLV and FCV. Cats infected solely with FIV appeared to have a greater prevalence of oral cavity infections and their oral cavity disease tended to be more severe than cats without FIV infection. FIV-infected cats that were coinfected with either FCV, or with FCV and FeLV, had the highest prevalence of oral cavity infections and the most severe oral lesions.

  7. Awareness and prevention of patient gag reflex among pedodontists in India: A web-based survey

    Directory of Open Access Journals (Sweden)

    Soumik Roy

    2016-01-01

    Full Text Available Aim: The aim of this study is to devise a reliable and valid web-based survey to predict the awareness level and prevention of patient's gag reflex among Indian pedodontists. Materials and Methods: An 11-question predictive gagging survey was created, refined, and tested on 377 pedodontists. The questions focused on age group, common procedure associated with gag reflex and the most common technique adapted by dentists in their clinics to prevent gag. Results: There was no statistically significant difference in gagging reflex among age groups with 53.5% of patients reported anxiety and fear as a main cause of gag; behavioral modification technique was considered as the most reliable method for gagging prevention in 68.5% of patients and there was no statistically significant difference in gagging severity index among patients irrespective of age, causes, and methods used to prevent it. Conclusion: The web-based gagging survey established that level of awareness regarding management of patient's gag is significantly low among pedodontists in India and hence is a major hindrance in the clinical practice.

  8. The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

    Science.gov (United States)

    Waddington, Lynne; Hijnen, Marcel; Velkov, Tony; McKinstry, William J.

    2017-01-01

    The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔGenthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly. PMID:28222188

  9. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

    Science.gov (United States)

    Lucas, Carolina G D O; Matassoli, Flavio L; Peçanha, Ligia M T; Santillo, Bruna Tereso; Oliveira, Luanda Mara da Silva; Oshiro, Telma Miyuki; Marques, Ernesto T D A; Oxenius, Annette; de Arruda, Luciana B

    2016-08-01

    The decline in number and function of T cells is a hallmark of HIV infection, and preservation or restoration of HIV-specific cellular immune response is a major goal of AIDS treatment. Dendritic cells (DCs) play a key role in the initiation and maintenance of the immune response, and their use as a vaccine vehicle is a promising strategy for enhancing vaccine efficacy. We evaluated the potential of DC-mediated immunization with a DNA vaccine consisting of HIV-1-p55gag (gag, group-specific antigen) associated to lysosomal associated protein (LAMP) sequence (LAMP/gag vaccine). Immunization of mice with mouse DCs transfected with LAMP/gag (Lg-mDCs) stimulated more potent B- and T-cell responses than naked DNA or DCs pulsed with inactivated HIV. Anti-Gag antibody levels were sustained for at least 3 mo after immunization, and recall T-cell responses were also strongly detected at this time point. Human DCs transfected with LAMP/gag (Lg-hDCs) were also activated and able to stimulate greater T-cell response than native gag-transfected DCs. Coculture between Lg-hDCs and T lymphocytes obtained from patients with HIV resulted in upregulation of CD38, CD69, HLA-DR, and granzyme B by CD4(+) and CD8(+) T cells, and increased IFN-γ and TNF-α production. These results indicate that the use of LAMP/gag-DC may be an efficient strategy for enhancing immune function in patients with HIV.-Lucas, C. G. D. O., Matassoli, F. L., Peçanha, L. M. T., Santillo, B. T., Oliveira, L. M. D. S., Oshiro, T. M., Marques, E. T. D. A., Jr., Oxenius, A., de Arruda, L. B. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

  10. Exosomes carring gag/env of ALV-J possess negative effect on immunocytes.

    Science.gov (United States)

    Wang, Guihua; Wang, Zhenzhen; Zhuang, Pingping; Zhao, Xiaomin; Cheng, Ziqiang

    2017-09-12

    J subgroup avian leukosis virus (ALV-J) is an exogenous retrovirus of avian. A key feature of ALV-J infection is leading to severe immunosuppressive characteristic of diseases. Viral components of retrovirus were reported closely associated with immunosuppression, and several similarities between exosomes and retrovirus preparations have lead to the hypotheses of retrovirus hijacker exosomes pathway. In this study, we purified exosomes from DF-1 cells infected and uninfected by ALV-J. Electron microscopy and mass spectrometry (MS) analysis showed that ALV-J not only increased the production of exosomes from ALV-J infected DF-1 cells (Exo-J) but also stimulated some proteins expression, especially ALV-J components secreted in exosomes. Immunosuppressive domain peptide (ISD) of envelope subunit transmembrane (TM) and gag of ALV-J were secreted in Exo-J. It has been reported that HIV gag was budded from endosome-like domains of the T cell plasma membrane. But env protein was first detected in exosomes from retrovirus infected cells. We found that Exo-J caused negative effects on splenocytes in a dose-dependant manner by flow cytometric analysis. And low dose of Exo-J activated immune activity of splenocytes, while high dose possessed immunosuppressive properties. Interestingly, Exo-J has no significant effects on the immunosuppression induced by ALV-J, and the immunosuppressive effects induced by Exo-J lower than that by ALV-J. Taken together, our data indicated that Exo-J supplied a microenvironment for the replication and transformation of ALV-J. Copyright © 2017. Published by Elsevier Ltd.

  11. Resultados de FIV/ICSI en relación con la edad de la mujer. Unidad de Reproducción Humana Asistida del HUCA

    OpenAIRE

    2012-01-01

    El trabajo analiza los resultados de los ciclos de Fecundación In Vitro (FIV) e Inyección Intracitoplasmática de Espermatozoides (ICSI)en relación con la edad de la mujer. Se obtienen unas conclusiones que nos dan una información relevante en la práctica diaria de la Unidad.

  12. Effects of preactivated MC540 in the treatment of lymphocytic plasmacytic stomatitis in feline leukemia virus and feline immunodeficiency virus positive cats.

    Science.gov (United States)

    Wiggs, R B; Lobprise, H B; Matthews, J L; Gulliya, K S

    1993-03-01

    Photoactive compounds and drugs are used therapeutically as antibacterial, antiviral and antitumor agents. This report examines the use of a photoactive compound, preactivated merocyanine 540 (pMC540), in the treatment of stomatitis in two cats that are both feline immunodeficiency virus (FIV) positive. One of the cats was also feline leukemia virus (FeLV) positive. Dramatic short term improvement is reported with the dosage regimen and complications.

  13. Regulation of Gag- and Env-Specific CD8+ T Cell Responses in ART-Naive HIV-Infected Patients: Potential Implications for Individualized Immunotherapy.

    Directory of Open Access Journals (Sweden)

    Christian Prebensen

    Full Text Available Strategies to develop a functional cure for HIV infection will likely require boosting of effector T cell responses to eliminate reactivated, latently infected cells. We have recently explored an assay for assessing antigen-specific regulation of T cell proliferation, which was related to clinical progression in untreated patients and to vaccine efficacy in two trials of therapeutic Gag-based vaccines. We here expand the same assay to further investigate regulation mediated by various inhibitory pathways. Peripheral blood mononuclear cells from 26 asymptomatic HIV-infected, antiretroviral therapy-naïve patients were stimulated with Gag and Env overlapping peptide panels for 5 days. Monoclonal antibodies (mAbs blocking inhibitory mediators interleukin (IL 10, transforming growth factor (TGF β, programmed death ligand (PD-L 1 and herpes virus entry mediator (HVEM were added to parallel cultures. Functional T cell regulation (FTR was defined as the difference in proliferation between stimulated cultures with and without blocking mAbs. FTR was detected in 54% of patients. Blockade of IL-10/PD-L1 and IL10/TGF-β detected all cases with Gag- and Env-associated FTR, respectively. In accordance with previous findings, isolated Env FTR was associated with higher plasma HIV RNA and lower CD4 counts, while patients with both Gag and Env FTR also had higher Gag- and Env-specific proliferative CD8+ T cell responses. There was no association between FTR and frequencies of activated regulatory T cells. In conclusion, we observed substantial heterogeneity in FTR between patients, inhibitory pathways and HIV antigens. FTR may help to individualize immunomodulation and warrants further assessment in clinical immunotherapy trials.

  14. Regulation of Gag- and Env-Specific CD8+ T Cell Responses in ART-Naïve HIV-Infected Patients: Potential Implications for Individualized Immunotherapy.

    Science.gov (United States)

    Prebensen, Christian; Lind, Andreas; Dyrhol-Riise, Anne-Ma; Kvale, Dag

    2016-01-01

    Strategies to develop a functional cure for HIV infection will likely require boosting of effector T cell responses to eliminate reactivated, latently infected cells. We have recently explored an assay for assessing antigen-specific regulation of T cell proliferation, which was related to clinical progression in untreated patients and to vaccine efficacy in two trials of therapeutic Gag-based vaccines. We here expand the same assay to further investigate regulation mediated by various inhibitory pathways. Peripheral blood mononuclear cells from 26 asymptomatic HIV-infected, antiretroviral therapy-naïve patients were stimulated with Gag and Env overlapping peptide panels for 5 days. Monoclonal antibodies (mAbs) blocking inhibitory mediators interleukin (IL) 10, transforming growth factor (TGF) β, programmed death ligand (PD-L) 1 and herpes virus entry mediator (HVEM) were added to parallel cultures. Functional T cell regulation (FTR) was defined as the difference in proliferation between stimulated cultures with and without blocking mAbs. FTR was detected in 54% of patients. Blockade of IL-10/PD-L1 and IL10/TGF-β detected all cases with Gag- and Env-associated FTR, respectively. In accordance with previous findings, isolated Env FTR was associated with higher plasma HIV RNA and lower CD4 counts, while patients with both Gag and Env FTR also had higher Gag- and Env-specific proliferative CD8+ T cell responses. There was no association between FTR and frequencies of activated regulatory T cells. In conclusion, we observed substantial heterogeneity in FTR between patients, inhibitory pathways and HIV antigens. FTR may help to individualize immunomodulation and warrants further assessment in clinical immunotherapy trials.

  15. T-body formation precedes virus-like particle maturation in S. cerevisiae

    DEFF Research Database (Denmark)

    Malagon, Francisco; Jensen, Torben Heick

    2011-01-01

    T-bodies are localized S. cerevisiae RNPs containing Ty1 retroviral components and speculated to play a role in the assembly of virus-like particles (VLPs). Mapping requirements for T-body formation, we demonstrate that ectopic expression of immature TyA1/Gag (Gag-p49), a structural component of ...

  16. Comparison and interpretation of diagnostic tests for feline immunodeficiency virus infection.

    Science.gov (United States)

    Barr, M C; Pough, M B; Jacobson, R H; Scott, F W

    1991-11-15

    Feline sera were submitted to the Cornell Feline Health Center (n = 497) or to the New York State Diagnostic Laboratory (n = 1,565) for feline immunodeficiency virus (FIV) testing. Some sera (n = 166) were submitted for confirmation of previous FIV-positive results; 151 of these sera had been tested at the referring veterinary practice or laboratory, using an in-house ELISA. Excluding the samples submitted for confirmation, a total of 173 samples (9.1%) were FIV-positive; 11.6% of the clinically ill or high-risk cats and 0.49% of the healthy, low risk cats were positive for FIV antibody. A commercially available ELISA for detection of antibody to FIV was evaluated in relation to the immunofluorescent antibody (IFA) test and the immunoblot assay. The ELISA was interpreted according to the manufacturer's instructions, with the ratio of sample optical density to positive control optical density (S/P) determining a positive or negative result. The ELISA results based on the S/P interpretation were compared with a kinetics-based (KELA) interpretation of the ELISA. The KELA values were reported as positive, negative, or equivocal. Using the immunoblot as the standard, ELISA (S/P interpretation) had sensitivity of 0.93 and specificity of 0.98, whereas the IFA test had sensitivity of 0.95 and specificity of 0.98. However, the sensitivity and specificity of the ELISA (S/P interpretation) were markedly reduced for sample results falling in the KELA equivocal range, indicating that equivocal results were valid interpretations for some sera. A high number (22.5%) of the samples submitted for confirmation of a positive result from use of the in-house ELISA were determined to be negative for FIV antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. A mouse model based on replication-competent Tiantan vaccinia expressing luciferase/HIV-1 Gag fusion protein for the evaluation of protective efficacy of HIV vaccine

    Institute of Scientific and Technical Information of China (English)

    HUANG Yang; QIU Chao; LIU Lian-xing; FENG Yan-meng; ZHU Ting; XU Jian-qing

    2009-01-01

    Background Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy. Methods We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy. Results Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (≤106 relative luciferase units (RLU)/mg protein) and HIV-1 Gag (>3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r2=0.71, P <0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006+3141) RLU/mg protein in control group to (1538±463) RLU/mg protein in vaccine group (P=0.1969). Conclusions The luciferase activity in ovary could represent viral replication in vivo;, this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.

  18. Seroepidemiological and clinical survey of feline immunodeficiency virus infection in northern Italy.

    Science.gov (United States)

    Peri, E V; Ponti, W; Dall'ara, P; Rocchi, M; Zecconi, A; Bonizzi, L

    1994-04-01

    Four hundred and thirty-nine feline serum samples from cats with different living conditions in the north of Italy were tested for antibodies to feline immunodeficiency virus (FIV) and for antigen of Feline Leukemia Virus by enzyme-linked immunosorbent assay. A Western blot technique was also used on the positive sera in order to confirm the presence of specific antibodies to FIV. The Western blot enabled the detection of a false positive serum. The prevalence of FIV infection in this population was 12.5% and among the seropositive cats a greater proportion was male (74.5%) than female (25.5%). A correlation between the clinical status and the evolution of the pathology is described together with a score based on the severity of the stomatitis in infected cats. The Western blot patterns of positive samples were then compared with the stage of the pathology. Statistical analysis on the distribution of FIV in stray cats, cats with garden and courtyard access and strictly house-confined cats showed a highly significant risk of the infection in the first group.

  19. First case of peritoneal cystic echinococcosis in a domestic cat caused by Echinococcus granulosus sensu stricto (genotype 1) associated to feline immunodeficiency virus infection.

    Science.gov (United States)

    Armua-Fernandez, Maria Teresa; Castro, Oscar F; Crampet, Alejandro; Bartzabal, Álvaro; Hofmann-Lehmann, Regina; Grimm, Felix; Deplazes, Peter

    2014-04-01

    A new cystic echinococcosis case in a cat in Uruguay is reported herein. The cat was taken to a veterinary clinic in Rocha city, Uruguay, due to dyspnea, constipation and abdominal enlargement. During surgery a large quantity of cysts was retrieved from the abdominal cavity. The cysts were morphologically studied and confirmed as Echinococcus granulosus sensu stricto (genotype 1) by molecular tools using cytochrome oxidase submit 1 and small subunit ribosomal RNA gene as target genes. Moreover, for the first time a coinfection with feline immunodeficiency virus (FIV) was detected. FIV-induced immunosuppression could be a determining factor in the development of cystic echinococcosis in cats.

  20. Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

    DEFF Research Database (Denmark)

    Molle, Dorothée; Segura-Morales, Carollna; Camus, Gregory

    2009-01-01

    HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the ...

  1. The Use of an Alternative Extraoral Periapical Technique for Patients with Severe Gag Reflex

    Science.gov (United States)

    e Silva, Mauro Henrique Chagas; Santos, Mariane Floriano Lopes; de Lima, Carolina Oliveira; Campos, Celso Neiva

    2016-01-01

    Gag reflex is a physiologic mechanism that promotes contraction of the muscles of the tongue and pharyngeal walls. Different factors, including intraoral radiographic films and sensors, may trigger this reflex. Patients with severe gag reflex may not be able to tolerate the presence of intraoral radiographic films or sensors during root canal therapy (RCT). This factor may prevent an appropriate intraoral radiograph, which is important in RCT. Different approaches have been used to facilitate dental procedures in patients suffering from severe gag reflex. The use of an extraoral radiographic technique is an alternative method to obtain working length confirmation in patients with severe gag reflex. In this report of 2 cases, the use of an extraoral radiographic technique as an alternative approach during RCT in patients with severe gag reflex associated with phobic behavior and trismus was successfully demonstrated. PMID:27547474

  2. How Can Hypnodontics Manage Severe Gag Reflex for Root Canal Therapy? A Case Report

    Science.gov (United States)

    Ramazani, Mohsen; zarenejad, Nafiseh; Parirokh, Masoud; Zahedpasha, Samir

    2016-01-01

    In endodontics, severe involuntary gagging can have a severe impact on treatment procedure. There are many ways to ease the gag reflex, one of which is hypnosis. A 34-year-old male was referred for root canal treatment of a molar tooth. He had not received any dental treatments for the past nine years due to fear of severe gag reflex. Three hypnotic sessions based upon eye fixation, progressive muscle relaxation and guided imagery techniques were spent for psychosomatic management. The gag reflex was controlled and reduced to a normal level, and the required dental treatments including root canal therapy and restoration were performed successfully. This report shows that hypnosis can control gag reflex for dental treatments. PMID:27141226

  3. The Use of an Alternative Extraoral Periapical Technique for Patients with Severe Gag Reflex

    Directory of Open Access Journals (Sweden)

    Mauro Henrique Chagas e Silva

    2016-01-01

    Full Text Available Gag reflex is a physiologic mechanism that promotes contraction of the muscles of the tongue and pharyngeal walls. Different factors, including intraoral radiographic films and sensors, may trigger this reflex. Patients with severe gag reflex may not be able to tolerate the presence of intraoral radiographic films or sensors during root canal therapy (RCT. This factor may prevent an appropriate intraoral radiograph, which is important in RCT. Different approaches have been used to facilitate dental procedures in patients suffering from severe gag reflex. The use of an extraoral radiographic technique is an alternative method to obtain working length confirmation in patients with severe gag reflex. In this report of 2 cases, the use of an extraoral radiographic technique as an alternative approach during RCT in patients with severe gag reflex associated with phobic behavior and trismus was successfully demonstrated.

  4. Prevalence and risk factors for cats testing positive for feline immunodeficiency virus and feline leukaemia virus infection in cats entering an animal shelter in New Zealand.

    Science.gov (United States)

    Gates, M C; Vigeant, S; Dale, A

    2017-11-01

    AIMS To estimate the prevalence of cats testing positive for antibodies to feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) antigens in domestic cats entering a New Zealand animal shelter, based on a commercial point-of-care ELISA, to identify risk factors associated with cats testing positive, and to compare the results obtained from the ELISA with those obtained using PCR-based testing. METHOD A cross-sectional study was performed on 388 cats entering the Royal New Zealand Society for the Prevention of Cruelty to Animals animal shelter in Auckland, New Zealand between 7 February 2014 and 30 May 2014. Whole blood samples were collected from each cat and tested for FIV antibody and FeLV antigen using a commercial point-of-care ELISA. Information on the signalment and health status of the cat at the time of entry was also recorded. Blood and saliva samples from a subset of cats were tested for FIV and FeLV proviral DNA using a real-time PCR assay. RESULTS Of the 388 cats in the study sample, 146 (37.6%) had been relinquished by owners, 237 (62.4%) were strays, and 5 (1.3%) were of unknown origin. Overall, 53/388 (13.7%) cats tested positive for FIV antibodies and 4/388 (1.0%) were positive for FeLV antigen. Stray cats had a higher FIV seroprevalence than relinquished cats (42/237 (17.8%) vs. 11/146 (7.5%); p=0.008). Of 53 cats that were FIV-seropositive, 51 (96%) tested positive for FIV proviral DNA using PCR testing of blood. Of these 51 cats, 28 (55%) were positive by PCR testing of saliva. Of the four cats that were FeLV antigen-positive by ELISA, two (50%) were positive for FeLV proviral DNA by PCR testing of blood. The odds of a cat being seropositive for FIV were greater for intact compared to desexed cats (OR=3.3; 95% CI=1.6-7.4) and for male compared to female cats (OR=6.5; 95% CI=3.2-14.0). CONCLUSIONS AND CLINICAL RELEVANCE The seroprevalence for FIV was 14% among cats entering an animal shelter in Auckland, whereas the prevalence of

  5. Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

    Directory of Open Access Journals (Sweden)

    Christina S Mullins

    Full Text Available A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC.The expression of HERV-H Gag proteins (Gag-H was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium.Taken together, the Gag-H antibody clone(s present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

  6. The nucleocapsid region of HIV-1 Gag cooperates with the PTAP and LYPXnL late domains to recruit the cellular machinery necessary for viral budding.

    Directory of Open Access Journals (Sweden)

    Vincent Dussupt

    2009-03-01

    Full Text Available HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPX(nL, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC, which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Bro(i are sufficient to bind Gag. Bro(i interferes with HIV-1 release in an NC-dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V. Although only Bro1-V contains binding determinants for CHMP4, both Bro(i and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPX(nL/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1-CHMP4 complex required for LYPX(nL-mediated budding.

  7. Antibodies specific for hypervariable regions 3 to 5 of the feline immunodeficiency virus envelope glycoprotein are not solely responsible for vaccine-induced acceleration of challenge infection in cats

    NARCIS (Netherlands)

    W. Huisman (Willem); E.J.A. Schrauwen (Eefje); S.D. Pas (Suzan); J.A. Karlas (Jos); G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Albert)

    2004-01-01

    textabstractIn a previous vaccination study in cats, the authors reported on accelerated feline immunodeficiency virus (FIV) replication upon challenge in animals vaccinated with a candidate envelope subunit vaccine. Plasma transfer studies as well as antibody profiles in vaccinated cats indicated a

  8. Mechanisms of human immunodeficiency virus type 2 RNA packaging

    DEFF Research Database (Denmark)

    Ni, Na; Nikolaitchik, Olga A; Dilley, Kari A;

    2011-01-01

    that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles...... two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together......Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2...

  9. Palateless custom bar supported overdenture: A treatment modality to treat patient with severe gag reflex

    Directory of Open Access Journals (Sweden)

    Kunwarjeet Singh

    2012-01-01

    Conclusion: Palateless custom bar supported overdenture procedure can be successfully used for the management of patients with severe gag reflex with improved denture retention, stability, chewing efficiency and comfort of the patient.

  10. 10A1-MuLV but not the related amphotropic 4070A MuLV is highly neurovirulent: importance of sequences upstream of the structural Gag coding region.

    Science.gov (United States)

    Münk, Carsten; Prassolov, Vladimir; Rodenburg, Michaela; Kalinin, Viacheslav; Löhler, Jürgen; Stocking, Carol

    2003-08-15

    Recombinants of Moloney murine leukemia virus (MoMuLV) with either an amphotropic (MoAmphoV) or 10A1-tropic host range (Mo10A1V) induce a spongiform neurodegenerative disease in susceptible mice. To test whether MoMuLV -derived sequences are required for induction of neuropathology, mice were inoculated with either the original 10A1 or the amphotropic (4070A) MuLV isolate. Strikingly, wild-type 10A1 was more neurovirulent than Mo10A1V, inducing severe neurological clinical symptoms with a median latency of 99 days in 100% of infected mice. In contrast, no motor disturbances were detected in any of the 4070A-infected mice, although limited central nervous system lesions were observed. A viral determinant conferring high neurovirulence to 10A1 was mapped to a region encompassing the first 676 bases of the viral genome, including the U5 LTR and encoding the amino-terminus of glycosylated Gag (glycoGag). In contrast to studies with the highly neurovirulent CasFr(KP) virus, an inverse correlation between surface expression levels of glycoGag and neurovirulence was not observed; however, this does not rule out a common underlying mechanism regulating virus pathogenicity.

  11. Molecular and clinical study on prevalence of feline herpesvirus type 1 and calicivirus in correlation with feline leukemia and immunodeficiency viruses

    OpenAIRE

    Najafi, Hamideh; MADADGAR, Omid; Jamshidi, Shahram; Ghalyanchi Langeroudi, Arash; Darzi Lemraski, Mahdieh

    2014-01-01

    Upper respiratory tract diseases (URTD) are common clinical problem in cats worldwide. Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the main primary pathogens. Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) are also among the most common infectious diseases of cats which suppress the immunity. Oropharyngeal and conjunctival swabs and blood samples were taken from 16 cats with clinical signs of URTD and 26 clinically healthy cats. PCR and RT-PCR were...

  12. Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice

    Science.gov (United States)

    Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

    2016-01-01

    Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction. PMID:26305669

  13. Emerging Viruses in the Felidae: Shifting Paradigms

    Directory of Open Access Journals (Sweden)

    Meredith A. Brown

    2012-02-01

    Full Text Available The domestic cat is afflicted with multiple viruses that serve as powerful models for human disease including cancers, SARS and HIV/AIDS. Cat viruses that cause these diseases have been studied for decades revealing detailed insight concerning transmission, virulence, origins and pathogenesis. Here we review recent genetic advances that have questioned traditional wisdom regarding the origins of virulent Feline infectious peritonitis (FIP diseases, the pathogenic potential of Feline Immunodeficiency Virus (FIV in wild non-domestic Felidae species, and the restriction of Feline Leukemia Virus (FeLV mediated immune impairment to domestic cats rather than other Felidae species. The most recent interpretations indicate important new evolutionary conclusions implicating these deadly infectious agents in domestic and non-domestic felids.

  14. Emerging viruses in the Felidae: shifting paradigms.

    Science.gov (United States)

    O'Brien, Stephen J; Troyer, Jennifer L; Brown, Meredith A; Johnson, Warren E; Antunes, Agostinho; Roelke, Melody E; Pecon-Slattery, Jill

    2012-02-01

    The domestic cat is afflicted with multiple viruses that serve as powerful models for human disease including cancers, SARS and HIV/AIDS. Cat viruses that cause these diseases have been studied for decades revealing detailed insight concerning transmission, virulence, origins and pathogenesis. Here we review recent genetic advances that have questioned traditional wisdom regarding the origins of virulent Feline infectious peritonitis (FIP) diseases, the pathogenic potential of Feline Immunodeficiency Virus (FIV) in wild non-domestic Felidae species, and the restriction of Feline Leukemia Virus (FeLV) mediated immune impairment to domestic cats rather than other Felidae species. The most recent interpretations indicate important new evolutionary conclusions implicating these deadly infectious agents in domestic and non-domestic felids.

  15. A novel minimal in vitro system for analyzing HIV-1 Gag mediated budding

    CERN Document Server

    Gui, Dong; Xu, Jun; Zandi, Roya; Gill, Sarjeet; Huang, I-Chueh; Rao, A L N; Mohideen, Umar

    2013-01-01

    A biomimetic minimalist model membrane is used to study the mechanism and kinetics of the in vitro HIV-1 Gag budding from a giant unilamellar vesicle (GUV). The real time interaction of the Gag, RNA and lipid leading to the formation of minivesicles is measured in real time using confocal microscopy. The Gag is found to lead to resolution limited punctae on the lipid membranes of the GUV. The introduction of the Gag to a GUV solution containing RNA led to the budding of minivesicles on the inside surface of the GUV. The diameter of the GUV decreased due to the bud formation. The corresponding rate of decrease of the GUV diameter was found to be linear in time. The bud formation and the decrease in GUV size were found to be proportional to the Gag concentration. The method is promising and will allow the systematic study of the dynamics of assembly of immature HIV and help classify the hierarchy of factors that impact the Gag protein initiated assembly of retroviruses such as HIV. The GUV system might also be ...

  16. Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

    Science.gov (United States)

    Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D

    2014-01-01

    The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

  17. Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Brad Lewis

    Full Text Available The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

  18. Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

    Directory of Open Access Journals (Sweden)

    Friew Yeshitila N

    2009-06-01

    Full Text Available Abstract Background Host restriction factor APOBEC3G (A3G blocks human immunodeficiency virus type 1 (HIV-1 replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP from its N- or C-terminal fragments. Results The results obtained with catalytic domain 1 and 2 (CD1 and CD2 mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22 to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity. Conclusion These studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules.

  19. Safety and immunogenicity of a live recombinant canarypox virus expressing HIV type 1 gp120 MN MN tm/gag/protease LAI (ALVAC-HIV, vCP205) followed by a p24E-V3 MN synthetic peptide (CLTB-36) administered in healthy volunteers at low risk for HIV infection. AGIS Group and L'Agence Nationale de Recherches sur Le Sida.

    Science.gov (United States)

    Salmon-Céron, D; Excler, J L; Finkielsztejn, L; Autran, B; Gluckman, J C; Sicard, D; Matthews, T J; Meignier, B; Valentin, C; El Habib, R; Blondeau, C; Raux, M; Moog, C; Tartaglia, J; Chong, P; Klein, M; Milcamps, B; Heshmati, F; Plotkin, S

    1999-05-01

    A live recombinant canarypox vector expressing HIV-1 gpl20 MN tm/gag/protease LAI (ALVAC-HIV, vCP205) alone or boosted by a p24E-V3 MN synthetic peptide (CLTB-36) was tested in healthy volunteers at low risk for HIV infection for their safety and immunogenicity. Both antigens were well tolerated. ALVAC-HIV (vCP205) induced low levels of neutralizing antibodies against HIV-1 MN in 33% of the volunteers. None of them had detectable neutralizing antibodies against a nonsyncytium-inducing HIV-1 clade B primary isolate (Bx08). After the fourth injection of vCP205, CTL activity was detected in 33% of the volunteers and was directed against Env, Gag, and Pol. This activity was mediated by both CD4+ and CD8+ lymphocytes. On the other hand, the CLTB-36 peptide was poorly immunogenic and induced no neutralizing antibodies or CTLs. Although the ALVAC-HIV (vCP205) and CLTB-36 prime-boost regimen was not optimal, further studies with ALVAC-HIV (vCP205) are warranted because of its clear induction of a cellular immune response and utility as a priming agent for other subunit antigens such as envelope glycoproteins, pseudoparticles, or new peptides.

  20. Frequent detection of papillomavirus DNA in clinically normal skin of cats infected and noninfected with feline immunodeficiency virus.

    Science.gov (United States)

    Munday, John S; Witham, Adrian I

    2010-06-01

    Feline cutaneous squamous cell carcinomas (SCCs) often contain felis domesticus papillomavirus type 2 (FdPV-2) DNA. While this may suggest FdPV-2 causes feline SCC development, the proportion of cats that are asymptomatically infected by this PV is unknown. Infection by feline immunodeficiency virus (FIV) is associated with high rates of cutaneous SCC development, possibly due to increased PV infection. This study examines the frequency of cutaneous asymptomatic FdPV-2 infections in cats and compares the rate of FdPV-2 infection in 22 FIV-positive cats with that in 22 FIV-negative cats. FdPV-2 sequences were detected in 39% of skin swabs. One or both swabs contained FdPV-2 DNA from 52% of the cats. FIV status, age or sex of the cat did not significantly influence FdPV-2 infection. Cats that shared a household with a PV-infected cat could remain uninfected suggesting infection depends more on host factors than exposure to the PV. These results indicate that asymptomatic FdPV-2 infections are common in cats, but do not provide evidence that FdPV-2 causes feline SCC development.

  1. Acompanhamento clínico-laboratorial de gatos naturalmente infectados com vírus da imunodeficiência (FIV) tratados com zidovudina(AZT)

    OpenAIRE

    Paparotto, Telma [UNESP

    2007-01-01

    O presente trabalho objetivou o acompanhamento clínico-laboratorial de doze felinos naturalmente infectados com o vírus da imunodeficiência Felina(FIV) durante o tratamento com o antiretroviral zidovudina (AZT). Para tanto, foram colhidas amostras sanguíneas de trezentos animais oriundos de diferentes cidades do Estado de São Paulo e encaminhadas para o laboratório de virologia do instituto de Biociência, Departamento de Microbiologia e Imunologia da Universidade Estadual Paulista para identi...

  2. Vírus da artrite encefalite caprina: isolamento e caracterização de parte do gene gag

    Directory of Open Access Journals (Sweden)

    P.P. Lima

    2004-04-01

    Full Text Available Amostras de sangue de 12 animais soropositivos pelo teste de imunodifusão em gel de agarose e que não apresentavam sinais clínicos sugestivos de infecção pelo vírus da artrite-encefalite caprina (CAEV foram coletadas para isolamento viral. Mácrofagos derivados de monócitos foram co-cultivados com células de membrana sinovial caprina (MSC, resultando em cinco amostras que apresentaram efeito citopático característico do tipo persistente, semelhante ao observado para o CAEV. Uma técnica de reação em cadeia de polimerase (PCR foi padronizada para amplificar parte do gene gag do genoma pró-viral, codificante para a proteína do capsídeo viral (p25. As cinco amostras foram amplificadas pela PCR e três delas, BR-UFMG/PL1, BR-UFMG/PL2 e BR-UFMG/PL3, foram seqüenciadas diretamente dos seus produtos de PCR. O alinhamento múltiplo das seqüências obtidas com outras de lentivírus de pequenos ruminantes (LVPR, obtidas no GenBank, e o dendrograma revelaram que as novas amostras de CAEV são únicas e distintas das demais amostras de LVPR, possuindo maior identidade de nucleotídeos e aminoácidos entre si e com as amostras de CAEV do que com a do vírus maedi-visna.Blood samples from 12 seropositive animals by agar gel immunodifusion test (AGID showing no evident clinical signs of disease were taken to attempt caprine arthritis-encephalitis virus (CAEV isolation. Monocyte-derived macrophages were co-cultured with goat synovial membrane cells (GSM resulting in five virus isolations, which presented cytophatic effects of the persistent type, resembling those observed for CAEV. A polymerase chain reaction (PCR assay was designed to amplify a portion of the gag proviral gene coding for the major core protein (p25. All of the five isolates were amplified by this PCR and three of them named BR-UFMG/PL1, BR-UFMG/PL2 and BR-UFMG/PL3, were sequenced directly from their PCR products. Multiple sequence analysis and a dendrogram including other

  3. Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

    Energy Technology Data Exchange (ETDEWEB)

    Tress, E.; Pierotti, M.; DeLeo, A.B.; O' Donnell, P.V.; Fleissner, E.

    1982-02-01

    To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulsechase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65/sup gag/, was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95/sup gag/, or its precursor, Pr75/sup gag/. No evidence was found for synthesis of gag-host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instances a monoclonal antibody, 35/56, which is specific for the NuLV G/sub IX/ antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same 35/56 phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.

  4. Studies on molluscan glycosaminoglycans (GAG) from backwater clam Donax cuneatus (Linnaeus)

    Institute of Scientific and Technical Information of China (English)

    P Vijayabaskar; ST Somasundaram

    2012-01-01

    Objective: To investigate the potent and specific anticoagulant activity of molluscan glycosaminoglycans (GAGs) isolated from whole clam tissue Donax cuneatus (D. cuneatus). Methods: Purification of few milligram quantities of GAGs from this tissue sample permitted a thorough examination of its anticoagulant activity characterization, which was partially purified by fractionation by anion exchange chromatography using DEAE cellulose column. The isolated crude and partially purified fractionated sample was showing metachromatic shift while using azure-A. The sample also exhibited prominent of biological and anti-fXa anticoagulant activity assays. Mobility was analyzed by two different buffer systems using agarose gel electrophoresis.Results:The fractionated molluscan GAG was also found to have similar peaks as that of standard heparin when assessed by the FT-IR spectrum. Finally molecular weight was determined by the gradient PAGE for crude and fractionated-II GAG, which were found to be 65 000 Da and 50 000 Da, respectively. Conclusions: The bivalve GAG was subjected to fractionation for further purification and its chemical components were analyzed. The fractionated clam heparin also showed substantial in vitro anticoagulant activity than that of commercial heparins.

  5. Polymorphism, recombination, and mutations in HIV type 1 gag-infecting Peruvian male sex workers.

    Science.gov (United States)

    Yabar, Carlos Augusto; Salvatierra, Javier; Quijano, Eberth

    2008-11-01

    HIV genetic diversity in female sex workers (FSW) has been previously described in Peru; however this information is not yet available for male sex workers (MSW). Therefore, purified peripheral blood mononuclear cell DNA from 147 HIV-infected subjects identified as MSW and FSW was used to amplify a 460-bp fragment corresponding to the p24-p7 region of the gag gene. The PCR product was digested with restriction enzymes to identify genetic polymorphism. Later, a random group of samples (n = 19) was sequenced to perform phylogenetic analysis, intragenic recombination analysis, and deleterious mutations leading to a nonfunctional protein in conservative regions of the Gag protein. RFLP analysis revealed 11 genetic variants for AluI and five for MspI. A group of nonsex workers (NSW) used for comparison showed different RFLP genetic variant distributions. Of interest, nine cases of mixed genetic variants were observed for MSW, one case for FSW, and none for NSW. Phylogenetic analysis revealed that all HIV-1 species were subtype B. Intragenic recombination analysis showed a B/C recombination case from an FSW (boostrap = 1000; p value < 0.05). Of interest, deleterious mutations were observed in three cases of conservative D2 zinc domains for Gag 3/19 and one case of the high homology region (1/19). This study shows that gag of HIV circulating from MSW has high genetic polymorphism involving deleterious mutations in conserved domains from the p24-p7 gag region.

  6. Regulation of Chemokine Function: The Roles of GAG-Binding and Post-Translational Nitration.

    Science.gov (United States)

    Thompson, Sarah; Martínez-Burgo, Beatriz; Sepuru, Krishna Mohan; Rajarathnam, Krishna; Kirby, John A; Sheerin, Neil S; Ali, Simi

    2017-08-03

    The primary function of chemokines is to direct the migration of leukocytes to the site of injury during inflammation. The effects of chemokines are modulated by several means, including binding to G-protein coupled receptors (GPCRs), binding to glycosaminoglycans (GAGs), and through post-translational modifications (PTMs). GAGs, present on cell surfaces, bind chemokines released in response to injury. Chemokines bind leukocytes via their GPCRs, which directs migration and contributes to local inflammation. Studies have shown that GAGs or GAG-binding peptides can be used to interfere with chemokine binding and reduce leukocyte recruitment. Post-translational modifications of chemokines, such as nitration, which occurs due to the production of reactive species during oxidative stress, can also alter their biological activity. This review describes the regulation of chemokine function by GAG-binding ability and by post-translational nitration. These are both aspects of chemokine biology that could be targeted if the therapeutic potential of chemokines, like CXCL8, to modulate inflammation is to be realised.

  7. Power and politics in international funding for reproductive health: the US Global Gag Rule.

    Science.gov (United States)

    Crane, Barbara B; Dusenberry, Jennifer

    2004-11-01

    Since 2001, the US government has used its power as a leading donor to family planning programmes to pursue policies in conflict with global agreements on reproductive rights. Prominent among these policies is the Mexico City Policy (or Global Gag Rule), which restricts non-governmental organisations (NGOs) in developing countries that receive USAID family planning funding from engaging in most abortion-related activities, even with their own funds. This paper reviews the history and political origins of the Gag Rule under several Republican party presidents. The Gag Rule has not achieved an overall reduction in abortions; rather, where it has disrupted family planning services, the policy is more likely to have increased the number of abortions. This paper concludes that the Gag Rule is a radical intrusion on the rights and autonomy of recipients of US funding. Regardless of whether or not it is rescinded in the future, the underlying issues in the politics of US reproductive health assistance are likely to persist. NGOs that wish to free themselves from the constraints it imposes must find the means to end their dependence on USAID funding, including turning to other donors. NGOs should also take the lead in opposing policies such as the Gag Rule that violate global agreements.

  8. T-body formation precedes virus-like particle maturation in S. cerevisiae.

    Science.gov (United States)

    Malagon, Francisco; Jensen, Torben Heick

    2011-01-01

    T-bodies are localized S. cerevisiae RNPs containing Ty1 retroviral components and speculated to play a role in the assembly of virus-like particles (VLPs). Mapping requirements for T-body formation, we demonstrate that ectopic expression of immature TyA1/Gag (Gag-p49), a structural component of the Ty1 capsid, is sufficient for T-body formation both under normal conditions as well as in a strain background devoid of endogenous Gag. Moreover, T-bodies are readily formed when Ty1 transposition is blocked. Thus, T-bodies represent an early stage in the Ty1 life cycle, preceding VLP maturation.

  9. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1 enhance Gag-specific immune response.

    Directory of Open Access Journals (Sweden)

    Rodrigo Maciel da Costa Godinho

    Full Text Available We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1 elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  10. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    Science.gov (United States)

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  11. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    Science.gov (United States)

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  12. Molecular and clinical study on prevalence of feline herpesvirus type 1 and calicivirus in correlation with feline leukemia and immunodeficiency viruses.

    Science.gov (United States)

    Najafi, Hamideh; Madadgar, Omid; Jamshidi, Shahram; Ghalyanchi Langeroudi, Arash; Darzi Lemraski, Mahdieh

    2014-01-01

    Upper respiratory tract diseases (URTD) are common clinical problem in cats worldwide. Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the main primary pathogens. Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) are also among the most common infectious diseases of cats which suppress the immunity. Oropharyngeal and conjunctival swabs and blood samples were taken from 16 cats with clinical signs of URTD and 26 clinically healthy cats. PCR and RT-PCR were used to detect FHV/FIV or FCV/FeLV infections, respectively. Feline calicivirus was detected in all cats with URTD and 87.00% and 93.00% of them were positive for FIV and FeLV, respectively. Feline herpesvirus rate of infection was 43.00% in sick cats. In clinically normal cats, prevalence rates of FCV and FHV were about 50.00%, but FIV and FeLV rates (42.00% and 65.00% respectively) were higher compared to other studies. Stomatitis was observed in 50.00% of cats with URTD. The main causative agent of corneal ulcers is FHV-1, but in 50.00% of cats with corneal ulcers, FCV was detected alone. It seems new variants of Caliciviruses are the main causative agents to attack uncommon tissues like cornea, although retroviral infections may be in the background of these various signs. The high retroviral prevalence may be due to existence of large population of stray cats. This is the first molecular study of FeLV and FCV in Iran and seems that FCV and FHV prevalence rates in FIV or FeLV infected cats is more than other non-infected ones.

  13. The use of low-level laser therapy for controlling the gag reflex in children during intraoral radiography.

    Science.gov (United States)

    Elbay, Mesut; Tak, Önjen; Şermet Elbay, Ülkü; Kaya, Can; Eryılmaz, Kubilay

    2016-02-01

    The current literature suggests that low-level laser stimulation of the PC 6 acupuncture points may prevent gagging. This study aimed to determine if low-level laser therapy (LLLT) can reduce the gag reflex in children undergoing intraoral maxillary radiography. This randomized, controlled, double-blind clinical trial was conducted with 25 children with moderate-to-very severe gag reflexes who required bilateral periapical radiographic examination of the maxillary molar region. Children's anxiety levels were initially evaluated using Corah's Dental Anxiety Scale (DAS) to identify any possible relationship between gagging and anxiety. A control radiograph was taken of one randomly selected side in each patient after simulated laser application so that the patient was blinded to the experimental conditions (control group). Laser stimulation was then performed for the experimental side. A laser probe was placed on the Pericardium 6 (PC 6) acupuncture point on each wrist, and laser energy was delivered for 14 s (300 mW, energy density 4 J/cm(2)) at a distance of 1 cm from the target tissue. Following laser stimulation, the experimental radiograph was taken (experimental group). Gagging responses were measured using the Gagging Severity Criteria for each group. Data were analyzed using Spearman's rho correlations and Mann-Whitney U tests. Both mean and median gagging scores were higher in the control group than in the experimental group. Patients who were unable to tolerate the intraoral control radiography were able to tolerate the procedure after LLLT. Differences between gagging scores of the control and experimental groups were statistically significant (P = .000). There was no significant correlation between gagging severity and anxiety score (P > .05). A negative correlation was found between age and gagging score in the control group (P ˂ .05). Within the limitations of this study, LLLT of the PC 6 acupuncture points appears to be a useful technique

  14. Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblasts.

    Science.gov (United States)

    Friso, Adelaide; Tomanin, Rosella; Alba, Sabrina; Gasparotto, Nicoletta; Puicher, Elisabetta Piller; Fusco, Mariella; Hortelano, Gonzalo; Muenzer, Joseph; Marin, Oriano; Zacchello, Franco; Scarpa, Maurizio

    2005-11-01

    Hunter syndrome, mucopolysaccharidosis type II (MPS II), is a X-linked inherited disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS), involved in the lysosomal catabolism of the glycosaminoglycans (GAG) dermatan and heparan sulfate. Such a deficiency leads to the intracellular accumulation of undegraded GAG and eventually to a progressive severe clinical pattern. Many attempts have been made in the last two to three decades to identify possible therapeutic strategies for the disorder, including gene therapy and somatic cell therapy. In this study we evaluated the intraperitoneal implantation of allogeneic myoblasts over-expressing IDS, enclosed in alginate microcapsules, in the MPS II mouse model. Animals were monitored for 8 weeks post-implantation, during which plasma and tissue IDS levels, as well as tissue and urinary GAG contents, were measured. Induced enzyme activity occurred both in the plasma and in the different tissues analyzed. A significant decrease in urinary undegraded GAG between the fourth and the sixth week of treatment was observed. Moreover, a biochemical reduction of GAG deposits was measured 8 weeks after treatment in the liver and kidney, on average 30 and 38%, respectively, while in the spleen GAG levels were almost normalized. Finally, the therapeutic effect was confirmed by histolochemical examination of the same tissues. Such effects were obtained following implantation of about 1.5 x 10(6) recombinant cells/animal. Taken together, these results represent a clear evidence of the therapeutic efficacy of this strategy in the MPS II mouse model, and encourage further evaluation of this approach for potential treatment of human beings. Copyright (c) 2005 John Wiley & Sons, Ltd.

  15. Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct.

    Science.gov (United States)

    Bergqvist, Ann-Sofi; Rodríguez-Martínez, Heriberto

    2006-06-01

    In vivo, bull sperm capacitation seems to occur mainly in the oviduct. Capacitation of bull spermatozoa can be triggered in vitro by exposure to heparin, a heavily sulphated glycosaminoglycan (S-GAG). We determined the concentration of S-GAGs in oviductal fluid from dairy heifers, collected over the course of several oestrous cycles via surgically implanted intraluminal catheters. We also investigated the presence of syndecans, i.e. heparan sulphate proteoglycans, in the bovine oviductal epithelium of Swedish dairy cattle during standing oestrus and the luteal phase of the oestrous cycle, using immunohistochemistry for three different polyclonal antibodies raised against human syndecan-2 and rat syndecan-1 and syndecan-2, respectively. The concentration of S-GAGs in oviductal fluid obtained from the ampullar segment of the oviduct was significantly higher (P=0.0026) than it was in fluid from the isthmic segment during the functional period, i.e. from prooestrus to metaoestrus (73.5+/-10.49 mg/L in ampullar ODF, compared to 43.2+/-10.74 mg/L in isthmic ODF); least square mean (L.S.M.)+/-standard error of the mean (S.E.M.). There was also a significantly higher concentration of S-GAGs in the fluid from the oviduct ipsilateral to the ovulation side 73.5+/-10.54 mg/L on the ovulation side, compared to 43.1+/-10.71 mg/L in the oviduct on the contralateral side (L.S.M.+/-S.E.M., P=0.0026) during this period. Both syndecan-1 and syndecan-2 were present in the epithelial cells lining all studied segments of the bovine oviduct, i.e. the UTJ, isthmus and ampulla, during both standing oestrus and dioestrus. The syndecans and S-GAGs found may influence the gametes, while they reside in the oviduct; the amounts of S-GAGs found in the bovine oviduct seem sufficient to act as capacitating factors in vivo.

  16. Importance of Basic Residues in Binding of Rous Sarcoma Virus Nucleocapsid to the RNA Packaging Signal

    OpenAIRE

    Lee, Eun-Gyung; Alidina, Annie; May, Cynthia; Linial, Maxine L.

    2003-01-01

    In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging. We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation. In this study, using both site-directed mutagenesis and in vivo random screening in the yeast three-hybr...

  17. Timing constraints of in vivo gag mutations during primary HIV-1 subtype C infection.

    Directory of Open Access Journals (Sweden)

    Vladimir Novitsky

    Full Text Available BACKGROUND: Aiming to answer the broad question "When does mutation occur?" this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. METHODS: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s. Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. RESULTS: Reverse mutations to the wild type appeared at a median (IQR of 62 (44;139 days p/s, while escape mutations from the wild type appeared at 234 (169;326 days p/s (p<0.001. Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78 days p/s and 104 (47;198 days p/s, respectively (p<0.001. Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78 days p/s and 90 (44;196 days p/s, respectively (p=0.006. Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105 days p/s and 219 (90;326 days p/s, respectively (p<0.001. Time of completeness for reverse and escape mutations was 152 (100;176 days p/s and 243 (101;370 days p/s, respectively (p=0.001. Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR: 2.6; 95% CI: 2.3-3.0, dominance (4.8 (3.4-6.8, and completeness (3.6 (2.3-5.5 of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. CONCLUSIONS: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the

  18. In vivo interference of Rous sarcoma virus budding by cis expression of a WW domain.

    Science.gov (United States)

    Patnaik, Akash; Wills, John W

    2002-03-01

    For all enveloped viruses, the actual mechanism by which nascent virus particles separate or "pinch off" from the cell surface is largely unknown. In the case of retroviruses, the Gag protein drives the budding process, and the virus release step is directed by the late (L) assembly domain within Gag. A PPPPY motif within the L domain of Rous sarcoma virus (RSV) was previously characterized as being critical for the release of virions and shown to interact in vitro with the WW domain of Yes-associated protein (Yap). To determine whether WW domain-L domain interactions can occur in vivo, we attempted to interfere with the host cell machinery normally recruited to the site of budding by inserting this WW domain in different locations within Gag. At a C-terminal location, the WW(Yap) domain had no effect on budding, suggesting that the intervening I domains (which provide the major region of Gag-Gag interaction) prevent its access to the L domain. When positioned on the other side of the I domains closer to the L domain, the WW(Yap) domain resulted in a dramatic interference of particle release, and confocal microscopy revealed a block to budding on the plasma membrane. Budding was restored by attachment of the heterologous L domain of human immunodeficiency virus type 1 Gag, which does not bind WW(Yap). These findings suggest that cis expression of WW domains can interfere with RSV particle release in vivo via specific, high-affinity interactions at the site of assembly on the plasma membrane, thus preventing host factor accessibility to the L domain and subsequent virus-cell separation. In addition, they suggest that L domain-specific host factors function after Gag proteins begin to interact.

  19. Sequencing of Gag/Env association with HIV genotyping resolution and HIV-related epidemiologic studies of HIV in China.

    Science.gov (United States)

    Ren, L; Wang, H W; Xu, Y; Feng, Y; Zhang, H F; Wang, K H

    2016-10-24

    HIV genotyping has led to conflicting results between laboratories. Therefore, identifying the most accurate gene combinations to sequence remains a priority. Datasets of Chinese HIV subtypes based on several markers and deposited in PubMed, Metstr, CNKI, and VIP databases between 2000 and 2015 were studied. In total, 9177 cases of amplification-positive samples from 26 provinces of China were collected and used to classify HIV subtypes based on eight individual genes or a combination thereof. CRF01_AE, CRF07_BC, CRF08_BC and B were the prevalent HIV subtypes in China, accounting for 84.07% of all genotypes. Gag/Env sequencing classified a greater number of HIV subtypes compared to other genes or combination of gene fragments. The geographical distribution of Gag and Gag/Env genotypes was similar to that observed with all genetic markers. Further principal component analysis showed a significantly different geographical distribution pattern of HIV in China for HIV genotypes detected with Gag/Env, which was in line with the distribution of all HIV genotypes in China. Gag/Env sequences had the highest diversity of the eight markers studied, followed by Gag and Gag/Pol/Env; Pol/Env polymorphisms were the least divergent. Gag/Env can serve as a high-resolution marker for HIV genotyping.

  20. Human Immunodeficiency Virus Type 1 Protease and the Emergence of Drug Resistance

    DEFF Research Database (Denmark)

    Poulsen, Nina Rødtness

    in the virus life cycle has made it a major target for drug development and active site competitive inhibitors have been successful in the battle against HIV. Unfortunately, the massive drug pressure along with high-level replication and lack of proofreading by the viral reverse transcriptase have resulted......Human immunodeficiency virus type-1 (HIV-1) protease (PR) is responsible for cleaving ten different sites in the viral Gag and Gag-pol polyproteins, thereby releasing the structural proteins and enzymes necessary for the maturation and infectivity of the HIV-1 virus. The vital role of HIV-1 PR...

  1. X-ray crystallographic characterization of rhesus macaque MHC Mamu-A*02 complexed with an immunodominant SIV-Gag nonapeptide

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Youjun [Laboratory of Molecular Immunology and Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate School, Chinese Academy of Sciences, Beijing (China); Qi, Jianxun [Graduate School, Chinese Academy of Sciences, Beijing (China); Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Zhang, Huimin; Wang, Jinzi [Laboratory of Molecular Immunology and Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080 (China); Liu, Jinhua [College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Jiang, Fan [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Gao, Feng, E-mail: gaofeng@im.ac.cn [Laboratory of Molecular Immunology and Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080 (China); College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China)

    2006-01-01

    X-ray crystallographic characterization of rhesus macaque MHC Mamu-A*02 complexed with an immunodominant SIV-Gag nonapeptide. Simian immunodeficiency virus (SIV) in the rhesus macaque is regarded as a classic animal model, playing a crucial role in HIV vaccine strategies and therapeutics by characterizing various cytotoxic T-lymphocyte (CTL) responses in macaque monkeys. However, the availability of well documented structural reports focusing on rhesus macaque major histocompatibility complex class I (MHC I) molecules remains extremely limited. Here, a complex of the rhesus macaque MHC I molecule (Mamu-A*02) with human β{sub 2}m and an immunodominant SIV-Gag nonapeptide, GESNLKSLY (GY9), has been crystallized. The crystal diffracts X-rays to 2.7 Å resolution and belongs to space group C2, with unit-cell parameters a = 124.11, b = 110.45, c = 100.06 Å, and contains two molecules in the asymmetric unit. The availability of the structure, which is being solved by molecular replacement, will provide new insights into rhesus macaque MHC I (Mamu-A*02) presenting pathogenic SIV peptides.

  2. Inhibition of activity of the protease from bovine leukemia virus.

    Science.gov (United States)

    Ménard, A; Leonard, R; Llido, S; Geoffre, S; Picard, P; Berteau, F; Precigoux, G; Hospital, M; Guillemain, B

    1994-06-13

    In view of the close similarity between bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I) we investigated the possibility of developing specific inhibitors of the proteases of these retroviruses using the purified enzyme from BLV. We tested the ability of this protease to specifically cleave various short oligopeptide substrates containing cleavage sites of BLV and HTLV-I proteases, as well as a recombinant BLV Gag precursor. The best substrate, a synthetic decapeptide bearing the natural cleavage site between the matrix and the capsid proteins of BLV Gag precursor polyprotein, was used to develop an inhibition assay. We determined the relative inhibitory effect of synthetic Gag precursor-like peptides in which the cleavable site was replaced by a non-hydrolyzable moiety. The encouraging inhibitory effect of these compounds indicates that potent non-peptidic inhibitors for retroviral proteases are not unattainable.

  3. Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

    Institute of Scientific and Technical Information of China (English)

    XIN LEI LIU; SHUNAG QING YU; XIA FENG; XIAO LI WANG; HONG MEI LIU; XIAO MEI ZHANG; HONG XIA LI; LING ZHOU; YI ZENG

    2006-01-01

    The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod. gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod. gag, rAd5-mod. gag or DNA and were boosted after 3 weeks.To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIVspecific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.

  4. Ethanol suppression of peripheral blood mononuclear cell trafficking across brain endothelial cells in immunodeficiency virus infection

    Directory of Open Access Journals (Sweden)

    Lola C Hudson

    2010-01-01

    Full Text Available Lola C Hudson1, Brenda A Colby1, Rick B Meeker21Department of Molecular Biosciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; 2Department of Neurology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAAbstract: Earlier studies suggested that the combination of alcohol use and immunodeficiency virus infection resulted in more severe neurologic disease than either condition individually. These deleterious interactions could be due to increased immune cell and virus trafficking or may result from interactions between ethanol and human immunodeficiency virus (HIV-associated toxicity within the brain. To determine the extent to which increased trafficking played a role, we examined the effect of ethanol on the migration of different peripheral blood mononuclear cell (PBMCs subsets across a brain endothelial cell monolayer. We utilized combinations of feline brain endothelial cells with astrocytes, and/or microglia with either acute exposure to 0.08 g/dL ethanol, a combination of ethanol and feline immunodeficiency virus (FIV, or FIV alone. Adherence of PBMCs to endothelium was increased in all combinations of cells with the addition of ethanol. Despite increased PBMC adhesion with ethanol treatment, transmigration of B cells, monocytes, CD4 T cells and CD8 T cells was not increased and was actually decreased in the presence of astrocytes. Expression of three common adhesion molecules, intercellular adhesion molecule-1 (ICAM1, ICAM2, and vascular cell adhesion molecule, was unchanged or slightly decreased by ethanol. This indicated that although adherence is increased by ethanol it is not due to an increased expression of adhesion molecules. RANTES, MIP1α, MIP1β, and MCP-1 mRNA expression was also studied in brain endothelial cells, astrocytes and microglia by reverse transcriptase-polymerase chain reaction. Ethanol treatment of astrocytes resulted in modest changes of

  5. First Complete Genome Sequence of a Simian Foamy Virus Isolate from a Cynomolgus Macaque

    Science.gov (United States)

    Sakai, Koji; Ami, Yasushi; Suzaki, Yuriko

    2016-01-01

    We report here the first complete proviral genome sequence (DDBJ/ENA/GenBank accession no. LC094267) of a simian foamy virus, SFVmfa/Cy5061, isolated from a cynomolgus macaque (Macaca fascicularis). This proviral genome consists of 12,965 nucleotides and has five open reading frames, gag, pol, env, tas, and bet, as with other foamy viruses. PMID:27908992

  6. Fisiologia e metabolismo placentário por canulação cordonal em gestações de bovinos normais, FIV e clonados

    OpenAIRE

    2010-01-01

    O desenvolvimento dos sistemas de produção de embriões por fecundação in vitro (FIV) ou transferência nuclear com células somáticas (TNCS) em diversas espécies animais, em especial a bovina, acarretou na manifestação de anormalidades de desenvolvimento in vivo, com a placenta sendo considerada o fator determinante no aparecimento destes distúrbios durante a gestação. A hipótese geral deste trabalho é de que alterações na formação da placenta, decorrentes das manipulações embrionárias in vitro...

  7. Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

    Directory of Open Access Journals (Sweden)

    Williamson Anna-Lise

    2009-06-01

    Full Text Available Abstract Background Recombinant Salmonella vaccine vectors may potentially be used to induce specific CD4+ T cell responses against foreign viral antigens. Such immune responses are required features of vaccines against pathogens such as human immunodeficiency virus type 1 (HIV-1. The aim of this study was to investigate the induction of systemic HIV-1-specific CD4+ T helper (Th responses in mice after oral immunization with a live attenuated Salmonella vaccine vector that expressed HIV-1 subtype C Gag. Groups of BALB/c mice were vaccinated orally three times (4 weeks apart with this recombinant Salmonella. At sacrifice, 28 days after the last immunization, systemic CD4+ Th1 and Th2 cytokine responses were evaluated by enzyme-linked immunospot assay and cytometric bead array. HIV-1 Gag-specific IgG1 and IgG2a humoral responses in the serum were determined by enzyme-linked immunosorbent assay. Results Mice vaccinated with the recombinant Salmonella elicited both HIV-1-specific Th1 (interferon-gamma (IFN-γ and tumour necrosis factor-alpha (TNF-α and Th2 (interleukin-4 (IL-4 and interleukin-5 (IL-5 cytokine responses. The vaccine induced 70 (IFN-γ spot-forming units (SFUs/10e6 splenocytes and 238 IL-4 SFUs/10e6 splenocytes. Splenocytes from vaccinated mice also produced high levels of Th1 and Th2 cytokines upon stimulation with a Gag CD4 peptide. The levels of IFN-γ, TNF-α, IL-4 and IL-5 were 7.5-, 29.1-, 26.2- and 89.3-fold above the background, respectively. Both HIV-1 Gag-specific IgG1 and IgG2a antibodies were detected in the sera of vaccinated mice. Conclusion The study highlights the potential of orally-delivered attenuated Salmonella as mucosal vaccine vectors for HIV-1 Subtype C Gag to induce Gag-specific CD4+ Th1 and Th2 cellular immune responses and antibodies which may be important characteristics required for protection against HIV-1 infection.

  8. Oral Health Related Quality of Life in Young Individuals with Dental Anxiety and Exaggerated Gag Reflex.

    Science.gov (United States)

    Almoznino, Galit; Zini, Avraham; Aframian, Doron J; Kaufman, Eliezer; Lvovsky, Alex; Hadad, Avraham; Levin, Liran

    2015-01-01

    To measure the oral health-related quality of life (OHRQoL) among young individuals presenting with dental anxiety and exaggerated gag reflex and to compare it to the OHRQoL among young individuals attending restorative dental treatment. Patients with dental anxiety and/or exaggerated gag reflex who were referred to the Department of Oral Medicine were included. Clinical examinations included DMFT index (Decayed, Missing and Filled Teeth) according to the WHO criteria and oral hygiene was assessed with the plaque index (PI). The survey included the validated Hebrew version of the Oral Health Impact Profile (OHIP-14). Overall, 322 patients completed the study. The dental anxiety group consisted of 68 patients, the exaggerated gag reflex group of 54 patients and the control group of 200 patients. Control group patients presented with lower PI and DMFT values. Patients with dental anxiety and/or exaggerated gag exhibited worse OHIP-14 scores overall as well as on each separate subscale. PI and DMFT were found to be in strong relation to the OHIP-14. Multivariable logistic regression analysis of factors influencing the OHIP-14 scores revealed a significant influence of the DMFT score. Patients with dental anxiety and exaggerated gag reflex were shown to suffer considerably from impaired oral health-related quality of life. This impairment may be attributed to a higher prevalence of caries and avoidance of dental treatment. Those populations should be identified and monitored carefully as well as encouraged to seek the appropriate behavioural treatment in order to improve their dental heath and oral health related quality of life.

  9. Demographic profile, plaque index and DMFT scores of young individuals with dental anxiety and exaggerated gag reflex.

    Science.gov (United States)

    Almoznino, Galit; Zini, Avraham; Aframian, Doron J; Kaufman, Eliezer; Lvovsky, Alex; Hadad, Avraham; Levin, Liran

    2015-01-01

    To characterise demographic and clinical parameters among individuals with dental anxiety and exaggerated gag reflex compared to a control group and to analyse the associations between the various parameters. Sixty-eight patients with dental anxiety and 54 patients with exaggerated gag reflex were compared to a control group of 200 individuals undergoing dental treatment. The collected data included demographic parameters, health status, smoking habits, Decayed, Missing and Filled Teeth (DMFT) and plaque index (PI) scores. PI was significantly higher among patients with exaggerated gag reflex (1.91 ± 0.95) and dental anxiety patients (1.82 ± 0.89) compared to the control group (1.27 ± 0.74; P anxiety (13.64 ± 7.57) compared to patients with exaggerated gag reflex (10.52 ± 5.42; P = 0.033), and between both groups compared to the control group (4.09 ± 4.034; P anxiety compared to the control group. DMFT was the only significant parameter positively associated with dental anxiety compared to exaggerated gag reflex. DMFT and PI scores were higher among patients with dental anxiety and exaggerated gag reflex. Clinicians should consider additional oral hygiene measures and education, maintenance meetings and recall visits in those patients, as well as using supplementary aids, such as fluoride mouthwash and fluoride varnish applications, to maintain oral hygiene without triggering the exaggerated gag reflex.

  10. Comparative study of the plasma globulin level, CD21(-) B-cell counts and FOXP3 mRNA expression level in CD4(+) T-cells for different clinical stages of feline immunodeficiency virus infected cats.

    Science.gov (United States)

    Takano, Tomomi; Hosoya, Shinobu; Shibao, Akari; Nagasaki, Bunpei; Yoshioka, Hisao; Satoh, Ryoichi; Hohdatsu, Tsutomu

    2012-02-01

    Feline immunodeficiency virus (FIV) infection leads to hypergammaglobulinemia through mechanisms that remain poorly understood. We investigated changes in plasma globulin level, B cells, and T cells with progression of the clinical stage of FIV-infected cats. We classified FIV-infected cats into the stage of Asymptomatic carrier (AC) and AIDS-related complex (ARC) based on the clinical symptoms, and measured the plasma globulin level, the CD4(+) T-cell counts, and analyzed surface markers of B cells. We investigated the relationship between the plasma globulin level and regulatory T cells (Tregs) using the Forkhead box P3 (FOXP3) mRNA expression level. In FIV-infected cats, the plasma globulin level and the surface immunoglobulin (sIg)(+) CD21(-) B-cell counts were increased, whereas the CD4(+) T-cell counts were decreased compared with specific-pathogen free (SPF) cats. The mRNA expression of Blimp-1 (master gene of plasma cells) was increased in peripheral blood, and the FOXP3 mRNA expression level was decreased in CD4(+) T-cells. These immunological changes were marked in the ARC stage. These data indicate that the decrease of Tregs and the increase of plasma cells lead to hypergammaglobulinemia.

  11. Collagen-GAG Scaffolds Grafted Onto Myocardial Infarcts in a Rat Model:A Delivery Vehicle for Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Z.; XIANG; R.; LIAO; M.; KELLY; M.; SPECTOR

    2005-01-01

    1 IntroductionThe objective of the present study was to investigate the response of rat myocardial scar tissue to type I collagen-glycosaminoglycan (GAG) tissue engineering scaffolds, and to assess the feasibility of using a collagen-GAG scaffold as a delivery vehicle for bone marrow-derived mesenchymal stem cells. The benefits of employing the collagen-GAG scaffold for this application include the following:(1) the large surface area of the three-dimensional sponge-like material allows for the delivery of ...

  12. Feline immunodeficiency virus and puma lentivirus in Florida panthers (Puma concolor coryi): epidemiology and diagnostic issues.

    Science.gov (United States)

    Miller, D L; Taylor, S K; Rotstein, D S; Pough, M B; Barr, M C; Baldwin, C A; Cunningham, M; Roelke, M; Ingram, D

    2006-04-01

    This study documents the seroprevalence of feline immunodeficiency virus (FIV) and puma lentivirus (PLV) in free-ranging and captive Florida panthers (Puma concolor coryi) (n = 51) and translocated Texas cougars (P. concolor stanleyana) (n = 10) from 1985 to 1998. The sera were tested for anti-FIV antibodies by enzyme-linked immunosorbent assay (ELISA) and Western blot tests. The ELISAs were read kinetically (KELA) and the sera were retrospectively examined by PLV peptide ELISA. Eleven panthers and one cougar were positive by KELA; 4 panthers and 4 cougars were equivocal; 35 panthers and 5 cougars were negative; and 1 panther had no data. Seven of the 11 KELA-positive panthers were also positive by Western blot tests and all but one were positive by PLV peptide ELISA. Ten KELA-negative and Western blot-negative cats, were positive by PLV peptide ELISA. KELA results varied within cats from one sample period to the next, but PLV peptide ELISA results were consistent. Territorial sympatry and mating behaviour, noted from radiotelemetry location data on the cats, may have contributed to viral transmission between seropositive animals. These findings suggest that Florida panthers and the introduced Texas cougars have been exposed to FIV and/or PLV.

  13. Evidence that Vpu modulates HIV-1 Gag-envelope interaction towards envelope incorporation and infectivity in a cell type dependent manner.

    Directory of Open Access Journals (Sweden)

    Archana Gautam

    Full Text Available The HIV-1 Vpu is required for efficient virus particle release from the plasma membrane and intracellular CD4 degradation in infected cells. In the present study, we found that the loss of virus infectivity as a result of envelope (Env incorporation defect caused by a Gag matrix (MA mutation (L30E was significantly alleviated by introducing a start codon mutation in vpu. Inactivation of Vpu partially restored the Env incorporation defect imposed by L30E substitution in MA. This effect was found to be comparable in cell types such as 293T, HeLa, NP2 and GHOST as well as in peripheral blood mononuclear cells (PBMC and monocyte-derived macrophages (MDM. However, in HeLa cells BST-2 knockdown was found to further alleviate the effect of Vpu inactivation on infectivity of L30E mutant. Our data demonstrated that the impaired infectivity of virus particles due to Env incorporation defect caused by MA mutation was modulated by start codon mutation in Vpu.

  14. Characterization of HIV-1 Gag-specific T cell responses in chronically infected Indian population

    Science.gov (United States)

    Kaushik, S; Vajpayee, M; Wig, N; Seth, P

    2005-01-01

    India is at the epicentre of the global HIV/AIDS epidemic in South-east Asia, predominated by subtype C infections. It is important to characterize HIV-1-specific T cell responses in this particular population with the aim of identifying protective correlates of immunity to control HIV-1 infection. In this study, we performed a comprehensive analysis of the breadth and magnitude of T cell responses directed at HIV-1 subtype C Gag, one of the most conserved HIV-1 proteins. The study population consisted of antiretroviral naive, chronic HIV-1 subtype C-infected individuals at various stages of infection. We used recent advanced techniques such as enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining to quantify the total CD4+ and CD8+ T cell response to HIV-1 gag at single peptide level, regardless of HLA haplotype of the infected individual. The p24-Gag was identified as the most frequently recognized subunit protein with the greatest magnitude of CD4+ and CD8+ T cell responses. Stronger and broader CD8 T cell responses were recognized, contrasting with the weaker and narrower CD4 T cell responses with regard to Gag protein subunits. The magnitude of the HIV-specific interferon (IFN)-γ responses was observed to be higher than the corresponding interleukin (IL)-2 response, indicating the persistence of antigenic load in chronically infected Indian population due to the probable dysfunction of HIV-specific, IFN-γ-secreting CD8 T cells in absence of IL-2 help. PMID:16232229

  15. Application of mouth gag and temporomandibular joint pain and trismus in tonsillectomy.

    Science.gov (United States)

    Kundi, Nasir Akram; Mehmood, Talat; Abid, Omair

    2015-04-01

    To determine the effect of duration of application of mouth gag on Temporomandibular (TM) joint pain and trismus after tonsillectomy. Descriptive study. Department of ENT and Head and Neck Surgery, Combined Military Hospital, Nowshera, from February to July 2012. A total of 40 patients undergoing tonsillectomy, in mouth opening prior to surgery was measured as inter incisor distance in cms. A stop watch was used to calculate the time of application of mouth gag. Mouth opening was again measured 06 hours after the surgery. Difference between the two readings was considered as trismus score and categorized as mild (1 cm), moderate (2 cm) and severe (3 cm). Patient was asked to score pain on a visual analogue scale (0 - 9). Score 0 was categorized as no pain; 1 - 3 as mild pain; 4 - 6 as moderate pain; 7 - 9 as severe pain. Spearman's rank correlation was used for finding correlation between time of mouth gag application and study outcome (pain and trismus). Trismus as observed by difference in inter incisor distance was mild in 11 patients; moderate in 15 patients and severe in 14 patients 06 hours after the surgery. Eleven (27.5%) had mild pain over temporomandibular joint, 15 (37.5%) had moderate and 14 (35%) had severe pain 06 hours after the surgery. Direct relationship was observed between duration of application of mouth gag with postoperative pain and trismus. Significant strong correlation was observed between length of mouth opening to severity of pain and trismus (rs = 0.738; p trismus in early postoperative period in tonsillectomy.

  16. Non-immunogenicity of overlapping gag peptides pulsed on autologous cells after vaccination of HIV infected individuals.

    Directory of Open Access Journals (Sweden)

    Henrik N Kløverpris

    Full Text Available BACKGROUND: HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation. METHODOLOGY: We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach 'OPAL-HIV-Gag(c'. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6, 24 mg (n = 7, 48 mg (n = 2 or matching placebo (n = 8 with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS. RESULTS: The OPAL-HIV-Gag(c peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c, 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours in OPAL-HIV-Gag(c but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001, compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16. CONCLUSION

  17. Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.

    Science.gov (United States)

    Nermut, M V; Wallengren, K; Pager, J

    1999-07-20

    Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.

  18. Genetic structure of avian myeloblastosis virus, released from transformed myeloblasts as a defective virus particle

    Science.gov (United States)

    Duesberg, Peter H.; Bister, Klaus; Moscovici, Carlo

    1980-01-01

    Chicken myeloblasts transformed by avian myeloblastosis virus (AMV) in the absence of nondefective helper virus (termed nonproducer cells) were found to release a defective virus particle (DVP) that contains avian tumor viral gag proteins but lacks envelope glycoprotein and a DNA polymerase. Nonproducer cells contain a Pr76 gag precursor protein and also a protein that is indistinguishable from the Pr180 gag-pol protein of nondefective viruses. The RNA of the DVP is 7.5 kilobases (kb) long and is 0.7 kb shorter than the 8.2-kb RNAs of the helper viruses of AMV, MAV-1 and MAV-2. Comparisons based on RNA·cDNA hybridization and mapping of RNase T1-resistant oligonucleotides indicated that DVP RNA shares with MAV RNAs nearly isogenic 5′-terminal gag and pol-related sequences of 5.3 kb and a 3′-terminal c-region of 0.7 kb that is different from that found in other avian tumor viruses. Adjacent to the c-region, DVP RNA contains a contiguous specific sequence of 1.5 kb defined by 14 specific oligonucleotides. Except for two of these oligonucleotides that map at its 5′ end, this sequence is unrelated to any sequences of nondefective avian tumor viruses of four different envelope subgroups as well as to the specific sequences of fibroblast-transforming avian acute leukemia and sarcoma viruses of four different RNA subgroups. The specific sequence of the DVP RNA is present in infectious stocks of AMV from this and other laboratories in an AMV-transformed myeloblast line from another laboratory, and it is about 70% related to nucleotide sequences of E26 virus, an independent isolate of an AMV-like virus. Preliminary experiments show DVP to be leukemogenic if fused into susceptible cells in the presence of helper virus. We conclude that DVP RNA is the leukemogenic component of infectious AMV and that its specific sequence, termed AMV, may carry genetic information for oncogenicity. Thus we have found here a transformation-specific RNA sequence, unrelated to helper virus

  19. Relevance of feline calicivirus, feline immunodeficiency virus, feline leukemia virus, feline herpesvirus and Bartonella henselae in cats with chronic gingivostomatitis.

    Science.gov (United States)

    Belgard, Sylvia; Truyen, Uwe; Thibault, Jean-Christophe; Sauter-Louis, Carola; Hartmann, Katrin

    2010-01-01

    Despite its common occurrence, the aetiology of chronic gingivostomatitis in cats remains uncertain. Aetiology is likely multifactorial, and several infectious agents may be associated with chronic gingivostomatitis. The purpose of this study was to investigate the prevalence of feline calicivirus (FCV), feline immunodeficiency virus (FIV), feline leukemia virus (FeLV), feline herpesvirus (FHV), and Bartonella henselae (B. henselae) in cats with chronic gingivostomatitis and in an age-matched control group. In addition, other factors, e. g., environmental conditions were investigated. In 52 cats with chronic gingivostomatitis and 50 healthy age-matched control cats, the presence of FCV ribonucleic acid (RNA), and FHV deoxyribonucleic acid (DNA) (polymerase chain reaction [PCR] from oropharyngeal swabs), and B. henselae DNA (PCR from oropharyngeal swabs and blood), as well as FeLV antigen (serum), and antibodies against FCV, B. henselae, and FIV (serum) were examined. FCV RNA was significantly more common in cats with chronic gingivostomatitis (53.8%, p gingivostomatitis (78.8%, p = 0.023) and controls (58.0%). Of the other infectious agents investigated, there was no significant difference in the prevalence between the cats with chronic gingivostomatitis and the controls. The results of this study allow the conclusion that FCV, but no other infectious agents, is commonly associated with chronic gingivostomatitis in cats.

  20. Escalating morphine exposures followed by withdrawal in feline immunodeficiency virus-infected cats: a model for HIV infection in chronic opiate abusers.

    Science.gov (United States)

    Barr, Margaret C; Huitron-Resendiz, Salvador; Sanchez-Alavez, Manuel; Henriksen, Steven J; Phillips, Tom R

    2003-11-24

    Opiate abuse is a risk factor for human immunodeficiency virus (HIV) infection. Because the direct effects of opiates on HIV infection are difficult to determine epidemiologically, animal models of lentivirus infection are relied upon to study the effects of opiates in the absence of confounding factors. Morphine, the predominant metabolite of heroin, is used in most experimental systems examining heroin abuse. In this study, morphine treatment of feline immunodeficiency virus (FIV)-infected cats modeled a typical pattern of escalating drug use interspersed with withdrawals. Plasma cortisol levels were measured for evidence of stress associated with morphine withdrawal. In the morphine-treated cats, cortisol levels peaked at time points corresponding to morphine withdrawal and returned to baseline levels during treatment and several weeks after the final withdrawal. Morphine-treated cats displayed clear behavioral and physical signs of opiate exposure and evidence of withdrawal when the drug was stopped. Morphine-exposed cats did not experience enhanced severity of FIV-related disease; in fact, morphine demonstrated a protective effect on FIV-associated changes in brainstem auditory evoked potentials. Our research suggests that opiate exposure is unlikely to adversely affect the progression of acute lentivirus infection and might be beneficial in controlling associated neurological disease.

  1. The Use of Recombinant Feline Interferon Omega Therapy as an Immune-Modulator in Cats Naturally Infected with Feline Immunodeficiency Virus: New Perspectives

    Directory of Open Access Journals (Sweden)

    Rodolfo Oliveira Leal

    2016-10-01

    Full Text Available Type I interferons (IFNs are well-known cytokines that, among their main functions, are key components of the host immune response against viral infections. Due to its immune modulation properties, they are commonly used in the therapeutic approach of various retroviral infections, namely human immunodeficiency virus (HIV and feline immunodeficiency virus (FIV. In HIV infection, it has been shown that IFN therapy limits early viral replication, particularly useful on post-exposure prophylaxis. In veterinary medicine, recombinant feline interferon omega (rFeIFN-ω was the first interferon licensed for use in cats. Several studies have recently shown that this compound seems to stimulate the innate immunity, decreasing clinical signs and co-infections in naturally FIV-infected cats. More than summarizing the main conclusions about rFeIFN-ω in cats, this review emphasizes the immune-modulation properties of IFN therapy, opening new perspectives for its use in retroviral infections. Either in FIV-infected cats or in HIV individuals, type I IFNs seem to induce an innate immune-modulation and should not be overlooked as a therapeutic option in retroviral infections.

  2. Efficacy of the acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against feline immunodeficiency virus.

    Science.gov (United States)

    Hartmann, K; Kuffer, M; Balzarini, J; Naesens, L; Goldberg, M; Erfle, V; Goebel, F D; De Clercq, E; Jindrich, J; Holy, A; Bischofberger, N; Kraft, W

    1998-02-01

    The acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were evaluated for their efficacy and side effects in a double-blind placebo-controlled trial using naturally occurring feline immunodeficiency virus (FIV)-infected cats. This natural retrovirus animal model is considered highly relevant for the pathogenesis and chemotherapy of HIV in humans. Both PMEA and FPMPA proved effective in ameliorating the clinical symptoms of FIV-infected cats, as measured by several clinical parameters including the incidence and severity of stomatitis, Karnofsky's score, immunologic parameters such as relative and absolute CD4+ lymphocyte counts, and virologic parameters including proviral DNA levels in peripheral blood mononuclear cells (PBMC) of drug-treated animals. In contrast with PMEA, FPMPA showed no hematologic side effects at a dose that was 2.5-fold higher than PMEA.

  3. Tracheal granuloma because of infection with a novel mycobacterial species in an old FIV-positive cat.

    Science.gov (United States)

    De Lorenzi, D; Solano-Gallego, L

    2009-03-01

    A 15-year-old domestic shorthair feline immunodeficiency virus-positive cat was presented with a five day history of productive cough and acute respiratory distress. Physical examination revealed inspiratory dyspnoea and diffuse gingivostomatitis. Radiographs showed an intratracheal mass located at the level of the sixth and the seventh cervical vertebrae. Bronchoscopy revealed a unique intratracheal mass occluding about 85 per cent of the tracheal lumen. The tracheal mass was removed bronchoscopically. A diagnosis of pyogranulomatous inflammation referable to a mycobacterial infection was made based on cytological and histopathological findings. 16S rRNA polymerase chain reaction testing and sequence analysis identified a novel mycobacterial species, likely a slow grower, with 95 per cent identity with Mycobacterium xenopi. To our knowledge, this is the first description of a tracheal mycobacterial granuloma in a cat, and the first time, a mycobacterium with this sequence has been identified.

  4. Leishmaniosis due to Leishmania infantum in a FIV and FeIV positive cat with a squamous cell carcinoma diagnosed with histological, serological and isoenzymatic methods

    Directory of Open Access Journals (Sweden)

    Grevot A.

    2005-09-01

    Full Text Available Leishmaniosis caused by Leishmania infantum is an endemic zoonosis present in the Mediterranean area. Canidae (dog and fox constitute the main reservoir hosts for the parasite, whilst wild rodents or the cat can be carriers of the protozoan and are considered as secondary potential reservoirs. This paper describes a case of disseminated feline leishmaniosis with cutaneous (ulcerative, visceral (spleen and lymph nodes and blood involvement in a FIV-FelV positive cat. The microscopic identification of the Leishmania infection was initially made on a skin biopsy of the temporal area, where a squamous cell carcinoma was diagnosed. The diagnosis of the disease was achieved by several serological techniques (ELISA, IFAT and Western-blot. The strain was obtained by blood culture, characterized by electrophoresis of isoenzymes and identified as Leishmania infantum zymodeme MON-1. Since the infection due to L. infantum is a zoonosis, the potential feline reservoir should be more investigated. Serological analysis by Western blot on domestic cats provides a useful tool. In veterinary practice, feline leishmaniosis should be systematically included in the differential diagnosis when compatible cutaneous lesions are present, especially in the endemic areas of canine leishmaniosis.

  5. Capsid proteins from human immunodeficiency virus type 1 and simian immunodeficiency virus SIVmac can coassemble into mature cores of infectious viruses.

    Science.gov (United States)

    Chen, Jianbo; Pathak, Vinay K; Peng, Weiqun; Hu, Wei-Shau

    2008-09-01

    We have recently shown that the Gag polyproteins from human immunodeficiency virus type 1 (HIV-1) and HIV-2 can coassemble and functionally complement each other. During virion maturation, the Gag polyproteins undergo proteolytic cleavage to release mature proteins including capsid (CA), which refolds and forms the outer shell of a cone-shaped mature core. Less than one-half of the CA proteins present within the HIV-1 virion are required to form the mature core. Therefore, it is unclear whether the mature core in virions containing both HIV-1 and HIV-2 Gag consists of CA proteins from a single virus or from both viruses. To determine whether CA proteins from two different viruses can coassemble into mature cores of infectious viruses, we exploited the specificity of the tripartite motif 5alpha protein from the rhesus monkey (rhTRIM5alpha) for cores containing HIV-1 CA (hCA) but not the simian immunodeficiency virus SIV(mac) CA protein (sCA). If hCA and sCA cannot coassemble into the same core when equal amounts of sCA and hCA are coexpressed, the infectivities of such virus preparations in cells should be inhibited less than twofold by rhTRIM5alpha. However, if hCA and sCA can coassemble into the same core structure to form a mixed core, rhTRIM5alpha would be able to recognize such cores and significantly restrict virus infectivity. We examined the restriction phenotypes of viruses containing both hCA and sCA. Our results indicate that hCA and sCA can coassemble into the same mature core to produce infectious virus. To our knowledge, this is the first demonstration of functional coassembly of heterologous CA protein into the retroviral core.

  6. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Robert J Scarborough

    2014-01-01

    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  7. Detection, molecular characterization and phylogenetic analysis of full-length equine infectious anemia (EIAV) gag genes isolated from Shackleford Banks wild horses.

    Science.gov (United States)

    Capomaccio, S; Willand, Z A; Cook, S J; Issel, C J; Santos, E M; Reis, J K P; Cook, R F

    2012-06-15

    The genetically distinct wild horse herds inhabiting Shackleford Banks, North Carolina are probably the direct descendents of Spanish stock abandoned after failed attempts to settle mid-Atlantic coastal regions of North America in the Sixteenth Century. In a 1996 island survey, 41% of the gathered horses were discovered seropositive for Equine Infectious Anemia Virus (EIAV) with additional cases identified in 1997 and 1998. As a result of their unique genetic heritage, EIAV seropositive individuals identified in the two latter surveys were transferred to a quarantine facility on the mainland. In September 2008 two of the horses SB1 and SB2 after 10 and 11 years in quarantine respectively, developed clinical signs of EIA. In the case of SB2 these were so severe that the only humane option was euthanasia. Although SB1, survived it experienced a second clinical episode one month later. In May 2009, a third horse in quarantine, SB3, developed extremely severe clinical EIA and was euthanized. This demonstrates naturally infected long-term inapparent carriers can experience recrudescence of very severe disease many years after initial exposure to EIAV. Phylogenetic analysis of complete EIAV gag gene sequences obtained from each of three Shackleford horses demonstrated they were infected with very closely related viruses. Although these were distinguishable from all other strains examined, they belong to a monophyletic group comprising almost exclusively of New World isolates that is distinct from a number of recently characterized Central European EIAV strains. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. HIV-specific CD8~+ T cell responses to HXB2 Gag and Nef peptide pools in Chinese HIV/AIDS patients

    Institute of Scientific and Technical Information of China (English)

    ZHANG HongWei; QIU ZhiFeng; JIAO Yang; WANG AiXia; LI TaiSheng

    2009-01-01

    HXB2 is primarily used as a template strain in developing HIV vaccines in Europe and the US. However, it is not yet known whether the strain can induce strong HIV-specific CD8~+ T cell responses in Chinese HIV/AIDS patients. In the present study, two groups of subjects were investigated: 9 AIDS patients and 7 long-term nonprogressors (LTNPs). HIV-specific CD8~+ T cell responses were examined in all patients through the ELISPOT assay. CD4~+ T cell counts, CD8~+ T cell counts, viral load and HIV subtype of each patient were also measured. Thailand B virus strain was identified among all the patients. The breadth and magnitude of HIV-specific CD8~+ T cell responses in the LTNPs group are greater than those in the AIDS group (P<0.01). There is a positive correlation between magnitude of HIV-specific CD8~+ T cell responses and CD4~+ T cells, and a negative correlation between HIV-specific CD8~+ T cell responses and mean viral load. In summary, the HIV-specific CD8~+ T cell responses to the HXB2 Gag and Nef peptide pools are considerable in Chinese HIV/AIDS patients infected with Thailand B virus strain. HIV-1 vaccines based on HXB2 strain that can induce extensive immunity may be helpful for Chinese.

  9. HIV-specific CD8~+ T cell responses to HXB2 Gag and Nef peptide pools in Chinese HIV/AIDS patients

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    HXB2 is primarily used as a template strain in developing HIV vaccines in Europe and the US. However,it is not yet known whether the strain can induce strong HIV-specific CD8+ T cell responses in Chinese HIV/AIDS patients. In the present study,two groups of subjects were investigated:9 AIDS patients and 7 long-term nonprogressors (LTNPs). HIV-specific CD8+ T cell responses were examined in all patients through the ELISPOT assay. CD4+ T cell counts,CD8+ T cell counts,viral load and HIV subtype of each patient were also measured. Thailand B virus strain was identified among all the patients. The breadth and magnitude of HIV-specific CD8+ T cell responses in the LTNPs group are greater than those in the AIDS group (P<0.01). There is a positive correlation between magnitude of HIV-specific CD8+ T cell responses and CD4+ T cells,and a negative correlation between HIV-specific CD8+ T cell responses and mean viral load. In summary,the HIV-specific CD8+ T cell responses to the HXB2 Gag and Nef peptide pools are considerable in Chinese HIV/AIDS patients infected with Thailand B virus strain. HIV-1 vaccines based on HXB2 strain that can induce extensive immunity may be helpful for Chinese.

  10. Detection of horses infected naturally with equine infectious anemia virus by nested polymerase chain reaction.

    Science.gov (United States)

    Nagarajan, M M; Simard, C

    2001-05-01

    A nested polymerase chain reaction (PCR) amplifying a region of the gag gene of equine infectious anemia virus (EIAV) was developed for the rapid and direct detection of proviral DNA from the peripheral blood of naturally infected horses and was compared with the Coggins test. DNA prepared from white blood cells of 122 field horses from 15 stables with reported cases of EIAV and one seronegative stable were analysed. Amplifications of expected size fragments were obtained by nested PCR for 88 horses using two different sets of primers targeting the gag region. The specificity of the amplified products was confirmed by hybridization using a digoxigenin-labeled probe. Gag-nested PCR-restriction fragment length polymorphism analysis distinguished two different subtypes of gag gene, A and B. Subtype A was found to be the most prevalent among the infected horses that were tested. The PCR-gag amplified sequence of subtype A shared 84.6% nucleotide and 93% deduced amino acid sequence identities with the prototype Wyoming strain whereas subtype B sequence was almost 100% identical to the prototype. Sequence analysis of gag subtype A suggests the presence of a novel EIAV variant among infected horses in Canada. The nested PCR assay developed in the present study detected more EIAV positive animals and was found as specific as the agar gel immunodiffusion (Coggins) assay and offers great potential a diagnostic test for the detection of EIAV infections in field horses.

  11. Effect of bovine lactoferrin on functions of activated feline peripheral blood mononuclear cells during chronic feline immunodeficiency virus infection.

    Science.gov (United States)

    Kobayashi, Saori; Sato, Reeko; Aoki, Takako; Omoe, Katsuhiko; Inanami, Osamu; Hankanga, Careen; Yamada, Yuichi; Tomizawa, Nobuyuki; Yasuda, Jun; Sasaki, Juso

    2008-05-01

    Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum gamma-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIV-infected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConA-induced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection.

  12. Ultrastructure and morphogenesis of human immunodeficiency virus.

    Science.gov (United States)

    Nakai, M; Goto, T

    1996-08-01

    The ultrastructure and morphogenesis of human immunodeficiency virus (HIV) were elucidated by observation with several techniques including immunoelectron microscopy and cryo-microscopy. The virus particle consists of an envelope, a core and matrix. The virus particles were observed extracellularly as having one of three profiles: (1) a centric or an eccentric electron-dense core, (2) rod-shaped electron-dense core, and (3) doughnut-shaped. HIV-1 particles in the hydrated state were observed by high resolution electron cryo-microscopy to be globular, and the lipid membrane was clearly resolved as a bilayer. Many projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially head parts, were found to vary in each projection. By isolation with Nonidet P40 and glutaraldehyde, HIV-1 cores were confirmed to consist of p24 protein by immunogold labeling. When the virus enters the cell, two entry modes were found: membrane fusion and endocytosis. No structures resembling virus particles could be seen in the cytoplasm after viral entry. In HIV-1-infected cells, positive reactions by immuno-labeling suggest that HIV-1 Gag may be produced in membrane-bound structures and transported to the cell surface by cytoskeletons. Then a crescent electron-dense layer was first formed underneath the cell membrane. Finally, the virus particle was released from the cell surface. Several cell clones producing defective particles were isolated from MT-4/HIV-1 cells. Among them, doughnut-shaped or teardrop-shaped particles were seen to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins faced each other against the inner electron dense ring, suggesting that the inner ring consists of a precursor Gag protein.

  13. The life-cycle of human immunodeficiency virus type 1.

    Science.gov (United States)

    Goto, T; Nakai, M; Ikuta, K

    1998-01-01

    The life-cycle of human immunodeficiency virus type 1 (HIV-1) has been studied using several techniques including immunoelectron microscopy and cryomicroscopy. The HIV-1 particle consists of an envelope, a core and the region between the core and the envelope (matrix). Virus particles in the extracellular space are observed as having various profiles: a central or an eccentric round electron-dense core, a bar-shaped electron-dense core, and immature doughnut-shaped particle. HIV-1 particles in the hydrated state were observed by high-resolution electron cryomicroscopy to be spherical and the lipid membrane was clearly resolved as a bilayer. Projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially the head parts, were found to vary with each projection. HIV-1 cores were isolated with a mixture of Nonidet P40 and glutaraldehyde, and were confirmed to consist of HIV-1 Gag p24 protein by immunogold labelling. On infection, the HIV-1 virus was found to enter the cell in two ways: membrane fusion and endocytosis. After viral entry, no structures resembling virus particles could be seen in the cytoplasm. In the infected cells, positive reactions by immunolabelling suggest that HIV-1 Gag is produced in membrane-bound structures and transported to the cell surface by the cytoskeletons. A crescent electron-dense layer is then formed underneath the cell membrane. Finally, the virus particle is released from the cell surface and found extracellularly to be a complete virus particle with an electron-dense core. However, several cell clones producing defective mature, doughnut-shaped (immature) or teardrop-shaped particles were found to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins exist facing each other against an inner electron-dense ring, suggesting that the inner ring consists of a precursor Gag protein showing a defect at the viral proteinase.

  14. Efficient production of HIV-1 virus-like particles from a mammalian expression vector requires the N-terminal capsid domain.

    Directory of Open Access Journals (Sweden)

    Pascal Jalaguier

    Full Text Available It is now well accepted that the structural protein Pr55(Gag is sufficient by itself to produce HIV-1 virus-like particles (VLPs. This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s is essential to the production of VLPs when Pr55(Gag is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55(Gag sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55(Gag-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development.

  15. Transmission of new CRF07_BC Strains with 7 amino acid deletion in Gag p6

    Directory of Open Access Journals (Sweden)

    Jianxin Lu

    2011-02-01

    Full Text Available Abstract A 7 amino acid deletion in Gag p6 (P6delta7 emerged in Chinese prevalent HIV-1 strain CRF07_BC from different epidemic regions. It is important to determine whether this mutation could be transmitted and spread. In this study, HIV-1 Gag sequences from 5 different epidemic regions in China were collected to trace the transmission linkage and to analyze genetic evolution of P6delta7 strains. The sequence analysis demonstrated that P6delta7 is a CRF07_BC specific deletion, different P6delta7 strains could be originated from different parental CRF07_BC recombinants in different epidemic regions, and the transmission of P6delta7 strain has occurred in IDU populations. This is for the first time to identify the transmission linkage for P6delta7 strains and serves as a wake-up call for further monitoring in the future; In addition, P6delta7 deletion may represent an evolutionary feature which might exert influence on the fitness of CRF07_BC strain.

  16. Inhibition of oncostatin M in osteoarthritic synovial fluid enhances GAG production in osteoarthritic cartilage repair

    Directory of Open Access Journals (Sweden)

    M Beekhuizen

    2013-09-01

    Full Text Available Mediators in the synovial fluid are thought to play a major role in osteoarthritic cartilage turnover. The purpose of the current study was to investigate the role of oncostatin M (OSM in osteoarthritis (OA by evaluating the presence of the cytokine and its receptors in the OA joint and interfering with its activity in synovial fluid co-cultured with cartilage explants. OSM levels were increased in the synovial fluid of osteoarthritic patients compared to healthy donors. Immunohistochemistry confirmed the presence of both the leukaemia inhibitory factor (LIF and OSM receptors for OSM throughout the whole depth of osteoarthritic cartilage and synovial tissue, whereas in healthy cartilage their presence seemed more restricted to the superficial zone. Blocking OSM activity, using an activity inhibiting antibody, in 25 % osteoarthritic synovial fluid added to OA cartilage explant cultures increased glycosaminoglycan (GAG content from 18.6 mg/g to 24.3 mg/g (P < 0.03 and total production from 7.0 mg/g to 11.9 mg/g (P < 0.003. However, OSM exogenously added to cartilage explant cultures reflecting low and high concentrations in the synovial fluid (5 and 50 pg/mL did not affect cartilage matrix turnover, suggesting that factors present in the synovial fluid act in concert with OSM to inhibit GAG production. The current study indicates the potential to enhance cartilage repair in osteoarthritis by modulating the joint environment by interfering with OSM activity.

  17. Collagen-GAG Scaffolds Grafted Onto Myocardial Infarcts in a Rat Model: A Delivery Vehicle for Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Z. XIANG; R. LIAO; M. KELLY; M. SPECTOR

    2005-01-01

    @@ 1 Introduction The objective of the present study was to investigate the response of rat myocardial scar tissue to type Ⅰ collagen-glycosaminoglycan (GAG) tissue engineering scaffolds, and to assess the feasibility of using a collagenGAG scaffold as a delivery vehicle for bone marrow-derived mesenchymal stem cells. The benefits of employing the collagen-GAG scaffold for this application include the following: (1) the large surface area of the three-dimensional sponge-like material allows for the delivery of a high cell density to the infarct site; (2) the scaffold allows for the localization and retention of the cells at the site of implantation; (3) the tissue response to the scaffold may promote angiogenesis at the implant site.

  18. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA.

    Science.gov (United States)

    Pasternak, Alexander O; DeMaster, Laura K; Kootstra, Neeltje A; Reiss, Peter; O'Doherty, Una; Berkhout, Ben

    2015-11-11

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. Here, we demonstrate that in HIV-infected patients on suppressive combination antiretroviral therapy, such host-derived transcripts do not significantly contribute to the HIV gag RNA level.

  19. Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

    Directory of Open Access Journals (Sweden)

    Irena Zurnic

    2016-08-01

    Full Text Available Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H screen with prototype FV (PFV Gag protein as bait we identified human polo-like kinase 2 (hPLK2, a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells.

  20. Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

    Science.gov (United States)

    Zurnic, Irena; Hütter, Sylvia; Rzeha, Ute; Stanke, Nicole; Reh, Juliane; Müllers, Erik; Hamann, Martin V.; Kern, Tobias; Gerresheim, Gesche K.; Serrao, Erik; Lesbats, Paul; Engelman, Alan N.; Cherepanov, Peter; Lindemann, Dirk

    2016-01-01

    Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. PMID:27579920

  1. Fujinami sarcoma virus: An avian RNA tumor virus with a unique transforming gene

    Science.gov (United States)

    Lee, Wen-Hwa; Bister, Klaus; Pawson, Anthony; Robins, Terry; Moscovici, Carlo; Duesberg, Peter H.

    1980-01-01

    The oncogenic properties and RNA of the Fujinami avian sarcoma virus (FSV) and the protein it encodes were investigated and compared to those of other avian tumor viruses with sarcomagenic properties such as Rous sarcoma virus and the acute leukemia viruses MC29 and erythroblastosis virus. Cloned stocks of FSV caused sarcomas in all chickens inoculated and were found to contain a 4.5-kilobase (kb) and an 8.5-kb RNA species. The 4.5-kb RNA was identified as the genome of defective FSV because it was absent from nondefective FSV-associated helper virus and because the titer of focus-forming units increased with the ratio of 4.5-kb to 8.5-kb RNA in virus preparations. This is, then, the smallest known tumor virus RNA with a transforming function. Comparisons with other viral RNAs, based on oligonucleotide mapping and molecular hybridization, indicated that 4.5-kb FSV RNA contains a 5′ gag gene-related sequence of 1 kb, an internal specific sequence of about 3 kb that is unrelated to Rous sarcoma virus, MC29, and erythroblastosis virus, and a 3′-terminal sequence of about 0.5 kb related to the conserved C region of avian tumor viruses. The lack of some or all nucleotide sequences of the essential virion genes, gag, pol, and env, and the isolation of FSV-transformed nonproducer cell clones indicated that FSV is replication defective. A 140,000-dalton, gag-related non-structural protein was found in FSV-transformed producer and nonproducer cells and was translated in vitro from full-length FSV RNA. This protein is expected to have a transforming function both because its intracellular concentration showed a positive correlation with the percentage of transformed cells in a culture and because FSV is unlikely to code for major additional proteins since the genetic complexities of FSV RNA and the FSV protein are almost the same. It is concluded that the transforming onc gene of FSV is distinct from that of Rous sarcoma virus and other avian tumor viruses with

  2. Mutations in the Primer Grip of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Impair Proviral DNA Synthesis and Virion Maturation

    Science.gov (United States)

    Yu, Qiang; Ottmann, Michele; Pechoux, Christine; Le Grice, Stuart; Darlix, Jean-Luc

    1998-01-01

    This report describes the effects of mutating highly conserved residues in the primer grip domain of human immunodeficiency virus type 1 reverse transcriptase (RT) on virus formation and infectivity. Among a series of RT mutant viruses, three (M230A, L234D, and W239A) were found to be noninfectious or very poorly infectious. Our data indicate that these mutations in RT caused severe defects in proviral DNA synthesis. Interestingly, assembly and maturation of mutant virus M230A were similar to those of the wild type, while mutants L234D and W239A showed impaired maturation. The immature morphology of RT mutants L234D and W239A is due at least in part to premature cleavage of the gag-pol precursor, prior to virion budding, indicating that intracellular stability of Pr160gag-pol is of key importance during virus assembly. PMID:9696874

  3. Failure to demonstrate human T cell lymphotropic virus type I in multiple sclerosis patients

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P;

    1990-01-01

    The polymerase chain reaction (PCR) technique was employed in searching for human T cell lymphotropic virus type I (HTLV-I) gag, env and pol sequences in samples of DNA prepared from two HTLV-I seropositive patients with tropical spastic paraparesis (TSP), the Swedish multiple sclerosis (MS...... and detection probes. In MS patients and healthy individuals, no signals were obtained with gag and env. In occasional experiments, weak signals were seen for the pol segment for a single MS patient and/or healthy individuals, but these signals were not reproducible in subsequent experiments. Thus, the present...

  4. Preparation and Characterization of Collagen-GAGs Bioactive Matrices for Tissue Engineering

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Collagen materials were crosslinked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in the presence of chondroitin sulfate (CS), one of glycosaminoglycans (GAGs). PVA and chitosan were also blended with collagen. The physical and chemical properties of the matrices were characterized by SEM, DSC, and ESCA. L929 cells were implanted on the matrices to show the cytotoxic and the biological characters of the materials. The results indicate that EDC is an effective and non-cytotoxic cross-link reagent, which can replace the common dialdehyde reagent. The attachment of CS can improve the stability of collagen and accelerate cell growth.The addition of PVA can prepare porous matrices with smaller bore size. There are reactions between the chitosan and collagen, and the composite has good biological character. The presence of chitosan can also increase the amount of incorporated CS.

  5. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA

    NARCIS (Netherlands)

    Pasternak, A.O.; DeMaster, L.K.; Kootstra, N.A.; Reiss, P.; O'Doherty, U.; Berkhout, B.

    2016-01-01

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent

  6. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA

    NARCIS (Netherlands)

    Pasternak, A.O.; DeMaster, L.K.; Kootstra, N.A.; Reiss, P.; O'Doherty, U.; Berkhout, B.

    2016-01-01

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent

  7. Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for ‑1 ribosomal frameshifting stimulation

    Science.gov (United States)

    Zhong, Zhensheng; Yang, Lixia; Zhang, Haiping; Shi, Jiahao; Vandana, J. Jeya; Lam, Do Thuy Uyen Ha; Olsthoorn, René C. L.; Lu, Lanyuan; Chen, Gang

    2016-12-01

    Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The ‑1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between ‑1 frameshifting efficiency and unfolding rate at forces of 15–35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and ‑1 frameshifting efficiency.

  8. A cis-acting element present within the Gag open reading frame negatively impacts on the activity of the HIV-1 IRES.

    Directory of Open Access Journals (Sweden)

    Fernando Valiente-Echeverría

    Full Text Available Translation initiation from the human immunodeficiency virus type-1 (HIV-1 mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS-1, a cis-acting regulatory element present within the gag open reading frame (ORF. In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1.

  9. Ebola Virus and Marburg Virus

    Science.gov (United States)

    Diseases and Conditions Ebola virus and Marburg virus By Mayo Clinic Staff Ebola virus and Marburg virus are related viruses that cause hemorrhagic ... Africa, where sporadic outbreaks have occurred for decades. Ebola virus and Marburg virus live in animal hosts, ...

  10. Ebola Virus and Marburg Virus

    Science.gov (United States)

    Ebola virus and Marburg virus Overview By Mayo Clinic Staff Ebola virus and Marburg virus are related viruses that ... Africa, where sporadic outbreaks have occurred for decades. Ebola virus and Marburg virus live in animal hosts, ...

  11. Molecular characterization and virus neutralization patterns of severe, non-epizootic forms of feline calicivirus infections resembling virulent systemic disease in cats in Switzerland and in Liechtenstein.

    Science.gov (United States)

    Willi, Barbara; Spiri, Andrea M; Meli, Marina L; Samman, Ayman; Hoffmann, Karolin; Sydler, Titus; Cattori, Valentino; Graf, Felix; Diserens, Kevin A; Padrutt, Isabelle; Nesina, Stefanie; Berger, Alice; Ruetten, Maja; Riond, Barbara; Hosie, Margaret J; Hofmann-Lehmann, Regina

    2016-01-15

    Feline calicivirus (FCV) infections are associated with oral ulceration, chronic stomatitis and a limping syndrome. Epizootic outbreaks of virulent systemic disease (VSD) have been reported in the USA and Europe. Here, the molecular characterization and neutralization patterns of FCV isolates from cases of severe, non-epizootic infection associated with skin ulceration and edema are presented. Samples from eleven symptomatic cats, four in-contact cats and 27 cats with no contact with symptomatic cats were collected and tested for FCV, feline herpesvirus-1 (FHV-1), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). Phylogenetic analyses based on the capsid (VP1) gene of FCV and virus neutralization with antisera raised against four FCV vaccine strains were performed. Nine kittens and two adult cats in two shelters and two veterinary clinics in four geographically distinct locations in Switzerland and Liechtenstein were affected. The cats showed fever, tongue and skin ulceration, head and paw edema, and occasionally jaundice, generalized edema and dyspnea. All symptomatic cats tested FCV-positive but were negative for FHV-1, FeLV and FIV, with the exception of one FIV-positive kitten. All kittens of one litter and both adult cats died. The disease did not spread to cats in the environment. Cats in the environment displayed phylogenetically distinct, but related, FCV strains. Virus neutralization patterns suggested that some cases might have been potentially prevented by vaccination with the optimal vaccine strain. In conclusion, clinicians should be aware of severe, non-epizootic forms of FCV infections with initial clinical presentations similar to VSD.

  12. Enhanced inhibition of Avian leukosis virus subgroup J replication by multi-target miRNAs

    Directory of Open Access Journals (Sweden)

    Meng Qing-Wen

    2011-12-01

    Full Text Available Abstract Background Avian leukosis virus (ALV is a major infectious disease that impacts the poultry industry worldwide. Despite intensive efforts, no effective vaccine has been developed against ALV because of mutations that lead to resistant forms. Therefore, there is a dire need to develop antiviral agents for the treatment of ALV infections and RNA interference (RNAi is considered an effective antiviral strategy. Results In this study, the avian leukosis virus subgroup J (ALV-J proviral genome, including the gag genes, were treated as targets for RNAi. Four pairs of miRNA sequences were designed and synthesized that targeted different regions of the gag gene. The screened target (i.e., the gag genes was shown to effectively suppress the replication of ALV-J by 19.0-77.3%. To avoid the generation of escape variants during virus infection, expression vectors of multi-target miRNAs were constructed using the multi-target serial strategy (against different regions of the gag, pol, and env genes. Multi-target miRNAs were shown to play a synergistic role in the inhibition of ALV-J replication, with an inhibition efficiency of viral replication ranging from 85.0-91.2%. Conclusion The strategy of multi-target miRNAs might be an effective method for inhibiting ALV replication and the acquisition of resistant mutations.

  13. The feline lymphoid cell line MBM and its use for feline immunodeficiency virus isolation and quantitation.

    Science.gov (United States)

    Matteucci, D; Mazzetti, P; Baldinotti, F; Zaccaro, L; Bendinelli, M

    1995-05-01

    We report on the development of a feline T lymphoblastoid cell line obtained from the peripheral blood mononuclear cells (PBMC) of a specific pathogen free cat and designated MBM. The cells are pan-T+, CD4- and CD8- and remained interleukin-2-dependent and concanavalin A-dependent throughout the period of observation. MBM cells have proved at least as sensitive as fresh blasts to infection with cell-free stocks of three feline immunodeficiency virus (FIV) isolates. Upon infection, they exhibit a lytic cytopathic effect. Repeated attempts to establish a chronic infection have failed. Using a limiting cell dilution method, it has been shown that MBM cells may be more sensitive than fresh blasts as substrate for isolating FIV from the PBMC of infected cats. These studies have also shown that considerable individual variations exist in the virus loads present in the PBMC of naturally infected cats, and that load size does not appear to correlate with cat age, clinical status, CD4/CD8 ratio and titer of serum neutralizing antibody.

  14. Failure to demonstrate human T cell lymphotropic virus type I in multiple sclerosis patients

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1990-01-01

    The polymerase chain reaction (PCR) technique was employed in searching for human T cell lymphotropic virus type I (HTLV-I) gag, env and pol sequences in samples of DNA prepared from two HTLV-I seropositive patients with tropical spastic paraparesis (TSP), the Swedish multiple sclerosis (MS......) patients who recently have been reported to be PCR-positive for HTLV-I gag and env sequences, and eight healthy individuals. Precautions were taken in order to reduce the risk of cross-contamination in the PCR. In the two TSP patients strong signals were obtained with gag, env and pol amplification primers...... data do not confirm the presence of HTLV-I sequences in MS patients....

  15. Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens.

    Science.gov (United States)

    Pushko, Peter; Tretyakova, Irina; Hidajat, Rachmat; Zsak, Aniko; Chrzastek, Klaudia; Tumpey, Terrence M; Kapczynski, Darrell R

    2017-01-15

    Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine. Copyright © 2016. Published by Elsevier Inc.

  16. Sulfated Glycans and Related Digestive Enzymes in the Zika Virus Infectivity: Potential Mechanisms of Virus-Host Interaction and Perspectives in Drug Discovery

    Science.gov (United States)

    2017-01-01

    As broadly reported, there is an ongoing Zika virus (ZIKV) outbreak in countries of Latin America. Recent findings have demonstrated that ZIKV causes severe defects on the neural development in fetuses in utero and newborns. Very little is known about the molecular mechanisms involved in the ZIKV infectivity. Potential therapeutic agents are also under investigation. In this report, the possible mechanisms of action played by glycosaminoglycans (GAGs) displayed at the surface proteoglycans of host cells, and likely in charge of interactions with surface proteins of the ZIKV, are highlighted. As is common for the most viruses, these sulfated glycans serve as receptors for virus attachment onto the host cells and consequential entry during infection. The applications of (1) exogenous sulfated glycans of different origins and chemical structures capable of competing with the virus attachment receptors (supposedly GAGs) and (2) GAG-degrading enzymes able to digest the virus attachment receptors on the cells may be therapeutically beneficial as anti-ZIKV. This communication attempts, therefore, to offer some guidance for the future research programs aimed to unveil the molecular mechanisms underlying the ZIKV infectivity and to develop therapeutics capable of decreasing the devastating consequences caused by ZIKV outbreak in the Americas. PMID:28203251

  17. The Cholestanol-Conjugated Sulfated Oligosaccharide PG545 Disrupts the Lipid Envelope of Herpes Simplex Virus Particles

    Science.gov (United States)

    Trybala, Edward; Görander, Staffan; Ekblad, Maria; Liljeqvist, Jan-Åke; Jennische, Eva; Lange, Stefan

    2015-01-01

    Herpes simplex virus (HSV) and many other viruses, including HIV, initiate infection of host cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans. Although GAG mimetics, such as sulfated oligo- and polysaccharides, exhibit potent antiviral activities in cultured cells, the prophylactic application of these inhibitors as vaginal microbicides failed to protect women upon their exposure to HIV. A possible explanation for this failure is that sulfated oligo- and polysaccharides exhibit no typical virucidal activity, as their interaction with viral particles is largely electrostatic and reversible and thereby vulnerable to competition with GAG-binding proteins of the genital tract. Here we report that the cholestanol-conjugated sulfated oligosaccharide PG545, but not several other sulfated oligosaccharides lacking this modification, exhibited virucidal activity manifested as disruption of the lipid envelope of HSV-2 particles. The significance of the virus particle-disrupting activity of PG545 was also demonstrated in experimental animals, as this compound, in contrast to unmodified sulfated oligosaccharide, protected mice against genital infection with HSV-2. Thus, PG545 offers a novel prophylaxis option against infections caused by GAG-binding viruses. PMID:26643323

  18. Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine.

    Science.gov (United States)

    Kanagavelu, Saravana K; Snarsky, Victoria; Termini, James M; Gupta, Sachin; Barzee, Suzanne; Wright, Jacqueline A; Khan, Wasif N; Kornbluth, Richard S; Stone, Geoffrey W

    2012-01-17

    DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP

  19. Profound Differences in Virus Population Genetics Correspond to Protection from CD4 Decline Resulting from Feline Lentivirus Coinfection

    Directory of Open Access Journals (Sweden)

    Abinash Padhi

    2010-12-01

    Full Text Available CD4 decline is a hallmark of disease onset in individuals infected with Feline Immunodeficiency Virus (FIV or Human Immunodeficiency Virus type 1 (HIV-1. Cats that are infected with a poorly replicating, apathogenic FIV (PLV prior to exposure to a virulent FIV strain (FIVC maintain CD4 numbers by mechanisms that are not correlated with a measurable adaptive immune response or reduction in circulating viral load. We employed population genetic approaches based on the 3' portion of the viral genome to estimate the population structure of FIVC from single and dual infected cats. In dual infected cats, FIVC effective population size was decreased during the initial viral expansion phase, and after three weeks of infection, the population declined sharply. The FIVC population recovered to pre-bottleneck levels approximately seven weeks post-FIVC infection. However, the population emerging from the bottleneck in dual infected cats was distinct based on estimates of temporal population structure and substitution profiles. The transition to transversion rate ratio (k increased from early to late phases in dual infected cats due primarily to a decrease in transversions whereas in single infected cats, k declined over time. Although one clone with extensive G to A substitutions, indicative of host cytidine deaminase editing, was recovered from a dual infected cat during the bottleneck, the post bottleneck population had an overall reduction in G to A substitutions. These data are consistent with a model of PLV-induced host restriction, putatively involving host DNA editing, that alters the dynamics of FIVC throughout the course of infection leading to disease attenuation.

  20. DNA encoding an HIV-1 Gag/human lysosome-associated membrane protein-1 chimera elicits a broad cellular and humoral immune response in Rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Priya Chikhlikar

    Full Text Available Previous studies of HIV-1 p55Gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type II (MHC II complex molecules by use of a DNA antigen formulation encoding Gag as a chimera with the mouse lysosome-associated membrane protein (mLAMP/gag. In the present study, we have analyzed the magnitude and breadth of Gag-specific T-lymphocyte and antibody responses elicited in Rhesus macaques after immunization with DNA encoding a human LAMP/gag (hLAMP/gag chimera. ELISPOT analyses indicated that the average Gag-specific IFN-gamma response elicited by the hLAMP/gag chimera was detectable after only two or three naked DNA immunizations in all five immunized macaques and reached an average of 1000 spot-forming cells (SFC/10(6 PBMCs. High IFN-gamma ELISPOT responses were detected in CD8(+-depleted cells, indicating that CD4(+ T-cells play a major role in these responses. The T-cell responses of four of the macaques were also tested by use of ELISPOT to 12 overlapping 15-amino acids (aa peptide pools containing ten peptides each, encompassing the complete Gag protein sequence. The two Mamu 08 immunized macaques responded to eight and twelve of the pools, the Mamu B01 to six, and the other macaque to five pools indicating that the hLAMP/gag DNA antigen formulation elicits a broad T-cell response against Gag. Additionally, there was a strong HIV-1-specific IgG response. The IgG antibody titers increased after each DNA injection, indicating a strong amnestic B-cell response, and were highly elevated in all the macaques after three immunizations. Moreover, the serum of each macaque recognized 13 of the 49 peptides of a 20-aa peptide library covering the complete Gag amino acid sequence. In addition, HIV-1-specific IgA antibodies were present in the plasma and external secretions, including nasal washes. These data support the findings of increased

  1. HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins.

    Directory of Open Access Journals (Sweden)

    Zabrina L Brumme

    Full Text Available BACKGROUND: Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution. METHODOLOGY/PRINCIPAL FINDINGS: HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q< or =0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association, followed by Gag then Pol (where approximately 15-20% of codons exhibited HLA associations, confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q< or =0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations. CONCLUSIONS/SIGNIFICANCE: The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate

  2. The pp24 phosphoprotein of Mason-Pfizer monkey virus contributes to viral genome packaging

    Directory of Open Access Journals (Sweden)

    Weldon Robert A

    2005-11-01

    Full Text Available Abstract Background The Gag protein of Mason-Pfizer monkey virus, a betaretrovirus, contains a phosphoprotein that is cleaved into the Np24 protein and the phosphoprotein pp16/18 during virus maturation. Previous studies by Yasuda and Hunter (J. Virology. 1998. 72:4095–4103 have demonstrated that pp16/18 contains a viral late domain required for budding and that the Np24 protein plays a role during the virus life cycle since deletion of this N-terminal domain blocked virus replication. The function of the Np24 domain, however, is not known. Results Here we identify a region of basic residues (KKPKR within the Np24 domain that is highly conserved among the phosphoproteins of various betaretroviruses. We show that this KKPKR motif is required for virus replication yet dispensable for procapsid assembly, membrane targeting, budding and release, particle maturation, or viral glycoprotein packaging. Additional experiments indicated that deletion of this motif reduced viral RNA packaging 6–8 fold and affected the transient association of Gag with nuclear pores. Conclusion These results demonstrate that the Np24 domain plays an important role in RNA packaging and is in agreement with evidence that suggests that correct intracellular targeting of Gag to the nuclear compartment is an fundamental step in the retroviral life cycle.

  3. Intersubtype differences in the effect of a rare p24 gag mutation on HIV-1 replicative fitness.

    Science.gov (United States)

    Chopera, Denis R; Cotton, Laura A; Zawaira, Alexander; Mann, Jaclyn K; Ngandu, Nobubelo K; Ntale, Roman; Carlson, Jonathan M; Mlisana, Koleka; Woodman, Zenda; de Assis Rosa, Debra; Martin, Eric; Miura, Toshiyuki; Pereyra, Florencia; Walker, Bruce D; Gray, Clive M; Martin, Darren P; Ndung'u, Thumbi; Brockman, Mark A; Karim, Salim Abdool; Brumme, Zabrina L; Williamson, Carolyn

    2012-12-01

    Certain immune-driven mutations in HIV-1, such as those arising in p24(Gag), decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24(Gag) M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.

  4. Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G.

    Directory of Open Access Journals (Sweden)

    Isabel Uyttendaele

    Full Text Available The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.

  5. The Mexico City Policy: a "gag rule" that violates free speech and democratic values.

    Science.gov (United States)

    Cohen, S A

    1998-04-01

    With the support of the Republican leadership of the US House of Representatives, Representative Chris Smith of New Jersey continues to attempt to limit provision of US family planning (FP) funding to foreign nongovernmental organizations (NGOs) who agree to enforce absolute prohibitions on engaging in abortion-related activities. Despite the fact that the House is withholding funds to meet US obligations to the UN and to support the International Monetary Fund, US President Clinton refuses to give in to pressure to enforce what amounts to a "gag rule." The Secretary of State explains that Clinton has no intention of punishing organizations engaging in free-speech protected democratic activities in foreign countries. Smith has offered a self-styled "compromise" that would allow Clinton to waive disqualification for a foreign NGO offering abortion services with its own funds in compliance with the laws of its country. This waiver would penalize the international FP program by $44 million and would not apply to the broad prohibitions against abortion lobbying (including lobbying for changes in laws, sponsoring conferences and workshops on "alleged" defects in abortion laws, and drafting and distributing materials or public statements on "alleged defects"). Smith's emphasis on the "lobbying" ban has widened the issue from a debate on abortion to a debate on democracy and free speech. Opposition to Smith's proposal is, thus, rising in many quarters. Smith also endorses withholding US contributions to the UN Population Fund because the fund has resumed work in China.

  6. Short communication: high natural polymorphism in the gag gene cleavage sites of non-B HIV type 1 isolates from Gabon.

    Science.gov (United States)

    Liégeois, Florian; Reteno, Dorine Gaëlle Ikanga; Mouinga-Ondémé, Augustin; Sica, Jeanne; Rouet, François

    2013-08-01

    The main goal of the present study was to determine the frequency of substitutions in the cleavage sites (CS) of gag gene among non-B HIV-1 isolates from Gabon. Fifty plasma specimens, collected in 2010-2011, from HIV-1-infected patients failing first-line antiretroviral (ARV) regimens (constituted of two nucleoside reverse transcriptase inhibitors+one nonnucleoside reverse transcriptase inhibitor) (n=38) and from HIV-1-infected individuals untreated with ARV (n=12) were analyzed in the gag and gag-pol cleavage sites. Compared to HXB2 reference sequence, the total median number of substitutions in gag and gag-pol CS was 10 (range, 5-18). The cleavage site p2/NC was the most variable of the four gag CS with 100% (50/50) isolates carrying at least 1 substitution (range, 1-9). The two gag-pol TFP/p6pol and p6pol/PR CS sites were also highly variable (at least one substitution, 50/50, 100% in both cases). Substitutions at position G381 (p2/NC), L449 (p1/p6gag), and K444 (TFP/p6pol) were significantly more frequent in CRF02_AG strains, compared to other non-B strains (30.4% vs. 3.7%, p=0.03; 87.0% vs. 59.3%, p=0.03; and 91.3% vs. 59.3%, p=0.01, respectively). Other non-B subtypes were significantly more likely to harbor substitutions at position N487 (p6pol) (70.4%) than CRF02_AG (39.1%) (p=0.02). In Gabon, gag and gag-pol cleavage sites were highly polymorphic in protease inhibitor-naive patients harboring non-B HIV-1 strains. In sub-Saharan Africa, further studies are definitively required to better understand the impact of gag mutations among subjects receiving second-line LPV/r-containing regimens (monotherapy or triple combinations).

  7. Fabrication of a definitive obturator from a 3D cast with a chairside digital scanner for a patient with severe gag reflex: a clinical report.

    Science.gov (United States)

    Londono, Jimmy; Abreu, Amara; Baker, Philip S; Furness, Alan R

    2015-11-01

    Patient gagging is a common problem during dental procedures such as maxillary impression making. This clinical report describes the use of a chairside intraoral scanner for a patient with a hypersensitive gag reflex. The technique proved to be a more comfortable alternative for the patient and an accurate method for the clinician to capture both hard and soft tissue detail for the fabrication of a definitive obturator.

  8. Acute leukemia viruses E26 and avian myeloblastosis virus have related transformation-specific RNA sequences but different genetic structures, gene products, and oncogenic properties

    Science.gov (United States)

    Bister, Klaus; Nunn, Michael; Moscovici, Carlo; Perbal, Bernard; Baluda, Marcel A.; Duesberg, Peter H.

    1982-01-01

    Replication-defective acute leukemia viruses E26 and myeloblastosis virus (AMV) cause distinct leukemias although they belong to the same subgroup of oncogenic avian tumor viruses based on shared transformation-specific (onc) RNA sequences. E26 causes predominantly erythroblastosis in chicken and in quail, whereas AMV induces a myeloid leukemia. However, upon cultivation in vitro for >1 month, a majority of surviving hemopoietic cells of E26-infected animals bear myeloid markers similar to those of AMV-transformed cells. We have analyzed the genetic structure and gene products of E26 virus for a comparison with those of AMV. An E26/helper virus complex was found to contain two RNA species: a 5.7-kilobase (kb) RNA that hybridizes with cloned AMV-specific proviral DNA and hence is probably the E26 genome; and an 8.5-kb RNA that is unrelated to AMV and represents helper virus RNA. Thus, E26 RNA is smaller than 7.5-kb AMV RNA. Hybridization of size-selected poly(A)-terminating E26 RNA fragments with AMV-specific DNA indicated that the shared specific sequences are located in the 5′ half of the E26 genome as opposed to a 3′ location in AMV RNA. In nonproducer cells transformed in vitro by E26, a gag-related nonstructural 135,000-dalton protein (p135) was found. No gag(Pr76) or gag-pol (Pr180) precursors of essential virion proteins, which are present in AMV nonproducer cells, were observed. p135 was also found in cultured E26 virus producing cells of several leukemic chickens, and its intracellular concentration relative to that of the essential virion proteins encoded by the helper virus correlates with the ratio of E26 to helper RNA in virions released by these cells. p135 is phosphorylated but not glycosylated; antigenically it is not related to the pol or env gene products. It appears to be coded for by a partial gag gene and by E26-specific RNA sequences, presumably including those shared with AMV. Hence, AMV and E26 appear to use different strategies for the

  9. Characteristics and outcomes of initial virologic suppressors during analytic treatment interruption in a therapeutic HIV-1 gag vaccine trial.

    Directory of Open Access Journals (Sweden)

    Jonathan Z Li

    Full Text Available BACKGROUND: In the placebo-controlled trial ACTG A5197, a trend favoring viral suppression was seen in the HIV-1-infected subjects who received a recombinant Ad5 HIV-1 gag vaccine. OBJECTIVE: To identify individuals with initial viral suppression (plasma HIV-1 RNA set point <3.0 log(10 copies/ml during the analytic treatment interruption (ATI and evaluate the durability and correlates of virologic control and characteristics of HIV sequence evolution. METHODS: HIV-1 gag and pol RNA were amplified and sequenced from plasma obtained during the ATI. Immune responses were measured by flow cytometric analysis and intracellular cytokine expression assays. Characteristics of those with and without initial viral suppression were compared using the Wilcoxon rank sum and Fisher's exact tests. RESULTS: Eleven out of 104 participants (10.6% were classified as initial virologic suppressors, nine of whom had received the vaccine. Initial virologic suppressors had significantly less CD4+ cell decline by ATI week 16 as compared to non-suppressors (median 7 CD4+ cell gain vs. 247 CD4+ cell loss, P = 0.04. However, of the ten initial virologic suppressors with a pVL at ATI week 49, only three maintained pVL <3.0 log(10 copies/ml. HIV-1 Gag-specific CD4+ interferon-γ responses were not associated with initial virologic suppression and no evidence of vaccine-driven HIV sequence evolution was detected. Participants with initial virologic suppression were found to have a lower percentage of CD4+ CTLA-4+ cells prior to treatment interruption, but a greater proportion of HIV-1 Gag-reactive CD4+ TNF-α+ cells expressing either CTLA-4 or PD-1. CONCLUSIONS: Among individuals participating in a rAd5 therapeutic HIV-1 gag vaccine trial, initial viral suppression was found in a subset of patients, but this response was not sustained. The association between CTLA-4 and PD-1 expression on CD4+ T cells and virologic outcome warrants further study in trials of other

  10. Foamy Virus Protein—Nucleic Acid Interactions during Particle Morphogenesis

    Science.gov (United States)

    Hamann, Martin V.; Lindemann, Dirk

    2016-01-01

    Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches. PMID:27589786

  11. A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine

    NARCIS (Netherlands)

    Mehendale, Sanjay; van Lunzen, Jan; Clumeck, Nathan; Rockstroh, Jurgen; Vets, Eva; Johnson, Philip R.; Anklesaria, Pervin; Barin, Burc; Boaz, Mark; Kochhar, Sonali; Lehrman, Jennifer; Schmidt, Claudia; Peeters, Mathieu; Schwarze-Zander, Carolynne; Kabamba, Kabeya; Glaunsinger, Tobias; Sahay, Seema; Thakar, Madhuri; Paranjape, Ramesh; Gilmour, Jill; Excler, Jean-Louis; Fast, Patricia; Heald, A1lison E.

    2008-01-01

    A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman prim

  12. A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine

    NARCIS (Netherlands)

    Mehendale, Sanjay; van Lunzen, Jan; Clumeck, Nathan; Rockstroh, Jurgen; Vets, Eva; Johnson, Philip R.; Anklesaria, Pervin; Barin, Burc; Boaz, Mark; Kochhar, Sonali; Lehrman, Jennifer; Schmidt, Claudia; Peeters, Mathieu; Schwarze-Zander, Carolynne; Kabamba, Kabeya; Glaunsinger, Tobias; Sahay, Seema; Thakar, Madhuri; Paranjape, Ramesh; Gilmour, Jill; Excler, Jean-Louis; Fast, Patricia; Heald, A1lison E.

    A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman

  13. Stampidine prevents mortality in an experimental mouse model of viral hemorrhagic fever caused by lassa virus

    Directory of Open Access Journals (Sweden)

    Tibbles Heather E

    2004-01-01

    Full Text Available Abstract Background The potential use of microorganisms as agents of biological warfare (BW is a growing concern. Lassa virus, a member of the Arenavirus class of Hemorrhagic fever (HF viruses has emerged as a worldwide concern among public health officials. The purpose of the present study was to further elucidate the antiviral activity spectrum of stampidine, a novel nucleoside analog with potent anti-viral activity against the immunodeficiency viruses HIV-1, HIV-2, and FIV, by examining its effects on survival of mice challenged with Lassa virus. Methods We examined the therapeutic effect of Stampidine in CBA mice inoculated with intracerebral injections of the Josiah strain of Lassa virus. Mice were treated either with vehicle or nontoxic doses of stampidine administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. Results The probability of survival following the Lassa challenge was significantly improved for stampidine treated mice (Kaplan Meier, Chi-squared = 11.7, df = 2, Log-Rank p-value = 0.003. Conclusion Therefore, stampidine shows clinical potential as a new agent for treatment of viral hemorrhagic fevers caused by Lassa virus.

  14. Effect of HIV-1 envelope cytoplasmic tail on adenovirus primed virus encoded virus-like particle immunizations

    DEFF Research Database (Denmark)

    Andersson, Anne Marie C; Ragonnaud, Emeline; Seaton, Kelly E.

    2016-01-01

    /c mice followed by sequential boosting with chimpanzee type 63, and chimpanzee type 3 adenoviral vectors encoding SIVmac239 Gag and full length consensus Env. Both vaccine regimens induced increasing titers of binding antibody responses after each immunization, and significant differences in immune......The low number of envelope (Env) spikes presented on native HIV-1 particles is a major impediment for HIV-1 prophylactic vaccine development. We designed virus-like particle encoding adenoviral vectors utilizing SIVmac239 Gag as an anchor for full length and truncated HIV-1 M consensus Env....... Truncated Env overexpressed VRC01 and 17b binding antigen on the surface of transduced cells while the full length Env vaccine presented more and similar amounts of antigen binding to the trimer conformation sensitive antibodies PGT151 and PGT145, respectively. The adenoviral vectors were used to prime Balb...

  15. Recombinant Marek's Disease Virus as a Vector-Based Vaccine against Avian Leukosis Virus Subgroup J in Chicken.

    Science.gov (United States)

    Liu, Yongzhen; Li, Kai; Gao, Yulong; Gao, Li; Zhong, Li; Zhang, Yao; Liu, Changjun; Zhang, Yanping; Wang, Xiaomei

    2016-11-04

    Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1) env or gag+env genes into the US2 gene of the Marek's disease herpesviruses (MDV) by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection.

  16. Recombinant Marek’s Disease Virus as a Vector-Based Vaccine against Avian Leukosis Virus Subgroup J in Chicken

    Directory of Open Access Journals (Sweden)

    Yongzhen Liu

    2016-11-01

    Full Text Available Avian leukosis virus subgroup J (ALV-J is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1 env or gag+env genes into the US2 gene of the Marek’s disease herpesviruses (MDV by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection.

  17. Recombinant Marek’s Disease Virus as a Vector-Based Vaccine against Avian Leukosis Virus Subgroup J in Chicken

    Science.gov (United States)

    Liu, Yongzhen; Li, Kai; Gao, Yulong; Gao, Li; Zhong, Li; Zhang, Yao; Liu, Changjun; Zhang, Yanping; Wang, Xiaomei

    2016-01-01

    Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1) env or gag+env genes into the US2 gene of the Marek’s disease herpesviruses (MDV) by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection. PMID:27827933

  18. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

    Science.gov (United States)

    Schur, Florian K. M.; Hagen, Wim J. H.; Rumlová, Michaela; Ruml, Tomáš; Müller, Barbara; Kräusslich, Hans-Georg; Briggs, John A. G.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

  19. Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

    Directory of Open Access Journals (Sweden)

    Yadvinder S. Ahi

    2016-11-01

    Full Text Available Gammaretroviruses, such as murine leukemia viruses (MLVs, encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag. MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.

  20. Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

    Science.gov (United States)

    Tanaka, Motoko; Robinson, Bridget A; Chutiraka, Kasana; Geary, Clair D; Reed, Jonathan C; Lingappa, Jaisri R

    2015-12-09

    The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues

  1. Feline immunodeficiency virus and retrovirus-mediated adventitial ex vivo gene transfer to rabbit carotid artery using autologous vascular smooth muscle cells.

    Science.gov (United States)

    Kankkonen, Hanna M; Turunen, Mikko P; Hiltunen, Mikko O; Lehtolainen, Pauliina; Koponen, Jonna; Leppänen, Pia; Turunen, Anna-Mari; Ylä-Herttuala, Seppo

    2004-03-01

    We have developed an ex vivo gene transfer technique to rabbit arterial wall using autologous smooth muscle cells (SMCs). SMCs were harvested from rabbit ear artery, transduced in vitro with vesicular stomatitis virus G-glycoprotein pseudotyped retrovirus or feline immunodeficiency virus (FIV) and returned to the adventitial surface of the carotid artery using a periadventitial silicone collar or collagen sheet placed around the artery. Beta-galactosidase (lacZ) and human apolipoprotein E3 (apoE3) cDNAs were used as transgenes. After retrovirus-mediated gene transfer of lacZ the selected cells implanted with high efficiency and expressed lacZ marker gene at a very high level 7 and 14 days after the operation. The level of lacZ expression decreased thereafter but was still detectable 12 weeks after the gene transfer, and was exclusively localized to the site of cell implantation inside the collar. Utilizing FIV vector expressing apoE3, low levels of apoE were measured from serum collected from a low-density lipoprotein receptor deficient Watanabe heritable hyperlipidemic rabbits 1 month after the gene transfer. The physiological effect of apoE expression was detected as transiently elevated serum cholesterol levels. The results indicate that the model can be used for high efficiency local gene transfer in arteries, e.g. during vascular surgery. The model is also valuable for studying expression, stability and safety of new gene transfer vectors and their expression products in vivo.

  2. In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission.

    Science.gov (United States)

    Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

    2015-11-11

    Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell-cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105-106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT-BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.

  3. [Functional analysis of Grp and Iris, the gag and env domesticated errantivirus genes, in the Drosophila melanogaster genome].

    Science.gov (United States)

    Makhnovskii, P A; Kuzmin, I V; Nefedova, L N; Kima, A I

    2016-01-01

    Drosophila melanogaster is the only invertebrate that contains endogenous retroviruses, which are called errantiviruses. Two domesticated genes, Grp and Iris, which originate from errantivirus gag and env, respectively, have been found in the D. melanogaster genome. The functions performed by the genes in Drosophila are still unclear. To identify the functions of domesticated gag and env in the D. melanogaster genome, expression of Iris and Grp was studied in strains differing by the presence or absence of the functional gypsy errantivirus. In addition, the expression levels were measured after injection of gram-positive and gram-negative bacteria, which activate different immune response pathways, and exposure to various abiotic stress factors. The presence of functional D. melanogaster retrovirus gypsy was found to increase the Grp expression level in somatic tissues of the carcass, while exerting no effect on the Iris expression level. Activation of the immune response in D. melanogaster by bacteria Bacillus cereus increased the Grp expression level and did not affect Iris expression. As for the effects of abiotic stress factors (oxidative stress, starvation, and heat and cold stress), the Grp expression level increased in response to starvation in D. melanogaster females, and the Iris expression level was downregulated in heat shock and oxidative stress. Based on the findings, Grp was assumed to play a direct role in the immune response in D. melanogaster; Iris is not involved in immune responses, but and apparently performs a cell function that is inhibited in stress.

  4. [Molecular evolution of mobile elements of the gypsy group: a homolog of the gag gene in Drosophila].

    Science.gov (United States)

    Nefedova, L N; Kim, A I

    2009-01-01

    Retrotransposons of the gypsy group of Drosophila melanogaster that are structurally similar to retroviruses of vertebrates occupy an important place among retroelements of eukaryotes. The infectious abilities of some retrotransposons of this group (gypsy, ZAM, and Idefix) have been demonstrated experimentally, and therefore they are true retroviruses. It is supposed that retrotransposons can evolve acquiring new components, the sources of which remain to be elucidated. In this work, the CG4680 gene (Gag related protein, Grp) homologous to gag of retrotransposons of the gypsy group has been identified in the genome of D. melanogaster and characterized. The Grp gene product has a highly conserved structure in different species of the Drosophilidae family and is under of stabilizing selection, which suggests its important genomic function in Drosophila. In view of the earlier data, it can be concluded that homologous genes of all components of gypsy retrotransposons are present in the Drosophila genome. These genes can be both precursors and products of domestication of retrovirus genes.

  5. A process-based model for ammonia emission from urine patches, GAG (Generation of Ammonia from Grazing): description, validation and sensitivity analysis

    DEFF Research Database (Denmark)

    Móring, A; Vieno, M.; Doherty, R M;

    2015-01-01

    In this paper a new process-based, weather-driven model for ammonia (NH3) emission from a urine patch has been developed and its sensitivity to various factors assessed. This model, the GAG model (Generation of Ammonia from Grazing) was developed as a part of a suite of weather-driven NH3 exchange...... models, as a necessary basis for assessing the effects of climate change on NH3 related atmospheric processes. GAG is capable of simulating the TAN (Total Ammoniacal Nitrogen) content, pH and the water content of the soil under a urine patch. To calculate the TAN budget, GAG takes into account urea...... hydrolysis as a TAN input and NH3 volatilization as a loss. In the water budget, in addition to the water content of urine, precipitation and evaporation are also considered. In the pH module we assumed that the main regulating processes are the dissociation and dissolution equilibria related to the two...

  6. Identification of peptides from human pathogens able to cross-activate an HIV-1-gag-specific CD4+ T cell clone.

    Science.gov (United States)

    Venturini, Sara; Allicotti, Gina; Zhao, Yindong; Simon, Richard; Burton, Dennis R; Pinilla, Clemencia; Poignard, Pascal

    2006-01-01

    Antigen recognition by T cells is degenerate both at the MHC and the TCR level. In this study, we analyzed the cross-reactivity of a human HIV-1 gag p24-specific CD4(+) T cell clone obtained from an HIV-1-seronegative donor using a positional scanning synthetic combinatorial peptide library (PS-SCL)-based biometrical analysis. A number of decapeptides able to activate the HIV-1 gag-specific clone were identified and shown to correspond to sequences found in other human pathogens. Two of these peptides activated the T cell clone with the same stimulatory potency as the original HIV-1 gag p24 peptide. These findings show that an HIV-1-specific human T helper clone can react efficiently with peptides from other pathogens and suggest that cellular immune responses identified as being specific for one human pathogen (HIV-1) could arise from exposure to other pathogens.

  7. Interaction of Mason-Pfizer monkey virus matrix protein with plasma membrane.

    Directory of Open Access Journals (Sweden)

    Jan ePrchal

    2014-01-01

    Full Text Available Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane through its N-terminal domain, the matrix protein. However, single generas of Retroviridae family differ in the way how they interact with plasma membrane. While in case of lentiviruses (e.g. human immunodeficiency virus (HIV the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, betaretroviruses (Mason-Pfizer monkey virus (M-PMV first assemble their virus-like particles in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV matrix protein with plasma membrane with emphasis on the structural aspects of the interaction with single phospholipids.

  8. Differential Clade-Specific HLA-B*3501 Association with HIV-1 Disease Outcome Is Linked to Immunogenicity of a Single Gag Epitope

    Science.gov (United States)

    Matthews, Philippa C.; Koyanagi, Madoka; Harndahl, Mikkel; Stryhn, Anette; Akahoshi, Tomohiro; Gatanaga, Hiroyuki; Oka, Shinichi; Juarez Molina, Claudia; Valenzuela Ponce, Humberto; Avila Rios, Santiago; Cole, David; Carlson, Jonathan; Payne, Rebecca P.; Ogwu, Anthony; Bere, Alfred; Ndung'u, Thumbi; Gounder, Kamini; Chen, Fabian; Riddell, Lynn; Luzzi, Graz; Shapiro, Roger; Brander, Christian; Walker, Bruce; Sewell, Andrew K.; Reyes Teran, Gustavo; Heckerman, David; Hunter, Eric; Buus, Søren; Takiguchi, Masafumi; Goulder, Philip J. R.

    2012-01-01

    The strongest genetic influence on immune control in HIV-1 infection is the HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrated that whereas HLA-B*3501 is associated with a high viral set point in two further B-clade-infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade-infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8+ T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope NPPIPVGDIY (NY10, Gag positions 253 to 262) (P = 2 × 10−5). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response toward NY10 was associated with a significantly lower viral set point. Nonimmunogenicity of NY10 in B-clade-infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag NY10 epitope. These data suggest that in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8+ T-cell response, by the addition of even a single epitope, may be of overriding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8+ T-cell response in all individuals, irrespective of HLA type. PMID:22973023

  9. Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez, Silvia A.; Paladino, Monica G. [Laboratorio de Virologia, CONICET-Universidad de Belgrano (UB), Villanueva 1324 (C1426BMJ), Buenos Aires (Argentina); Affranchino, Jose L., E-mail: jose.affranchino@comunidad.ub.edu.ar [Laboratorio de Virologia, CONICET-Universidad de Belgrano (UB), Villanueva 1324 (C1426BMJ), Buenos Aires (Argentina)

    2012-06-20

    The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated.

  10. Proteoglycans act as cellular hepatitis delta virus attachment receptors.

    Science.gov (United States)

    Lamas Longarela, Oscar; Schmidt, Tobias T; Schöneweis, Katrin; Romeo, Raffaella; Wedemeyer, Heiner; Urban, Stephan; Schulze, Andreas

    2013-01-01

    The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.

  11. Mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease SAT type viruses.

    Science.gov (United States)

    Maree, Francois F; Blignaut, Belinda; de Beer, Tjaart A P; Visser, Nico; Rieder, Elizabeth A

    2010-10-01

    Foot-and-mouth disease virus (FMDV) infects host cells by adhering to the alpha(V) subgroup of the integrin family of cellular receptors in a Arg-Gly-Asp (RGD) dependent manner. FMD viruses, propagated in non-host cell cultures are reported to acquire the ability to enter cells via alternative cell surface molecules. Sequencing analysis of SAT1 and SAT2 cell culture-adapted variants showed acquisition of positively charged amino acid residues within surface-exposed loops of the outer capsid structural proteins. The fixation of positively charged residues at position 110-112 in the beta F-beta G loop of VP1 of SAT1 isolates is thought to correlate with the acquisition of the ability to utilise alternative glycosaminoglycan (GAG) molecules for cell entry. Similarly, two SAT2 viruses that adapted readily to BHK-21 cells accumulated positively charged residues at positions 83 and 85 of the beta D-beta E loop of VP1. Both regions surround the fivefold axis of the virion. Recombinant viruses containing positively charged residues at position 110 and 112 of VP1 were able to infect CHO-K1 cells (that expresses GAG) and demonstrated increased infectivity in BHK-21 cells. Therefore, recombinant SAT viruses engineered to express substitutions that induce GAG-binding could be exploited in the rational design of vaccine seed stocks with improved growth properties in cell cultures.

  12. Replication of biotinylated human immunodeficiency viruses.

    Science.gov (United States)

    Belshan, Michael; Matthews, John M; Madson, Christian J

    2011-01-01

    Previous work demonstrated recently the adaptation of the Escherichia coli biotin ligase BirA - biotin acceptor sequence (BAS) labeling system to produce human immunodeficiency virus type 1 viruses with biotinylated integrase (NLXIN(B)) and matrix (NLXMA(B)) proteins (Belshan et al., 2009). This report describes the construction of an HIV permissive cell line stably expressing BirA (SupT1.BirA). Consistent with the results in the previous report, NLXMA(B) replicated similar to wild-type levels and expressed biotinylated Gag and MA proteins in the SupT1.BirA cells, whereas the replication of NLXIN(B) was reduced severely. Three additional HIV type 2 (HIV-2) viruses were constructed with the BAS inserted into the vpx and vpr accessory genes. Two BAS insertions were made into the C-terminal half of the Vpx, including one internal insertion, and one at the N-terminus of Vpr. All three viruses were replication competent in the SupT1.BirA cells and their target proteins biotinylated efficiently and incorporated into virions. These results demonstrate the potential utility of the biotinylation system to label and capture HIV protein complexes in the context of replicating virus.

  13. L-A-lus, a New Variant of the L-A Totivirus Found in Wine Yeasts with Klus Killer Toxin-Encoding Mlus Double-Stranded RNA: Possible Role of Killer Toxin-Encoding Satellite RNAs in the Evolution of Their Helper Viruses

    Science.gov (United States)

    Rodríguez-Cousiño, Nieves; Gómez, Pilar

    2013-01-01

    Yeast killer viruses are widely distributed in nature. Several toxins encoded in double-stranded RNA (dsRNA) satellites of the L-A totivirus have been described, including K1, K2, K28, and Klus. The 4.6-kb L-A genome encodes the Gag major structural protein that forms a 39-nm icosahedral virion and Gag-Pol, a minor fusion protein. Gag-Pol has transcriptase and replicase activities responsible for maintenance of L-A (or its satellite RNAs). Recently we reported a new killer toxin, Klus. The L-A virus in Klus strains showed poor hybridization to known L-A probes, suggesting substantial differences in their sequences. Here we report the characterization of this new L-A variant named L-A-lus. At the nucleotide level, L-A and L-A-lus showed only 73% identity, a value that increases to 86% in the amino acid composition of Gag or Gag-Pol. Two regions in their genomes, however, the frameshifting region between Gag and Pol and the encapsidation signal, are 100% identical, implying the importance of these two cis signals in the virus life cycle. L-A-lus shows higher resistance than L-A to growth at high temperature or to in vivo expression of endo- or exonucleases. L-A-lus also has wider helper activity, being able to maintain not only Mlus but also M1 or a satellite RNA of L-A called X. In a screening of 31 wine strains, we found that none of them had L-A; they carried either L-A-lus or a different L-A variant in K2 strains. Our data show that distinct M killer viruses are specifically associated with L-As with different nucleotide compositions, suggesting coevolution. PMID:23728812

  14. Role of a GAG hinge in the nucleotide-induced conformational change governing nucleotide specificity by T7 DNA polymerase.

    Science.gov (United States)

    Jin, Zhinan; Johnson, Kenneth A

    2011-01-14

    A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.

  15. From shelf to shelf: assessing historical and contemporary genetic differentiation and connectivity across the Gulf of Mexico in Gag, Mycteroperca microlepis.

    Directory of Open Access Journals (Sweden)

    Nathaniel K Jue

    Full Text Available Describing patterns of connectivity among populations of species with widespread distributions is particularly important in understanding the ecology and evolution of marine species. In this study, we examined patterns of population differentiation, migration, and historical population dynamics using microsatellite and mitochondrial loci to test whether populations of the epinephelid fish, Gag, Mycteroperca microlepis, an important fishery species, are genetically connected across the Gulf of Mexico and if so, whether that connectivity is attributable to either contemporary or historical processes. Populations of Gag on the Campeche Bank and the West Florida Shelf show significant, but low magnitude, differentiation. Time since divergence/expansion estimates associated with historical population dynamics indicate that any population or spatial expansions indicated by population genetics would have likely occurred in the late Pleistocene. Using coalescent-based approaches, we find that the best model for explaining observed spatial patterns of contemporary genetic variation is one of asymmetric gene flow, with movement from Campeche Bank to the West Florida Shelf. Both estimated migration rates and ecological data support the hypothesis that Gag populations throughout the Gulf of Mexico are connected via present day larval dispersal. Demonstrating this greatly expanded scale of connectivity for Gag highlights the influence of "ghost" populations (sensu Beerli on genetic patterns and presents a critical consideration for both fisheries management and conservation of this and other species with similar genetic patterns.

  16. Targeting of conserved gag-epitopes in early HIV infection is associated with lower plasma viral load and slower CD4+ T cell depletion

    DEFF Research Database (Denmark)

    Perez, Carina L.; Milush, Jeffrey M.; Buggert, Marcus

    2013-01-01

    We aimed to investigate whether the character of the immunodominant HIV-Gag peptide (variable or conserved) targeted by CD8+ T cells in early HIV infection would influence the quality and quantity of T cell responses, and whether this would affect the rate of disease progression. Treatment-naive ...

  17. The use of the Molt mouth gag to assist in oral fiberoptic tracheal intubation of a developmentally challenged patient presenting with severe trismus.

    Science.gov (United States)

    Kamel, Ihab; Mulligan, Joseph; Luke, Jessica; Barnette, Rodger

    2015-02-15

    Airway management in developmentally challenged, and often uncooperative, patients presents difficulty for the anesthesiologist and may be further complicated by severe trismus. We describe a case wherein the use of the Molt mouth gag significantly facilitated airway control using fiberoptic tracheal intubation.

  18. Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Zachar, Vladimir; Nielsen, P.E.;

    1997-01-01

    We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs) was investi......We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs......) was investigated. We found that a bis-PNA (parallel antisense 10mer linked to antiparallel antisense 10mer) was superior to both the parallel antisense 10mer and antiparallel antisense 10mer in inhibiting reverse transcription of the gene, thus indicating triplex formation at the target sequence. A complete arrest...... that would indicate PNA-mediated RNase H activation of the tested RTs. In conclusion, PNA appears to have a potential to become a specific and efficient inhibitor of reverse transcription in vivo , provided sufficient intracellular levels are achievable....

  19. ECHO virus

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/001340.htm ECHO virus To use the sharing features on this page, please enable JavaScript. Enteric cytopathic human orphan (ECHO) viruses are a group of viruses that lead to ...

  20. Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus.

    Science.gov (United States)

    Cappelli, Katia; Capomaccio, Stefano; Cook, Frank R; Felicetti, Michela; Marenzoni, Maria Luisa; Coppola, Giacomo; Verini-Supplizi, Andrea; Coletti, Mauro; Passamonti, Fabrizio

    2011-01-01

    The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the "clades" described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.

  1. Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

    Science.gov (United States)

    Balagopal, Vidya; Beemon, Karen L.

    2017-01-01

    All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

  2. Detecção do provírus da Imunodeficiência Felina em gatos domésticos pela técnica de Reação em Cadeia da Polimerase Detection of feline immunodeficiency provirus in domestic cats by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Ana Paula Ferrary Caldas

    2000-03-01

    Full Text Available A infecção de gatos domésticos pelo Vírus da Imunodeficiência Felina (FIV é um dos modelos mais promissores para o estudo da infecção pelo vírus da imunodeficiência humana (HIV que causa a Síndrome de Imunodeficiência Adquirida (AIDS. O FIV causa, em gatos, uma enfermidade similar àquela observada em pacientes com AIDS, sobretudo no que diz respeito ao aumento da susceptibilidade a infecções oportunistas. No presente estudo, utilizou-se a Reação em Cadeia da Polimerase (PCR, com o objetivo de detectar o provírus do FIV em gatos com sinais clínicos de imunodeficiência. O fragmento de DNA escolhido como alvo para amplificação situa-se no gene gag do lentivírus felino, o qual é conservado entre as diferentes amostras do vírus. O DNA utilizado foi extraído a partir de amostras de sangue e de tecidos de animais com suspeita clínica de imunodeficiência. Das 40 amostras analisadas, 15 foram positivas, das quais 4 foram submetidas à hibridização, confirmando a especificidade dos fragmentos amplificados. Esses resultados demonstram a presença do FIV na população de gatos domésticos do Rio Grande do Sul, Brasil.Feline immunodeficiency virus (FIV infection of domestic cats is one of the most promising animal models for the infection by the human immunodeficiency virus (HIV which causes acquired immunodeficiency syndrome (AIDS. Infected cats may develop a disease similar to that observed in AIDS patients, with increased susceptibility to opportunistic infections. In this study we used the polymerase chain reaction (PCR to detect proviral DNA of feline immunodeficiency virus on the blood and tissue samples from cats with a clinical diagnosis of immunodeficiency. The PCR primers were used to amplify the gag gene, which is conserved among different isolates. From 40 samples analyzed, 15 were positive and 4 of them were submitted to hybridization to confirm the specificity of the amplified fragments. These results confirm the

  3. Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques.

    Science.gov (United States)

    Thippeshappa, Rajesh; Tian, Baoping; Cleveland, Brad; Guo, Wenjin; Polacino, Patricia; Hu, Shiu-Lok

    2015-12-30

    Human immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 10(8) PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.

  4. Molecular epidemiology of human immunodeficiency virus-1 in the state of Ceará, Northeast, Brazil

    Directory of Open Access Journals (Sweden)

    Sandra Rocha Gadelha

    2003-06-01

    Full Text Available We analyzed, by env and gag heteroduplex mobility assay, 149 human immunodeficiency virus (HIV-1 positive samples collected in Ceará during the year 2000. The prevalence of subtype B was 81.2% and the prevalence of subtype F and B/F recombinants were both 2.7%. Eight (5.4% and 12 (8% out of 149 samples showed indeterminate results in the env and gag analysis respectively. By FokI restriction fragment length polymorphism, 34% of the subtype B samples were identified as the typical Brazilian subtype B.In the present study, we identified HIV-1 subtype F and B/F in Ceará for the first time. Our results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates.

  5. Co-translational localization of an LTR-retrotransposon RNA to the endoplasmic reticulum nucleates virus-like particle assembly sites.

    Directory of Open Access Journals (Sweden)

    Jung H Doh

    2014-03-01

    Full Text Available The transcript of retrovirus-like transposons functions as an mRNA for synthesis of capsid and replication proteins and as the genomic RNA of virus-like particles (VLPs, wherein the genome is replicated. Retrotransposon RNA and proteins coalesce in a cytoplasmic focus, or retrosome, to initiate VLP assembly, but it is not known how the retrosome is nucleated. We determined how the RNA and Gag protein of the Saccharomyces cerevisiae Ty1 retrotransposon are directed to the retrosome. We found that Ty1 RNA is translated in association with signal recognition particle (SRP, a universally conserved chaperone that binds specific ribosome-nascent chain (RNC complexes and targets the nascent peptide to the endoplasmic reticulum (ER. Gag is translocated to the ER lumen; yet, it is also found in the cytoplasm, associated with SRP-RNC complexes. In the absence of ER translocation, Gag is synthesized but rapidly degraded, and Ty1 RNA does not coalesce in retrosomes. These findings suggest that Gag adopts a stable conformation in the ER lumen, is retrotranslocated to the cytoplasm, binds to Ty1 RNA on SRP-RNC complexes and multimerizes to nucleate retrosomes. Consistent with this model, we show that slowing the rate of co-translational ER translocation by limiting SRP increases the prevalence of retrosomes, while suppressing the translocation defect of srp hypomorphs by slowing translational elongation rapidly decreases retrosome formation. Thus, retrosomes are dynamic foci of Ty1 RNA-RNC complexes whose formation is modulated by the rate of co-translational ER translocation. Together, these findings suggest that translating Ty1 mRNA and the genomic RNA of VLPs originate in a single pool and moreover, that co-translational localization of Ty1 RNA nucleates the presumptive VLP assembly site. The separation of nascent Gag from its RNA template by transit through the ER allows Gag to bind translating Ty1 RNA without displaying a cis-preference for its encoding

  6. When cats' ways of life interact with their viruses: a study in 15 natural populations of owned and unowned cats (Felis silvestris catus).

    Science.gov (United States)

    Hellard, E; Fouchet, D; Santin-Janin, H; Tarin, B; Badol, V; Coupier, C; Leblanc, G; Poulet, H; Pontier, D

    2011-09-01

    In natural populations, virus circulation is influenced by host behavior and physiological characteristics. Cat populations exhibit a great variability in social and spatial structure, the existence of different ways of life within a same population may also result in different epidemiological patterns. To test this hypothesis, we used a logistic regression to analyze the risk factors of Feline immunodeficiency virus (FIV), feline herpes virus (FHV), feline calicivirus (FCV), and feline parvovirus (FPV) infection in owned (fed and sheltered) and unowned (neither fed nor sheltered, unsocialized) cats living in a rural environment in the North Eastern part of France. A serological survey was carried out in 492 non-vaccinated and non-sterilized individuals from 15 populations living in the same area. The prevalence of feline leukemia virus (FeLV) was also studied, but too few were infected to analyze the risk factors of this virus. For each virus, the epidemiological pattern was different in owned and unowned cats. Unowned cats were more frequently infected by directly transmitted viruses like FIV, FHV and FCV (21.22%, 67.66%, 86.52% in unowned cats vs 9.55%, 53.88%, 77.18% in owned cats, respectively), a difference that may be explained by a more solitary and more aggressive behavior in unowned adults, and/or possibly by a higher sensitivity related to a more stressful life. On the contrary, owned cats were more frequently infected with FPV (36.41% in owned cats vs 15.61% in unowned cats), possibly as a result of their concentration around human settlements. The present study showed that owned and unowned cats living in a same area have behavioral and physiological characteristics sufficiently different to influence virus circulation. Pooling different types of cats in a single sample without taking it into account could give a wrong picture of the epidemiology of their viruses. The conclusion of this work can be extended to any epidemiological studies led in

  7. Human Endogenous Retrovirus K(HML-2) Gag- and Env-Specific T-Cell Responses Are Infrequently Detected in HIV-1-Infected Subjects Using Standard Peptide Matrix-Based Screening

    Science.gov (United States)

    John, Vivek M.; Hunter, Diana V.; Martin, Eric; Mujib, Shariq; Mihajlovic, Vesna; Burgers, Peter C.; Luider, Theo M.; Gyenes, Gabor; Sheppard, Neil C.; SenGupta, Devi; Tandon, Ravi; Yue, Feng-Yun; Benko, Erika; Kovacs, Colin; Nixon, Douglas F.; Ostrowski, Mario A.

    2012-01-01

    T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15mer peptide mapping. PMID:22205657

  8. Addition of sub-anaesthetic dose of ketamine reduces gag reflex during propofol based sedation for upper gastrointestinal endoscopy: A prospective randomised double-blind study

    Directory of Open Access Journals (Sweden)

    Manish Tandon

    2014-01-01

    Full Text Available Background and Aims: Gag reflex is unwanted during upper gastrointestinal endoscopy (UGIE. Experimental studies have demonstrated that N-methyl-D-aspartate receptor antagonism prevents gag reflex. We conducted a study to determine if sub-anaesthetic doses of ketamine, added to propofol, reduce the incidence of gag reflex. Methods: This prospective, randomised, double-blind and placebo-controlled study was done in a tertiary care hospital. A total of 270 patients undergoing UGIE, were randomised to propofol (P group (n = 135 or propofol plus ketamine (PK group (n = 135. All patients received propofol boluses titrated to Ramsay sedation score of not <4. Patients in PK group in addition received ketamine, 0.15 mg/kg immediately before the first-propofol dose. Top-up doses of propofol were given as required. Stata 11 software (StataCorp. was used to calculate the proportion of patients with gag reflex and the corresponding relative risk. Propofol consumed and time to recovery in the two groups was compared using Student′s t-test and Cox proportional hazards regression respectively. Results: Significantly, fewer patients in the PK group had gag reflex compared to the P group (3 vs. 23, risk ratio = 0.214, 95% confidence interval [CI], 0.07-0.62; P = 0.005. The incidence of hypotension (6 vs. 16, risk ratio = 0.519, 95% CI = 0.25-1.038; P = 0.06, number of required airway manoeuvres (4 vs. 19, risk ratio = 0.32, 95% CI = 0.13-0.74; P = 0.014, median time to recovery (4 min vs. 5 min, hazard ratio = 1.311, 95% CI = 1.029-1.671; P = 0.028 and propofol dose administered (152 mg vs. 167 mg, 95% CI = 4.74-24.55; P = 0.004 was also less in the PK group compared to the P group. Conclusion: Ketamine in sub-anaesthetic dose decreases gag reflex during UGIE.

  9. The fitness landscape of HIV-1 gag: advanced modeling approaches and validation of model predictions by in vitro testing.

    Directory of Open Access Journals (Sweden)

    Jaclyn K Mann

    2014-08-01

    Full Text Available Viral immune evasion by sequence variation is a major hindrance to HIV-1 vaccine design. To address this challenge, our group has developed a computational model, rooted in physics, that aims to predict the fitness landscape of HIV-1 proteins in order to design vaccine immunogens that lead to impaired viral fitness, thus blocking viable escape routes. Here, we advance the computational models to address previous limitations, and directly test model predictions against in vitro fitness measurements of HIV-1 strains containing multiple Gag mutations. We incorporated regularization into the model fitting procedure to address finite sampling. Further, we developed a model that accounts for the specific identity of mutant amino acids (Potts model, generalizing our previous approach (Ising model that is unable to distinguish between different mutant amino acids. Gag mutation combinations (17 pairs, 1 triple and 25 single mutations within these predicted to be either harmful to HIV-1 viability or fitness-neutral were introduced into HIV-1 NL4-3 by site-directed mutagenesis and replication capacities of these mutants were assayed in vitro. The predicted and measured fitness of the corresponding mutants for the original Ising model (r = -0.74, p = 3.6×10-6 are strongly correlated, and this was further strengthened in the regularized Ising model (r = -0.83, p = 3.7×10-12. Performance of the Potts model (r = -0.73, p = 9.7×10-9 was similar to that of the Ising model, indicating that the binary approximation is sufficient for capturing fitness effects of common mutants at sites of low amino acid diversity. However, we show that the Potts model is expected to improve predictive power for more variable proteins. Overall, our results support the ability of the computational models to robustly predict the relative fitness of mutant viral strains, and indicate the potential value of this approach for understanding viral immune evasion

  10. Identification of ALV-J associated acutely transforming virus Fu-J carrying complete v-fps oncogene.

    Science.gov (United States)

    Wang, Yixin; Li, Jianliang; Li, Yang; Fang, Lichun; Sun, Xiaolong; Chang, Shuang; Zhao, Peng; Cui, Zhizhong

    2016-06-01

    Transduction of oncogenes by ALVs and generation of acute transforming viruses is common in natural viral infections. In order to understand the molecular basis for the rapid oncogenicity of Fu-J, an acutely transforming avian leukosis virus isolated from fibrosarcomas in crossbreed broilers infected with subgroup J avian leukosis virus (ALV-J) in China, complete genomic structure of Fu-J virus was determined by PCR amplification and compared with those of Fu-J1, Fu-J2, Fu-J3, Fu-J4, and Fu-J5 reported previously. The results showed that the genome of Fu-J was defective, with parts of gag gene replaced by the complete v-fps oncogene and encoded a 137 kDa Gag-fps fusion protein. Sequence analysis revealed that Fu-J and Fu-J1 to Fu-J5 were related quasi-species variants carrying different lengths of v-fps oncogenes generated from recombination between helper virus and c-fps gene. Comparison of virus carrying v-fps oncogene also gave us a glimpse of the molecular characterization and evolution process of the acutely transforming ALV.

  11. Risk of equine infectious anemia virus disease transmission through in vitro embryo production using somatic cell nuclear transfer.

    Science.gov (United States)

    Gregg, K; Polejaeva, I

    2009-08-01

    Prevention and regulation of equine infectious anemia virus (EIAV) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. Embryo production via the somatic cell nuclear transfer (SCNT) technology uses oocytes collected mainly from untested animals, which creates a potential risk of EIAV transmission through infected embryos. The current review examines the risk of EIAV disease transmission through SCNT embryo production and transfer. Equine infectious anemia virus is a lentivirus from the family Retroviridae. Because of a lack of direct reports on this subject, relevant information gathered from close relatives of EIAV, such as human immunodeficiency virus (HIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), and small ruminant lentiviruses (SRLVs), is summarized and used to predict the biological plausibility of EIAV disease transmission through transfers of the equine SCNT embryos. Based on published information regarding interaction of oocytes with lentiviruses and the sufficiency of oocyte and embryo washing procedures to prevent lentivirus transmission from in vitro-produced embryos of various species, we predicted the risk of EIAV transmission through SCNT embryo production and transfer to be very small or absent.

  12. Bovine leukemia virus: purification and characterization of the aspartic protease.

    Science.gov (United States)

    Menard, A; Mamoun, R Z; Geoffre, S; Castroviejo, M; Raymond, S; Precigoux, G; Hospital, M; Guillemain, B

    1993-04-01

    To develop efficient bovine leukemia virus (BLV) protease (PR) inhibitors, pure enzyme is required. For this, we have developed a two-step chromatographic nondenaturing purification protocol of PR from virions. As expected, the purified protein presents a molecular weight (14 kDa) and a NH2 terminal end fitting with previously reported data. The enzymatic activity of BLV PR was characterized using a synthetic peptide containing a potential cleavage site of the BLV gag-pro polypeptide precursor as substrate. The protease was most active at pH 6, 40 degrees, and high salt concentration (1-2 M NaCl or ammonium sulfate). In contrast, using a natural substrate such as a human T-cell leukemia virus recombinant gag precursor, BLV PR activity was higher at a low salt concentration (0.5 M NaCl). Besides, the use of different potentially cleavable molecules revealed that PR activity may be influenced by the substrate conformational structure around the cleavage site. Replacement of the two amino acids of a synthetic substrate cleavable site by a statin residue completely inhibited the enzymatic activity of the BLV PR.

  13. First determination of the incidence of the unique TOR1A gene mutation, c.907delGAG, in a Mediterranean population.

    Science.gov (United States)

    Frédéric, Mélissa; Lucarz, Estelle; Monino, Christine; Saquet, Céline; Thorel, Delphine; Claustres, Mireille; Tuffery-Giraud, Sylvie; Collod-Béroud, Gwenaelle

    2007-04-30

    The c.907delGAG mutation in the TOR1A gene (also named DYT1) is the most common cause of early-onset primary dystonia. The mutation frequency and prevalence have so far been only estimated from rare clinical epidemiological reports in some populations. The purpose of this study was to investigate the incidence at birth of the c.907delGAG mutation in a French-representative mixed population of newborn from South-Eastern France. We applied an automated high-throughput genotyping method to dried blood spot samples from 12,000 newborns registered in Hérault between 2004 and 2005. Only one allele was found to carry the mutation, which allows to determine its incidence at birth as 1/12,000 per year in this area.

  14. [Transcriptional analysis of the Grp gene, a genomic homolog of the retrotransposon gypsy gag gene, in Drosophila melanogaster].

    Science.gov (United States)

    Nefedova, L N; Kuz'min, I V; Burmistrova, D A; Rezazadekh, S; Kim, A I

    2011-08-01

    In the present work, we studied the Grp gene (CG4680, Gag related protein) expression at the transcriptional level. It was found that at the embryonic and larval stages of D. melanogaster development the Grp expression proceeds at a low level, but it significantly increases at the adult stage. Adult individuals display a tissue-specific expression: an eleveated level of transcription is observed in the gut tissues, but not in the chitin carcass, head, and gonads. Since the gut may potentially be a primary barrier for the penetration of a viral infection, we conducted a comparative analysis of Grp gene transcription in D. melanogaster strains differing in the presence of active copies of the gypsy errantivirus and in the status of the flamenco gene controlling sensitivity to errantiviral infections. No noticeable differences in the level of Grp gene transcription were revealed. Thus, the Grp gene is not a pseudogene, but it is a functional gene of the D. melanogaster genome whose role remains to be elucidated.

  15. Directly infected resting CD4+T cells can produce HIV Gag without spreading infection in a model of HIV latency.

    Directory of Open Access Journals (Sweden)

    Matthew J Pace

    Full Text Available Despite the effectiveness of highly active antiretroviral therapy (HAART in treating individuals infected with HIV, HAART is not a cure. A latent reservoir, composed mainly of resting CD4+T cells, drives viral rebound once therapy is stopped. Understanding the formation and maintenance of latently infected cells could provide clues to eradicating this reservoir. However, there have been discrepancies regarding the susceptibility of resting cells to HIV infection in vitro and in vivo. As we have previously shown that resting CD4+T cells are susceptible to HIV integration, we asked whether these cells were capable of producing viral proteins and if so, why resting cells were incapable of supporting productive infection. To answer this question, we spinoculated resting CD4+T cells with or without prior stimulation, and measured integration, transcription, and translation of viral proteins. We found that resting cells were capable of producing HIV Gag without supporting spreading infection. This block corresponded with low HIV envelope levels both at the level of protein and RNA and was not an artifact of spinoculation. The defect was reversed upon stimulation with IL-7 or CD3/28 beads. Thus, a population of latent cells can produce viral proteins without resulting in spreading infection. These results have implications for therapies targeting the latent reservoir and suggest that some latent cells could be cleared by a robust immune response.

  16. Deep and superficial skin scrapings from a feline immunodeficiency virus-positive cat.

    Science.gov (United States)

    Neel, Jennifer A; Tarigo, Jaime; Tater, Kathy C; Grindem, Carol B

    2007-03-01

    An 8-year-old, neutered male, domestic shorthair cat housed at the North Carolina State University, College of Veterinary Medicine, Laboratory Animal Research facility as part of a research colony was examined because of mulifocal skin lesions. The lesions consisted of patchy alopecia with mild crusting of the periauricular region, neck, and dorsum; periauricular excoriations; marked dorsal seborrhea and scaling; and generalized erythematous papules. A moderate amount of ceruminous exudate was present in both ear canals. Results of testing for feline immunodeficiency virus (FIV) were positive. An ear swab specimen and superficial and deep skin scrapings were obtained, mounted with oil on glass slides, and coverslipped for microscopic examination. Two populations of mites were observed: a large population of slender, long (approximately 200 microm), adult mites with long, tapering abdomens that comprised two-thirds of the total body length; and a smaller population of more translucent and shorter mites (approximately 100 microm) with wide, blunt abdomens that had prominent transverse ridges. The interpretation was demodicosis, with Demodex cati and D gatoi co-infection. Histologic sections of biopsies from skin lesions on the neck, dorsum, and periauricular area contained a mild perivascular and perifollicular inflammatory infiltrate composed predominantly of histiocytes, lymphocytes, and plasma cells. Diffusely within the follicular lumina and occasionally within the superficial keratin, a myriad of Demodex organisms were observed. Intrafollicular mites were compatible in appearance with D cati whereas those in the corneal layer were suggestive of D gatoi. Demodicosis is an uncommon disease of cats, and rare cases of dual infection have been documented, occasionally in FIV-infected cats. The dual infection emphasizes the importance of doing both superficial and deep skin scrapings and of recognizing the unique microscopic features of different Demodex mites.

  17. Apoptosis of the teratocarcinoma cell line Tera-1 leads to the cleavage of HERV-K10gag proteins by caspases and/or granzyme B.

    Science.gov (United States)

    Beyer, T D; Kolowos, W; Dumitriu, I E; Voll, R E; Heyder, P; Gaipl, U S; Kalden, J R; Herrmann, M

    2002-09-01

    Redistribution, post-translational modifications and coclustering with viral antigens contribute to the immunogenicity of apoptotic cell-derived autoantigens. Almost all known targets of the humoral autoimmune response in systemic lupus erythematosus (SLE) are cleaved by caspases or granzyme B during apoptosis. Antibodies against retroviral proteins can frequently be detected in the sera of SLE patients without overt retroviral infections. These antibodies may represent cross-reactive antibodies or may have been induced by proteins encoded by endogenous retroviral sequences. We used Tera-1 cells that abundantly express a group-specific antigen of human endogenous retroviruses, HERV-K10gag polyprotein, to investigate its processing during apoptosis. Tera-1 cells induced to undergo apoptosis showed an altered HERV-K10gag processing compared with viable cells. In addition, granzyme B was able to cleave HERV-K10gag isolated from viable Tera-1 cells. Similar to nuclear autoantigens, endogenous retroviral proteins are cleaved during the execution phase of apoptosis. These post-translational modifications may result in the generation of T-cell neoepitopes or a changed epitope hierarchy of retroviral proteins. Therefore, immunogenicity of retroviral antigens in SLE patients may result from a similar mechanism as described for nuclear autoantigens.

  18. A process-based model for ammonia emission from urine patches, GAG (Generation of Ammonia from Grazing): description and sensitivity analysis

    Science.gov (United States)

    Móring, Andrea; Vieno, Massimo; Doherty, Ruth M.; Laubach, Johannes; Taghizadeh-Toosi, Arezoo; Sutton, Mark A.

    2016-03-01

    In this paper a new process-based, weather-driven model for ammonia (NH3) emission from a urine patch has been developed and its sensitivity to various factors assessed. The GAG model (Generation of Ammonia from Grazing) is capable of simulating the TAN (total ammoniacal nitrogen) and the water content of the soil under a urine patch and also soil pH dynamics. The model tests suggest that ammonia volatilization from a urine patch can be affected by the possible restart of urea hydrolysis after a rain event as well as CO2 emission from the soil. The vital role of temperature in NH3 exchange is supported by our model results; however, the GAG model provides only a modest overall temperature dependence in total NH3 emission compared with the literature. This, according to our findings, can be explained by the higher sensitivity to temperature close to urine application than in the later stages and may depend on interactions with other nitrogen cycling processes. In addition, we found that wind speed and relative humidity are also significant influencing factors. Considering that all the input parameters can be obtained for larger scales, GAG is potentially suitable for field and regional scale application, serving as a tool for further investigation of the effects of climate change on ammonia emissions and deposition.

  19. Efeito do tratamento por interferão ómega de origem felina (rFeIFN-Ω) na evolução clínica de gatos naturalmente infectados com os vírus da imunodeficiência (FIV) e leucemia felinas (FeLV) e na excreção de vírus respiratórios concomitantes

    OpenAIRE

    Cravo, Joana Acciaioli Pena

    2011-01-01

    Dissertação de Mestrado Integrado em Medicina Veterinária O vírus da imunodeficiência felina (FIV) e o vírus da leucemia felina (FeLV) são retrovírus responsáveis por duas das doenças infecciosas mais comuns e que põem em risco o bem-estar e a vida dos gatos em todo o mundo. Os imunoestimuladores, tais como os interferões rHuIFN-α e rFeIFN-ω, são amplamente utilizados em Medicina Veterinária no tratamento de gatos com FIV e FeLV. No entanto, a administração de rHuIFN-α a felinos tem limita...

  20. A process-based model for ammonia emission from urine patches, GAG (Generation of Ammonia from Grazing: description, validation and sensitivity analysis

    Directory of Open Access Journals (Sweden)

    A. Móring

    2015-07-01

    Full Text Available In this paper a new process-based, weather-driven model for ammonia (NH3 emission from a urine patch has been developed and its sensitivity to various factors assessed. This model, the GAG model (Generation of Ammonia from Grazing was developed as a part of a suite of weather-driven NH3 exchange models, as a necessary basis for assessing the effects of climate change on NH3 related atmospheric processes. GAG is capable of simulating the TAN (Total Ammoniacal Nitrogen content, pH and the water content of the soil under a urine patch. To calculate the TAN budget, GAG takes into account urea hydrolysis as a TAN input and NH3 volatilization as a loss. In the water budget, in addition to the water content of urine, precipitation and evaporation are also considered. In the pH module we assumed that the main regulating processes are the dissociation and dissolution equilibria related to the two products of urea hydrolysis: ammonium and bicarbonate. Finally, in the NH3 exchange flux calculation we adapted a canopy compensation point model that accounts for exchange with soil pores and stomata as well as deposition to the leaf surface. We validated our model against measurements, and carried out a sensitivity analysis. The validation showed that the simulated parameters (NH3 exchange flux, soil pH, TAN budget and water budget are well captured by the model (r > 0.5 for every parameter at p 3 emission. In addition, our results suggested that more sophisticated simulation of CO2 emission in the model could potentially improve the modelling of pH. The sensitivity analysis highlighted the vital role of temperature in NH3 exchange; however, presumably due to the TAN limitation, the GAG model currently provides only a modest overall temperature dependence in total NH3 emission compared with the values in the literature. Since all the input parameters can be obtained for study at larger scales, GAG is potentially suitable for larger scale application, such as

  1. Preparation and test of type Ⅰ collagen-glycosaminoglycan (GAG) template%Ⅰ型胶原-糖胺聚糖模板的制备与检测

    Institute of Scientific and Technical Information of China (English)

    徐青镭; 吴海山; 周维江; 赵定麟

    2001-01-01

    Objective: To synthesize the collagen-GAG template and to evaluate its feasibility to be used as the MSCs vehicle for meniscal tissue engineering. Methods: The collagen-GAG template was synthesized from rat tail type Ⅰ collagen and GAG using Yannas method. Then the post-stimulated MSCs by bFGF and TGF-β1 were added in. The MSCs-enriched collagen sponges were cultured in vitro, two weeks later the histological and ultrastructure detection was performed. Results: The histological and ultrastructure of the collagen-GAG template remained intact after 2 weeks' culture, and the MSCs in it remained viable. Conclusion: The collagen-GAG template synthesized in this experiment is suitable for the meniscal tissue engineering reconstruction as the vehicle for MSCs seed cells.%目的:制备胶原-糖胺聚糖模板,并检测其作为半月板组织工程重建的相关种子细胞载体的可行性。方法:采用Yannas法以大鼠尾作为原料提取Ⅰ型胶原,加入糖胺聚糖后冻干成为Ⅰ型胶原-糖胺聚糖模板,体外加入经碱性成纤维细胞生长因子(bFGF)和转化生长因子-β1(TGF-β1)协同刺激后的兔骨髓间充质干细胞培养2周后,行组织学及电镜检测。结果:培养2周后所制备的胶原-糖胺聚糖模板显微及超微结构保持良好,对骨髓间充质干细胞无毒害作用。结论:本实验方法所制备的胶原-糖胺聚糖模板适合用作半月板组织工程重建的种子细胞生物载体。

  2. Zika Virus

    Science.gov (United States)

    Zika is a virus that is spread mostly by mosquitoes. A pregnant mother can pass it to ... through blood transfusions. There have been outbreaks of Zika virus in the United States, Africa, Southeast Asia, ...

  3. Chikungunya Virus

    Science.gov (United States)

    ... Gaines, PhD, MPH, MA, CHES Differentiating Chikungunya From Dengue: A Clinical Challenge For Travelers CDC Travelers' Health Chikungunya Virus Home Prevention Transmission Symptoms & Treatment Geographic Distribution Chikungunya virus in ...

  4. Chikungunya virus

    Science.gov (United States)

    Chikungunya virus infection; Chikungunya ... Where Chikungunya is found Before 2013, the virus was found in Africa, Asia, Europe, and the Indian and Pacific oceans. In late 2013, outbreaks occurred for the first time in the ...

  5. 河南地区HIV-1毒株Gag基因抗原表位变异特征及准种特点研究%Study of Gag gene antigen epitypes variation and the quasispecies group characteristics in Henan area HIV-1 strains

    Institute of Scientific and Technical Information of China (English)

    杜鹏; 陈国敏; 李泽琳; 曾毅

    2009-01-01

    Objective To study the characteristics of the variation of antigen epitopes and quasispecies group in the HIV-1 viruses in Henan province in China. Methods The region of the p17-p24 of the HIV-1 gag gene was amplified by nested polymerse chain resction(PCR), purified products were cloned into the vector, the obtained were analyzed by MEGA soft wares. Results B' subtype strains were predominant in Henan province, the mutations in antigen epitypes of the p17 region of the gag gene focus on E62g(55.8% ) ,Y79f(48.9% ) ,T84V (48.9), I44V(44.2%), the p24 region had not found the distinct mutation. Conclusion Both the PCR sequences and clone strains sequences demonstrated that four antigen epitopes mutations in p17 region of the HIV B' subtype gag gene, and the region of p24 was more conservative,which was suitable for development of the epitope vaccien.%目的 探讨中国河南地区人免疫缺陷病毒Ⅰ型(HIV-1)毒株GAG蛋白抗原表位变异特征,并对其准种特点加以分析.方法 套式聚合酶链反应(Nested-PCR)扩增确认HIV阳性样本gagp17~p24基因区段并测序,PCR产物纯化后克隆,挑选克隆株鉴定为阳性后测序,以MEGA(version 3.0)等软件进行分析.结果 河南HIV毒株为B'亚型;gag基因p17区段抗原表位突变有E62G(55.80%),Y79F(48.90%),T84V(48.90%),144V(44.20%),gag基因p24区段抗原表位未见明显变异.结论 HIV-1 B'亚型毒株gag基因p17区段的4个抗原表位,存在较大变异,p24区段较为保守,适合抗原表位疫苗的研制.

  6. Selective expansion of viral variants following experimental transmission of a reconstituted feline immunodeficiency virus quasispecies.

    Directory of Open Access Journals (Sweden)

    Brian J Willett

    Full Text Available Following long-term infection with virus derived from the pathogenic GL8 molecular clone of feline immunodeficiency virus (FIV, a range of viral variants emerged with distinct modes of interaction with the viral receptors CD134 and CXCR4, and sensitivities to neutralizing antibodies. In order to assess whether this viral diversity would be maintained following subsequent transmission, a synthetic quasispecies was reconstituted comprising molecular clones bearing envs from six viral variants and its replicative capacity compared in vivo with a clonal preparation of the parent virus. Infection with either clonal (Group 1 or diverse (Group 2 challenge viruses, resulted in a reduction in CD4+ lymphocytes and an increase in CD8+ lymphocytes. Proviral loads were similar in both study groups, peaking by 10 weeks post-infection, a higher plateau (set-point being achieved and maintained in study Group 1. Marked differences in the ability of individual viral variants to replicate were noted in Group 2; those most similar to GL8 achieved higher viral loads while variants such as the chimaeras bearing the B14 and B28 Envs grew less well. The defective replication of these variants was not due to suppression by the humoral immune response as virus neutralising antibodies were not elicited within the study period. Similarly, although potent cellular immune responses were detected against determinants in Env, no qualitative differences were revealed between animals infected with either the clonal or the diverse inocula. However, in vitro studies indicated that the reduced replicative capacity of variants B14 and B28 in vivo was associated with altered interactions between the viruses and the viral receptor and co-receptor. The data suggest that viral variants with GL8-like characteristics have an early, replicative advantage and should provide the focus for future vaccine development.

  7. Vectores recombinantes basados en el virus modificado de Ankara (MVA), con deleción en el gen C6L, como vacunas contra el VIH/SIDA y otras enfermedades

    OpenAIRE

    García-Arriaza, J.; Gómez, Carmen E.; Esteban, Mariano

    2011-01-01

    La presente invención se engloba dentro de los campos de la biología molecular y de la biotecnología. Específicamente se refiere a virus recombinantes basados en el virus modificado de Ankara (MVA) que expresan los antígenos gp120 y Gag-Pol-Nef del virus de la inmunodeficiencia humana (VIH-1) de subtipo B (MVA-B), sobre los que se ha delecionado el gen de vaccinia C6L, y que han sido diseñados para utilizarse como vacunas contra el VIH/SIDA y otras enfermedades.

  8. Vectores recombinantes basados en el virus modificado de ankara (MVA), con deleción en el gen C6L, como vacunas contra el VIH/SIDA y otras enfermedades

    OpenAIRE

    García-Arriaza, J.; Gómez, Carmen E.; Esteban, Mariano

    2011-01-01

    [ES] La presente invención engloba dentro de los campos de la biología molecular y de la biotecnología. Especificamente se refiere a virus recombinantes basados en el virus modificado de Ankara (MVA) que expresan los antigenos gp120 y Gag-Pol-Nef del virus de la inmunodeficienciahumana (VIH-1) de subtipo B (MVA-B), sobre los que see ha delecionado el gen de vaccinia C6L, y que han sido diseñados para utilizarse como vacunas contra el VIH/SIDA y otras enfermedades.

  9. Comparison of three feline leukaemia virus (FeLV) point-of-care antigen test kits using blood and saliva.

    Science.gov (United States)

    Westman, Mark E; Malik, Richard; Hall, Evelyn; Sheehy, Paul A; Norris, Jacqueline M

    2017-02-01

    Feline leukaemia virus (FeLV) can be a challenging infection to diagnose due to a complex feline host-pathogen relationship and occasionally unreliable test results. This study compared the accuracy of three point-of-care (PoC) FeLV p27 antigen test kits commonly used in Australia and available commercially worldwide (SNAP FIV/FeLV Combo, Witness FeLV/FIV and Anigen Rapid FIV/FeLV), using detection of FeLV provirus by an in-house real-time polymerase chain reaction (qPCR) assay as the diagnostic gold standard. Blood (n=563) and saliva (n=419) specimens were collected from a population of cats determined to include 491 FeLV-uninfected and 72 FeLV-infected individuals (45 progressive infections [p27 and qPCR positive], 27 regressive infections [p27 negative, qPCR positive]). Sensitivity and specificity using whole blood was 63% and 94% for SNAP Combo, 57% and 98% for Witness, and 57% and 98% for Anigen Rapid, respectively. SNAP Combo had a significantly lower specificity using blood compared to the other two kits (P=0.004 compared to Witness, P=0.007 compared to Anigen Rapid). False-positive test results occurred with all three kits using blood, and although using any two kits in parallel increased specificity, no combination of kits completely eliminated the occurrence of false-positive results. We therefore recommend FeLV proviral PCR testing for any cat that tests positive with a PoC FeLV antigen kit, as well as for any cat that has been potentially exposed to FeLV but tests negative with a FeLV antigen kit, before final assignment of FeLV status can be made with confidence. For saliva testing, sensitivity and specificity was 54% and 100%, respectively, for all three test kits. The reduced sensitivity of saliva testing compared to blood testing, although not statistically significant, suggests saliva testing with the current generation of PoC FeLV antigen kits is unsuitable for screening large populations of cats, such as in shelters. Copyright © 2016 Elsevier

  10. Lack of infection with XMRV or other MLV-related viruses in blood, post-mortem brains and paternal gametes of autistic individuals.

    Directory of Open Access Journals (Sweden)

    Carla Lintas

    Full Text Available BACKGROUND: Autistic spectrum disorder (ASD is characterized by impaired language, communication and social skills, as well as by repetitive and stereotypic patterns of behavior. Many autistic subjects display a dysregulation of the immune system which is compatible with an unresolved viral infection with prenatal onset, potentially due to vertical viral transmission. Recently, the xenotropic murine leukemia virus-related virus (XMRV has been implicated in chronic fatigue syndrome (CFS and in prostate cancer by several, though not all studies. METHODOLOGY/PRINCIPAL FINDINGS: We assessed whether XMRV or other murine leukemia virus (MLV-related viruses are involved in autistic disorder. Using nested PCR targeted to gag genomic sequences, we screened DNA samples from: (i peripheral blood of 102 ASD patients and 97 controls, (ii post-mortem brain samples of 20 ASD patients and 17 sex- and age-matched controls, (iii semen samples of 11 fathers of ASD children, 25 infertile individuals and 7 fertile controls. No XMRV gag DNA sequences were detected, whereas peripheral blood samples of 3/97 (3.1% controls were positive for MLV. CONCLUSIONS| SIGNIFICANCE: No MLV-related virus was detected in blood, brain, and semen samples of ASD patients or fathers. Hence infection with XMRV or other MLV-related viruses is unlikely to contribute to autism pathogenesis.

  11. Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults.

    Directory of Open Access Journals (Sweden)

    Spyros A Kalams

    Full Text Available BACKGROUND: DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques. METHODOLOGY/PRINCIPAL FINDINGS: We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37 DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug IL-12 DNA. However, after three doses, 44.4% (4/9 of vaccinees receiving gag DNA and intermediate dose (500 ug of IL-12 DNA demonstrated a detectable cellular immune response. CONCLUSIONS/SIGNIFICANCE: This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity. TRIAL REGISTRATION: Clinicaltrials.gov NCT00115960 NCT00111605.

  12. CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

    Institute of Scientific and Technical Information of China (English)

    罗坤; 金宁一; 郭志儒; 秦云龙; 郭炎; 方厚华; 安汝国; 殷震

    2001-01-01

    To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4+ T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

  13. Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

    Directory of Open Access Journals (Sweden)

    Smeulders Liesbeth

    2010-10-01

    Full Text Available Abstract Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1 reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM. Specific cytotoxicity was reverted by addition

  14. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    Science.gov (United States)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  15. Permanences GAG-EPA

    CERN Multimedia

    GAC-EPA

    2015-01-01

    Le GAC organise chaque mois des permanences avec entretiens individuels. La prochaine permanence se tiendra le : Mardi 5 mai de 13 h 30 à 16 h 00 Salle de réunion de l’Association du personnel Les permanences suivantes auront lieu les mardis 2 juin, 1er septembre, 6 octobre, 3 novembre et 1er décembre 2015. Les permanences du Groupement des Anciens sont ouvertes aux bénéficiaires de la Caisse de pensions (y compris les conjoints survivants) et à tous ceux qui approchent de la retraite. Nous invitons vivement ces derniers à s’associer à notre groupement en se procurant, auprès de l’Association du personnel, les documents nécessaires.

  16. A process-based model for ammonia emission from urine patches, GAG (Generation of Ammonia from Grazing): description and sensitivity analysis

    DEFF Research Database (Denmark)

    Móring, Andrea; Vieno, Massimo; M. Doherty, Ruth

    2016-01-01

    In this paper a new process-based, weather-driven model for ammonia (NH3) emission from a urine patch has been developed and its sensitivity to various factors assessed. The GAG model (Generation of Ammonia from Grazing) is capable of simulating the TAN (total ammoniacal nitrogen) and the water...... content of the soil under a urine patch and also soil pH dynamics. The model tests suggest that ammonia volatilization from a urine patch can be affected by the possible restart of urea hydrolysis after a rain event as well as CO2 emission from the soil. The vital role of temperature in NH3 exchange...

  17. A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability

    NARCIS (Netherlands)

    M. Lutzelberger; L.S. Reinert; A.T. Das; B. Berkhout; J. Kjems

    2006-01-01

    Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-c

  18. The 5' untranslated region and Gag product of Idefix, a long terminal repeat-retrotransposon from Drosophila melanogaster, act together to initiate a switch between translated and untranslated states of the genomic mRNA.

    Science.gov (United States)

    Meignin, Carine; Bailly, Jean-Luc; Arnaud, Frédérick; Dastugue, Bernard; Vaury, Chantal

    2003-11-01

    Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5' untranslated region (5'UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5'UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5'UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.

  19. Computer Virus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Computer viruses are small software programs that are designed to spread from one computerto another and to interfere with computer operation.A virus might delete data on your computer,use your e-mail program to spread itself to othercomputers,or even erase everything on your hard disk.Viruses are most easily spread by attach-ments in e-mail messages or instant messaging messages.That is why it is essential that you never

  20. Association of human mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol does not require other viral proteins

    Directory of Open Access Journals (Sweden)

    Lydia Kobbi

    2016-06-01

    Full Text Available In human, the cytoplasmic (cLysRS and mitochondrial (mLysRS species of lysyl-tRNA synthetase are encoded by a single gene. Following HIV-1 infection, mLysRS is selectively taken up into viral particles along with the three tRNALys isoacceptors. The GagPol polyprotein precursor is involved in this process. With the aim to reconstitute in vitro the HIV-1 tRNA3Lys packaging complex, we first searched for the putative involvement of another viral protein in the selective viral hijacking of mLysRS only. After screening all the viral proteins, we observed that Vpr and Rev have the potential to interact with mLysRS, but that this association does not take place at the level of the assembly of mLysRS into the packaging complex. We also show that tRNA3Lys can form a ternary complex with the two purified proteins mLysRS and the Pol domain of GagPol, which mimicks its packaging complex.

  1. Human Ubc9 is involved in intracellular HIV-1 Env stability after trafficking out of the trans-Golgi network in a Gag dependent manner.

    Directory of Open Access Journals (Sweden)

    Christopher R Bohl

    Full Text Available The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER and its trafficking to the trans-Golgi network (TGN were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.

  2. Phase I safety and immunogenicity evaluations of an alphavirus replicon HIV-1 subtype C gag vaccine in healthy HIV-1-uninfected adults.

    Science.gov (United States)

    Wecker, M; Gilbert, P; Russell, N; Hural, J; Allen, M; Pensiero, M; Chulay, J; Chiu, Ya-Lin; Abdool Karim, S S; Burke, D S

    2012-10-01

    On the basis of positive preclinical data, we evaluated the safety and immunogenicity of an alphavirus replicon HIV-1 subtype C gag vaccine (AVX101), expressing a nonmyristoylated form of Gag, in two double-blind, randomized, placebo-controlled clinical trials in healthy HIV-1-uninfected adults. Escalating doses of AVX101 or placebo were administered subcutaneously to participants in the United States and Southern Africa. Because of vaccine stability issues, the first trial was halted prior to completion of all dose levels and a second trial was implemented. The second trial was also stopped prematurely due to documentation issues with the contract manufacturer. Safety and immunogenicity were evaluated through assessments of reactogenicity, reports of adverse events, and assessment of replication-competent and Venezuelan equine encephalitis (VEE) viremia. Immunogenicity was measured using the following assays: enzyme-linked immunosorbent assay (ELISA), chromium 51 ((51)Cr)-release cytotoxic T lymphocyte (CTL), gamma interferon (IFN-γ) ELISpot, intracellular cytokine staining (ICS), and lymphoproliferation assay (LPA). Anti-vector antibodies were also measured. AVX101 was well tolerated and exhibited only modest local reactogenicity. There were 5 serious adverse events reported during the trials; none were considered related to the study vaccine. In contrast to the preclinical data, immune responses in humans were limited. Only low levels of binding antibodies and T-cell responses were seen at the highest doses. This trial also highlighted the difficulties in developing a novel vector for HIV.

  3. Cross-clade cytotoxic T cell response to human immunodeficiency virus type 1 proteins among HLA disparate North Americans and Thais.

    Science.gov (United States)

    Lynch, J A; deSouza, M; Robb, M D; Markowitz, L; Nitayaphan, S; Sapan, C V; Mann, D L; Birx, D L; Cox, J H

    1998-10-01

    A globally effective vaccine will need to elicit cytotoxic T lymphocytes (CTL) capable of recognizing diverse human immunodeficiency virus type 1 (HIV-1) clades. Study of the cellular immune responses of HIV-1-infected persons may allow predictions to be made regarding useful vaccine antigen components. The frequency and magnitude of CTL responses to clade E and B Gag, Pol-RT, Env, and Nef proteins were compared in 12 HLA-characterized, clade E-infected Thais and in 10 clade B-infected North Americans using vaccinia recombinant constructs for protein expression. While responses were detected against all proteins, they were most frequent and cross-reactive to Gag in both groups. Pol-RT was recognized less frequently in Thais than North Americans. Cross-clade protein recognition was common but not uniformly present among these HLA-disparate individuals. Population-specific CTL data are needed to adequately prepare for vaccine trials outside of North America and Europe.

  4. Phytophthora viruses.

    Science.gov (United States)

    Cai, Guohong; Hillman, Bradley I

    2013-01-01

    Phytophthora sp. is a genus in the oomycetes, which are similar to filamentous fungi in morphology and habitat, but phylogenetically more closely related to brown algae and diatoms and fall in the kingdom Stramenopila. In the past few years, several viruses have been characterized in Phytophthora species, including four viruses from Phytophthora infestans, the late blight pathogen, and an endornavirus from an unnamed Phytophthora species from Douglas fir. Studies on Phytophthora viruses have revealed several interesting systems. Phytophthora infestans RNA virus 1 (PiRV-1) and PiRV-2 are likely the first members of two new virus families; studies on PiRV-3 support the establishment of a new virus genus that is not affiliated with established virus families; PiRV-4 is a member of Narnaviridae, most likely in the genus Narnavirus; and Phytophthora endornavirus 1 (PEV1) was the first nonplant endornavirus at the time of reporting. Viral capsids have not been found in any of the above-mentioned viruses. PiRV-1 demonstrated a unique genome organization that requires further examination, and PiRV-2 may have played a role in late blight resurgence in 1980s-1990s.

  5. Computer Virus

    Institute of Scientific and Technical Information of China (English)

    高振桥

    2002-01-01

    If you work with a computer,it is certain that you can not avoid dealing, with at least one computer virus.But how much do you know about it? Well,actually,a computer virus is not a biological' one as causes illnesses to people.It is a kind of computer program

  6. Development of avian sarcoma and leukosis virus-based vector-packaging cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Stoker, A.W.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1988-03-01

    The authors have constructed an avian leukosis virus derivative with a 5{prime} deletion extending from within the tRNA primer binding site to a SacI site in the leader region. The aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1{Psi}{sup {minus}}, to develop vector-packaging cell lines. They show that RAV-1{Psi}{sup {minus}} can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, they have demonstrated that QT6-derived lines expressing RAV-1{Psi}{sup {minus}} can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-{Psi}{sup {minus}}-expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. They show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.

  7. The 5′ Untranslated Region and Gag product of Idefix, a Long Terminal Repeat-Retrotransposon from Drosophila melanogaster, Act Together To Initiate a Switch between Translated and Untranslated States of the Genomic mRNA

    OpenAIRE

    Meignin, Carine; Bailly, Jean-Luc; Arnaud, Frédérick; Dastugue, Bernard; Vaury, Chantal

    2003-01-01

    Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5′ untranslated region (5′UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5′UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be i...

  8. Infectivity of chimeric human T-cell leukemia virus type I molecular clones assessed by naked DNA inoculation.

    Science.gov (United States)

    Zhao, T M; Robinson, M A; Bowers, F S; Kindt, T J

    1996-06-25

    Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.

  9. Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity.

    Directory of Open Access Journals (Sweden)

    Dhiraj Acharya

    Full Text Available Chikungunya virus (CHIKV is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal or C6/36 cells (mosquito through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKV mos has slower replication than mammalian cell-derived CHIKV (CHIKV vero, when tested in both human and murine cell lines. Consistent with this, CHIKV mos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKV mos produces a lower level of viremia and less severe footpad swelling when compared with CHIKV vero. Interestingly, CHIKV mos has impaired ability to bind to glycosaminoglycan (GAG receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKV mos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKV vero after a single passage in mammalian cells. Furthermore, CHIKV vero and CHIKV mos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells.

  10. The receptors for gibbon ape leukemia virus and amphotropic murine leukemia virus are not downregulated in productively infected cells

    Directory of Open Access Journals (Sweden)

    Eiden Maribeth V

    2011-07-01

    Full Text Available Abstract Background Over the last several decades it has been noted, using a variety of different methods, that cells infected by a specific gammaretrovirus are resistant to infection by other retroviruses that employ the same receptor; a phenomenon termed receptor interference. Receptor masking is thought to provide an earlier means of blocking superinfection, whereas receptor down regulation is generally considered to occur in chronically infected cells. Results We used replication-competent GFP-expressing viruses containing either an amphotropic murine leukemia virus (A-MLV or the gibbon ape leukemia virus (GALV envelope. We also constructed similar viruses containing fluorescence-labeled Gag proteins for the detection of viral particles. Using this repertoire of reagents together with a wide range of antibodies, we were able to determine the presence and availability of viral receptors, and detect viral envelope proteins and particles presence on the cell surface of chronically infected cells. Conclusions A-MLV or GALV receptors remain on the surface of chronically infected cells and are detectable by respective antibodies, indicating that these receptors are not downregulated in these infected cells as previously proposed. We were also able to detect viral envelope proteins on the infected cell surface and infected cells are unable to bind soluble A-MLV or GALV envelopes indicating that receptor binding sites are masked by endogenously expressed A-MLV or GALV viral envelope. However, receptor masking does not completely prevent A-MLV or GALV superinfection.

  11. Computer viruses

    Science.gov (United States)

    Denning, Peter J.

    1988-01-01

    The worm, Trojan horse, bacterium, and virus are destructive programs that attack information stored in a computer's memory. Virus programs, which propagate by incorporating copies of themselves into other programs, are a growing menace in the late-1980s world of unprotected, networked workstations and personal computers. Limited immunity is offered by memory protection hardware, digitally authenticated object programs,and antibody programs that kill specific viruses. Additional immunity can be gained from the practice of digital hygiene, primarily the refusal to use software from untrusted sources. Full immunity requires attention in a social dimension, the accountability of programmers.

  12. Intradermal delivery of recombinant vaccinia virus vector DIs induces gut-mucosal immunity.

    Science.gov (United States)

    Yoshino, N; Kanekiyo, M; Hagiwara, Y; Okamura, T; Someya, K; Matsuo, K; Ami, Y; Sato, S; Yamamoto, N; Honda, M

    2010-08-01

    Antigen-specific mucosal immunity is generally induced by the stimulation of inductive mucosal sites. In this study, we found that the replication-deficient vaccinia virus vector, DIs, generates antigen-specific mucosal immunity and systemic responses. Following intradermal injection of recombinant DIs expressing simian immunodeficiency virus gag (rDIsSIVgag), we observed increased levels of SIV p27-specific IgA and IgG antibodies in faecal extracts and plasma samples, and antibody-forming cells in the intestinal mucosa and spleen of C57BL/6 mice. Antibodies against p27 were not detected in nasal washes, saliva, and vaginal washes. The enhanced mucosal and systemic immunity persisted for 1 year of observation. Induction of Gag-specific IFN-gamma spot-forming CD8(+) T cells in the spleen, small intestinal intraepithelial lymphocytes, and submandibular lymph nodes was observed in the intradermally injected mice. Heat-inactivated rDIsSIVgag rarely induced antigen-specific humoral and T-helper immunity. Moreover, rDIsSIVgag was detected in MHC class II IA antigen-positive (IA(+)) cells at the injection site. Consequently, intradermal delivery of rDIs effectively induces antigen-specific humoral and cellular immunity in gut-mucosal tissues of mice. Our data suggest that intradermal injection of an rDIs vaccine may be useful against mucosally transmitted pathogens.

  13. Human Immunodeficiency Virus type 1 in seronegative infants born to HIV-1-infected mothers

    Directory of Open Access Journals (Sweden)

    G Reyes-Terán

    2006-06-01

    Full Text Available Abstract Background Some individuals repeatedly exposed to Human Immunodeficiency Virus do not seroconvert and are resistant to HIV infection. Here, in a pediatric cohort of HIV seronegative infants born of HIV-infected mothers, we have studied eight non-breastfed children in whom viral DNA was detected in their PBMC. Our objective was to assess whether silent infection in these children can be explained by the presence of integrated viral DNA. Methods The presence of viral DNA was corroborated by nested PCR with primers for gag and the nef/LTR regions of HIV-1. Integration of HIV DNA into the host genome was assessed by an Alu-LTR PCR. Amplicons were sequenced and phylogenetic analyzes were done. Results HIV-1 DNA was detected in the earliest available PBMC sample from all eight infants, and two of them tested positive for HIV DNA at 2 years of age. Nested PCR resulted in the amplification of gag, nef/LTR and Alu-LTR fragments, which demostrated that HIV-1 DNA was integrated in the host cell genome. Each individual has a characteristic sequence pattern and is different from the LTR sequence of HXB2 prototype virus and other Mexican isolates. Conclusion HIV-1 DNA was observed in PBMC from HIV exposed seronegative children in this pediatric cohort.

  14. Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

    Directory of Open Access Journals (Sweden)

    Solbak Sara MØ

    2011-02-01

    Full Text Available Abstract Background The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L- domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX, is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively

  15. Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors

    DEFF Research Database (Denmark)

    Bahrami, Shervin; Pedersen, Finn Skou; Duch, Mogens R.

    2003-01-01

    envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random......The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV...... mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells...

  16. Oligoclonal CD8 lymphocytes from persons with asymptomatic human immunodeficiency virus (HIV) type 1 infection inhibit HIV-1 replication.

    Science.gov (United States)

    Toso, J F; Chen, C H; Mohr, J R; Piglia, L; Oei, C; Ferrari, G; Greenberg, M L; Weinhold, K J

    1995-10-01

    CD8 lymphocytes from asymptomatic human immunodeficiency virus (HIV) type 1-infected patients can suppress virus production from infected CD4 cells. Suppressive activity is separate and distinct from cytotoxic T lymphocyte (CTL) reactivities and is likely mediated by a soluble factor(s). The majority of HIV-1 suppression studies have been done in the context of bulk CD8 cell cultures. In this study, viral suppression was characterized by clonal populations of CD8 cells derived from HIV-1-infected patients. Most of the suppressive clones were devoid of detectable CTL reactivity against env-, gag-, pol-, and nef-expressing targets. Among the suppressive clones derived from an individual patient, a marked heterogeneity was evident with respect to phenotypic markers, cytokine production, and T cell receptor V beta expression. These results suggest that noncytolytic virus suppression is oligoclonal in nature. Clones provide tools for future studies aimed at understanding the mechanism of suppression and identifying the suppressive factor.

  17. Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

    Directory of Open Access Journals (Sweden)

    Natalia Makarova

    Full Text Available BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC. Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

  18. Hb S [β6(A3)Glu→Val, GAG>GTG] and β-globin gene cluster haplotype distribution in Mauritania.

    Science.gov (United States)

    Veten, Fatimetou M; Abdelhamid, Isselmou O; Meiloud, Ghlana M; Ghaber, Sidi M; Salem, Mohamed L; Abbes, Salem; Houmeida, Ahmed O

    2012-01-01

    Of 1050 Mauritanian blood donors screened from the two main racial groups, i.e., the Moors and Black Africans, 60 were found to carry Hb S [β6(A3)Glu→Val, GAG>GTG], giving a global frequency of 5.71%. The prevalence observed in the Black African Mauritanians (10.69%) is almost five times that found in the Moor group (2.25%). Four of the five main β(S) haplotypes were detected in this study: Senegal (77.8%), Benin (8.8%), Arab-Indian (5.5%) and Bantu (4.4%). These data showed that Hb S is a serious public health problem in Mauritania. They also confirm the ethnic heterogeneity of the Mauritanian population.

  19. A polyvalent Clade B virus-like particle HIV vaccine combined with partially protective oral preexposure prophylaxis prevents simian-human immunodeficiency virus Infection in macaques and primes for virus-amplified immunity.

    Science.gov (United States)

    Ross, Ted M; Pereira, Lara E; Luckay, Amara; McNicholl, Janet M; García-Lerma, J Gerardo; Heneine, Walid; Eugene, Hermancia S; Pierce-Paul, Brooke R; Zhang, Jining; Hendry, R Michael; Smith, James M

    2014-11-01

    Vaccination and preexposure prophylaxis (PrEP) with antiretrovirals have shown only partial protection from HIV-1 infection in human trials. Oral Truvada (emtricitabine/tenofovir disoproxil fumarate) is FDA approved as PrEP but partial adherence reduces efficacy. If combined as biomedical preventions (CBP), an HIV vaccine could protect when PrEP adherence is low and PrEP could prevent vaccine breakthroughs. The efficacy of combining oral PrEP with an HIV vaccine has not been evaluated in humans. We determined the efficacy of combining a DNA/virus-like particle (VLP) vaccine with partially effective intermittent PrEP in Indian rhesus macaques (RM). Eight RM received intramuscular inoculations of five DNA plasmids encoding four HIV-1 Clade B primary isolate Envs and SIVmac239 Gag (at weeks 0 and 4), followed by intramuscular and intranasal inoculations of homologous Gag VLPs and four Env VLPs (at weeks 12, 16, and 53). At week 61, we initiated weekly rectal exposures with heterologous SHIV162p3 (10 TCID50) along with oral Truvada (TDF, 22 mg/kg; FTC 20 mg/kg) dosing 2 h before and 22 h after each exposure. This PrEP regimen previously demonstrated 50% efficacy. Five controls (no vaccine, no PrEP) received weekly SHIV162p3. All controls were infected after a median of four exposures; the mean peak plasma viral load (VL) was 3.9×10(7) vRNA copies/ml. CBP protected seven of eight (87.5%) RM. The one infected CBP RM had a reduced peak VL of 8.8×10(5) copies/ml. SHIV exposures during PrEP amplified Gag and Env antibody titers in protected RM. These results suggest that combining oral PrEP with HIV vaccines could enhance protection against HIV-1 infection.

  20. Human endogenous retrovirus K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected in HIV-1-infected subjects using standard peptide matrix-based screening

    NARCIS (Netherlands)

    R.B. Jones (R. Brad); V.M. John (Vivek); D.V. Hunter (Diana); E. Martin (Eric); S. Mujib (Shariq); V. Mihajlovic (Vesna); P.C. Burgers (Peter); T.M. Luider (Theo); G. Gyenes (Gabor); N.C. Sheppard (Neil); D. SenGupta (Devi); R. Tandon (Ravi); F.-Y. Yue (Feng-Yun); W.S. Benko (William); C. Kovacs (Carrie); R. Nixon; M.A. Ostrowski (Mario)

    2012-01-01

    textabstractT-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific

  1. Intra- and inter-clade cross-reactivity by HIV-1 Gag specific T-cells reveals exclusive and commonly targeted regions: implications for current vaccine trials.

    Directory of Open Access Journals (Sweden)

    Lycias Zembe

    Full Text Available The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Af