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Sample records for plasma virus isolated

  1. Viruses isolated from Panamanian sloths.

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    Seymour, C; Peralta, P H; Montgomery, G G

    1983-11-01

    Seven virus strains were isolated in Vero cells from whole blood samples from 80 wild-caught sloths, Bradypus variegatus and Choloepus hoffmanni, from Central Panamá. Four strains of at least two different serotypes are related to Changuinola virus; two of these were associated with prolonged or recrudescent viremias. One strain is an antigenic subtype of Punta Toro virus, and another, described here as Bradypus-4 virus, is a new, antigenically ungrouped virus. A second new virus from sloths, Utive virus, forms an antigenic complex within the Simbu serogroup with Utinga and Pintupo viruses. Tests on sequential plasma samples from radio-marked free-ranging sloths and from recently captured animals maintained in captivity showed that both species develop neutralizing antibodies following naturally acquired virus infections. Antibodies against the Changuinola and Simbu serogroup viruses are widespread in both sloth species and are especially prevalent in Choloepus, but are virtually absent in all other wild vertebrate species tested.

  2. Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

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    Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

    2015-10-01

    We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  3. Decode Fish Lymphocystis Virus Isolated from China

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    @@ Aresearch group headed by Prof. Zhang Qiya from the CAS Institute of Hydrobiology (IHB) has succeeded in sequencing the complete genome of lymphocystis disease virus isolated from China (LCDV-C), a virus isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. Their work has been published in the recent issue of Journal of Virology (2004, 78 (13): 6982- 6994).

  4. Detection of dengue virus in platelets isolated from dengue patients.

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    Noisakran, Sansanee; Gibbons, Robert V; Songprakhon, Pucharee; Jairungsri, Aroonroong; Ajariyakhajorn, Chuanpis; Nisalak, Ananda; Jarman, Richard G; Malasit, Prida; Chokephaibulkit, Kulkanya; Perng, Guey Chuen

    2009-03-01

    Though thrombocytopenia or dysfunction of platelets is common in dengue virus infection, the role of platelets has not been established. We enrolled 33 hospitalized children with serologically confirmed dengue virus infection. Blood specimens were collected during hospitalization. Platelets and plasma were isolated from the whole blood. Detection of dengue virus in plasma and platelets was carried out by RT-PCR with primers that can differentiate different dengue serotypes simultaneously, and by electron transmission microscopy (EM). Dengue viral RNA was detected in the platelets and plasma by conventional RT-PCR. A significantly higher percentage of dengue viral RNA was detected in platelets than in plasma (p = 0.03). Platelets isolated 5 days after onset of fever were most likely positive for viral RNA. Concurrent infection or co-circulation with multiple dengue serotypes was observed in 12% of patients. Infrequently, negative-stranded dengue viral RNA was detected in platelets and in plasma. Importantly, EM confirmed the presence of dengue viral-like particles inside platelets prepared from dengue patients. Our findings suggest the presence of dengue virus in platelets may be associated with the dysfunction of platelets observed in dengue patients.

  5. A Multicomponent Animal Virus Isolated from Mosquitoes.

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    Ladner, Jason T; Wiley, Michael R; Beitzel, Brett; Auguste, Albert J; Dupuis, Alan P; Lindquist, Michael E; Sibley, Samuel D; Kota, Krishna P; Fetterer, David; Eastwood, Gillian; Kimmel, David; Prieto, Karla; Guzman, Hilda; Aliota, Matthew T; Reyes, Daniel; Brueggemann, Ernst E; St John, Lena; Hyeroba, David; Lauck, Michael; Friedrich, Thomas C; O'Connor, David H; Gestole, Marie C; Cazares, Lisa H; Popov, Vsevolod L; Castro-Llanos, Fanny; Kochel, Tadeusz J; Kenny, Tara; White, Bailey; Ward, Michael D; Loaiza, Jose R; Goldberg, Tony L; Weaver, Scott C; Kramer, Laura D; Tesh, Robert B; Palacios, Gustavo

    2016-09-14

    RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. Although multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health.

  6. Adaptive evolution of simian immunodeficiency viruses isolated from two conventional progressor macaques with neuroaids

    Energy Technology Data Exchange (ETDEWEB)

    Foley, Brian T [Los Alamos National Laboratory; Korber, Bette T [Los Alamos National Laboratory

    2008-01-01

    Simian immunodeficiency virus infection of macaques may result in neuroAIDS, a feature more commonly observed in macaques with rapid progressive disease than in those with conventional disease. This is the first report of two conventional progressors (H631 and H636) with encephalitis in rhesus macaques inoculated with a derivative of SIVsmES43-3. Phylogenetic analyses of viruses isolated from the cerebral spinal fluid (CSF) and plasma from both animals demonstrated tissue compartmentalization. Additionally, virus from the central nervous system (CNS) was able to infect primary macaque monocyte-derived macrophages more efficiently than virus from plasma. Conversely, virus isolated from plasma was able to replicate better in peripheral blood mononuclear cells than virus from CNS. We speculate that these viruses were under different selective pressures in their separate compartments. Furthermore, these viruses appear to have undergone adaptive evolution to preferentially replicate in their respective cell targets. Analysis of the number of potential N-linked glycosylation sites (PNGS) in gp160 showed that there was a statistically significant loss of PNGS in viruses isolated from CNS in both macaques compared to SIVsmE543-3. Moreover, virus isolated from the brain in H631, had statistically significant loss of PNGS compared to virus isolated from CSF and plasma of the same animal. It is possible that the brain isolate may have adapted to decrease the number of PNGS given that humoral immune selection pressure is less likely to be encountered in the brain. These viruses provide a relevant model to study the adaptations required for SIV to induce encephalitis.

  7. [Isolation and purification of virus damaging sunflower].

    Science.gov (United States)

    Zakusilo, A O; Didenko, L F; Kniazieva, N A; Boĭko, A L

    1994-01-01

    A procedure has been developed for purifying intact virus's isolate particles evoking yellow spot mosaic disease in sunflower. Purification of pathogen in 0.1 M sodium phosphate buffer, pH 8.0 containing 0.05 M Na3SO3 and 0.2% 2-mercaptoethanol is used. After first clarification extract was exposed to two cycles of high-speed centrifugation and fractionated in linear 10-40% (wt vol-1) sucrose density gradient. Virus was recovered from appropriate fractions after dialysis against 0.01 M Na2SO3.

  8. The first bluetongue virus isolation in Yugoslavia.

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    Djuricić, B; Jermolenko, G; Milosević, B; Radojicić, S; Debeljak, Z; Tomić, A

    2004-01-01

    Catarrhal fever in sheep or bluetongue (BT) has not been recorded in Yugoslavia until recently. During the first incidence of BT disease in Serbia and Montenegro in 2001, the authors conducted field studies on suspected cases of the disease and collected samples for laboratory diagnosis. BT virus (BTV) was isolated and identified as serotype 9 by the Institute for Animal Health in Pirbright, United Kingdom (the Office International des Epizooties BT reference laboratory).

  9. CCR5, GPR15, and CXCR6 are major coreceptors of human immunodeficiency virus type 2 variants isolated from individuals with and without plasma viremia.

    NARCIS (Netherlands)

    H. Blaak (Hetty); P.H.M. Boers (Patrick); R.A. Gruters (Rob); H. Schuitemaker; M.E. van der Ende (Marchina); A.D.M.E. Osterhaus (Albert)

    2005-01-01

    textabstractHuman immunodeficiency virus type 2 (HIV-2) is generally considered capable of using a broad range of coreceptors. Since HIV-2 variants from individuals with nonprogressive infection were not studied previously, the possibility that broad coreceptor usage is a property of variants associ

  10. Cytopathological characteristics of tomato spotted wilt virus isolates

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    Anna Rudzińska-Langwald

    2014-01-01

    Full Text Available The electron microscopy study revealed that four examined virus isolates in the cells of the infected host plant produced different inclusions depending on the virus isolate and the time of passaging by mechanical transmission. Numerous virus particle inclusions as well as viroplasm and filamentous inclusions typical for TSWV were present in the plant cells infected with TSWV isolate (PPR. This isolate was kept in N. rustica by 4 mechanical transmissions. A similar virus isolate but maintained for 2 years by mechanical transmission in Nicotiana plants (TI produced virus particle inclusions as well as amorphous inclusions typical for defective isolates. In plant cells infected with the same isolate but maintained by mechanical transmission one year longer (T2 no virus particle inclusions were produced. In the amorphous inclusions produced by this isolate virus particles were seen, but they were not surrounded by additional membrane. The isolate G induced only amorphous inclusions dispersed within the cytoplasm of infected cells. No virus particles were seen in the amorphous inclusions. The mechanical transmission of TSWV isolates in N. rustica plants reduced the number of virus particles present in the cytoplasm. The defectivenes of the isolate cause also the appearance of a new type of inclusion - the amorphous inclusions.

  11. Molecular characterization of an Akabane virus isolate from West Java, Indonesia.

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    Purnomo Edi, Suryo; Ibrahim, Afif; Sukoco, Rinto; Bunali, Lukman; Taguchi, Masaji; Kato, Tomoko; Yanase, Tohru; Shirafuji, Hiroaki

    2017-04-08

    We isolated an arbovirus from bovine blood in Indonesia. The arbovirus was obtained from the plasma of a cow showing no clinical symptoms in West Java in February 2014, and was identified as Akabane virus (AKAV) by AKAV-specific RT-PCR and subsequent sequence analysis. Phylogenetic analysis based on partial S segment indicated the AKAV isolate, WJ-1SA/P/2014, was most closely related with two isolates from Israel and Turkey reported in 2001 and 2015, respectively, and that WJ-1SA/P/2014 isolate belongs to AKAV genogroup Ib. This is the first isolation of AKAV from Indonesia.

  12. Mayaro virus isolated from a Trinidadian mosquito, Mansonia venezuelensis.

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    AITKEN, T H; DOWNS, W G; ANDERSON, C R; SPENCE, L; CASALS, J

    1960-04-01

    A strain of Mayaro virus has been isolated in Trinidad from the mosquito Mansonia venezuelensis. This is the first record of isolation of this agent from naturally infected mosquitoes, caught in the wild.

  13. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates.

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    Fenyö, E M; Morfeldt-Månson, L; Chiodi, F; Lind, B; von Gegerfelt, A; Albert, J; Olausson, E; Asjö, B

    1988-01-01

    According to their capacity to replicate in vitro, human immunodeficiency virus (HIV) isolates can be divided into two major groups, rapid/high and slow/low. Rapid/high viruses can easily be transmitted to a variety of cell lines of T-lymphoid (CEM, H9, and Jurkat) and monocytoid (U937) origin. In contrast, slow/low viruses replicate transiently, if at all, in these cell lines. Except for a few isolates, the great majority of slow/low viruses replicate in peripheral blood mononuclear cells and Jurkat-tatIII cells constitutively expressing the tatIII gene of HIV-1. The viruses able to replicate efficiently cause syncytium formation and are regularly isolated from immunodeficient patients. Poorly replicating HIV isolates, often obtained from individuals with no or mild disease, show syncytium formation and single-cell killing simultaneously or, with some isolates, cell killing only. Images PMID:2459416

  14. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats.

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    Towner, Jonathan S; Amman, Brian R; Sealy, Tara K; Carroll, Serena A Reeder; Comer, James A; Kemp, Alan; Swanepoel, Robert; Paddock, Christopher D; Balinandi, Stephen; Khristova, Marina L; Formenty, Pierre B H; Albarino, Cesar G; Miller, David M; Reed, Zachary D; Kayiwa, John T; Mills, James N; Cannon, Deborah L; Greer, Patricia W; Byaruhanga, Emmanuel; Farnon, Eileen C; Atimnedi, Patrick; Okware, Samuel; Katongole-Mbidde, Edward; Downing, Robert; Tappero, Jordan W; Zaki, Sherif R; Ksiazek, Thomas G; Nichol, Stuart T; Rollin, Pierre E

    2009-07-01

    In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

  15. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats.

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    Jonathan S Towner

    2009-07-01

    Full Text Available In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus based on detection of Marburg virus RNA in 31/611 (5.1% bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

  16. Isolation of surface tubules of fowlpox virus.

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    Carter, J K; Cheville, N F

    1981-01-01

    Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.

  17. Tobacco streak virus isolated from lettuce.

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    Abtahi, F S; Khodai Motlagh, M

    2009-05-01

    Tobacco streak virus (TSV) is an ilarvirus with a worldwide distribution. This virus infects many plants and causes significant yield losses. In this study, 300 samples of lettuce were collected from lettuce fields in Tehran Province. Infected plants show symptoms such as: mosaic, vein clearing, vein necrosis, yellowing and leaf distortion. DAS-ELISA (Double Antibody Sandwich-ELISA) was used with a polyclonal antiserum against TSV. Five isolates (T1, T2, T3, T4 and T5), which are collected, respectively from Mohammad Abad (Karaj), Malek Abad (Karaj), Hashtgerd (Karaj), Tarand Balla (Varamin) and Deh mah sin (Pishva) were inoculated on 29 species of Cucurbitaceae, Amaranthaceae, Solanacea, Compositae, Leguminosae and Chenopodiacea. Chenopodium quinoa 6 days after inoculation showed necrotic local lesions. Gomphrena globosa 10 days after inoculation developed chlorotic local lesions. Systemic symptoms were produced in Datura stramonium. Phaseolus vulgaris cv. Red Kidney 5 days after inoculation developed necrotic local lesions. Nicotiana tabacum 7 days after inoculation showed necrotic and chlorotic local lesions. Nicotiana clevelandii 15 days after inoculation developed leaf distortion and vein necrosis. Lactuca sativa 10-15 days after inoculation developed leaf istortion and mosaic. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed using one primer pairs designed by DSMZ. An approximately 710 bp fragment was amplified with a specific primer.

  18. An Improved Culture System for Virus Isolation and Detection

    Institute of Scientific and Technical Information of China (English)

    Yu-chen XIA; Zhi-hong HU; Zhi-juan QIU; Zhong-bin MA; Hua-lin WANG; Fei DENG

    2008-01-01

    Cell culture plays an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology based studies of viruses. In the present work, a new system that could permits multiple (different) cell lines to be simultaneously cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called an isolated co-culture system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus.

  19. Rapid centrifugation assay standarization for dengue virus isolation

    OpenAIRE

    Palomino, Miryam; Escuela de Biología, facultad de Ciencias Naturales y Matemáticas, Universidad Nacional federico Villarreal. Lima, Perú. Biólogo.; Gutierrez, Victoria; Centro Nacional de Salud Pública, Instituto Nacional de Salud. Lima, Perú. Biólogo.; Salas, Ramses; Laboratorio de Biotecnología, facultad de Ciencias Naturales y Matemáticas, Universidad Nacional federico Villarreal. Lima, Perú. Biólogo.

    2010-01-01

    The plate centrifugation assay was standardized for dengue virus isolation from serum samples. C6/36-HT cells were used determining the optimal values for centrifugation spin speed, inoculum, sera dilution, and incubation time. Then, 22 positive serum samples with viral isolation and viral strains of the four reference dengue virus serotypes were tested simultaneously by the standardized plate centrifugation method and the conventional tube culture. The isolations were typified by indirec...

  20. High sequence conservation among cucumber mosaic virus isolates from lily.

    Science.gov (United States)

    Chen, Y K; Derks, A F; Langeveld, S; Goldbach, R; Prins, M

    2001-08-01

    For classification of Cucumber mosaic virus (CMV) isolates from ornamental crops of different geographical areas, these were characterized by comparing the nucleotide sequences of RNAs 4 and the encoded coat proteins. Within the ornamental-infecting CMV viruses both subgroups were represented. CMV isolates of Alstroemeria and crocus were classified as subgroup II isolates, whereas 8 other isolates, from lily, gladiolus, amaranthus, larkspur, and lisianthus, were identified as subgroup I members. In general, nucleotide sequence comparisons correlated well with geographic distribution, with one notable exception: the analyzed nucleotide sequences of 5 lily isolates showed remarkably high homology despite different origins.

  1. Isolation of Japanese encephalitis virus from clinical specimens using a continuous mosquito cell line.

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    Leake, C J; Burke, D S; Nisalak, A; Hoke, C H

    1986-09-01

    During the 1983 Japanese encephalitis (JE) epidemic in northern Thailand, we systematically attempted to isolate JE virus (JEV) from clinical specimens collected from 49 consecutive JE patients at 1 provincial hospital. Fresh acute plasma and cerebrospinal fluid (CSF) samples and postmortem brain samples were immediately inoculated onto cultured monolayers of Aedes pseudoscutellaris (LSTM-AP-61) cells which had been shipped to the epidemic site. None of 49 plasma samples yielded virus. None of 30 fresh CSF samples from nonfatal cases yielded virus, but 5 of 15 (33%) CSF samples from fatal cases did. Inoculation of fresh brain specimens obtained at autopsy yielded virus in every case attempted (7 of 7), whereas postmortem needle biopsy specimens of brain yielded virus in only 1 of 4 cases. Isolates were most frequently successful using thalamic tissue (6 of 7 cases), but isolates were also commonly obtained from frontal cortex (4/7), occipital cortex (4/7), cerebellum (4/7), medulla (4/7) and pons (2/7).

  2. High sequence conservation among cucumber mosaic virus isolates from Lily

    NARCIS (Netherlands)

    Chen, Y.K.; Derks, A.F.L.M.; Langeveld, S.; Goldbach, R.; Prins, M.

    2001-01-01

    For classification of Cucumber mosaic virus (CMV) isolates from ornamental crops of different geographical areas, these were characterized by comparing the nucleotide sequences of RNAs 4 and the encoded coat proteins. Within the ornamental-infecting CMV viruses both subgroups were represented. CMV i

  3. Complete Genome Sequences of Five Zika Virus Isolates.

    Science.gov (United States)

    Ladner, Jason T; Wiley, Michael R; Prieto, Karla; Yasuda, Chadwick Y; Nagle, Elyse; Kasper, Matthew R; Reyes, Daniel; Vasilakis, Nikolaos; Heang, Vireak; Weaver, Scott C; Haddow, Andrew; Tesh, Robert B; Sovann, Ly; Palacios, Gustavo

    2016-05-12

    Zika virus is an emerging human pathogen of great concern due to putative links to microcephaly and Guillain-Barre syndrome. Here, we report the complete genomes, including the 5' and 3' untranslated regions, of five Zika virus isolates, one from the Asian lineage and four from the African lineage.

  4. Characterization of a Zika Virus Isolate from Colombia.

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    Lahon, Anismrita; Arya, Ravi P; Kneubehl, Alexander R; Vogt, Megan B; Dailey Garnes, Natalie J M; Rico-Hesse, Rebecca

    2016-09-01

    Zika virus (Flavivirus genus) is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments. We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015). Here, we describe the patient's clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3' un-translated region), to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains. We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas.

  5. Characterization of a Zika Virus Isolate from Colombia

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    Lahon, Anismrita; Arya, Ravi P.; Kneubehl, Alexander R.; Vogt, Megan B.; Dailey Garnes, Natalie J. M.; Rico-Hesse, Rebecca

    2016-01-01

    Background Zika virus (Flavivirus genus) is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments. Methodology/Clinical findings We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015). Here, we describe the patient’s clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3’ un-translated region), to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains. Conclusions/Significance We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas. PMID:27654889

  6. Isolation and characterization of cidofovir resistant vaccinia viruses

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    Prichard Mark N

    2008-05-01

    Full Text Available Abstract Background The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. One mode of action for CDV has been demonstrated to be the inhibition of the viral DNA polymerase. Results We have isolated several CDV resistant (CDVR vaccinia viruses through a one step process, two of which have unique single mutations within the DNA polymerase. An additional resistant virus isolate provides evidence of a second site mutation within the genome involved in CDV resistance. The CDVR viruses were 3–7 fold more resistant to the drug than the parental viruses. The virulence of the CDVR viruses was tested in mice inoculated intranasally and all were found to be attenuated. Conclusion Resistance to CDV in vaccinia virus can be conferred individually by at least two different mutations within the DNA polymerase gene. Additional genes may be involved. This one step approach for isolating resistant viruses without serial passage and in the presence of low doses of drug minimizes unintended secondary mutations and is applicable to other potential antiviral agents.

  7. Persistent parainfluenza virus shedding during isolation at the South Pole.

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    Muchmore, H G; Parkinson, A J; Humphries, J E; Scott, E N; McIntosh, D A; Scott, L V; Cooney, M K; Miles, J A

    1981-01-15

    Persistent parainfluenza virus shedding in healthy young adults occurred throughout the 8 1/2-month winter isolation period at Amundsen-Scott South Pole Station during 1978. Two episodes of respiratory illness were observed after 10 and 29 weeks of complete social isolation. Throat swabs collected both routinely, and during each outbreak of respiratory illness, were directly inoculated into cell cultures. Parainfluenza virus types 1 and 3 were recovered from both symptomatic and asymptomatic subjects throughout the winter. No other viruses were obtained by these efforts. The presence of parainfluenza virus in these subjects long after the accepted incubation period for viral upper respiratory illness, and when the introduction of new virus to this community was impossible, suggests its persistence in man.

  8. Chikungunya virus isolation using simplified cell culture technique in Mauritius.

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    Pyndiah, M N; Pursem, V; Meetoo, G; Daby, S; Ramuth, V; Bhinkah, P; Chuttoo, R; Paratian, U

    2012-03-01

    During the chikungunya outbreak of 2005 - 2006, the only laboratory facilities available in Mauritius were virus isolation in cell culture tubes and serology. The laboratory was submerged with large numbers of blood samples. Comparative isolation was made in human embryonic lung (HEL) and VERO cells grown in 96-well plate. Culture on HEL cells was found to be more sensitive and presence of cytopathic effect (CPE) was observed earlier than in VERO cells. Out of the 18 300 blood samples inoculated on HEL, 11 165 were positive. This virus isolation method was of great help for the surveillance and control of the vectors. In cases of an outbreak a cheap, rapid and simple method of isolating chikungunya virus is described.

  9. Isolation of influenza viruses in MDCK 33016PF cells and clearance of contaminating respiratory viruses.

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    Roth, Bernhard; Mohr, Hannah; Enders, Martin; Garten, Wolfgang; Gregersen, Jens-Peter

    2012-01-11

    This paper summarizes results obtained by multiplex PCR screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in MDCK 33016PF cells. Using the ResPlexII v2.0 (Qiagen) multiplex PCR, 393 positive results were obtained in 468 clinical samples collected during an influenza season in Germany. The overall distribution of positive results was influenza A 42.0%, influenza B 38.7%, adenovirus 1.5%, bocavirus 0.5%, coronavirus 3.3%, enterovirus 5.6%, metapneumovirus 1.0%, parainfluenza virus 0.8%, rhinovirus 4.1%, and respiratory syncytial virus (RSV) 2.5%. Double infections of influenza virus together with another virus were found for adenovirus B and E, bocavirus, coronavirus, enterovirus and for rhinovirus. These other viruses were rapidly lost upon passages in MDCK 33016PF cells and under conditions as applied to influenza virus passaging. Clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in MDCK 33016PF cells. Using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼10(4)), the positive results for the different viruses turned negative already after 2 or 3 passages in MDCK 33016PF cells. These results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in MDCK cells. In combination with their superior isolation efficiency, MDCK cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses.

  10. Gene detection, virus isolation, and sequence analysis of avian leukosis viruses in Taiwan country chickens.

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    Chang, Shu-Wei; Hsu, Meng-Fang; Wang, Ching-Ho

    2013-06-01

    Avian leukosis virus (ALV) infection in Taiwan Country chickens (TCCs) was investigated by using gene detection, virus isolation, and sequence analysis. The blood samples of 61 TCC flocks at market ages from a slaughter house were screened for exogenous ALVs using polymerase chain reaction to investigate the ALV infection status. The buffy coats from three breeder and four commercial chicken flocks were cocultured with DF-1 cells to isolate the virus. The full proviral DNA genomes of two ALV isolates were sequenced, analyzed, and compared with reference ALV strains. The gene detection results showed that 60 and 43 of the 61 flocks were infected with subgroup A of ALV (ALV-A) and subgroup J of ALV (ALV-J), respectively. Virus isolation results showed that five ALV-As and two ALV-Js were isolated from those seven TCC flocks. The full sequences of the isolates showed that isolate TW-3577 possessed a myeloblastosis-associated virus 1 gp85 coding region and an ALV-J 3'-untranslated region (3'UTR) and was similar to ordinary ALV-A. However, TW-3593 was unique. The 3'UTR of this isolate displayed high identity to endogenous counterpart sequence and its gp85 was different from all subgroups. This unique ALV is common in Taiwan.

  11. Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type 1 isolation from vertically infected pediatric patients.

    Science.gov (United States)

    Paul, M O; Tetali, S; Pahwa, S

    1994-05-01

    In this study, we examined variables related to human immunodeficiency virus (HIV) isolation utilizing samples from 51 HIV-infected (153 plasma and 122 peripheral blood mononuclear cell [PBMC] samples) and 57 uninfected (182 plasma and 163 PBMC samples) infants. Our chief observation was that cryopreservation of donor PBMCs does not significantly alter their sensitivity or specificity for isolation of HIV from patient PBMCs or plasma.

  12. Isolation of sindbis virus from a hooded crow in Germany.

    Science.gov (United States)

    Eiden, Martin; Ziegler, Ute; Keller, Markus; Müller, Kerstin; Granzow, Harald; Jöst, Hanna; Schmidt-Chanasit, Jonas; Groschup, Martin H

    2014-03-01

    Sindbis virus (SINV) is an arbovirus that causes clinical symptoms, including arthritis, rash, and fever during acute human infections. In Europe, SINV outbreaks are largely restricted to northern Europe. Intrigued by the isolation of SINV from mosquitoes in southwestern Germany in 2009, we initiated a passive arbovirus-monitoring program in birds and analyzed a total of 685 samples. By this approach, we were able to detect a SINV in a Hooded Crow in Germany for the first time. It was possible to isolate SINV virus in cell cultures and even to visualize virus particles by electron microscopy. After the determination of the complete SINV genome sequence, the phylogenetic analysis revealed its close relationship to SINV genotype I sequences previously obtained from mosquitoes in Germany and Scandinavia. This first report on the isolation of viable SINV indicates the potential involvement of crows in an enzootic circulation of SINV in Germany and Central Europe.

  13. Isolation of biologically-active exosomes from human plasma.

    Science.gov (United States)

    Muller, Laurent; Hong, Chang-Sook; Stolz, Donna B; Watkins, Simon C; Whiteside, Theresa L

    2014-09-01

    Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies.

  14. Full Genomic Characterization of a Saffold Virus Isolated in Peru

    Directory of Open Access Journals (Sweden)

    Mariana Leguia

    2015-11-01

    Full Text Available While studying respiratory infections of unknown etiology we detected Saffold virus in an oropharyngeal swab collected from a two-year-old female suffering from diarrhea and respiratory illness. The full viral genome recovered by deep sequencing showed 98% identity to a previously described Saffold strain isolated in Japan. Phylogenetic analysis confirmed the Peruvian Saffold strain belongs to genotype 3 and is most closely related to strains that have circulated in Asia. This is the first documented case report of Saffold virus in Peru and the only complete genomic characterization of a Saffold-3 isolate from the Americas.

  15. Full Genomic Characterization of a Saffold Virus Isolated in Peru.

    Science.gov (United States)

    Leguia, Mariana; Loyola, Steev; Rios, Jane; Juarez, Diana; Guevara, Carolina; Silva, Maria; Prieto, Karla; Wiley, Michael; Kasper, Matthew R; Palacios, Gustavo; Bausch, Daniel G

    2015-11-20

    While studying respiratory infections of unknown etiology we detected Saffold virus in an oropharyngeal swab collected from a two-year-old female suffering from diarrhea and respiratory illness. The full viral genome recovered by deep sequencing showed 98% identity to a previously described Saffold strain isolated in Japan. Phylogenetic analysis confirmed the Peruvian Saffold strain belongs to genotype 3 and is most closely related to strains that have circulated in Asia. This is the first documented case report of Saffold virus in Peru and the only complete genomic characterization of a Saffold-3 isolate from the Americas.

  16. [Indications for fresh frozen plasma: evaluation of virus inactivating preparations].

    Science.gov (United States)

    Pindur, G; Kiesewetter, H; Seyfert, U T; Wenzel, E

    1993-01-01

    When no specific factor concentrate is available fresh-frozen plasma (FFP) is indicated in the treatment of clinically relevant hemorrhagic diathesis. These disorders include congenital factor V and XI deficiencies, multiple factor defects, as disseminated intravascular coagulation and severe liver disease, and patients receiving massive transfusions, when bleeding occurs and severe abnormalities on coagulation testing are evident. FFP is beneficial when used with plasma exchange in thrombotic thrombocytopenic purpura and related disorders. Various virucidal treatments including solvent-detergent (SD), photoactivated dyes (methylene blue) or pasteurization have been evolved to improve virus safety of human plasma. More extensive studies to demonstrate efficient virus inactivation in plasma have been performed with SD compared to other methods. On the other hand, the use of single-donor FFP in methylene blue treatment is possibly superior to pooled plasma which is processed according to the SD procedure. Pasteurization enables the inactivation not only of lipid-enveloped but also of non-lipid-enveloped viruses. Virucidal treatment of plasma may cause alterations in clotting factors, fibrinolysis and protease inhibitors; however, the currently achieved recovery of procoagulant activities is approximately comparable with that found in untreated FFP. The toxicity of virucidal additives is reported to be negligible since manufacturing includes a removal procedure (SD) or comparably low amounts (methylene blue) are used in inactivation treatment.

  17. Cold oxygen plasma technology efficiency against different airborne respiratory viruses.

    Science.gov (United States)

    Terrier, O; Essere, B; Yver, M; Barthélémy, M; Bouscambert-Duchamp, M; Kurtz, P; VanMechelen, D; Morfin, F; Billaud, G; Ferraris, O; Lina, B; Rosa-Calatrava, M; Moules, V

    2009-06-01

    Respiratory infections caused by viruses are major causes of upper and lower respiratory tract infections. They account for an important mortality and morbidity worldwide. Amongst these viruses, influenza viruses and paramyxoviruses are major pathogens. Their transmission is mainly airborne, by direct transmission through droplets from infected cases. In the context of an influenza pandemic, as well as for the reduction of nosocomial infections, systems that can reduce or control virus transmission will reduce the burden of this disease. It may also be part of the strategy for pandemic mitigation. A new system based on physical decontamination of surface and air has been developed. This process generates cold oxygen plasma (COP) by subjecting air to high-energy deep-UV light. To test its efficiency, we have developed an experimental device to assess for the decontamination of nebulized respiratory viruses. High titer suspensions of influenza virus type A, human parainfluenza virus type 3 and RSV have been tested. Different experimental conditions have been evaluated against these viruses. The use of COP with an internal device allowed the best results against all viruses tested. We recorded a reduction of 6.5, 3.8 and 4 log(10) TCID50/mL of the titre of the hPIV-3, RSV and influenza virus A (H5N2) suspensions. The COP technology is an efficient and innovative strategy to control airborne virus dissemination. It could successfully control nosocomial diffusion of respiratory viruses in hospital setting, and could be useful for the reduction of influenza transmission in the various consultation settings implemented for the management of cases during a pandemic.

  18. Differences among isolates of simian hemorrhagic fever (SHF) virus.

    Science.gov (United States)

    Gravell, M; London, W T; Leon, M E; Palmer, A E; Hamilton, R S

    1986-01-01

    Simian hemorrhagic fever (SHF) virus is a member of the Togaviridae family which currently is unclassified to genus. We have studied the relatedness of four different SHF virus isolates obtained from infected macaque or patas monkeys. Differences were found among isolates in type and severity of disease produced in patas monkeys, cell sensitivity to infection, viral antigens, and levels of specific antibody induced in patas monkeys. Based on these criteria, the four isolates have been grouped in two categories: those producing acute infections in patas monkeys (LVR, P-180) and those producing persistent infections (P-248, P-741). The P-180 isolate induced the most severe disease in experimentally infected patas monkeys, but only occasionally were their infections fatal. Persistently infected patas monkeys were viremic over a period of years, but showed no signs or symptoms of infection. All four isolates were found to be antigenically related by use of enzyme-linked immunosorbent assay (ELISA); the P-248 isolate showing the weakest antigenic relationship. However, none of the four isolates induced cross-neutralizing antibodies in infected patas monkeys. High titers of specific IgG antibody (up to 31,250 as determined by ELISA) were induced in acutely infected patas monkeys (LVR, P-180), but antibody was barely detectable (less than or equal to 50) in persistently infected patas monkeys (P-248, P-741). LVR lytically infected USU-104 cells, patas monkey peritoneal macrophages (PMAC), and rhesus monkey PMAC. The P-180 isolate lytically infected both patas monkey PMAC and rhesus monkey PMAC, but not USU-104 cells. The isolates producing persistent infections (P-248, P-741) lytically infected only rhesus monkey PMAC. These results show that marked differences exist among isolates of SHF virus from naturally infected animals. These differences should be useful in categorizing new isolates.

  19. Aphid Transmission of the Ontario Isolate of Plum Pox Virus.

    Science.gov (United States)

    Lowery, D Thomas; Vickers, Patricia M; Bittner, Lori A; Stobbs, Lorne W; Foottit, Robert G

    2015-10-01

    Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18-28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs.

  20. Isolation of Newcastle disease virus from birds of prey.

    Science.gov (United States)

    Chu, H P; Trow, E W; Greenwood, A G; Jennings, A R; Keymer, I F

    1976-01-01

    In the 4 year period 1971-74 11 isolations of Newcastle Disease Virus (NDV) were made from 44 birds of prey that died in captivity. Three species of Falconiformes were involved, including one red-headed falcon (Falco chicquera), 5 European kestrels (F. tinnunculus), and 2 secretary birds (Sagittarius serpentarius), also 2 species of Strigiformes, comprising 2 barn owls (Tyto alba) and one little owl (Athene noctua). All NDV isolates were of the velogenic type.

  1. The lipidomes of vesicular stomatitis virus, semliki forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry

    DEFF Research Database (Denmark)

    Kalvodova, Lucie; Sampaio, Julio L; Cordo, Sandra;

    2009-01-01

    kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid...

  2. [Therapeutic use of fresh plasma and "virus-safe" plasma].

    Science.gov (United States)

    Seyfert, U T; Lorenz, C; Espig, J; Heinrich, H; Grund, S; Rupp, K H; Wenzel, E

    1991-01-01

    The use of plasma and factor concentrates for the treatment of coagulation disorders is well established, but many questions remain. The indications for fresh frozen plasma are still not clearly established and excessive use is rampant. The safety of all blood products has not yet been firmly established. Peer review of transfusion practice in a hospital can be achieved by a Hospital Transfusion Committee. The review and analysis of the statistical reports of the transfusion service and strategies for enhancing quality of patient care will be presented.

  3. Complete Genome Sequence of Zika Virus Isolated from Semen

    Science.gov (United States)

    Graham, Victoria; Lewandowski, Kuiama; Dowall, Stuart D.; Pullan, Steven T.; Hewson, Roger

    2016-01-01

    Zika virus (ZIKV) is an emerging pathogenic flavivirus currently circulating in numerous countries in South America, the Caribbean, and the Western Pacific Region. Using an unbiased metagenomic sequencing approach, we report here the first complete genome sequence of ZIKV isolated from a clinical semen sample. PMID:27738033

  4. Increased virulence of Marek's disease virus field isolates.

    Science.gov (United States)

    Witter, R L

    1997-01-01

    The continuation of an apparent evolutionary trend of Marek's disease virus (MDV) towards greater virulence may explain recent increased losses from Marek's disease (MD) in vaccinated flocks. To address this question, the virulence of 31 isolates of serotype 1 MDV obtained from layer or broiler flocks between 1987 and 1995 were characterized. Each isolate was cultured in duck embryo fibroblasts for four to six passages, and ascertained to be free from contamination with avian retroviruses, chicken anemia virus, and MDVs of other serotypes. The viruses, along with prototype viruses JM/102W and Md5, were tested for virulence by inoculation at 6 days of age into laboratory strain 15I5 x 7(1) chickens of three types: nonvaccinated, vaccinated with turkey herpesvirus (HVT) and bivalent (HVT + SB-1)-vaccinated. The results showed that three isolates did not differ from JM/102W and were classified in the virulent (vMDV) pathotype. Twenty-one isolates produced significantly higher levels of MD in HVT-vaccinated chickens than did the JM/102W control and were classified in the very virulent (vvMDV) pathotype. Seven isolates, five of which were isolated in 1994 or 1995, produced significantly higher levels of MD in bivalent-vaccinated chickens than did the Md5 (vvMDV) control. These isolates, provisionally designated as the vv+MDV pathotype, appeared to be at the high end of a virulence continuum. Several MD response parameters, including lymphoma mortality, early mortality with bursal/thymic atrophy, and frequency of visceral lymphomas or ocular lesions in nonvaccinated chickens were positively correlated with virulence. These findings support the continued evolution of MDV towards greater virulence.

  5. Isolation and characterization of an H9N2 influenza virus isolated in Argentina

    Science.gov (United States)

    Xu, Kemin; Ferreri, Lucas; Rimondi, Agustina; Olivera, Valeria; Romano, Marcelo; Ferreyra, Hebe; Rago, Virgina; Uhart, Marcela; Chen, Hongjun; Sutton, Troy; Pereda, Ariel; Perez, Daniel R.

    2016-01-01

    As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) – herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale. PMID:22709552

  6. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  7. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  8. An evolutionary insight into Newcastle disease viruses isolated in Antarctica.

    Science.gov (United States)

    Soñora, Martin; Moreno, Pilar; Echeverría, Natalia; Fischer, Sabrina; Comas, Victoria; Fajardo, Alvaro; Cristina, Juan

    2015-08-01

    The disease caused by Newcastle disease virus (NDV) is a severe threat to the poultry industry worldwide. Recently, NDV has been isolated in the Antarctic region. Detailed studies on the mode of evolution of NDV strains isolated worldwide are relevant for our understanding of the evolutionary history of NDV. For this reason, we have performed Bayesian coalescent analysis of NDV strains isolated in Antarctica to study evolutionary rates, population dynamics, and patterns of evolution. Analysis of F protein cleavage-site sequences of NDV isolates from Antarctica suggested that these strains are lentogenic. Strains isolated in Antarctica and genotype I reference strain Ulster/67 diverged from ancestors that existed around 1958. The time of the most recent common ancestor (MRCA) was established to be around 1883 for all class II viruses. A mean rate of evolution of 1.78 × 10(-3) substitutions per site per year (s/s/y) was obtained for the F gene sequences of NDV strains examined in this study. A Bayesian skyline plot indicated a decline in NDV population size in the last 25 years. The results are discussed in terms of the possible role of Antarctica in emerging or re-emerging viruses and the evolution of NDV populations worldwide.

  9. Mayaro virus fever in French Guiana: isolation, identification, and seroprevalence.

    Science.gov (United States)

    Talarmin, A; Chandler, L J; Kazanji, M; de Thoisy, B; Debon, P; Lelarge, J; Labeau, B; Bourreau, E; Vié, J C; Shope, R E; Sarthou, J L

    1998-09-01

    This paper reports the first isolation of Mayaro (MAY) virus from a patient infected in French Guiana. The identification was initially performed using immunofluorescent antibody testing with specific mouse antibody, and confirmed by plaque-reduction neutralization testing and reverse transcription-polymerase chain reaction. To determine if MAY virus infection is widespread in French Guiana, a serosurvey was performed to determine the prevalence of antibody to this virus in various ethnic groups and areas of French Guiana. Human sera (n = 1,962) were screened using the hemagglutination inhibition (HI) test. To determine whether MAY virus circulates in the rain forest, a serosurvey in monkey populations was performed. Monkey sera (n = 150) were also screened for antibody to MAY virus using HI testing. Of the human sera tested, 6.3% were positive for anti-MAY virus antibodies. Significant differences in MAY virus seroprevalence between different age groups were observed. Seroprevalence rates increased with age, with a large increase in people 10-19 years of age in comparison with those less than 10 years of age. After adjustment for age, significant differences were also found between places of residence. The prevalence of anti-MAY virus antibody was higher in people living in contact with the forest, especially in the Haut Oyapock area (odds ratio [OR] = 97.7, 95% confidence interval [CI] = 48.2-197.9) and along the Maroni River (OR = 39.7, 95% CI = 20.6-76.6). The ethnic differences observed in this study were probably due to differences in residence. Among monkeys, higher seroprevalence rates were found in Alouatta seniculus (66.0%) than in Saguinus midas (18.2%). Among Alouatta, the seroprevalence increased significantly with weight (and therefore with age). This study indicates that MAY virus is present in French Guiana, and human infections occur in areas where people live near the tropical rain forest.

  10. Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals.

    Science.gov (United States)

    Konadu, Kateena Addae; Huang, Ming Bo; Roth, William; Armstrong, Wendy; Powell, Michael; Villinger, Francois; Bond, Vincent

    2016-01-05

    Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

  11. Molecular characterization of Duck Plague virus isolated from Bangladesh

    Directory of Open Access Journals (Sweden)

    Md. Mostakin Ahamed

    2015-09-01

    Full Text Available Duck plague (DP is the most feared duck disease in the world. For isolation, identification, molecular detection and characterization of DP virus (DPV, a total of 94 samples were collected from commercial farms (n=6 and households (n=13 from Rajshahi (n=37, Netrokona (n=35 and Mymensingh (n=22 districts of Bangladesh. The samples were processed and inoculated into 11-13 days old embryonated duck eggs for virus propagation. Virus was identified using agar gel immunodiffusion test (AGIT and passive hemagglutination (PHA test, and was confirmed by polymerase chain reaction (PCR targeting DNA polymerase and gC genes, followed by sequencing. Pathogenicity tests were performed using duck embryos, ducklings and ducks. Among the 94 samples, 17 isolates were confirmed as DPV by PCR amplification of partial DNA polymerase (446-bp and gC genes (78-bp, respectively. One of the isolates (Anatid herpes 1 BAU DMH was sequenced and found to be closely related with a Chinese variant of DPV (GenBank: JQ647509.1. Thus, we assume that both Bangladeshi and Chinese isolates of DPV may have a common ancestor. [J Adv Vet Anim Res 2015; 2(3.000: 296-303

  12. Guiding-center models for edge plasmas and numerical simulations of isolated plasma filaments

    DEFF Research Database (Denmark)

    Madsen, Jens

    The work presented in this thesis falls into two categories: development of reduced dynamical models applicable to edge turbulence in magnetically confined fusion plasmas and numerical simulations of isolated plasma filaments in the scrape-off layer region investigating the influence of finite...... models are presented that overcome some of the difficulties associated with the development of reduced dynamical models applicable to the edge. Second order guiding-center coordinates are derived using the phasespace Lie transform method. Using a variational principle the corresponding Vlasov......-Maxwell equations in a more tractable form, which could be relevant for direct numerical simulations of edge plasma turbulence. Finally, an investigation of the influence of finite Larmor radius effects on the radial transport of isolated plasma filaments (blobs) in the scrape-off region of fusion plasmas...

  13. Default in plasma and intestinal IgA responses during acute infection by simian immunodeficiency virus

    Directory of Open Access Journals (Sweden)

    Chaoul Nada

    2012-05-01

    Full Text Available Abstract Background Conflicting results regarding changes in mucosal IgA production or in the proportions of IgA plasma cells in the small and large intestines during HIV-infection have been previously reported. Except in individuals repeatedly exposed to HIV-1 but yet remaining uninfected, HIV-specific IgAs are frequently absent in mucosal secretions from HIV-infected patients. However, little is known about the organization and functionality of mucosal B-cell follicles in acute HIV/SIV infection during which a T-dependent IgA response should have been initiated. In the present study, we evaluated changes in B-cell and T-cell subsets as well as the extent of apoptosis and class-specific plasma cells in Peyer’s Patches, isolated lymphoid follicles, and lamina propria. Plasma levels of IgA, BAFF and APRIL were also determined. Results Plasma IgA level was reduced by 46% by 28 days post infection (dpi, and no IgA plasma cells were found within germinal centers of Peyer’s Patches and isolated lymphoid follicles. This lack of a T-dependent IgA response occurs although germinal centers remained functional with no sign of follicular damage, while a prolonged survival of follicular CD4+ T-cells and normal generation of IgG plasma cells is observed. Whereas the average plasma BAFF level was increased by 4.5-fold and total plasma cells were 1.7 to 1.9-fold more numerous in the lamina propria, the relative proportion of IgA plasma cells in this effector site was reduced by 19% (duodemun to 35% (ileum at 28 dpi. Conclusion Our data provide evidence that SIV is unable to initiate a T-dependent IgA response during the acute phase of infection and favors the production of IgG (ileum or IgM (duodenum plasma cells at the expense of IgA plasma cells. Therefore, an early and generalized default in IgA production takes place during the acute of phase of HIV/SIV infection, which might impair not only the virus-specific antibody response but also IgA responses

  14. Genetic diversity of Hungarian Maize dwarf mosaic virus isolates.

    Science.gov (United States)

    Gell, Gyöngyvér; Balázs, Ervin; Petrik, Kathrin

    2010-04-01

    The genetic diversity of the coat-protein (CP) region and the untranslated C-terminal region (3'UTR) of Maize dwarf mosaic virus (MDMV) was analyzed to evaluate the variability between isolates (inter-isolate sequence diversity). The results of inter-isolate sequence diversity analysis showed that the diversity of the MDMV CP gene is fairly high (p-distance: up to 0.136). During sequence analysis, a 13 amino-acid residue insertion and an 8 amino-acid residue deletion were found within the N-terminal region of the CP gene. The phylogenetic analysis showed that-unlike other potyvirus species in this subgroup-the MDMV isolates could not be distinguished on the basis of their host plants or geographic origins.

  15. Full Genomic Characterization of a Saffold Virus Isolated in Peru

    OpenAIRE

    Mariana Leguia; Steev Loyola; Jane Rios; Diana Juarez; Carolina Guevara; Maria Silva; Karla Prieto; Michael Wiley; Kasper, Matthew R; Gustavo Palacios; Daniel G Bausch

    2015-01-01

    While studying respiratory infections of unknown etiology we detected Saffold virus in an oropharyngeal swab collected from a two-year-old female suffering from diarrhea and respiratory illness. The full viral genome recovered by deep sequencing showed 98% identity to a previously described Saffold strain isolated in Japan. Phylogenetic analysis confirmed the Peruvian Saffold strain belongs to genotype 3 and is most closely related to strains that have circulated in Asia. This is the first do...

  16. Isolation of an influenza C virus introduced into Japan by a traveler from Malaysia.

    Science.gov (United States)

    Matsuzaki, Yoko; Sato, Katsuhiko; Sugawara, Kanetsu; Takashita, Emi; Muraki, Yasushi; Morishita, Takayuki; Kumagai, Norimichi; Suzuki, Sousuke; Hongo, Seiji

    2005-02-01

    An influenza C virus was isolated from a Japanese traveler who had visited Malaysia in April 1999. Phylogenetic analysis indicated that the genome composition of this virus was distinct from that of any other strain isolated in Japan. The possibility that a genetically unique influenza C virus was introduced into Japan by a traveler is shown.

  17. Interferon susceptibility of herpes simplex virus strains isolated from patients enrolled in clinical trials.

    OpenAIRE

    Armstrong, J. A.; Skicki-Mullen, M B; Breinig, M K; Ho, M

    1983-01-01

    Herpes simplex virus type 1 strains isolated from patients who had received interferon in a clinical trial were not more resistant to human leukocyte interferon than strains derived from recipients of a placebo. The susceptibility of herpes simplex virus type 2 strains isolated from herpes genitalis was slightly less than that of herpes simplex virus type 1 strains causing herpes genitalis.

  18. Identification of a strain of maize dwarf mosaic virus, related to sugarcane mosaic virus isolated from maize in Burundi

    Directory of Open Access Journals (Sweden)

    Verhoyen, M.

    1983-01-01

    Full Text Available A strain of maize dwarf mosaic virus related to sugarcane mosaic virus has been isolated from maize in Burundi. The properties (including electron microscopy and serology of the virus are described, and elements for a control strategy are reviewed.

  19. Antigenic and genetic characterization of rabies virus isolates from Uruguay.

    Science.gov (United States)

    Guarino, Helena; Castilho, Juliana Galera; Souto, Juanita; Oliveira, Rafael de Novaes; Carrieri, Maria Luiza; Kotait, Ivanete

    2013-05-01

    After 25 years without any reported cases of rabies in Uruguay, the northern region of the country experienced an epizootic of bovine paralytic rabies in October 2007. The outbreak affected bovines and equines, and the main source of infection was the bat Desmodus rotundus, the only hematophagous species in the country. From October 2007 to July 2008, 42 bovine, 3 equine and 120 chiropteran samples were submitted to the National Veterinary Diagnostic Laboratory for rabies testing. A total of 12 samples (7 bovine, 2 equine and 3 from D. rotundus) were positive by the fluorescent antibody test, and viruses were isolated by the mouse inoculation test. The objective of this study was to compare the antigenic and genetic characteristics of these isolates and three isolates from insectivorous bats from other regions. Antigenic typing using a panel of eight monoclonal antibodies identified all 12 viruses as variant 3 (AgV3), a variant associated with D. rotundus. Two isolates from insectivorous bats (Tadarida brasiliensis and Molossus sp.) were characterized as antigenic variant 4 (AgV4) while the third, from Myotis sp., could not be characterized using this panel as its reactivity pattern did not match that of any of the known antigenic variants. Partial N-gene sequences (nt 149-1420) of these isolates were aligned with homologous sequences derived from GenBank by the CLUSTAL/W method and used to build a neighbor-joining distance tree with the Kimura 2-parameter model. All 12 isolates were genetically grouped into the D. rotundus cluster as they shared 100% identity. In the phylogenetic analysis, the three isolates from insectivorous bats segregated into three clusters: one related to T. brasiliensis, one to Myotis sp. and the other to Lasiurus sp., although the isolate associated with the latter came from a Molossus sp. specimen. These results indicate that AgV3 was associated with the outbreak of bovine paralytic rabies in Uruguay. This is the first report of rabies

  20. Genome sequencing and characterization analysis of a Beijing isolate of chicken corona virus infectious bronchitis virus

    Institute of Scientific and Technical Information of China (English)

    JIN Weiwu; YU Jialin; LI Ning; GONG Yuanshi; SUN Qixin; CHEN Zhangliang; CHEN Chen; ZHANG Ying; ZHAO Yiqiang; FENG Jidong; CHEN Fuyong; WU Qingming; YANG Hanchun; WANG Ming

    2004-01-01

    Avian infectious bronchitis virus (AIBV) is lassified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 28 kilo-bases,which has a 5′ cap structure and 3′ polyadenylation tract.The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames: 5′-orfla-orflab-s-3a-3b-e-m- 6a-6b-n-3′. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2%similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N protein, also showed that AIBV Beijing isolate is lone virus in group Ⅲ and is distant from SARS coronavirus. In conclusion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.

  1. Genomic characterization of Zika virus isolated from Indonesia.

    Science.gov (United States)

    Yudhaputri, Frilasita A; Trimarsanto, Hidayat; Perkasa, Aditya; Yohan, Benediktus; Haryanto, Sotianingsih; Wiyatno, Ageng; Soebandrio, Amin; Myint, Khin Saw; Ledermann, Jeremy P; Rosenberg, Ronald; Powers, Ann M; Sasmono, R Tedjo

    2017-10-01

    Zika virus (ZIKV) JMB-185 strain was isolated from a febrile patient in Jambi, Indonesia in 2014. To understand its genetic characteristics, we performed whole genome sequencing using the Ion Torrent PGM platform on the supernatant of the first passage. The phylogenetic analysis showed that the isolate was not closely related to the Brazilian ZIKV associated with microcephaly or isolates from the recent Singapore Zika outbreak. Molecular evolution analysis indicated that JMB-185 strain may have been circulating in the Southeast Asia region, including Indonesia since 2000. We observed high nucleotide sequence identity between Indonesia, Thailand, Singapore, and American strains although unique amino acid substitutions were also observed. This report provides information on the genomic characteristics of Indonesian ZIKV which may be used for further studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Report of isolations of unusual lyssaviruses (rabies and Mokola virus identified retrospectively from Zimbabwe : short communication

    Directory of Open Access Journals (Sweden)

    J. Bingham

    2001-07-01

    Full Text Available Rabies isolates that had been stored between 1983 and 1997 were examined with a panel of anti-lyssavirus nucleocapsid monoclonal antibodies. Out of 56 isolates from cats and various wild carnivore species, 1 isolate of Mokola virus and 5 other non-typical rabies viruses were identified. The Mokola virus isolate was diagnosed as rabies in 1993 from a cat. Genetic analysis of this isolate suggests that it falls in a distinct subgroup of the Mokola virus genotype. The 5 non-typical rabies viruses were isolated from honey badgers (Mellivora capensis, African civets (Civettictis civetta and an unidentified mongoose (Herpestidae. These isolates are representatives of rarely-reported wildlife-associated strains of rabies, probably maintained by the slender mongoose (Galerella sanguinea. These findings indicate that both Mokola virus and the mongoose-associated variant may be more common in Zimbabwe than is apparent from routine surveillance.

  3. Characterization of RD-114 Virus Isolated from a Commercial Canine Vaccine Manufactured Using CRFK Cells ▿

    Science.gov (United States)

    Yoshikawa, Rokusuke; Sato, Eiji; Igarashi, Tatsuhiko; Miyazawa, Takayuki

    2010-01-01

    Recently, we found that several commercial pet vaccines were contaminated with an infectious endogenous retrovirus, RD-114-related virus. Here, we determined the entire nucleotide sequences of RD-114-related viruses isolated from CRFK cells and a vaccine manufactured using CRFK cells. These RD-114-related viruses were nearly identical to the authentic RD-114 virus. PMID:20631117

  4. Cedar virus: a novel Henipavirus isolated from Australian bats.

    Directory of Open Access Journals (Sweden)

    Glenn A Marsh

    Full Text Available The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV and Nipah virus (NiV for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV, which shares significant features with the known henipaviruses. The genome size (18,162 nt and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin-B2 for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.

  5. Molecular analysis of Korean isolate of barley yellow mosaic virus.

    Science.gov (United States)

    Lee, Kui Jae; Choi, Min Kyung; Lee, Wang Hyu; Rajkumar, Mani

    2006-04-01

    The complete sequences of both RNAs of an isolate of barley yellow mosaic virus (BaYMV) from Haenam, Korea, were determined. RNA1 is 7639 nucleotides long [excluding the 3'-poly(A)], and codes for a 270 kDa polyprotein of 2411 amino acids which contains the capsid protein (CP) at the C terminus and seven putative non-structural proteins. RNA2 is 3582 nucleotides long and codes for a polyprotein of 890 amino acids, which contains a 28 kDa putative proteinase (P1) and a 73 kDa polypeptide (P2). The whole sequences of Korean isolate (BaYMV-K) closely resembled those of an isolate from Japan (BaYMV-J) (99.6 identical nucleotides for RNA1; 99.4 for RNA2) and china (BaYMV-C) (96.7 and 96.2%, respectively) than from Germany (BaYMV-G) (93.6 and 90.4%, respectively). The greatest differences between the BaYMV-K and BaYMV-J isolates were in the 3'-NCRs of RNA1 and 5' NCRs of RNA2 and there were also some other regions of difference in Nib Pro (RNA1) and P1 (RNA2). Further, the phylogenetic analysis of CP region showed that Asian and European isolates formed distinct clusters. However, molecular variations between isolates could not be linked to earlier results showing differences in cultivar response.

  6. Isolasi, Identifikasi, Sifat Fisik, dan Biologi Virus Tetelo yang Diisolasi dari Kasus di Lapangan (ISOLATION, IDENTIFICATION, PHISICAL, AND BIOLOGICAL CHARACTER OF NEWCASTLE DISEASE VIRUS ISOLATED FROM FIELD CASES

    Directory of Open Access Journals (Sweden)

    Michael Haryadi Wibowo

    2013-07-01

    Full Text Available native chicken farm suspected to Newcastle disease (ND virus infection. Specimens were taken andcollected from the lung was further processed. Suspected materials were inoculated into allantoic sacc inspecific pathogenic free of 10 days embryonating egg chicken. The growth of the virus was determined withthe ability to agglutinate the chicken red blood cells or hemaglutination test. Positive hemaglutinationwas performed with hemaglutinatin inhibition test using specific antibody against ND virus. Method forND virus isolation, propagation and identification were based on the standard procedure of serologicalidentification for ND virus serological identification. 13 out of 34 samples were identified as ND viruses.Observation on the course and time of the virus to kill the chicken embryo could be differentiated intomoderate virus patho-type were 10 isolates and a virulent strains were 3 isolates. Further characterizationbased on the elution time observation indicated 11 isolates were not pathogenic strain and 2 isolates werenot virulent strain. Hemagglutinin stability study revealed that 11 isolates were sensitive being heated at560C for 30 minutes while 2 isolates were resistant. Biological characteristic of ND virus to hemagglutinateon various mammalian red blood cells indicating that most isolates were HA negative. Two isolates wereHA positive with cattle, horse and sheep red blood cell, and one isolate indicated positive HA test by usingsheep red blood cell. Control virus was lentogenic patho-type of La Sota strain showed HA and HI testpositive, elution time was 29 minutes, stability on the hemagglutinin after heating was 2 minutes and HApositive with cattle, horse and sheep red blood cell.

  7. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    Science.gov (United States)

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  8. Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses

    Directory of Open Access Journals (Sweden)

    Clavel François

    2008-02-01

    Full Text Available Abstract Background Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1. Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. Methods To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70, and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799, soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5 was also evaluated for a subset of the recombinant viruses (n = 20. Results Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. Conclusion These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in

  9. Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing.

    Science.gov (United States)

    Donis, Ruben O; Davis, C Todd; Foust, Angie; Hossain, M Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, Odewijk; Neumeier, Elisabeth; Ziegler, Thedi

    2014-11-12

    Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine

  10. Dengue-1 Virus Isolation during First Dengue Fever Outbreak on Easter Island, Chile

    OpenAIRE

    Perret, Cecilia; Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-01-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription–polymerase chain reaction as being dengue type 1.

  11. Dengue-1 virus isolation during first dengue fever outbreak on Easter Island, Chile.

    Science.gov (United States)

    Perret, Cecilia; Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-11-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription-polymerase chain reaction as being dengue type 1.

  12. PIXE analysis of human spermatozoa isolated from seminal plasma

    Science.gov (United States)

    Maeda, K.; Sasa, Y.; Kusuyama, H.; Yoshida, K.; Uda, M.

    1990-04-01

    PIXE has been applied to the multielemental and microanalysis of human spermatozoa. This is the first attempt to determine the chemical compositions of the motile spermatozoa free from contaminations of seminal plasma without loss of component elements during washing. The spermatozoa were isolated from semen by letting them swim into a kind of physiological saline, Tyrode's solution. Relative concentrations of P, K, Ca, Ti, Fe, Zn and Br in motile spermatozoa were determined by the use of the chlorine K X-ray peak intensity for evaluating the amount of Tyrode's solution contained in the sample targets. The concentrations of calcium and iron in spermatozoa were considerably higher than in seminal plasma. The concentrations of P, K, Zn and Br in spermatozoa were not so different from those in seminal plasma.

  13. Isolation, Genetic Characterization, and Seroprevalence of Adana Virus, a Novel Phlebovirus Belonging to the Salehabad Virus Complex, in Turkey

    Science.gov (United States)

    Alkan, Cigdem; Alwassouf, Sulaf; Piorkowski, Géraldine; Bichaud, Laurence; Tezcan, Seda; Dincer, Ender; Ergunay, Koray; Ozbel, Yusuf; Alten, Bulent; de Lamballerie, Xavier

    2015-01-01

    ABSTRACT A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. (Diptera; Psychodidae) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies. IMPORTANCE Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or

  14. Hepatitis B virus infection in isolated Afro-Brazilian communities.

    Science.gov (United States)

    Motta-Castro, Ana R C; Martins, Regina M B; Yoshida, Clara F T; Teles, Sheila A; Paniago, Anamaria M; Lima, Kátia M B; Gomes, Selma A

    2005-10-01

    The prevalence and genotypes of hepatitis B virus (HBV) have distinct geographical distribution. In Brazil, some African-descendants have been maintained as small isolated communities since the slavery period. In this study, HBV infection among these communities of African origin was examined. Individuals (1,058) living in 12 communities were interviewed and serum samples screened for the presence of HBV markers. HBsAg-positive sera were tested for HBV DNA by PCR and positive samples were genotyped by restriction fragment length polymorphism (RFLP). The overall prevalence of HBV infection was 19.8% (95% CI: 17.5-22.3), ranging from 5.5% to 42.4%, depending on the communities studied. Multivariate analysis of risk factors showed that increasing age, family history of hepatitis, and sexual activity were associated significantly with this infection. HBsAg was detected in 23/1,058 (2.2%) individuals. HBV DNA was present in 2/2 of HBeAg-positive serum samples and in 18/21 (85.7%) anti-HBe-positive samples. All HBV isolates belonged to genotype A, subtype Aa. Three RFLP patterns were identified: AI (17 isolates), AIV (1 isolate), and AVI (2 isolates). These findings suggest a common introduction of HBV during the slave trade from Africa to Brazil.

  15. First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis

    Directory of Open Access Journals (Sweden)

    Mondher Boughalmi

    2013-11-01

    Full Text Available Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3 and France (2. The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection.

  16. Use of convalescent plasma in Ebola virus infection.

    Science.gov (United States)

    Garraud, Olivier

    2017-02-01

    The recent Ebola virus epidemics which threatened three West African countries (Dec.2014-Apr.2016) has urged global collaborative health organizations and countries to set up measures to stop the infection and to treat patients, near half of them being at risk of death. Convalescent plasma-recovered from rescued West Africans-was considered a feasible therapeutic option. Efficacy was difficult to evaluate because of numerous unknowns (especially evolution of neutralizing antibodies), prior to the cessation of active transmission. This raises a large body of questions spanning epidemiological, virological, immunological but also ethical, sociological and anthropological aspects, alongside with public health concerns, in order to be better prepared to the next outbreak. This essay summarizes efforts made by a large number of groups worldwide, and attempts to address still unanswered questions on the benefit of specific versus non-specific plasma on altered-leaking-vascular endothelia in Ebola infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Characterization of viruses infecting potato plants from a single location in Shetland, an isolated scottish archipelago

    DEFF Research Database (Denmark)

    Mortensen, R.J.; Shen, Xinyi; Reid, Alex

    2010-01-01

    Sequence data were obtained from 29 isolates of Potato virus A (PVA), Potato virus S (PVS), Potato virus V (PVV) and Potato virus X (PVX) infecting nine tubers from Shetland, one of the most remote inhabited islands in the United Kingdom. These isolates were sequenced in the coat protein region...... production. Phylogenetic homogeneity of the Shetland isolates of PVS and PVV was apparent. PVX was more heterogeneous, and Shetland isolates cluster with the Scottish isolates in a group which includes Asian and European isolates. For PVA, the majority of the Shetland and Scottish mainland isolates formed...... a predominantly Scottish grouping, with the remaining Shetland and Scottish mainland isolates clustering with a previously characterized Scottish isolate. There were three main groups of PVA, of which the Scottish grouping was the only one which did not have a fully characterized representative. To extend...

  18. First isolation of West Nile virus from a dromedary camel.

    Science.gov (United States)

    Joseph, Sunitha; Wernery, Ulrich; Teng, Jade Ll; Wernery, Renate; Huang, Yi; Patteril, Nissy Ag; Chan, Kwok-Hung; Elizabeth, Shyna K; Fan, Rachel Yy; Lau, Susanna Kp; Kinne, Jörg; Woo, Patrick Cy

    2016-06-08

    Although antibodies against West Nile virus (WNV) have been detected in the sera of dromedaries in the Middle East, North Africa and Spain, no WNV has been isolated or amplified from dromedary or Bactrian camels. In this study, WNV was isolated from Vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in Dubai. Complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. There was no clustering of the present WNV with other WNVs isolated in other parts of the Middle East. Within lineage 1a, the dromedary WNV occupied a unique position, although it was most closely related to other WNVs of cluster 2. Comparative analysis revealed that the putative E protein encoded by the genome possessed the original WNV E protein glycosylation motif NYS at E154-156, which contained the N-linked glycosylation site at N-154 associated with increased WNV pathogenicity and neuroinvasiveness. In the putative NS1 protein, the A70S substitution observed in other cluster 2 WNVs and P250, which has been implicated in neuroinvasiveness, were present. In addition, the foo motif in the putative NS2A protein, which has been implicated in neuroinvasiveness, was detected. Notably, the amino-acid residues at 14 positions in the present dromedary WNV genome differed from those in most of the closely related WNV strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative E and NS5 proteins. The present study is the first to demonstrate the isolation of WNV from dromedaries. This finding expands the possible reservoirs of WNV and sources of WNV infection.

  19. First isolation of West Nile virus from a dromedary camel

    Science.gov (United States)

    Joseph, Sunitha; Wernery, Ulrich; Teng, Jade LL; Wernery, Renate; Huang, Yi; Patteril, Nissy AG; Chan, Kwok-Hung; Elizabeth, Shyna K; Fan, Rachel YY; Lau, Susanna KP; Kinne, Jörg; Woo, Patrick CY

    2016-01-01

    Although antibodies against West Nile virus (WNV) have been detected in the sera of dromedaries in the Middle East, North Africa and Spain, no WNV has been isolated or amplified from dromedary or Bactrian camels. In this study, WNV was isolated from Vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in Dubai. Complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. There was no clustering of the present WNV with other WNVs isolated in other parts of the Middle East. Within lineage 1a, the dromedary WNV occupied a unique position, although it was most closely related to other WNVs of cluster 2. Comparative analysis revealed that the putative E protein encoded by the genome possessed the original WNV E protein glycosylation motif NYS at E154–156, which contained the N-linked glycosylation site at N-154 associated with increased WNV pathogenicity and neuroinvasiveness. In the putative NS1 protein, the A70S substitution observed in other cluster 2 WNVs and P250, which has been implicated in neuroinvasiveness, were present. In addition, the foo motif in the putative NS2A protein, which has been implicated in neuroinvasiveness, was detected. Notably, the amino-acid residues at 14 positions in the present dromedary WNV genome differed from those in most of the closely related WNV strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative E and NS5 proteins. The present study is the first to demonstrate the isolation of WNV from dromedaries. This finding expands the possible reservoirs of WNV and sources of WNV infection. PMID:27273223

  20. Simple filter microchip for rapid separation of plasma and viruses from whole blood

    Directory of Open Access Journals (Sweden)

    Wang SQ

    2012-09-01

    Full Text Available ShuQi Wang,1 Dusan Sarenac,1 Michael H Chen,1 Shih-Han Huang,1 Francoise F Giguel,2 Daniel R Kuritzkes,3 Utkan Demirci1,41Bio-acoustic MEMS in Medicine Laboratory, Department of Medicine, Division of Biomedical Engineering, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; 2Infectious Diseases Unit, Massachusetts General Hospital, Boston, MA, USA; 3Section of Retroviral Therapeutics, Brigham and Women's Hospital, Boston, MA, USA; 4Harvard-MIT Health Sciences and Technology, Cambridge, MA, USAAbstract: Sample preparation is a significant challenge for detection and sensing technologies, since the presence of blood cells can interfere with the accuracy and reliability of virus detection at the nanoscale for point-of-care testing. To the best of our knowledge, there is not an existing on-chip virus isolation technology that does not use complex fluidic pumps. Here, we presented a lab-on-a-chip filter device to isolate plasma and viruses from unprocessed whole blood based on size exclusion without using a micropump. We demonstrated that viruses (eg, HIV can be separated on a filter-based chip (2-µm pore size from HIV-spiked whole blood at high recovery efficiencies of 89.9% ± 5.0%, 80.5% ± 4.3%, and 78.2% ± 3.8%, for viral loads of 1000, 10,000 and 100,000 copies/mL, respectively. Meanwhile, 81.7% ± 6.7% of red blood cells and 89.5% ± 2.4% of white blood cells were retained on 2 µm pore–sized filter microchips. We also tested these filter microchips with seven HIV-infected patient samples and observed recovery efficiencies ranging from 73.1% ± 8.3% to 82.5% ± 4.1%. These results are first steps towards developing disposable point-of-care diagnostics and monitoring devices for resource-constrained settings, as well as hospital and primary care settings.Keywords: microchip, filtration, virus isolation, plasma separation, point-of-care

  1. An experimental study of the pathogenicity of a duck hepatitis A virus genotype C isolate in specific pathogen free ducklings.

    Science.gov (United States)

    Zhang, Huanrong; Pi, JinKui; Tang, Cheng; Yue, Hua; Yang, Falong

    2012-12-01

    Duck hepatitis A virus genotype C (DHAV-C), recognized recently, is one of the pathogens causing fatal duck viral hepatitis in ducklings, especially in Asia. To demonstrate the pathogenesis of the DHAV-C isolate, 3-day-old specific pathogen free ducklings were inoculated subcutaneously with a DHAV-C isolate and the clinical signs were observed. Virus distribution, histological and apoptotic morphological changes of various tissues were examined at different times post inoculation. The serial, characteristic changes included haemorrhage and swelling of the liver. Apoptotic cells and virus antigen staining were found in all of the tissues examined. Where more virus antigen staining was detected, there were more severe histopathological and apoptotic changes. The amount of virus antigen and the histological and apoptotic morphological changes agreed with each other and became increasingly severe with length of time after infection. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages and monocytes in immune organs such as the bursa of Fabricius, thymus and spleen, and in liver, kidney and cerebral cells. Necrosis was also observed within 72 h post inoculation in all organs examined, except the cerebrum, and was characterized by cell swelling and collapsed plasma membrane. These results suggest that the recent outbreak of disease caused by DHAV-C virus is pantropic, causing apoptosis and necrosis of different organs. The apoptosis and necrosis caused by the DHAV-C field strain in this study is associated with pathogenesis and DHAV-C-induced lesions.

  2. Isolation of saint louis encephalitis virus from a horse with neurological disease in Brazil.

    Directory of Open Access Journals (Sweden)

    Roberta Rosa

    2013-11-01

    Full Text Available St. Louis encephalitis virus (SLEV is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil.

  3. Isolation of Saint Louis Encephalitis Virus from a Horse with Neurological Disease in Brazil

    Science.gov (United States)

    Rosa, Roberta; Costa, Erica Azevedo; Marques, Rafael Elias; Oliveira, Taismara Simas; Furtini, Ronaldo; Bomfim, Maria Rosa Quaresma; Teixeira, Mauro Martins; Paixão, Tatiane Alves; Santos, Renato Lima

    2013-01-01

    St. Louis encephalitis virus (SLEV) is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil. PMID:24278489

  4. Isolation & molecular characterization of human parainfluenza virus in Chennai, India

    Directory of Open Access Journals (Sweden)

    C P Indumathi

    2015-01-01

    Full Text Available Background & objectives: Human parainfluenza virus (HPIV accounts for a significant proportion of lower respiratory tract infections in children as well as adults. This study was done to detect the presence of different subtypes of HPIV from patients having influenza like illness (ILI. Methods: Throat and nasal swabs from 232 patients with ILI who were negative for influenza viruses were tested by multiplex reverse transcription polymerase chain reaction(mRT-PCR for the detection of human parainfluenza virus. All samples were inoculated in rhesus monkey kidney (LLC-MK2 cell line. Results: Of the 232 samples, 26(11.2% were positive by mRT-PCR and nine (34.6% showed cytopathic effect with syncytium formation for HPIV and all were HPIV-3 serotype, other serotypes like 1,2,4 were negative. The HPIV-3 strains (HN gene were sequenced and analysed. Two novel mutations were identified at amino acid residues 295 and 297. Interpretation & conclusions: The mRT-PCR assay offers a rapid, sensitive and accurate diagnostic method for detection of HPIV which enables early detection and control. In our study there was a predominance of HPIV among 1-5 yr age group and the school going age group was less affected. Further studies need to be done to characterize HPIV isolated from different parts of the country.

  5. Isolation of eastern equine encephalitis virus in A549 and MRC-5 cell cultures.

    Science.gov (United States)

    Sotomayor, E A; Josephson, S L

    1999-07-01

    Eastern equine encephalitis (EEE) has been diagnosed either serologically or by virus isolation. Until now, the recovery of EEE virus has been delegated to reference laboratories with the expertise and resources needed to amplify the virus in a susceptible vertebrate host and/or to isolate and identify the virus in cell culture. We report a case in which EEE virus was recovered directly from a patient's cerebrospinal fluid in A549 and MRC-5 cell cultures. Many clinical virology laboratories routinely use these cells to recover adenovirus, herpes simplex virus, and enterovirus. To the best of our knowledge, this is the first report of isolation of EEE virus in A549 cell culture. This report demonstrates the possibility of recovery of EEE virus in cell culture without the necessity of bioamplification or maintaining unusual cell lines.

  6. Isolation of Zika Virus Imported from Tonga into Australia

    Science.gov (United States)

    Pyke, Alyssa T.; Moore, Peter R.; Hall-Mendelin, Sonja; McMahon, Jamie L.; Harrower, Bruce J; Constantino, Tanya R; van den Hurk, Andrew F

    2016-01-01

    Introduction: The globally emergent Zika virus (ZIKV) is a threat to Australia, given the number of imported cases from epidemic regions and the presence of competent mosquito vectors. We report the isolation of ZIKV from a female traveler who recently returned from Tonga to Brisbane, Queensland, Australia in 2016. Methods: A specific TaqMan real-time reverse transcriptase polymerase chain reaction assay (RT-PCR) assay was used to detect ZIKV in serum and urine samples. Conventional cell culture techniques and suckling mice were employed in an attempt to isolate ZIKV from serum and urine. Results: A ZIKV isolate (TS17-2016) was recovered from the serum sample after one passage in suckling mouse brains and harvested 11 days post inoculation. Phylogenetic analysis of complete envelope (E) gene sequences demonstrated TS17-2016 shared 99.9% nucleotide identity with other contemporary sequences from Tonga 2016, Brazil 2015 and French Polynesia 2013 within the Asian lineage. Discussion: This is the first known report of successful isolation of ZIKV from a human clinical sample in Australia and the first from a traveler from Tonga. This study highlights the potential difficulties in isolating ZIKV from acute clinical samples using conventional cell culture techniques, particularly in non-endemic countries like Australia where access to samples of sufficient viral load is limited. The successful isolation of TS17-2016 will be essential for continued investigations of ZIKV transmission and pathogenicity and will enable the advancement of new preventative control measures extremely relevant to the Australian and Pacific region. PMID:27679739

  7. West Nile virus in Tunisia, 2014: First isolation from mosquitoes.

    Science.gov (United States)

    Wasfi, F; Dachraoui, K; Cherni, S; Bosworth, A; Barhoumi, W; Dowall, S; Chelbi, I; Derbali, M; Zoghlami, Z; Beier, J C; Zhioua, E

    2016-07-01

    Several outbreaks of human West Nile virus (WNV) infections were reported in Tunisia during the last two decades. Serological studies on humans as well as on equine showed intensive circulation of WNV in Tunisia. However, no virus screening of mosquitoes for WNV has been performed in Tunisia. In the present study, we collected mosquito samples from Central Tunisia to be examined for the presence of flaviviruses. A total of 102 Culex pipiens mosquitoes were collected in September 2014 from Central Tunisia. Mosquitoes were pooled according to the collection site, date and sex with a maximum of 5 specimens per pool and tested for the presence of flaviviruses by conventional reverse transcription heminested PCR and by a specific West Nile virus real time reverse transcription PCR. Of a total of 21 pools tested, 7 were positive for WNV and no other flavivirus could be evidenced in mosquito pools. In addition, WNV was isolated on Vero cells. Phylogenetic analysis showed that recent Tunisian WNV strains belong to lineage 1 WNV and are closely related to the Tunisian strain 1997 (PAH 001). This is the first detection and isolation of WNV from mosquitoes in Tunisia. Some areas of Tunisia are at high risk for human WNV infections. WNV is likely to cause future sporadic and foreseeable outbreaks. Therefore, it is of major epidemiological importance to set up an entomological surveillance as an early alert system. Timely detection of WNV should prompt vector control to prevent future outbreaks. In addition, education of people to protect themselves from mosquito bites is of major epidemiological importance as preventive measure against WNV infection.

  8. Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis

    NARCIS (Netherlands)

    Wahyuningsih, Retno; SahBandar, Ivo N.; Theelen, Bart; Hagen, Ferry; Poot, Ge; Meis, Jacques F.; Rozalyani, Anna; Sjam, Ridhawati; Widodo, Djoko; Djauzi, Samsuridjal; Boekhout, Teun

    2008-01-01

    Candida nivariensis was isolated from an Indonesian human immunodeficiency virus-infected patient who suffered from oropharyngeal candidiasis and was identified with molecular tools. Our isolate demonstrated low MICs to amphotericin B, flucytosine, posaconazole, caspofungin, and isavueonazole and wa

  9. Virus isolations from mosquitoes collected during the 1982 Japanese encephalitis epidemic in northern Thailand.

    Science.gov (United States)

    Leake, C J; Ussery, M A; Nisalak, A; Hoke, C H; Andre, R G; Burke, D S

    1986-01-01

    From 16 June to 15 August, 1982 CDC light traps were used to collect mosquitoes in the province of Kamphaengphet, N. Thailand. 353,042 mosquitoes comprising 59 species were collected and identified, and 345,173 were placed in pools for attempted virus isolation by inoculation of C6/36 Aedes albopictus mosquito cell cultures. Viruses were isolated from 63 mosquito pools. These comprised 56 flaviviruses, identified as 35 isolates of Japanese encephalitis (JE) virus strains, 18 strains of Tembusu (TEM) virus and three untyped flaviviruses (FLA); three alphaviruses, identified as the first isolates of Getah (GET) virus to have been made in Thailand; and four viruses which are still unidentified. Most virus isolates were from Culex tritaeniorhynchus mosquitoes collected in carbon dioxide baited light traps. JE virus was isolated only over a ten-day period and the last isolate was obtained one week before the peak of admission of human encephalitis cases at Kamphaengphet Provincial Hospital. Rapid screening of isolates grown on Ae. pseudoscutellaris (LSTM-AP-61) mosquito cells by indirect immunofluorescence using flavivirus group-specific and JE-specific monoclonal antibodies showed a high degree of correlation with plaque reduction neutralization tests. An antigen capture enzyme immunoassay (EIA) test successfully identified about 50% of the JE virus positive pools, but the method saved considerable processing time.

  10. Genomic characterization of Indian isolates of egg drop syndrome 1976 virus.

    Science.gov (United States)

    Raj, G D; Sivakumar, S; Sudharsan, S; Mohan, A C; Nachimuthu, K

    2001-02-01

    Five Indian isolates of egg drop syndrome (EDS) 1976 virus and the reference strain 127 were compared by restriction enzyme analysis of viral DNA, and the hexon gene amplified by polymerase chain reaction. Using these techniques, no differences were seen among these viruses. However, partial sequencing of the hexon gene revealed major differences (4.6%) in one of the isolates sequenced, EDS Kerala. Phylogenetic analysis also placed this isolate in a different lineage compared with the other isolates. The need for constant monitoring of the genetic nature of the field isolates of EDS viruses is emphasized.

  11. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene.

    Science.gov (United States)

    Kim, W-S; Oh, M-J; Nishizawa, T; Park, J-W; Kurath, G; Yoshimizu, M

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan.

  12. Molecular studies of the Brazilian infectious bronchitis virus isolates

    Directory of Open Access Journals (Sweden)

    JT Abreu

    2010-06-01

    Full Text Available Avian infectious bronchitis virus (IBV isolates have been widely characterized by reverse transcription followed by polymerase chain reaction and DNA sequencing. In present study, these techniques were applied to three viral genomic regions comprising the complete and/or a partial S1 segment, S2 and nucleocapsid genes. DNA sequences from viral isolates obtained from 1972 to 1989 and from 2006 to 2008 were compared. High similarity (>90% was observed among some of the genomic segments, including S1 hypervariable region, which could suggest a common origin or ancestry. DNA sequences from S2 and N protein genes obtained from different infected tissues of the same flock were analyzed, and a clear segregation between respiratory and intestinal tract was observed. Therefore, these data suggest co-circulation of more than one viral strain in the same flock. 57.1% of DNA sequences from the S1 complete segment samples, 53.3% from the S2 fragment and 62.5% from the partial N gene were found to be different from analyzed sequences from reference strains leading to the conclusion that parte of viral isolates included in this study may be considered region specific. Considering the simultaneous analysis of the three genes, a large IBV genetic profile was observed in both old and recent isolates groups. However, most prominent diversity between viral isolates was obtained in the period from 1972 and 1989, showing the presence of a large number of variants in the state of Minas Gerais before the official approval of vaccination (1980.

  13. Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to human influenza C viruses.

    Science.gov (United States)

    Hause, Ben M; Ducatez, Mariette; Collin, Emily A; Ran, Zhiguang; Liu, Runxia; Sheng, Zizhang; Armien, Anibal; Kaplan, Bryan; Chakravarty, Suvobrata; Hoppe, Adam D; Webby, Richard J; Simonson, Randy R; Li, Feng

    2013-02-01

    Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

  14. Complete Genome Sequences of New Emerging Newcastle Disease Virus Strains Isolated from China

    Science.gov (United States)

    Liu, Hualei; Lv, Yan; Afonso, Claudio L.; Ge, Shengqiang; Zheng, Dongxia; Zhao, Yunling

    2013-01-01

    Five Newcastle disease virus strains isolated from geese were classified into a new genotype, designated genotype XII. The complete genome sequences of two strains indicated that these viruses were distinct from viruses of genotype VII. More investigations need to be conducted for us to understand the origin of these new strains. PMID:23469337

  15. Complete Genome Sequence of a Street Rabies Virus Isolated from a Dog in Nigeria

    Science.gov (United States)

    Zhou, Ming; Zhou, Zutao; Kia, Grace S. N.; Gnanadurai, Clement W.; Leyson, Christina M.; Umoh, Jarlath U.; Kwaga, Jacob P.; Kazeem, Haruna M.

    2013-01-01

    A canine rabies virus (RABV) was isolated from a trade dog in Nigeria. Its entire genome was sequenced and found to be closely related to canine RABVs circulating in Africa. Sequence comparison indicates that the virus is closely related to the Africa 2 RABV lineage. The virus is now termed DRV-NG11. PMID:23469344

  16. The Dynamics of an Isolated Plasma Filament at the Edge of a Toroidal Device, Rev. 1

    Energy Technology Data Exchange (ETDEWEB)

    Ryutov, D D

    2006-09-28

    The dynamics of an isolated plasma filament (an isolated blob) in the far scrape-off layer (SOL) of a toroidal device is described, with a proper averaging of the geometrical parameters as well as plasma parameters along the filament. The analysis is limited to the magnetohydrodynamic description. The effects of the anchored ends and finite plasma resistivity are also discussed.

  17. Guiding-center models for edge plasmas and numerical simulations of isolated plasma filaments

    Energy Technology Data Exchange (ETDEWEB)

    Madsen, Jens

    2010-09-15

    The work presented in this thesis falls into two categories: development of reduced dynamical models applicable to edge turbulence in magnetically confined fusion plasmas and numerical simulations of isolated plasma filaments in the scrape-off layer region investigating the influence of finite Larmor radius effects on the radial plasma transport. The coexistence of low-frequency fluctuations, having length scales comparable to the ion gyroradius, steep pressure gradients and strong E x B flows in the edge region of fusion plasmas violates the standard gyrokinetic ordering. In this thesis two models are presented that overcome some of the difficulties associated with the development of reduced dynamical models applicable to the edge. Second order guiding-center coordinates are derived using the phasespace Lie transform method. Using a variational principle the corresponding Vlasov-Maxwell equations expressed in guiding-center coordinates are derived including a local energy theorem. The second order terms describe lowest order finite Larmor radius effects. This set of equations might be relevant for edge plasmas due to the capability of capturing strong E x B flows and lowest order finite Larmor radius effects self-consistently. Next, an extension of the existing gyrokinetic formalism with strong flows is presented. In this work the background electric fields is dynamical, whereas earlier contributions did only incorporate a stationary electric field. In an ordering relevant for edge plasma turbulence, fully electromagnetic second order gyrokinetic coordinates and the corresponding gyrokinetic Vlasov-Maxwell equations are derived, including a local energy theorem. By taking the polarization and magnetization densities in the drift kinetic limit, we present the gyrokinetic Vlasov-Maxwell equations in a more tractable form, which could be relevant for direct numerical simulations of edge plasma turbulence. Finally, an investigation of the influence of finite Larmor

  18. Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

    Science.gov (United States)

    Psikal, I; Smíd, B; Rodák, L; Valícek, L; Bendová, J

    2003-08-01

    Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

  19. Comparison of mosquito densoviruses: two clades of viruses isolated from indigenous mosquitoes.

    Science.gov (United States)

    Sangdee, Kusavadee; Pattanakitsakul, Sa-Nga

    2013-07-04

    We analyzed the phylogenetic tree of densoviruses isolated from indigenous mosquitoes and mosquito cell lines. Our findings suggest two distinct clades of densovirus. The viruses in the first clade were isolated from an indigenous mosquito which had the Aedes aegypti densovirus (AaeDNV) as a representative virus. The other clade of viruses was isolated from mosquito indigenous cell line which had the Aedes albopictus densovirus (AalDNV) as the representative virus. The origin of the two clades of DNVs is unclear but the phylogenetic trees were significantly different from each other. The two major densoviruses, AaeDNV and AalDNV, that infect mosquitoes that are known to carry viruses responsible for dengue hemorrhagic fever and yellow fever. Understanding the evolution of these two clades of densoviruses is important for studying the distribution of these viruses in mosquito cell lines and the information gained may be applied to understanding other viruses in various mosquito cell lines.

  20. Studies on antigenic and genomic properties of Brazilian rabies virus isolates

    NARCIS (Netherlands)

    Schaefer, R.; Batista, H.B.; Franco, A.C.; Rijsewijk, F.A.M.; Roehe, P.M.

    2005-01-01

    Despite the recognized stability of rabies virus, differences among isolates from different species have been found. This work was carried out with the aim to identify antigenic and genomic differences in Brazilian rabies virus isolates and to verify whether such alterations would bear any relations

  1. Molecular-genetic analysis of field isolates of Avian Leucosis Viruses in the Russian Federation

    Science.gov (United States)

    Commercial poultry farms in 14 regions of Russian Federation were monitored for avian leukosis virus (ALV) infection using virus isolation tests and serology. Results indicated the presence of two subgroups of ALV in farms located in 11 of 14 regions. Analysis of the genomes of 12 field isolates of...

  2. Complete Genome Sequence of an African Swine Fever Virus Isolate from Sardinia, Italy

    Science.gov (United States)

    Torresi, Claudia; Oggiano, Annalisa; Malmberg, Maja; Iscaro, Carmen; De Mia, Gian Mario; Belák, Sándor

    2016-01-01

    Previous genetic characterization of African swine fever virus isolates from the Italian island of Sardinia, where the virus has been present since 1978, has largely been limited to a few selected genomic regions. Here, we report the complete genome sequence of the isolate 47/Ss/08 collected during an outbreak in 2008. PMID:27856577

  3. First Complete Genome Sequence of a Chikungunya Virus Strain Isolated from a Patient Diagnosed with Dengue Virus Infection in Malaysia

    Science.gov (United States)

    Gan, Han Ming; Rohani, Ahmad

    2016-01-01

    Here, we report the complete genome sequence of a chikungunya virus coinfection strain isolated from a dengue virus serotype 2-infected patient in Malaysia. This coinfection strain was determined to be of the Asian genotype and contains a novel insertion in the nsP3 gene. PMID:27563048

  4. Full-length clone and characterization of a human immunodeficiency virus type 1 subtype B' isolated from Hubei Province, China

    Institute of Scientific and Technical Information of China (English)

    TAN Jian-xin; KANG Xian-jiang; ZHANG Wei; LIU Ping-ping; TONG Xiao; YANG Rong-ge

    2007-01-01

    @@ There are two types of Human Immunodeficiency Virus (HIV): HIV-1 and HIV-2. HIV-1 dominates epidemics in many different parts of the world, and HIV-2 is principally responsible for the epidemic in western Africa. In China, Zeng et al1 have reported the first individual infected with HIV-1 in 1985. And in the 1990s,there was a severe epidemic involving the HIV-1 B' strain among people who sold blood and plasma in Henan,Hubei and adjacent provinces.2 To further study in HIV/AIDS vaccines and HIV-1 drug resistance for people in these regions, we need to construct an infectious HIV-1 B' molecular clone which is representative of the virus in these areas.3 To this end, we have isolated a HIV-1 B' virus from a child who was infected with HIV-1 from his mother in Hubei province, and have constructed a full-length clone from this genome.

  5. GENOTYPING OF ISOLATED VIRUSES FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS) IN CROATIA

    OpenAIRE

    Irena Vardić; Damir Kapetanović; Damir Valić; Božidar Kurtović; Zlatica Teskeredžić; Emin Teskeredžić

    2007-01-01

    Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout aquaculture. Detection of these viruses in Croatia initiated investigation of their genetic relatedness to the worldwide IHNV and IPNV isolates. For this purpose, determination of nucleotide sequences of G and NV genes for IHNV and VP2/NS region for IPNV was performed. Phylogenetic analyses revealed that Croatian IHNV isolate was clustering within European cl...

  6. Integration of antibody by surface functionalization of graphite-encapsulated magnetic beads using ammonia gas plasma technology for capturing influenza A virus.

    Science.gov (United States)

    Sakudo, Akikazu; Chou, Han; Ikuta, Kazuyoshi; Nagatsu, Masaaki

    2015-05-01

    Antibody-integrated magnetic beads have been functionalized for influenza A virus capture. First, ammonia plasma produced by a radio frequency power source was reacted with the surface of graphite-encapsulated magnetic beads to introduce amino groups. Anti-influenza A virus hemagglutinin antibody was then anchored by its surface sulfide groups to the amino groups on the beads via N-succinimidyl 3-(2-pyridyldithio) propionate. After incubation with influenza A virus, adsorption of the virus to the beads was confirmed by immunochromatography, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and inoculation of chicken embryonated eggs, indicating that virus infectivity is maintained and that the proposed method is useful for the enhanced detection and isolation of influenza A virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Isolation and Identification of Virus dsRNA from Strawberry Plants

    Institute of Scientific and Technical Information of China (English)

    LI He; DAI Hong-yan; ZHANG Zhi-hong; GAO Xiu-yan; DU Guo-dong; ZHANG Xin-yu

    2007-01-01

    The analysis of virus genome is based on nucleic acid isolation. The aims of this study were to develop a method for isolation and identification of virus double-stranded ribonucleic acid (dsRNA) and to elucidate the nucleotide sequences of strawberry virus. Using the modified method, virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants and detected using agarose gel electrophoresis with ethidium bromide staining and reverse transcription-polymerase chain reaction (RT-PCR). The quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from in vitro plantlets was higher than that from the young leaves of field plants. For the field-grown plants, there was more dsRNA in the young leaves. Virus dsRNA extracted from strawberry plants was resistant to deoxyribonuclease Ⅰ (DNase Ⅰ ), but evidently, it became resistant to ribonuclease A (RNase A) only in the presence of 0.5 M NaCl. Its bands in agarose gel could be readily recycled using an agarose gel DNA purification kit. With RT-PCR, the segments of both strawberry mottle virus and Strawberry mild yellow edge virus genomes were amplified by using the virus dsRNA recycled from gel or treated with DNase Ⅰ /RNase A as templates. The system developed for dsRNA isolation and identification in strawberry plants laid a sound foundation for the work on genome analysis of strawberry virus isolates in China.

  8. Acylated simian virus 40-specific proteins in the plasma membrane of HeLa cells infected with adenovirus 2-simian virus 40 hybrid virus Ad2+ND2

    Energy Technology Data Exchange (ETDEWEB)

    Klockmann, U.; Deppert, W.

    1983-04-30

    HeLa cells infected with the adenovirus 2-simian virus 40 (Ad2+SV40) hybrid virus Ad2+ND2 were labeled with either (/sup 35/S)methionine or (/sup 3/H)palmitate and fractionated into cytoplasmic, nuclear, and plasma membrane fractions. Analysis of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the SV40-specific proteins in the plasma membrane fraction were specificially acylated.

  9. Application of polymerase chain reaction and virus isolation techniques for the detection of viruses in aborted and newborn foals.

    Science.gov (United States)

    Hornyák, A; Bakonyi, T; Kulik, Mónika; Kecskeméti, S; Rusvai, M

    2006-06-01

    The occurrence of two important pathogens, equine herpesvirus 1 (EHV1) and equine arteritis virus (EAV) causing abortions, perinatal foal mortality and respiratory disease, was investigated by polymerase chain reaction (PCR) and virus isolation to demonstrate the presence of abortigenic viruses in samples from 248 horse fetuses in Hungary. We found 26 EHV1- and 4 EAV-positive aborted or prematurely born foals from 16 and 4 outbreaks, respectively, proving that despite the widely applied vaccination, EHV1 is a far more important cause of abortions in the studs than EAV. We compared the virus content of different organs of the fetuses by PCR and isolation to identify the organ most suitable for virus demonstration. Our investigations indicate that the quantity of both viruses is highest in the lungs; therefore, according to our observations, in positive cases the probability of detection is highest from lung samples of aborted or newborn foals. Both the PCR and the virus isolation results revealed that the liver, though widely used, is not the best organ to sample either for EHV1 or for EAV detection. From the analysis of the epidemiological data, we tried to estimate the importance of the two viruses in the Hungarian horse population.

  10. The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses

    NARCIS (Netherlands)

    Souza Richards, R.; Adams, I.P.; Kreuze, J.F.; Souza, de J.; Cuellar, W.; Dullemans, A.M.; Vlugt, van der R.A.A.; Glover, R.; Hany, U.; Dickinson, M.; Boonham, N.

    2014-01-01

    The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), whi

  11. Isolation of recombinant field strains of Marek's disease virus integrated with reticuloendotheliosis virus genome fragments

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Zhi; CUI; Zhizhong

    2005-01-01

    Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.

  12. Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

    Directory of Open Access Journals (Sweden)

    Botton S.A.

    1998-01-01

    Full Text Available Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs (Corapi WV, Donis RO and Dubovi EJ (1990 American Journal of Veterinary Research, 55: 1388-1394. Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3% reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R was calculated, 49 of 66 comparisons (74.24% between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

  13. Isolation of new Brazilian giant viruses from environmental samples using a panel of protozoa

    Science.gov (United States)

    Dornas, Fábio P.; Khalil, Jacques Y. B.; Pagnier, Isabelle; Raoult, Didier; Abrahão, Jônatas; La Scola, Bernard

    2015-01-01

    The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation. PMID:26500630

  14. New Brazilian giant viruses isolation from environmental samples using a panel of protozoa.

    Directory of Open Access Journals (Sweden)

    Fábio Pio Dornas

    2015-10-01

    Full Text Available The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini and Vermamoeba vermiformis as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in Acanthamoeba polyphaga (46.38% and the lowest in Vermamoeba vermiformis (0%. Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation.

  15. Characterization of emerging Newcastle disease virus isolates in China.

    Science.gov (United States)

    Wang, Jing-Yu; Liu, Wan-Hua; Ren, Juan-Juan; Tang, Pan; Wu, Ning; Wu, Hung-Yi; Ching, Ching-Dong; Liu, Hung-Jen

    2015-08-07

    Newcastle disease (ND) is a devastating worldwide disease of poultry characterized by increased respiration, circulatory disturbances, hemorrhagic enteritis, and nervous signs. Sequence analysis shows several amino acid residue substitutions at neutralizing epitopes on the F and HN proteins of recent Shaanxi strains. Both Cross protection and cross serum neutralization tests revealed that the traditional vaccine strains were unable to provide full protection for the flocks. To better understand the epidemiology of Newcastle disease outbreak, a portion of the F gene and the full-length HN gene were amplified from Shaanxi isolates by reverse transcription-polymerase chain reaction (RT-PCR) and then conducted sequence and phylogenetic analyzes. In pathogenicity analysis, both high intra-cerebral pathogenicity index (ICPI) and mean death time (MDT) tests of chicken embryo were carried out. Furthermore, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with a LaSota vaccine strain were challenged by the recent Shaanxi strain was also performed. Nine Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken flocks in China were genotypically and pathotypically characterized. Amino acid sequence analysis revealed that all the recent Shaanxi-isolated NDVs have (112)R-R-Q-K-R-F(117) for the C-terminus of the F2 protein and exhibit high ICPI and MDT of chicken embryos, suggesting that they were all classified as velogenic type of NDVs. Phylogenetic analysis of these isolates showed that they belong to subgenotype VIId that have been implicated in the recent outbreaks in northwestern China. The percentage of amino acid sequence identity of F protein between recent Shaanxi stains and five vaccine strains was in the range of 81.9 %-88.1 %, while the percentage of amino acid sequence identity of HN protein between recent Shaanxi strains and vaccine strains was in the range of 87.4 %-91.2 %. Furthermore, a number

  16. Isolation of a new herpes virus from human CD4 sup + T cells

    Energy Technology Data Exchange (ETDEWEB)

    Frenkel, N.; Schirmer, E.C.; Wyatt, L.S.; Katsafanas, G.; Roffman, E.; Danovich, R.M. (National Institutes of Health, Rockville, MD (USA)); June, C.H. (Naval Medical Research Institute, Bethesda, MD (USA))

    1990-01-01

    A new human herpes virus has been isolated from CD4{sup +} T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpes virus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpes virus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hydridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. The authors conclude that the RK virus is distinct from previously characterized human herpesviruses. The authors propose to designate it as the prototype of a new herpes virus, the seventh human herpes virus identified to date.

  17. Expression of Bovine Leukemia Virus Genome is Blocked by a Nonimmunoglobulin Protein in Plasma from Infected Cattle

    Science.gov (United States)

    Gupta, P.; Ferrer, J. F.

    1982-01-01

    Plasma of cattle infected with bovine leukemia virus contains a soluble factor that blocks the expression of the viral genome in cultured lymphocytes. The blocking factor is not present in plasma of bovine leukemia virus-free cattle or of cattle infected with common bovine viruses. Blocking of bovine leukemia virus expression by the plasma factor is reversible, and seems to be mediated by a nonimmunoglobulin protein molecule.

  18. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    OpenAIRE

    Kim, W. -S.; Oh, M. -J.; Nishizawa, T; Park, J. -W.; Kurath, G; Yoshimizu, M

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogen...

  19. Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

    Science.gov (United States)

    Turell, M J; O'Guinn, M L; Jones, J W; Sardelis, M R; Dohm, D J; Watts, D M; Fernandez, R; Travassos da Rosa, A; Guzman, H; Tesh, R; Rossi, C A; Ludwig, V; Mangiafico, J A; Kondig, J; Wasieloski, L P; Pecor, J; Zyzak, M; Schoeler, G; Mores, C N; Calampa, C; Lee, J S; Klein, T A

    2005-09-01

    As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

  20. Phylogenetic analysis of Newcastle disease viruses isolated from commercial poultry in Mozambique (2011-2016).

    Science.gov (United States)

    Mapaco, Lourenço P; Monjane, Iolanda V A; Nhamusso, Antonieta E; Viljoen, Gerrit J; Dundon, William G; Achá, Sara J

    2016-10-01

    The complete sequence of the fusion (F) protein gene from 11 Newcastle disease viruses (NDVs) isolated from commercial poultry in Mozambique between 2011 and 2016 has been generated. The F gene cleavage site motif for all 11 isolates was (112)RRRKRF(117) indicating that the viruses are virulent. A phylogenetic analysis using the full F gene sequence revealed that the viruses clustered within genotype VIIh and showed a higher similarity to NDVs from South Africa, China and Southeast Asia than to viruses previously described in Mozambique in 1994, 1995 and 2005. The identification of these new NDVs has important implications for Newcastle disease management and control in Mozambique.

  1. Isolation and Characterization of Metallosphaera turreted icosahedral virus (MTIV), a founding member of a new family of archaeal viruses.

    Science.gov (United States)

    Wagner, Cassia; Reddy, Vijay; Asturias, Francisco; Khoshouei, Maryam; Johnson, John E; Manrique, Pilar; Munson-McGee, Jacob; Baumeister, Wolfgang; Lawrence, C Martin; Young, Mark J

    2017-08-02

    Our understanding of archaeal virus diversity and structure is just beginning to emerge. Here we describe a new archaeal virus, tentatively named Metallosphaera turreted icosahedral virus (MTIV), that was isolated from an acidic hot spring in Yellowstone National Park, USA. Two strains of the virus were identified and found to replicate in an archaeal host species closely related to Metallosphaera yellowstonensis Each strain encodes for a 9.8-9.9 kb, linear dsDNA genome with large inverted terminal repeats. Each genome encodes for 21 ORFs. Between the strains the ORFs display high homology, but they are quite distinct from other known viral genes. The 70-nm diameter virion is built upon on a T=28 icosahedral lattice. Both single particle cryo-electron microscopy and cryo-tomography reconstructions reveal an unusual structure that has 42 turret-like projections: 12 from each of the 5-fold axes and 30 hexameric units positioned on icosahedral 2-fold axes. Both the virion structural properties and genome content support MTIV as the founding member of a new family of archaeal viruses.Importance: Many archaeal viruses are quite different than viruses infecting bacteria and eukaryotes. Initial characterization of MTIV reveals a virus distinct from other known bacterial, eukaryotic, and archaeal viruses; this finding suggests that viruses infecting Archaea are still an understudied group of viruses. As the first known virus infecting the Metallosphaera, MTIV provides a new system for exploring archaeal virology by examining host-virus interactions and the unique features of MTIV structure-function relationships. These studies will likely expand our understanding of virus ecology and evolution. Copyright © 2017 American Society for Microbiology.

  2. The isolation of Gurnbiro virus from larvae and darkling Ivelles (Carcinops pumilin

    Directory of Open Access Journals (Sweden)

    Lies Parede

    1996-03-01

    Full Text Available Gumboro (infectious bursal disease, IBD virus was isolated from darkling beetles (Carrinaps pumilin and their larvae in a commercial pulletchicken farm with repeated outbreaks incidence of Gumboro disease in Tangertng, West Java. In addition, these over populated beetles and their larvae were suspected to be infected and then shed the virus or acted as vectors. Isolation was done by repeated passages of virus using chicken embryo fibroblast cells as prime media, which then showed the evidence of cylop: ihic effecis. The isolation was followed by antigen detection by means of ELISA test.

  3. Emergence of a new arbovirus disease in Brazil. I. Isolation and characterization of the etiologic agent, Rocio virus.

    Science.gov (United States)

    de Souza Lopes, O; Coimbra, T L; de Abreu Sacchetta, L; Calisher, C H

    1978-05-01

    In April, 1975, an epidemic of human encephalitis was detected in several counties in the State of São Paulo, Brazil; the epidemic continued into 1976. A virus was isolated from central nervous system (CNS) tissues of a 39-year-old male who died on December 8, 1975; the virus was found to be a new flavivirus for which the name Rocio virus is proposed. Nine further isolations of Rocio virus were obtained from CNS tissues of 17 patients who died with clinical symptoms of encephalitis. Isolations of virus and serologic evidence of Rocio virus infection in a significant proportion of the encephalitis patients suggested that Rocio virus was the etiologic agent of the epidemic. Rocio virus was isolated only from patients who died within 5 days of onset of illness. The virus was isolated from two sentinel mice exposed in the epidemic zone and from a rufous collared sparrow (Zonotrichia capensis) collected in the area.

  4. [Subtypes of dengue virus serotypes 2, 3 and 4 isolated in Santander District, Colombia].

    Science.gov (United States)

    Cortés, Fabián M; Gómez, Sergio Y; Ocazionez, Raquel E

    2007-01-01

    Virus serotypes 2, 3 and 4 that had circulated in Santander District, Colombia in the period 1998-2004 were analyzed. Identifying the subtype of a dengue virus serotype is a useful tool for surveillance of severe risk factors because the strain potential to cause hemorrhagic dengue makes the difference among them. Simultaneous sequence amplification technique known as restriction site specific-polymerase chain reaction (RSS-PCR) was used to determine the subtype by comparing the electrophoretic pattern of the local isolate to the reference virus. Virus serotype 2 corresponded to subtype A similar to the one isolated in Thailand (1996) and to the other isolated in Porto Rico (1986); virus serotypes 3 were of subtype C like the virus found in Sri Lanka (1990), Honduras (1995) and Porto Rico (2000); virus serotypes 4 were a variant of subtype B similar to a virus from Porto Rico (1987) and to another virus from Tahiti (1985). The study confirmed the presence in Colombia of dengue virus subtypes circulating now in the Americas.

  5. Isolation of Tacaribe virus, a Caribbean arenavirus, from host-seeking Amblyomma americanum ticks in Florida.

    Directory of Open Access Journals (Sweden)

    Katherine A Sayler

    Full Text Available Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573, with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2% of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

  6. Isolation of Tacaribe virus, a Caribbean arenavirus, from host-seeking Amblyomma americanum ticks in Florida.

    Science.gov (United States)

    Sayler, Katherine A; Barbet, Anthony F; Chamberlain, Casey; Clapp, William L; Alleman, Rick; Loeb, Julia C; Lednicky, John A

    2014-01-01

    Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV) is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks) collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573), with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2%) of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

  7. Isolation and characterization of two new herpes-like viruses from capuchin monkeys.

    Science.gov (United States)

    Lewis, M A; Frye, L D; Gibbs, C J; Chou, S M; Cutchins, E C; Gajdusek, D C; Ward, G

    1976-09-01

    Two herpes-like viruses were isolated from capuchin monkey (Cebus apella) brain and (Cebus albifrons) spleen cell cultures, respectively. Both isolates induced similar cytopathic effects consisting of rounded and ballooned cells in the original monkey cell cultures and in a wide range of permissive cell types. Neutralizing antibody to each virus was present in serum from the capuchin monkey from which it was isolated, but the two viruses did not cross-react by neutralization. Fluorescein isothiocyanate conjugates of hyperimmune rabbit serum to one of the isolates showed an antigenic cross relationship between the two isolates. By electron microscopy, herpes-like virus particles were observed in the nucleus and cytoplasm of infected human diploid fibroblast cell cultures. Virus-infected cell cultures stained with acridine orange revealed small deoxyribonucleic acid-containing intranuclear inclusion bodies. Both viruses were inhibited by 5-fluorodeoxyuridine and inactivated by chloroform or exposure to 56 degrees C for 30 min. Antisera prepared against 16 prototype herpesviruses and cytomegaloviruses did not neutralize approximately 100 50% tissue culture infective doses of either capuchin isolate. Neutralizing antibody to the capuchin isolates was detected in sera from 8 of 17 capuchin monkeys but not in sera from 16 humans, 15 chimpanzees, and 10 spider, 6 rhesus, and 5 squirrel monkeys.

  8. Dengue virus serotype 2 from a sylvatic lineage isolated from a patient with dengue hemorrhagic fever.

    Directory of Open Access Journals (Sweden)

    Jane Cardosa

    Full Text Available Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF with thrombocytopenia (12,000/ul and a raised hematocrit (29.5% above baseline in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975.

  9. Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus

    Directory of Open Access Journals (Sweden)

    J. A. Swanstrom

    2016-07-01

    Full Text Available Zika virus (ZIKV is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73% failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], 1:100 serum dilution; 9% levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV.

  10. Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes.

    Science.gov (United States)

    Liang, Xiaozhen; Collins, Christopher M; Mendel, Justin B; Iwakoshi, Neal N; Speck, Samuel H

    2009-11-01

    Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68) gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency) account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners) that do not directly participate in virus replication, but rather facilitate virus reactivation by

  11. Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes.

    Directory of Open Access Journals (Sweden)

    Xiaozhen Liang

    2009-11-01

    Full Text Available Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68 gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners that do not directly participate in virus replication, but rather facilitate virus

  12. Virus excretion and antibody dynamics in goats inoculated with a field isolate of peste des petits ruminants virus.

    Science.gov (United States)

    Liu, W; Wu, X; Wang, Z; Bao, J; Li, L; Zhao, Y; Li, J

    2013-11-01

    A field isolate of peste des petits ruminants virus (PPRV) from an outbreak in Tibet, China, was inoculated into goats to investigate the dynamics of virus excretion and antibody production. Further, animals received PPRV vaccine strain Nigeria 75/1. Ocular, nasal and oral samples were tested for the presence of virus antigen by one-step real-time qualitative RT-PCR (qRT-PCR); competitive ELISA (c-ELISA) was used for the measurement of specific antibodies against PPRV. Virus particles could be detected as early as day 3 post-inoculation (pi) and virus excretion lasted for up to day 26 pi. All four goats inoculated with the PPRV field isolate were seropositive as early as day 10 pi. In animals inoculated with the vaccine strain, antibody was detected at day 14 pi, and levels of neutralizing antibodies remained above the protection threshold level (1 : 8) for 8 months. Both virus particles and neutralizing antibodies were detected earlier in goats challenged with the field isolate than in those receiving the vaccine strain. © 2013 Blackwell Verlag GmbH.

  13. 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

  14. Genome Sequence of Lassa Virus Isolated from the First Domestically Acquired Case in Germany

    Science.gov (United States)

    Wolff, Svenja; Schultze, Tilman; Fehling, Sarah Katharina; Mengel, Jan Philipp; Kann, Gerrit; Wolf, Timo; Eickmann, Markus; Becker, Stephan

    2016-01-01

    Lassa virus (LASV) is a zoonotic, hemorrhagic fever-causing virus endemic in West Africa, for which no approved vaccines or specific treatment options exist. Here, we report the genome sequence of LASV isolated from the first case of acquired Lassa fever disease outside of Africa. PMID:27660771

  15. Characterization of Recombinant H9N2 Influenza Viruses Isolated from Wild Ducks in China

    Science.gov (United States)

    Zhu, Guangjian; Wang, Renjie; Xuan, Fujun; Daszak, Peter; Anthony, Simon J.; Zhang, Shuyi; Zhang, Libiao; He, Guimei

    2013-01-01

    Wild birds are considered to be the natural reservoirs for avian influenza A viruses (AIV). During active influenza surveillance in Poyang Lake of southeast China, we isolated and characterized eleven H9N2 viruses from two species of wild ducks. Phylogenetic analysis showed that the eleven isolates were almost identical with 99.3% to 100% nucleotide homology in their entire genome, and they all closely related in whole eight genes (95.6-99.4% homology) to human H9N2 isolates (HK/33982/2009) and clustered in the same sublineage. The isolates belonged to triple reassortant H9N2 genotype viruses containing Ck/Bei-like NA genes, Y439-like PA genes and six other G1-like genes. We also found that the subtype of virus replicated efficiently in the lungs and tracheas of BALB/c mice and caused mortality in 20-40% of infected groups after 3-6 days, which indicates that the subtype of virus is capable of establishing lethal mammalian infections. However, whether or not the virus has features transmittable from wild ducks to humans is not known. This study showed that the subtype of virus was detected for the first time in wild birds, and also suggested that wild birds may carry the virus for a long time and spread it over long distances along migratory routes, so more attention should be paid to the continued surveillance of wild birds. PMID:23830774

  16. Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico

    Science.gov (United States)

    Rodríguez-Castillo, Araceli; Ortiz-Alcántara, Joanna María; Gonzalez-Durán, Elizabeth; Segura-Candelas, José Miguel; Pérez-Agüeros, Sandra Ivette; Escobar-Escamilla, Noé; Méndez-Tenorio, Alfonso; Diaz-Quiñonez, José Alberto

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico. PMID:26159533

  17. Dengue-1 Virus Isolation during First Dengue Fever Outbreak on Easter Island, Chile

    Science.gov (United States)

    Abarca, Katia; Ovalle, Jimena; Ferrer, Pablo; Godoy, Paula; Olea, Andrea; Aguilera, Ximena; Ferrés, Marcela

    2003-01-01

    Dengue virus was detected for the first time in Chile, in an outbreak of dengue fever on Easter Island. The virus was isolated in tissue culture and characterized by reverse transcription–polymerase chain reaction as being dengue type 1. PMID:14718094

  18. Two biologically distinct isolates of Zucchini yellow mosaic virus lack seed transmissibility in cucumber.

    Science.gov (United States)

    Glasa, M; Kollerova, E

    2007-01-01

    The seed transmission of the Zucchini yellow mosaic virus (ZYMV) was studied in cucumber using two isolates unrelated in their biological characteristics. Although the virus could be readily detected in mature seeds harvested from infected cucumbers, the seedlings obtained from infected germinated seeds tested negative for ZYMV using both ELISA and RT-PCR assays. No evidence was obtained for transmission of two ZYMV isolates through seeds.

  19. Genetic analysis of influenza B viruses isolated in Uganda during the 2009–2010 seasons

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    Byarugaba Denis K

    2013-01-01

    Full Text Available Abstract Background Influenza B viruses can cause morbidity and mortality in humans but due to the lack of an animal reservoir are not associated with pandemics. Because of this, there is relatively limited genetic sequences available for influenza B viruses, especially from developing countries. Complete genome analysis of one influenza B virus and several gene segments of other influenza B viruses isolated from Uganda from May 2009 through December 2010 was therefore undertaken in this study. Methods Samples were collected from patients showing influenza like illness and screened for influenza A and B by PCR. Influenza B viruses were isolated on Madin-Darby Canine Kidney cells and selected isolates were subsequently sequenced and analyzed phylogenetically. Findings Of the 2,089 samples collected during the period, 292 were positive by PCR for influenza A or B; 12.3% of the PCR positives were influenza B. Thirty influenza B viruses were recovered and of these 25 that grew well consistently on subculture were subjected to further analysis. All the isolates belonged to the B/Victoria-lineage as identified by hemagglutination inhibition assay and genetic analysis except one isolate that grouped with the B-Yamagata-lineage. The Ugandan B/Victoria-lineage isolates grouped in clade 1 which was defined by the N75K, N165K and S172P substitutions in hemagglutinin (HA protein clustered together with the B/Brisbane/60/2008 vaccine strain. The Yamagata-like Ugandan strain, B/Uganda/MUWRP-053/2009, clustered with clade 3 Yamagata viruses such as B/Bangladesh/3333/2007 which is characterized by S150I and N166Y substitutions in HA. Conclusion In general there was limited variation among the Ugandan isolates but they were interestingly closer to viruses from West and North Africa than from neighboring Kenya. Our isolates closely matched the World Health Organization recommended vaccines for the seasons.

  20. Characterization of an H10N8 influenza virus isolated from Dongting lake wetland

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    Chen Jianjun

    2011-01-01

    Full Text Available Abstract Background Wild birds, especially those in wetlands and aquatic environments, are considered to be natural reservoirs of avian influenza viruses. It is accepted that water is an important component in the transmission cycle of avian influenza virus. Monitoring the water at aggregation and breeding sites of migratory waterfowl, mainly wetland, is very important for early detection of avian influenza virus. The epidemiology investigation of avian influenza virus was performed in Dongting lake wetland which is an international important wetland. Results An H10N8 influenza virus was isolated from Dongting Lake wetland in 2007. Phylogenetic analysis indicated that the virus was generated by multiple gene segment reassortment. The isolate was lowly pathogenic for chickens. However, it replicated efficiently in the mouse lung without prior adaptation, and the virulence to mice increased rapidly during adaptation in mouse lung. Sequence analysis of the genome of viruses from different passages showed that multiple amino acid changes were involved in the adaptation of the isolates to mice. Conclusions The water might be an important component in the transmission cycle of avian influenza virus, and other subtypes of avian influenza viruses (other than H5, H7 and H9 might evolve to pose a potential threat to mammals and even humans.

  1. Isolation of Madre de Dios Virus (Orthobunyavirus; Bunyaviridae), an Oropouche Virus Species Reassortant, from a Monkey in Venezuela.

    Science.gov (United States)

    Navarro, Juan-Carlos; Giambalvo, Dileyvic; Hernandez, Rosa; Auguste, Albert J; Tesh, Robert B; Weaver, Scott C; Montañez, Humberto; Liria, Jonathan; Lima, Anderson; Travassos da Rosa, Jorge Fernando Soares; da Silva, Sandro P; Vasconcelos, Janaina M; Oliveira, Rodrigo; Vianez, João L S G; Nunes, Marcio R T

    2016-08-03

    Oropouche virus (OROV), genus Orthobunyavirus, family Bunyaviridae, is an important cause of human illness in tropical South America. Herein, we report the isolation, complete genome sequence, genetic characterization, and phylogenetic analysis of an OROV species reassortant, Madre de Dios virus (MDDV), obtained from a sick monkey (Cebus olivaceus Schomburgk) collected in a forest near Atapirire, a small rural village located in Anzoategui State, Venezuela. MDDV is one of a growing number of naturally occurring OROV species reassortants isolated in South America and was known previously only from southern Peru.

  2. Isolation of Madre de Dios Virus (Orthobunyavirus; Bunyaviridae), an Oropouche Virus Species Reassortant, from a Monkey in Venezuela

    Science.gov (United States)

    Navarro, Juan-Carlos; Giambalvo, Dileyvic; Hernandez, Rosa; Auguste, Albert J.; Tesh, Robert B.; Weaver, Scott C.; Montañez, Humberto; Liria, Jonathan; Lima, Anderson; da Rosa, Jorge Fernando Soares Travassos; da Silva, Sandro P.; Vasconcelos, Janaina M.; Oliveira, Rodrigo; Vianez, João L. S. G.; Nunes, Marcio R. T.

    2016-01-01

    Oropouche virus (OROV), genus Orthobunyavirus, family Bunyaviridae, is an important cause of human illness in tropical South America. Herein, we report the isolation, complete genome sequence, genetic characterization, and phylogenetic analysis of an OROV species reassortant, Madre de Dios virus (MDDV), obtained from a sick monkey (Cebus olivaceus Schomburgk) collected in a forest near Atapirire, a small rural village located in Anzoategui State, Venezuela. MDDV is one of a growing number of naturally occurring OROV species reassortants isolated in South America and was known previously only from southern Peru. PMID:27215299

  3. Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient

    Science.gov (United States)

    Bordi, Licia; Castilletti, Concetta; Colavita, Francesca; Quartu, Serena; Nicastri, Emanuele; Lauria, Francesco Nicola; Petrosillo, Nicola; Lanini, Simone; Kobinger, Gary; Zumla, Alimuddin; Di Caro, Antonino; Ippolito, Giuseppe; Capobianchi, Maria Rosaria; Lalle, Eleonora

    2017-01-01

    An unprecedented Ebola virus (EBOV) epidemic occurred in 2013–2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW) infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA) and the presence of positive sense RNA (pos-RNA), including both replication intermediate (antigenomic-RNA) and messenger RNA (mRNA) molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI), Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract. PMID:28056096

  4. Genetic characterization and evolutionary analysis of Newcastle disease virus isolated from domestic duck in South Korea.

    Science.gov (United States)

    Gaikwad, Satish; Kim, Ji-Ye; Lee, Hyun-Jeong; Jung, Suk Chan; Choi, Kang-Seuk

    2016-03-15

    Domestic ducks are considered a potential reservoir of Newcastle disease virus. In the study, a Newcastle disease virus (NDV) isolated from a domestic duck during surveillance in South Korea was characterized. The complete genome of the NDV isolate was sequenced, and the phylogenetic relationship to reference strains was studied. Phylogenetic analysis revealed that the strain clustered in genotype I of Class II ND viruses, has highly phylogenetic similarity to NDV strains isolated from waterfowl in China, but was distant from the viruses isolated in chickens and vaccine strains used in South Korea. Pathogenicity experiment in chickens revealed it to be a lentogenic virus. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the avirulent motif (112)GKQGRL(117) at the cleavage site and caused no apparent disease in chickens and ducks. With phylogeographic analysis based on fusion gene, we estimate the origin of an ancestral virus of the isolate and its sister strain located in China around 1998. It highlights the need of continuous surveillance to enhance current understanding of the molecular epidemiology and evolution of the pathogenic strains.

  5. Characterization of Dak Nong virus, an insect nidovirus isolated from Culex mosquitoes in Vietnam.

    Science.gov (United States)

    Kuwata, Ryusei; Satho, Tomomitsu; Isawa, Haruhiko; Yen, Nguyen Thi; Phong, Tran Vu; Nga, Phan Thi; Kurashige, Tomokazu; Hiramatsu, Yukihiro; Fukumitsu, Yuki; Hoshino, Keita; Sasaki, Toshinori; Kobayashi, Mutsuo; Mizutani, Tetsuya; Sawabe, Kyoko

    2013-11-01

    In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

  6. Isolation and characterization of a virus infecting the freshwater algae Chrysochromulina parva

    Energy Technology Data Exchange (ETDEWEB)

    Mirza, S.F. [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Staniewski, M.A. [Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 (Canada); Short, C.M. [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Long, A.M. [Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 (Canada); Chaban, Y.V. [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Short, S.M., E-mail: steven.short@utoronto.ca [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 (Canada); Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 (Canada)

    2015-12-15

    Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145 nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485 kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva's abundance in Lake Ontario was relatively stable, yet CpV-BQ1's abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history. - Highlights: • A virus infecting the algae C. parva was isolated from Lake Ontario. • Virus characteristics demonstrated that this novel virus is an NCLDV. • The virus's polB sequence suggests taxonomic affiliation with the Phycodnaviridae. • The virus's capsid protein sequences also suggest Mimiviridae ancestry. • Surveys of host and virus natural abundances revealed complex host–virus dynamics.

  7. Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

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    Abdel-Moneim Ahmed S

    2012-08-01

    Full Text Available Abstract Background Avian influenza virus H9N2 is a panzootic pathogen that affects poultry causing mild to moderate respiratory distress but has been associated with high morbidity and considerable mortality. Interspecies transmission of H9N2 from avian species to mammalian hosts does occur. The virus possesses human virus-like receptor specificity and it can infect humans producing flu-like illness. Methods Recently, mild influenza like symptoms were detected in H5N1 vaccinated flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus evolution was investigated by sequencing the viral genes to screen the possible virus recombination. The viral amino acid sequences from the isolated H9N2 strains were compared to other related sequences from the flu data base that were used to assess the robustness of the mutation trend. Changes in the species-associated amino acid residues or those that enabled virulence to mammals were allocated. Results Phylogenetic analyses of haemagglutinin and neuraminidase genes showed that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that prevails in Israel. The six internal segments of the isolated virus were found to be derived from the same sub-lineage with no new evidence of reassortment. The results demonstrated conserved genetic and biological constitution of H9N2 viruses in the Middle East. The recently isolated H9N2 virus from chicken in Egypt possessed amino acids that could enable the virus to replicate in mammals and caused severe disease in domestic chickens. Conclusion The study highlights the importance of continuous monitoring of the mutations evolved in avian influenza viruses and its impact on virulence to avian species in addition to its importance in the emergence of new strains with the capacity to be a pandemic candidate.

  8. Sequence analysis of a soil-borne wheat mosaic virus isolate from Italy shows that it is the same virus as European wheat mosaic virus and Soil-borne rye mosaic virus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The complete sequence of the two RNAs of a furovirus isolate fromdurum wheat in Italy was determined. Sequence comparisons and phylogenetic analysis were done to compare the Italian virus with Soil-borne wheat mosaic virus (SBWMV) from the USA and with furovirus sequences recently published as European wheat mosaic virus (EWMV), from wheat in France, and Soil-borne rye mosaic virus (SBRMV), from rye and wheat in Germany. Over the entire genome, the Italian isolate RNA1 and RNA2 had respectively 97.5% and 98.6% nucleotide identity with EWMV, 95.5% and 85.8% with SBRMV-G and 70.6% and 64.5% with SBWMV. The Italian isolate was therefore clearly distinct from SBWMV. The European isolates all appear to belong to the same virus and the name Soil-borne cereal mosaic virus may resolve earlier ambiguities.

  9. CPm gene diversity in field isolates of Citrus tristeza virus from Colombia.

    Science.gov (United States)

    Oliveros-Garay, Oscar Arturo; Martinez-Salazar, Natalhie; Torres-Ruiz, Yanneth; Acosta, Orlando

    2009-01-01

    The nucleotide sequence diversity of the CPm gene from 28 field isolates of Citrus tristeza virus (CTV) was assessed by SSCP and sequence analyses. These isolates showed two major shared haplotypes, which differed in distribution: A1 was the major haplotype in 23 isolates from different geographic regions, whereas R1 was found in isolates from a discrete region. Phylogenetic reconstruction clustered A1 within an independent group, while R1 was grouped with mild isolates T30 from Florida and T385 from Spain. Some isolates contained several minor haplotypes, which were very similar to, and associated with, the major haplotype.

  10. Genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in Hokkaido, Japan.

    Science.gov (United States)

    Abe, Yuri; Tamura, Tomokazu; Torii, Shiho; Wakamori, Shiho; Nagai, Makoto; Mitsuhashi, Kazuya; Mine, Junki; Fujimoto, Yuri; Nagashima, Naofumi; Yoshino, Fumi; Sugita, Yukihiko; Nomura, Takushi; Okamatsu, Masatoshi; Kida, Hiroshi; Sakoda, Yoshihiro

    2016-01-01

    In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5'-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.

  11. Identification of hepatotropic viruses from plasma using deep sequencing: a next generation diagnostic tool.

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    John Law

    Full Text Available We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH, non-alcoholic steatohepatitis (NASH, and patients without liver disease (control. RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV infection that contained multiple sequences matching GB virus C (GBV-C. Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be

  12. The antigenic property of the H5N1 avian influenza viruses isolated in central China

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    Zou Wei

    2012-08-01

    Full Text Available Abstract Background Three influenza pandemics outbroke in the last century accompanied the viral antigen shift and drift, resulting in the change of antigenic property and the low cross protective ability of the existed antibody to the newly emerged pandemic virus, and eventually the death of millions of people. The antigenic characterizations of the viruses isolated in central China in 2004 and 2006–2007 were investigated in the present study. Results Hemagglutinin inhibition assay and neutralization assay displayed differential antigenic characteristics of the viruses isolated in central China in two periods (2004 and 2006–2007. HA genes of the viruses mainly located in two branches in phylogeny analysis. 53 mutations of the deduced amino acids of the HA genes were divided into 4 patterns. Mutations in pattern 2 and 3 showed the main difference between viruses isolated in 2004 and 2006–2007. Meanwhile, most amino acids in pattern 2 and 3 located in the globular head of the HA protein, and some of the mutations evenly distributed at the epitope sites. Conclusions The study demonstrated that a major antigenic drift had occurred in the viruses isolated in central China. And monitoring the antigenic property should be the priority in preventing the potential pandemic of H5N1 avian influenza virus.

  13. Isolation, Identification and Molecular Characterization of Highly Pathogenic Newcastle Disease Virus From Field Outbreaks

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    Asma Ashraf

    Full Text Available ABSTRACT Newcastle disease (ND is a major infectious disease of the poultry caused by a virulent strain of Avian Paramyxovirus - 1, that is a single strand non-segmented negative sense RNA virus. ND virus is major threat to the poultry industry in many countries of the world. The study was aimed to isolate and identify Newcastle disease virus (NDV by using a haemagglutination inhibition (HI test and reverse transcription-polymerase chain reaction (RT-PCR assay. A total 100 samples of infected and dead birds were collected from different poultry farms. The weight of the birds was ranged 1000-1200g. The birds were divided into 3 groups. Haemagglutination assay (HA was performed to detect the presence of NDV in suspension of infected homogenized tissues and it was found that HA is not the best method to detect the virus when it is in trace amounts. RT-PCR using NDV specific primers analyzed different clinical and postmortem samples. Reverse transcriptase polymerase chain reaction and specific primers was used for determining the presence of viruses. It was found that the virus was present in most of the infected samples except the serum of infected birds. During multiple sequence alignment (MSA it was found that, our isolates have high homology (98% with other reported NDV isolates. Phylogenetic analysis revealed that our isolate was closely related with viscerotropic velogenic types of NDV, which are highly pathogenic Newcastle disease virus.

  14. Genomic 3' terminal sequence comparison of three isolates of rabbit haemorrhagic disease virus.

    Science.gov (United States)

    Milton, I D; Vlasak, R; Nowotny, N; Rodak, L; Carter, M J

    1992-05-15

    Comparison of sequence data is necessary in older to investigate virus origins, identify features common to virulent strains, and characterize genomic organization within virus families. A virulent caliciviral disease of rabbits recently emerged in China. We have sequenced 1100 bases from the 3' ends of two independent European isolates of this virus, and compared these with previously determined calicivirus sequences. Rabbit caliciviruses were closely related, despite the different countries in which isolation was made. This supports the rapid spread of a new virus across Europe. The capsid protein sequences of these rabbit viruses differ markedly from those determined for feline calicivirus, but a hypothetical 3' open reading frame is relatively well conserved between the caliciviruses of these two different hosts and argues for a functional role.

  15. Genetic characterization of a noncytopathic bovine viral diarrhea virus 2b isolated from cattle in China.

    Science.gov (United States)

    Wang, Wei; Shi, Xinchuan; Chen, Chaoyang; Wu, Hua

    2014-10-01

    In January 2013, several clinical signs of cattle with diarrhea, cough, nasal discharge, and fever were reported in Jilin province, China. One virus named SD1301 was isolated and identified. Complete genome of the virus is 12258nt in length and contains a 5'UTR, one open reading frame encoding a polyprotein of 3,897 amino acids and a 3'UTR. Phylogenetic analysis of 5'UTR, N(pro), E1 and E2 gene demonstrated the virus belonged to BVDV 2b, and genetically related to the BVDV strain Hokudai-Lab/09 from Japan in 2010. This bovine viral diarrhea virus displays a unique genetic signature with 27-nucleotide deletion in the 5'UTR, which is similar to the bovine viral diarrhea virus C413 (AF002227). This was the first confirmed isolation of ncp BVDV2b circulating in bovine herd of China.

  16. [Genome sequencing and analysis of a peste des petits ruminants virus isolate, China/Tib/07].

    Science.gov (United States)

    Liu, Wen-Hua; Bao, Jing-Yue; Wu, Xiao-Dong; Wang, Zhi-Liang

    2010-07-01

    Peste des petits ruminants virus is a member of Morbillivirus Paramyxoviridae. The complete genome of a Peste des petits ruminants virus (PPRV) isolate, China/Tib/07 was sequenced and molecular characteristics was analyzed. The internal sequences of the virus genome were amplified by RT-PCR with primers designed according to the published data in GenBank, while the sequences of the 3' and 5' ends of the genome were determined by RACE. Amplification products were directly sequenced,assembled and analyzed with DNAStar4.0. Results showed that China/Tib/07 genome consisted of 15 948 nucleotides in length, encoding six structural proteins and two non-structural proteins just like other known PPRV genomes. Phylogenetically, the virus genome shared homology of 91.6%-98.1% with Southwest Asian isolates among PPRV strains and the highest homology of 64.3% with rinderpest virus among morbillivirus members.

  17. Phylogenetic analysis of recent isolates of classical swine fever virus from Colombia.

    Science.gov (United States)

    Sabogal, Zonia Yubyll; Mogollón, José Darío; Rincón, Maria Antonia; Clavijo, Alfonso

    2006-01-01

    The ability to discriminate between different classical Swine fever virus (CSFV) isolates is a prerequisite for identifying the possible origin of an outbreak. To determine the relatedness between Colombian isolates from different geographical regions, genetic sequences of the glycoprotein E2 and the 5'UTR of CSFV were amplified by PCR, sequenced and compared with reference strains of different genetic grouping. The viruses originated from classical swine fever (CSF) outbreaks in Colombia during 1998-2002. All viruses characterized belonged to genogroup 1 and were members of the subgroup 1.1. The results indicate that the outbreaks from the year 2002 are caused by a strain related to the virus CSF/Santander, isolated in 1980, suggesting that the current CSF outbreaks are the consequence of a single strain that continues to circulate in the field. For the first time, an association between isolates from outbreaks in Colombia in the 1990s was established with a virus isolate from Brazil, indicating a possible origin of the virus causing the outbreak.

  18. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses.

    Science.gov (United States)

    Kuno, G; Gubler, D J; Vélez, M; Oliver, A

    1985-01-01

    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability.

  19. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses*

    Science.gov (United States)

    Kuno, G.; Gubler, D. J.; Vélez, M.; Oliver, A.

    1985-01-01

    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability. PMID:2861916

  20. Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil.

    Science.gov (United States)

    Pauvolid-Corrêa, Alex; Solberg, Owen; Couto-Lima, Dinair; Kenney, Joan; Serra-Freire, Nicolau; Brault, Aaron; Nogueira, Rita; Langevin, Stanley; Komar, Nicholas

    2015-01-01

    We describe the isolation of a novel flavivirus, isolated from a pool of mosquitoes identified as Culex (Culex) chidesteri collected in 2010 in the Pantanal region of west-central Brazil. The virus is herein designated Nhumirim virus (NHUV) after the name of the ranch from which the mosquito pool was collected. Flavivirus RNA was detected by real-time RT-PCR of homogenized mosquitoes and from the corresponding C6/36 culture supernatant. Based on full-genome sequencing, the virus isolate was genetically distinct from but most closely related to Barkedji virus (BJV), a newly described flavivirus from Senegal. Phylogenetic analysis demonstrated that NHUV grouped with mosquito-borne flaviviruses forming a clade with BJV. This clade may be genetically intermediate between the Culex-borne flaviviruses amplified by birds and the insect-only flaviviruses.

  1. Biological characterization and complete nucleotide sequence of a Tunisian isolate of Moroccan watermelon mosaic virus.

    Science.gov (United States)

    Yakoubi, S; Desbiez, C; Fakhfakh, H; Wipf-Scheibel, C; Marrakchi, M; Lecoq, H

    2008-01-01

    During a survey conducted in October 2005, cucurbit leaf samples showing virus-like symptoms were collected from the major cucurbit-growing areas in Tunisia. DAS-ELISA showed the presence of Moroccan watermelon mosaic virus (MWMV, Potyvirus), detected for the first time in Tunisia, in samples from the region of Cap Bon (Northern Tunisia). MWMV isolate TN05-76 (MWMV-Tn) was characterized biologically and its full-length genome sequence was established. MWMV-Tn was found to have biological properties similar to those reported for the MWMV type strain from Morocco. Phylogenetic analysis including the comparison of complete amino-acid sequences of 42 potyviruses confirmed that MWMV-Tn is related (65% amino-acid sequence identity) to Papaya ringspot virus (PRSV) isolates but is a member of a distinct virus species. Sequence analysis on parts of the CP gene of MWMV isolates from different geographical origins revealed some geographic structure of MWMV variability, with three different clusters: one cluster including isolates from the Mediterranean region, a second including isolates from western and central Africa, and a third one including isolates from the southern part of Africa. A significant correlation was observed between geographic and genetic distances between isolates. Isolates from countries in the Mediterranean region where MWMV has recently emerged (France, Spain, Portugal) have highly conserved sequences, suggesting that they may have a common and recent origin. MWMV from Sudan, a highly divergent variant, may be considered an evolutionary intermediate between MWMV and PRSV.

  2. Complete Genome Sequence of a Tomato Isolate of Parietaria Mottle Virus from Italy.

    Science.gov (United States)

    Martínez, Carolina; Aramburu, José; Rubio, Luis; Galipienso, Luis

    2015-12-17

    We report here the complete genome sequence of isolate T32 of parietaria mottle virus (PMoV) infecting tomato plants in Turin, Italy, obtained by Sanger sequencing. T32 shares 90.48 to 96.69% nucleotide identity with other two PoMV isolates, CR8 and Pe1, respectively, whose complete genome sequences are available.

  3. Genetic detection and isolation of crimean-congo hemorrhagic fever virus, Kosovo, Yugoslavia.

    Science.gov (United States)

    Papa, Anna; Bozovi, Bojana; Pavlidou, Vassiliki; Papadimitriou, Evangelia; Pelemis, Mijomir; Antoniadis, Aantonis

    2002-08-01

    Crimean-Congo hemorrhagic fever virus (C-CHFV) strains were isolated from a fatal case and the attending physician in Kosovo, Yugoslavia. Early, rapid diagnosis of the disease was achieved by reverse transcription-polymerase chain reaction. The physician was successfully treated with oral ribavirin. These cases yielded the first genetically studied C-CHFV human isolates in the Balkans.

  4. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.

    Science.gov (United States)

    Kalra, Hina; Adda, Christopher G; Liem, Michael; Ang, Ching-Seng; Mechler, Adam; Simpson, Richard J; Hulett, Mark D; Mathivanan, Suresh

    2013-11-01

    Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.

  5. Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal

    Science.gov (United States)

    Karlsson, Erik A.; Ip, Hon S.; Hall, Jeffrey S.; Yoon, Sun W.; Johnson, Jordan; Beck, Melinda A.; Webby, Richard J.; Schultz-Cherry, Stacey

    2014-01-01

    The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells. Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

  6. Genetic diversity and relationships among Venezuelan equine encephalitis virus field isolates from Colombia and Venezuela.

    Science.gov (United States)

    Moncayo, A C; Medina, G M; Kalvatchev, Z; Brault, A C; Barrera, R; Boshell, J; Ferro, C; Freier, J E; Navarro, J C; Salas, R; De Siger, J; Vasquez, C; Walder, R; Weaver, S C

    2001-12-01

    During field studies of enzootic Venezuelan equine encephalitis (VEE) viruses associated with epizootic emergence, a large number of virus isolates were made in sylvatic foci of Venezuela and Colombia. To rapidly characterize these isolates, antigenic subtypes were determined by means of immunofluorescence and by single-strand conformational polymorphism (SSCP) analysis by use of an 856-bp fragment from the P62 gene, which we used to distinguish genetic variants. Representative isolates were sequenced to assess the sensitivity of SSCP to detect genetic differences. The SSCP analysis distinguished isolates differing by as little as 1 nucleotide; overall, differences of > or = 1 nucleotide were recognized 89% of the time, and the sensitivity to distinguish strains that differed by only 1 or 4 nucleotides was 17 and 57%, respectively. Phylogenetic analyses of representative sequences showed that all recent isolates from the Catatumbo region of western Venezuela and the middle Magdalena Valley of Colombia were closely related to epizootic subtype IAB and IC strains; strains from Yaracuy and Miranda States were more distantly related. Cocirculation of the same virus genotype in both Colombian and Venezuelan foci indicated that these viruses are readily transported between enzootic regions separated by > 300 km. The SSCP analysis appears to be a simple, fast, and relatively efficient method of screening VEE virus isolates to identify meaningful genetic variants.

  7. Complete nucleotide sequence analysis of a Dengue-1 virus isolated on Easter Island, Chile.

    Science.gov (United States)

    Cáceres, C; Yung, V; Araya, P; Tognarelli, J; Villagra, E; Vera, L; Fernández, J

    2008-01-01

    Dengue-1 viruses responsible for the dengue fever outbreak in Easter Island in 2002 were isolated from acute-phase sera of dengue fever patients. In order to analyze the complete genome sequence, we designed primers to amplify contiguous segments across the entire sequence of the viral genome. RT-PCR products obtained were cloned, and complete nucleotide and deduced amino acid sequences were determined. This report constitutes the first complete genetic characterization of a DENV-1 isolate from Chile. Phylogenetic analysis shows that an Easter Island isolate is most closely related to Pacific DENV-1 genotype IV viruses.

  8. Molecular characterisation of Newcastle disease virus isolates from different geographical regions in Mozambique in 2005

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    Raul Fringe

    2012-02-01

    Full Text Available Newcastle disease (ND is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. Viral determinants for Newcastle disease virus (NDV virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. The prevalence of Newcastle disease in village poultry in Mozambique is not well documented and strains of NDV involved in yearly outbreaks are unknown. The fusion (F protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. Newcastle disease viruses from various geographical regions of Mozambique were sequenced and compared genetically to published sequences obtained from GenBank. Samples were collected in three different areas of Mozambique and NDV was isolated by infection of embryonated chicken eggs. Sequence analysis of the F-protein encoding gene was used to classify 28 isolates from Mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in GenBank. The isolates obtained from Mozambique grouped mainly into two clades. In the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from Mozambique that were previously described. In the second clade, 16 isolates group together with sequences obtained from GenBank originating from Australia, China, South Africa and the USA. Eleven of these isolates showed a high similarity with sequences from South Africa. The number of samples sequenced (n = 28, as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in Mozambique in 2005. Viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. From this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in

  9. Molecular characterisation of Newcastle disease virus isolates from different geographical regions in Mozambique in 2005.

    Science.gov (United States)

    Fringe, Raul; Bosman, Anna-Mari; Ebersohn, Karen; Bisschop, Shahn; Abolnik, Celia; Venter, Estelle

    2012-08-31

    Newcastle disease (ND) is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. Viral determinants for Newcastle disease virus (NDV) virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. The prevalence of Newcastle disease in village poultry in Mozambique is not well documented and strains of NDV involved in yearly outbreaks are unknown. The fusion (F) protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. Newcastle disease viruses from various geographical regions of Mozambique were sequenced and compared genetically to published sequences obtained from GenBank. Samples were collected in three different areas of Mozambique and NDV was isolated by infection of embryonated chicken eggs. Sequence analysis of the F-protein encoding gene was used to classify 28 isolates from Mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in GenBank. The isolates obtained from Mozambique grouped mainly into two clades. In the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from Mozambique that were previously described. In the second clade, 16 isolates group together with sequences obtained from GenBank originating from Australia, China, South Africa and the USA. Eleven of these isolates showed a high similarity with sequences from South Africa. The number of samples sequenced (n = 28), as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in Mozambique in 2005. Viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. From this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in Mozambique.

  10. Characterization of West Nile viruses isolated from captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia.

    Science.gov (United States)

    Osorio, Jorge E; Ciuoderis, Karl A; Lopera, Juan G; Piedrahita, Leidy D; Murphy, Darby; Levasseur, James; Carrillo, Lina; Ocampo, Martha C; Hofmeister, Erik

    2012-09-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

  11. Characterization of West Nile viruses isolated form captive American flamingoes (Phoenicopterus ruber) in Medellin, Colombia.

    Science.gov (United States)

    Osorio, Jorge E.; Ciuoderis, Karl A.; Lopera, Juan G.; Piedrahita, Leidy D.; Murphy, Darby; LeVasseur, James; Carrillo, Lina; Ocampo, Martha C.; Hofmeister, Erik

    2012-01-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

  12. GENOTYPING OF ISOLATED VIRUSES FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS IN CROATIA

    Directory of Open Access Journals (Sweden)

    Irena Vardić

    2007-07-01

    Full Text Available Infectious hematopoietic necrosis virus (IHNV and infectious pancreatic necrosis virus (IPNV are important pathogens in rainbow trout aquaculture. Detection of these viruses in Croatia initiated investigation of their genetic relatedness to the worldwide IHNV and IPNV isolates. For this purpose, determination of nucleotide sequences of G and NV genes for IHNV and VP2/NS region for IPNV was performed. Phylogenetic analyses revealed that Croatian IHNV isolate was clustering within European clade most closely related to the North American M genogroup. Croatian IPNV isolate appeared in the cluster of genogroup III, together with French, English, Danish and Norwegian isolates. These results are important for further epidemiological studies of IHNV and IPNV outbreaks in Croatia.

  13. Construction and identification of reverse genetics system of Dengue type 2 virus isolated in China

    Institute of Scientific and Technical Information of China (English)

    ZHU Wuyang; CHEN Shuiping; QIN Chenggeng; YU Man; JIANG Tao; DENG Yongqiang; QIN Ede

    2006-01-01

    To construct infectious full-length cDNA clone of dengue virus type 2 isolated in China (DEN2-43), according to the published nucleotide sequence of the virus strain, the approximately 11 kb full-length cDNAs of DEN2-43 were amplified by long RT-PCR and fusion PCR. Full-length cDNA clones were constructed by inserting the full-length cDNA into a low copy vector pWSK29, from which rescued virus D212 was acquired by transcription in vitro and electroporation. The full-length cDNA clone pD212 was infectious, and rescued virus acquired in C6/36 cells was indistinguishable from DEN2-43 virus in biological properties including suckling mice neurovirulence. The reverse genetics system helps elucidate the mechanism of pathogenesis of dengue virus and develop novel vaccine against dengue.

  14. Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    OpenAIRE

    Nishizawa, T; Kinoshita, S; Kim, W. -S.; Higashi, S.; Yoshimizu, M

    2006-01-01

    Infectious hematopoietic necrosis virus (IHNV), a member of the genus Novirhabdovirus, causes a highly lethal disease of salmonid fish. In the present study, G gene nucleotide sequences of 9 Japanese IHNV isolates obtained from 1971 to 1996 were analyzed to evaluate the genetic diversity and compared with IHNV isolates from North America and Europe. A radial phylogenetic tree revealed 5 major clusters including 3 genogroups (U, M and L) for North American isolates and 1 genogroup for European...

  15. The spectrum of viruses isolated from Pulsatilla pratensis (Ranunculaceae a native plant of Ukraine

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    О. А. Shydlovska

    2016-03-01

    Full Text Available The article is devoted to virus screening of wild plants of Ukraine’s flora. The object of the research is the Red Book plant Pulsatilla pratensis (L. Mill., which grows on the territory of Kanev Nature Reserve. Isolated isometric infectious virus-like particles with diameters of 34, 36, 43, 47, 50 and 57 nm were isolated from selected plants of P. pratensis. In our research, determination of the infectious nature of the pathogen, host range, concentration of viruses in plants, species identity and virus isolation from the mixture in mixed viral infections were carried with using indicator plants. The typical viral symptoms were observed on indicator plants: browning of the leaf plate, mottling, chlorosis and necrosis. All symptoms were systemic and could be caused by a variety of viruse species. Virions with sizes from 34 to 43 nm produced the necrotic and chlorotic spotting on Chenopodium amaranticolor Coste and Reyn. On the other hand, virions with sizes from 47 to 57 nm produced the necrosis, chlorosis and deformation of the leaf plates on Cucumis sativus L. That is not typical for viruses previously discovered on P. pratensis. The viruses isolated in these plants viruses were cumulated in small concentrations and rapidly lost their infectivity. The number of isolated viruses was insufficient for their identification. Four bacteriophage isolates with long phage tails of different size were isolated from P. pratensis roots and radical soil. The biological (lytic activity towards the tracer bacteria, the morphology of negative colonies, and bacteriophage protein structure were characterized. According to our research, it is possible to divide phages into three subgroups that probably correspond to three different types of viruses. Results of the polypeptide analysis may reflect an evolutionary process in a population of phages that had a common ancestor. Comparison of phage proteins of different hosts shows a variety of molecular weights of

  16. Molecular characterization of Belgian pseudorabies virus isolates from domestic swine and wild boar.

    Science.gov (United States)

    Verpoest, Sara; Cay, Ann Brigitte; De Regge, Nick

    2014-08-06

    Aujeszky's disease is an economically important disease in domestic swine caused by suid herpesvirus 1, also called pseudorabies virus (PRV). In several European countries, including Belgium, the virus has successfully been eradicated from the domestic swine population. The presence of PRV in the wild boar population however poses a risk for possible reintroduction of the virus into the domestic pig population. It is therefore important to assess the genetic relatedness between circulating strains and possible epidemiological links. In this study, nine historical Belgian domestic swine isolates that circulated before 1990 and five recent wild boar isolates obtained since 2006 from Belgium and the Grand Duchy of Luxembourg were genetically characterized by restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis. While all wild boar isolates were characterized as type I RFLP genotypes, the RFLP patterns of the domestic swine isolates suggest that a shift from genotype I to genotype II might have occurred in the 1980s in the domestic population. By phylogenetic analysis, Belgian wild boar isolates belonging to both clade A and B were observed, while all domestic swine isolates clustered within clade A. The joint phylogenetic analysis of both wild boar and domestic swine strains showed that some isolates with identical sequences were present within both populations, raising the question whether these strains represent an increased risk for reintroduction of the virus into the domestic population.

  17. Characterization of influenza A viruses isolated from wild waterfowl in Zambia.

    Science.gov (United States)

    Simulundu, Edgar; Ishii, Akihiro; Igarashi, Manabu; Mweene, Aaron S; Suzuki, Yuka; Hang'ombe, Bernard M; Namangala, Boniface; Moonga, Ladslav; Manzoor, Rashid; Ito, Kimihito; Nakamura, Ichiro; Sawa, Hirofumi; Sugimoto, Chihiro; Kida, Hiroshi; Simukonda, Chuma; Chansa, Wilbroad; Chulu, Jack; Takada, Ayato

    2011-06-01

    Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008-2009), 12 viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowl. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. Whilst some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the inter-regional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harboured particular residues predominantly observed in human influenza viruses. Interestingly, the isolates with human-associated residues exhibited higher levels of virus replication in the lungs of infected mice and caused more morbidity as measured by weight loss than an isolate lacking such residues. This study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to gain a better understanding of the emergence of strains with the potential to infect mammals.

  18. Genetic characterization and pathogenicity assessment of Newcastle disease virus isolated from wild peacock.

    Science.gov (United States)

    Khulape, Sagar A; Gaikwad, Satish S; Chellappa, Madhan Mohan; Mishra, Bishnu Prasad; Dey, Sohini

    2014-12-01

    The continued spread and occurrence of Newcastle disease virus (NDV) has posed potential threat to domestic poultry industry around the globe. Mainly, wild avian species has always been implicated for the natural reservoir for virus and spread of the disease. In the present study, we report the isolation of Newcastle disease virus (NDV/Peacock/India/2012) in necropsy brain tissue sample of wild peacock from North India. Complete genome of the virus was found to be 15,186 nucleotides (nts) with six genes in order of 3'-N-P-M-F-HN-L-5', which was limited by 55-nts leader region at the 3' end and a 114-nts trailer sequence at 5' end. Sequence analysis of fusion protein revealed the dibasic amino acid cleavage site (112)R-R-Q-K-R-F(117), a characteristic motif of virulent virus. Phylogenetic analysis placed the isolate in genotype II of Newcastle disease virus showing the lowest mean percent divergence (6 %) with other genotype II counterparts. The isolate was characterized as mesogenic (intermediate pathotype) based on the mean death time (63 h) in embryonated chicken eggs and the intra-cerebral pathogenicity index (1.40) in day-old chicks. The report emphasizes the dynamic ecology of NDV strains circulating in a wild avian host during the outbreak of 2012 in North India. Further the genotypic and pathotypical characterizations of the isolate could help in development of homologous vaccine against NDV strain circulating in avian population.

  19. Characterization of influenza A (H7N9 viruses isolated from human cases imported into Taiwan.

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    Ji-Rong Yang

    Full Text Available A novel avian influenza A (H7N9 virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave. The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses.

  20. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts, progress report

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P L

    1993-01-01

    Our goal is to provide a mechanistic understanding of the cellular and molecular aspects of freezing injury and cold acclimation from a perspective of the structural and functional integrity of the plasma membrane -- the primary site of freezing injury in winter cereals. We have utilized protoplasts isolated from leaves of winter rye (Secale cereale L. cv Puma) to study the cryobehavior of the plasma membrane during a freeze/thaw cycle. The focus of our current studies is on lesions in the plasma membrane that result from severe freeze-induced dehydration and result in the alteration of the semipermeable characteristics of the plasma membrane so that the protoplasts are osmotically unresponsive. In protoplasts isolated from non-acclimated rye leaves (NA protoplasts), injury is associated with the formation of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal II phase transitions in the plasma membrane and the subtending lamellae. However, lamellar-to-hexagonal II phase transitions are not observed following severe dehydration of protoplasts isolated from cold-acclimated rye leaves (ACC protoplasts). Rather, injury is associated with the fracture-jump lesion,'' which, in freeze-fracture electron microscopy studies, is manifested as localized deviations in the fracture face of the plasma membrane. The fracture plane jumps'' from the plasma membrane to either subtending aparticulate lamellae or aparticulate regions of various endomembranes (predominantly chloroplast envelopes) that are in close apposition with the plasma membrane.

  1. Molecular Characteristics of S1 Gene of Infectious Bronchitis Virus Isolated from Chicken Proventriculus

    Institute of Scientific and Technical Information of China (English)

    CHENG Li-qin; ZHOU Ji-yong; John Dikki; SHEN Xing-yan; CHEN Ji-gang; ZHANG De-yong

    2003-01-01

    Infectious bronchitis virus was isolated from swollen proventriculi of clinically ill chicken. Thesuspected virus samples (2/97, 3/97, 1/98) were adapted in SPF chicken embryos for virus isolation andidentification. All the virus isolates were able to agglutinate chicken erythrocytes after treatment with trypsin,and interfer with the reproduction of Newcastle disease virus in chicken embryos, and have low antigenic relat-edness values with reference positive IBV. The isolates 2/97, 3/97, 1/98 RNAs extracted from the allantoicfluid of inoculated embryonated eggs were converted to cDNA by reverse transcription with 3'-primer of S1gene of (IBV). Polymerase chain reaction (PCR) was performed with two primers which span the S1 gene.Amplified product of 1.93 kb was subjected to EcoR Ⅰ and BamH Ⅰ digestion and the fragments obtainedwere the same as expected size. The PCR product was ligated to pBlueScript-SK (+) vector, and its nucleotidesequence was determined by the dideoxy-mediated chain termination method. Nucleotide sequence analysisshowed 73.6 - 99.7 % homology between the isolated IBV and the IBV strains in GenBank. The homology ofamino acid was 71.4 - 99.4 %.

  2. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

    Science.gov (United States)

    2016-01-01

    Avian leukosis virus (ALV) belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate. PMID:27597865

  3. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

    Directory of Open Access Journals (Sweden)

    Faruku Bande

    2016-01-01

    Full Text Available Avian leukosis virus (ALV belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate.

  4. Genetic diversity of Japanese encephalitis virus isolates obtained from the Indonesian archipelago between 1974 and 1987.

    Science.gov (United States)

    Schuh, Amy J; Guzman, Hilda; Tesh, Robert B; Barrett, Alan D T

    2013-07-01

    Five genotypes (GI-V) of Japanese encephalitis virus (JEV) have been identified, all of which have distinct geographical distributions and epidemiologies. It is thought that JEV originated in the Indonesia-Malaysia region from an ancestral virus. From that ancestral virus GV diverged, followed by GIV, GIII, GII, and GI. Genotype IV appears to be confined to the Indonesia-Malaysia region, as GIV has been isolated in Indonesia from mosquitoes only, while GV has been isolated on three occasions only from a human in Malaysia and mosquitoes in China and South Korea. In contrast, GI-III viruses have been isolated throughout Asia and Australasia from a variety of hosts. Prior to this study only 13 JEV isolates collected from the Indonesian archipelago had been studied genetically. Therefore the sequences of the envelope (E) gene of 24 additional Indonesian JEV isolates, collected throughout the archipelago between 1974 and 1987, were determined and a series of molecular adaptation analyses were performed. Phylogenetic analysis indicated that over a 14-year time span three genotypes of JEV circulated throughout Indonesia, and a statistically significant association between the year of virus collection and genotype was revealed: isolates collected between 1974 and 1980 belonged to GII, isolates collected between 1980 and 1981 belonged to GIV, and isolates collected in 1987 belonged to GIII. Interestingly, three of the GII Indonesian isolates grouped with an isolate that was collected during the JE outbreak that occurred in Australia in 1995, two of the GIII Indonesian isolates were closely related to a Japanese isolate collected 40 years previously, and two Javanese GIV isolates possessed six amino acid substitutions within the E protein when compared to a previously sequenced GIV isolate collected in Flores. Several amino acids within the E protein of the Indonesian isolates were found to be under directional evolution and/or co-evolution. Conceivably, the tropical climate

  5. Plasma Epstein–Barr virus and Hepatitis B virus in non-Hodgkin lymphomas: Two lymphotropic, potentially oncogenic, latently occurring DNA viruses

    Directory of Open Access Journals (Sweden)

    Mahua Sinha

    2016-01-01

    Full Text Available Context: There is a need to study potential infective etiologies in lymphomas. Lymphocyte-transforming viruses can directly infect lymphocytes, disrupt normal cell functions, and promote cell division. Epstein–Barr virus (EBV is known to be associated with several lymphomas, especially Hodgkin lymphomas (HLs. And recently, the lymphocyte-transforming role of hepatitis B virus (HBV has been emphasized. Aims: The aim of this study was to elucidate the association of two potentially oncogenic, widely prevalent latent DNA viruses, EBV and HBV, in non-HL (NHL. Settings and Design: In this prospective study, we estimated plasma EBV and HBV DNA in NHL patients. Materials and Methods: Peripheral blood was obtained from newly diagnosed, treatment na ïve, histologically confirmed NHL patients. Plasma EBV DNA was quantified by real-time polymerase chain reaction (PCR targeting Epstein–Barr Nucleic acid 1 while the plasma HBV DNA was detected using nested PCR targeting HBX gene. In a small subset of patients, follow-up plasma samples post-anticancer chemotherapy were available and retested for viral DNA. Results: Of the 110 NHL patients, ~79% were B-cell NHL and ~21% were T-cell NHL. Plasma EBV-DNA was detected in 10% NHLs with a higher EBV association in Burkitt lymphoma (33.3% than other subtypes. Pretherapy HBV DNA was detected in 21% NHLs; most of them being diffuse large B-cell lymphoma (DLBCL. Moreover, 42% of DLBCL patients had HBV DNA in plasma. Since all patients were HBV surface antigen seronegative at diagnosis, baseline plasma HBV-DNAemia before chemotherapy was indicative of occult hepatitis B infection. Conclusions: Our findings indicate a significant association of HBV with newly diagnosed DLBCL.

  6. Receptor specificity and erythrocyte binding preferences of avian influenza viruses isolated from India

    Directory of Open Access Journals (Sweden)

    Pawar Shailesh D

    2012-10-01

    Full Text Available Abstract Introduction Hemagglutination (HA and hemagglutination inhibition (HI assays are conventionally used for detection and identification of influenza viruses. HI assay is also used for detection of antibodies against influenza viruses. Primarily turkey or chicken erythrocytes [red blood cells (RBCs] are used in these assays, as they are large, nucleated, and sediment fast, which makes it easy to determine the titer. Human influenza viruses agglutinate RBCs from chicken, human, and guinea pig, but not from horse. Human influenza viruses bind preferentially to sialic acid (SA linked to galactose (Gal by α 2, 6 linkage (SA α 2, 6-Gal, whereas avian influenza (AI viruses bind preferentially to SA α 2, 3-Gal linkages. With this background, the present study was undertaken to study erythrocyte binding preferences and receptor specificities of AI viruses isolated from India. Materials and methods A total of nine AI virus isolates (four subtypes from India and three reference AI strains (three subtypes were tested in HA and HI assays against mammalian and avian erythrocytes. The erythrocytes from turkey, chicken, goose, guinea pig and horse were used in the study. The receptor specificity determination assays were performed using goose and turkey RBCs. The amino acids present at 190 helix, 130 and 220 loops of the receptor-binding domain of the hemagglutinin protein were analyzed to correlate amino acid changes with the receptor specificity. Results All tested highly pathogenic avian influenza (HPAI H5N1 viruses reacted with all five types of RBCs in the HA assay; AI H9N2 and H5N2 viruses did not react with horse RBCs. For H5N1 viruses guinea pig and goose RBCs were best for both HA and HI assays. For H9N2 viruses, guinea pig, fowl and turkey RBCs were suitable. For other tested AI subtypes, avian and guinea pig RBCs were better. Eight isolates of H5N1, one H4N6 and one H7N1 virus showed preference to avian sialic acid receptors. Importantly

  7. Nucleotide sequencing and serologic analysis of Cache Valley virus isolates from the Yucatan Peninsula of Mexico.

    Science.gov (United States)

    Blitvich, Bradley J; Loroño-Pino, Maria A; Garcia-Rejon, Julian E; Farfan-Ale, Jose A; Dorman, Karin S

    2012-08-01

    Nucleotide sequencing was performed on part of the medium and large genome segments of 17 Cache Valley virus (CVV) isolates from the Yucatan Peninsula of Mexico. Alignment of these sequences to all other sequences in the Genbank database revealed that they have greatest nucleotide identity (97-98 %) with the equivalent regions of Tlacotalpan virus (TLAV), which is considered to be a variety of CVV. Next, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice that had been inoculated with a representative isolate from the Yucatan Peninsula (CVV-478) or the prototype TLAV isolate (61-D-240). The PRNT titers exhibited a twofold difference in one direction and no difference in the other direction suggesting that CVV-478 and 61-D-240 belong to the same CVV subtype. In conclusion, we demonstrate that the CVV isolates from the Yucatan Peninsula of Mexico are genetically and antigenically similar to the prototype TLAV isolate.

  8. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen;

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membran...

  9. IDENTIFICATION AND EFFECTS OF MIXED INFECTION OF Potyvirus ISOLATES WITH Cucumber mosaic virus IN CUCURBITS

    Directory of Open Access Journals (Sweden)

    GRAZIELA DA SILVA BARBOSA

    2016-01-01

    Full Text Available Mixed infections in cucurbits are frequently observed in natural conditions between viruses from the Potyvirus genus and Cucumber mosaic virus (CMV, which significantly decreases productivity. The objectives of the present study was to compare the host range of PRSV - W, WMV, and ZYMV isolates and evaluate the effects of mixed infections with CMV in zucchini plants ( Cucurbita pepo L.. Host range studies comprising 23 plant species confirmed some similarities and biological differences among the isolates of PRSV - W, ZYMV, and WMV. RT - PCR confirmed the amplification of DNA fragments of the PRSV - W, WMV, and ZYMV coat protein gene ( cp and cytoplasm inclusion gene ( ci . The virus interaction studies in zucchini Caserta plants indicated synergistic interactions, particularly among species from the Potyvirus genus, and some CMV interference with some virus combinations.

  10. Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate.

    Science.gov (United States)

    Benchimol, Marlene; Chang, T-H; Alderete, John F

    2002-10-08

    Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.

  11. Isolation of Hepatitis C Virus in Norjizac Vials

    Directory of Open Access Journals (Sweden)

    Zary Nokhodian

    2010-01-01

    locations within four months, were tested for HCV. Two-hundred micro-liters from the solution in each vial were obtained and using a high pure viral nucleic acid kit (Roche Diagnostics GmbH, Germany, RNA extraction was done. Thereafter, using Moloney Murine Leukemia Virus (M-MuLV reverse transcriptase (Fermentas, Life Science, C-DNA was produced. Using specific primers and a probe for the detection of HCV (Tagman probe, PCR was done (Corrbette Research 6000. All laboratory staff was unaware of the nature of the drug under analysis. Overall, two (14%; 95% confidence interval [CI]: 0%-32% of the 14 tested vials were found positive for HCV with a viral load >104/mL. We found that Norjizak, itself, can be contaminated with HCV and that avoiding shared needles and syringes is not enough for being infected by HCV in Norjizak abusers. The same thing may be true for other countries that manufacture this drug.Although conduction of "harm reduction programs," has resulted in significant decrease in preventing HIV infection among IDUs, the prevalence and incidence of hepatitis C still remains high among this group (12-14. One of the most important points about the survival of HCV is its prolonged stay in the environment. For instance, HCV RNA has been found to be stable in plasma or serum at 40 ºC for seven days (15. It is believed that HCV can live outside the body for a long time-estimates vary from 7-10 days to weeks or months (16. Hepatitis C is common among IDUs and needs longterm and expensive treatment courses. In Iran, establishment of the "harm reduction program" has been accepted by the higher governmental authorities and we adapted it to our religious and traditional culture. Harm reduction can work effectively only if it is genuinely supported by an enabling policy and legislative environment (17. Formerly, we thought that HCV was transmitted only through needle and syringe sharing among the IDUs, but in this study, we found that a new and potentially very dangerous route

  12. Mechanism and scaling for convection of isolated structures in nonuniformly magnetized plasmas

    DEFF Research Database (Denmark)

    Garcia, O.E.; Bian, N.H.; Naulin, V.

    2005-01-01

    Large-scale radial advection of isolated structures in nonuniformly magnetized plasmas is investigated. The underlying mechanism considered is due to the nonlinear evolution of interchange motions, without any presumption of plasma sheaths. Theoretical arguments supported by numerical simulations...... of the structures, compares favorably with recent experimental measurements of radially propagating blob structures in the scrape-off layer of magnetically confined plasmas. (C) 2005 American Institute of Physics....

  13. Heterogeneity in pepper isolates of cucumber mosaic virus

    Science.gov (United States)

    Rodriguez-Alvarado, G.; Kurath, G.; Dodds, J.A.

    1995-01-01

    Twenty-four cucumber mosaic cucumovirus (CMV) field isolates from pepper crops in Cali-fornia were characterized and compared by nucleic acid hybridization subgrouping, virion electrophoresis, and biological effects in several hosts. Isolates, belonging to subgroup I or subgroup II, were found that induced severe symptoms in mechanically inoculated bell pep-pers. Only two isolates, both from subgroup II, were mild. A group of 19 isolates collected from a single field were all in subgroup II and appeared identical by virion electrophoresis, but they exhibited varying degrees of symptom severity in peppers. As a more detailed indicator of heterogeneity, these 19 isolates were examined by RNase protection assays to delect sequence variation in the coat protein gene region of their genomes. The patterns of bands observed were complex and a high degree of genomic heterogeneity was detected between isolates, with no apparent correlation to symptomatology in bell pepper.

  14. Existence of variant strains Fowlpox virus integrated with Reticuloendotheliosis virus in its genome in field isolates in Tanzania.

    Science.gov (United States)

    Mzula, Alexanda; Masola, Selemani N; Kasanga, Christopher J; Wambura, Philemon N

    2014-06-01

    Fowlpox virus (FPV) is one example of poultry viruses which undergoes recombination with Reticuloendotheliosis virus (REV). Trepidation had been raised, and it was well established on augmented pathogenicity of the FPV upon integration of the full intact REV. In this study, we therefore intended at assessing the integration of REV into FPV genome of the field isolates obtained in samples collected from different regions of Tanzania. DNA extraction of 85 samples (scabs) was performed, and FPV-specific PCR was done by the amplification of the highly conserved P4b gene. Evaluation of FPV-REV recombination was done to FPV-specific PCR positively identified samples by amplifying the env gene and REV long terminal repeats (5' LTR). A 578-bp PCR product was amplified from 43 samples. We are reporting for the first time in Tanzania the existence of variant stains of FPV integrated with REV in its genome as 65 % of FPV identified isolates were having full intact REV integration, 21 % had partial FPV-REV env gene integration and 5 % had partial 5' LTR integration. Despite of the fact that FPV-REV integrated stains prevailed, FPV-REV-free isolates (9 %) also existed. In view of the fact that full intact REV integration is connected with increased pathogenicity of FPV, its existence in the FPV genome of most field isolates could have played a role in increased endemic, sporadic and recurring outbreaks in selected areas in Tanzania.

  15. Molecular characterization of the complete genome of a street rabies virus WH11 isolated from donkey in China.

    Science.gov (United States)

    Xie, Tingbo; Yu, Hua; Wu, Jie; Ming, Pinggang; Huang, Sijia; Shen, Zhijun; Xu, Gelin; Yan, Jiaxin; Yu, Bin; Zhou, Dunjin

    2012-12-01

    The complete genomic sequence of a rabies virus isolate WH11, isolated from brain tissue of a rabid donkey in China, was determined and compared with other rabies viruses. This is the first Chinese street strain which was isolated from donkey and the entire length and organization of the virus was similar to that of other rabies viruses. Multiple alignments of amino acid sequences of the nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and large protein of WH11 with those of other rabies viruses were undertaken to examine the conservative degree of functional regions. Phylogenetic analysis using the complete genomic sequence of WH11 determined that this isolate is most closely related with rabies viruses previously isolated in China and the attenuated Chinese vaccine strain CTN181.

  16. Molecular characterization and experimental host range of an isolate of Wissadula golden mosaic St. Thomas virus.

    Science.gov (United States)

    Collins, A M; Mujaddad-ur-Rehman, Malik; Brown, J K; Reddy, C; Wang, A; Fondong, V; Roye, M E

    2009-12-01

    Partial genome segments of a begomovirus were previously amplified from Wissadula amplissima exhibiting yellow-mosaic and leaf-curl symptoms in the parish of St. Thomas, Jamaica and this isolate assigned to a tentative begomovirus species, Wissadula golden mosaic St. Thomas virus. To clone the complete genome of this isolate of Wissadula golden mosaic St. Thomas virus, abutting primers were designed to PCR amplify its full-length DNA-A and DNA-B components. Sequence analysis of the complete begomovirus genome obtained, confirmed that it belongs to a distinct begomovirus species and this isolate was named Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). The genome of WGMSTV-[JM:Alb:05] is organized similar to that of other bipartite Western Hemisphere begomoviruses. Phylogenetic analyses placed the genome components of WGMSTV-[JM:Alb:05] in the Abutilon mosaic virus clade and showed that the DNA-A component is most closely related to four begomovirus species from Cuba, Tobacco leaf curl Cuba virus, Tobacco leaf rugose virus, Tobacco mottle leaf curl virus, and Tomato yellow distortion leaf virus. The putative Rep-binding-site motif in the common region of WGMSTV-[JM:Alb:05] was observed to be identical to that of Chino del tomate virus-Tomato [Mexico:Sinaloa:1983], Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005], and Tomato leaf curl Sinaloa virus-[Nicaragua:Santa Lucia], suggesting that WGMSTV-[JM:Alb:05] is capable of forming viable pseudo-recombinants with these begomoviruses, but not with other members of the Abutilon mosaic virus clade. Biolistic inoculation of test plant species with partial dimers of the WGMSTV-[JM:Alb:05] DNA-A and DNA-B components showed that the virus was infectious to Nicotiana benthamiana and W. amplissima and the cultivated species Phaseolus vulgaris (kidney bean) and Lycopersicon esculentum (tomato). Infected W. amplissima plants developed symptoms similar to symptoms observed under field

  17. Isolation of a virulent Newcastle disease virus from confiscated LaSota vaccine.

    Science.gov (United States)

    Pedersen, Janice C; Hines, Nichole L; Killian, Mary Lea; Predgen, Ann S; Schmitt, Beverly J

    2013-06-01

    Vials of Newcastle disease vaccine labeled as LaSota were confiscated by the Arizona Division of Customs and Border Protection officials. Two different avian type 1 paramyxoviruses were isolated from all three vials of vaccine submitted to the National Veterinary Services Laboratories. The LaSota strain of avian paramyxovirus type 1 virus was isolated from all three vials and analyzed by nucleotide sequence analysis. A virulent Newcastle disease virus was also present in all three vials, but in low concentration. The virulence of the Newcastle disease virus was characterized by the intracerebral chicken pathogenicity index chicken inoculation assay but could not be determined by nucleotide sequence analysis from the virus isolated from embryonating chicken eggs. The intracerebral chicken pathogenicity index value for the isolated Newcastle disease virus was 1.55. Strains of Newcastle disease virus with intracerebral pathogenicity indexes significantly above 1.0 have been found to selectively kill many types of cancer cells while not affecting normal nonneoplastic cells and are considered to be a viable option for cancer treatment in humans by alternative medical researchers; however, the treatment is not approved for use in the United States by the Food and Drug Administration. Customs and Border Protection officials have been notified of an increased risk of Newcastle disease virus entering the United States for use as a nonapproved cancer treatment. Illegal importation of Newcastle disease vaccine for vaccination of backyard poultry is also a threat. This case report emphasizes the importance of conducting chicken inoculation for complete virus pathotyping and demonstrates the need for stringent security procedures at U.S. borders to detect known livestock pathogens that may be smuggled in for use in animal agriculture and reasons unrelated to animal agriculture.

  18. Marek's disease virus isolates with unusual tropism and virulence for ocular tissues: clinical findings, challenge studies and pathological features.

    Science.gov (United States)

    Ficken, M D; Nasisse, M P; Boggan, G D; Guy, J S; Wages, D P; Witter, R L; Rosenberger, J K; Nordgren, R M

    1991-09-01

    Outbreaks of Marek's disease (MD) were diagnosed in two flocks from the same company. Clinical signs, mainly blindness (>90%), but also depression, mild paralysis, and 11 to 12% mortality by 20 weeks of age were observed. MD virus, serotype 1 was isolated. The isolates were designated NC-1 (flock 1) and NC-2 (flock 2). Challenge experiments were conducted with these isolates and with two reference MD virus strains (JM/102W and Md5) in unvaccinated, turkey herpesvirus- (HVT) vaccinated and bivalent- (HVT and SB-1) vaccinated chickens. Blindness, gross ocular lesions and tumour formation were observed in a high proportion of all groups challenged with NC-1 and NC-2 when compared with chickens challenged with JM/102W and Md5. In chickens challenged with isolates NC-1 and NC-2, corneal changes included oedema, midstromal cellular infiltration consisting of macrophages, lymphocytes, plasma cells and lesser numbers of heterophils, collagen degeneration and keratic precipitates consisting primarily of macrophages covering the central endothelium. Eosinophilic intranuclear inclusion bodies were present in mononuclear cells infiltrating the cornea. Changes in the uveal tract consisted of inflammatory cell infiltrates similar to those present in the cornea. Retinal lesions included disruption of the retinal pigmented epithelium, inflammatory cell infiltration in the subretinal space, photoreceptor degeneration and in severely affected eyes, necrosis of retinal cellular elements. Pecten changes consisted of necrosis and mononuclear cell infiltration. Intranuclear inclusion bodies were abundantly present in cells of the retina's ganglion and inner nuclear cell layers. The unusual clinical manifestation of MD, the unusual tropism and virulence of NC-1 and NC-2 for ocular tissues and the incomplete protection afforded by conventional vaccination suggest that these isolates may be new pathotypes.

  19. Isolation of capsid proteins of foot-and-mouth disease virus by chromatofocusing.

    Science.gov (United States)

    Murdin, A D; Doel, T R; Spier, R E

    1983-10-01

    A method for the isolation of foot-and-mouth disease virus (FMDV) capsid proteins was developed. The FMDV capsid proteins VP1, VP2, VP3 and VP0 were isolated from sucrose gradient purified virus by chromatofocusing in a pH 7.4-4.0 gradient on Polybuffer exchanger PBE 94. Under the conditions used the proteins eluted in the sequence VP1, VP2, VP0 (when present) and VP3. Capsid protein VP4 did not elute and could not be isolated by this method. Protein concentration in the eluate was monitored by the use of a radiolabelled marker and recoveries of approximately 50% of the input marker could be achieved when using up to 15 mg of virus and a 30-ml column. The high capacity and relative simplicity of chromatofocusing make it a useful alternative to other methods of purifying proteins.

  20. The complete sequence of a sugarcane mosaic virus isolate causing maize dwarf mosaic disease in China

    Institute of Scientific and Technical Information of China (English)

    程晔; 陈炯; 陈剑平

    2002-01-01

    The complete sequence of a potyvirus from maize in Zhejiang Province was determined. The RNA was 9596 nucleotides long, excluding the 3′-poly (A) tail, and there was a single long open reading frame (ORF) of 9192 nts encoding a 346.1 ku polyprotein. The polyprotein had substantial amino acid sequence homology with those encoded by the RNAs of a Chinese isolate of sorghum mosaic virus (SrMV-C) and a Bulgarian isolate of maize dwarf mosaic virus, but it was most closely related to sugarcane mosaic virus (SCMV) isolates, for which only partial sequences have been published. According to the published criteria for distinguishing potyviruses, the sequence reported here is clearly a strain of SCMV, but it also showed a surprisingly high amino acid homology with SrMV-C in the HC-Pro, P3 and CI proteins.

  1. Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation.

    Science.gov (United States)

    Dornas, Fábio P; Silva, Lorena C F; de Almeida, Gabriel M; Campos, Rafael K; Boratto, Paulo V M; Franco-Luiz, Ana P M; La Scola, Bernard; Ferreira, Paulo C P; Kroon, Erna G; Abrahão, Jônatas S

    2014-01-01

    Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

  2. Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation.

    Directory of Open Access Journals (Sweden)

    Fábio P Dornas

    Full Text Available Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water and hospital (ventilator plastic device tube substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

  3. Resistance breaking tomato spotted wilt virus isolates on resistant pepper varieties in Italy.

    Science.gov (United States)

    Crescenzi, A; Viggiano, A; Fanigliulo, A

    2013-01-01

    In spring 2012, resistance breaking (RB) isolates of tomato spotted wilt virus (TSWV) that overcome the resistance conferred by the Tsw gene in different pepper hybrids have been recovered in different locations in southern Italy (Campania and Apulia regions) in protected cultivation, about one month after transplant. The percentage of symptomatic plants was 5-10% and, only in particular cases of advanced stage of cultivation, it reached 30-50% at the end of cycle. All TSWV isolates induced similar systemic symptoms in all resistant infected pepper hybrids: yellowing or browning of apical leaves, which later become necrotic, long necrotic streakson stems, extending to the terminal shoots, complete necrosis of younger fruits and large necrotic streaks and spots on fruits formed after infection. On ripe fruits, yellow spots with concentric rings or necrotic streaks could be observed. Leaf extracts of these samples were tested in ELISA for the detection of TSWV, Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Impatiens necrotic spot virus (INSV), Potato virus Y (PVY), Alfalfa mosaic virus (AMV), Pepper mild mottle virus (PMMoV) and Pepper Mottle Virus (PepMoV). Only TSWV was detected in all the field samples tested. The correspondent virus isolates were inoculated mechanically and by Frankliniella occidentalis on to a set of different pepper and tomato hybrids, as well as on some herbaceous test plants, in order to investigate for their ability to overcome the resistance genes Tsw and Sw5, respectively. Tomato hybrids carrying the Sw5 gene were uninfected by all RB isolates, whereas all resistant pepper hybrids became systemically infected. RB isolates did not differ noticeably in transmission efficiency when they were tested with the thrips F. occidentalis. Obtained results demonstrate that evolved strains of TSWV have emerged, that they are able to overcome the Tsw resistance gene in pepper plants experimentally inoculated both

  4. Sequence analysis and genetic diversity of five new Indian isolates of cucumber mosaic virus.

    Science.gov (United States)

    Kumar, S; Gautam, K K; Raj, S K

    2015-12-01

    Cucumber mosaic virus (CMV) is an important virus since it causes severe losses to many economically important crops worldwide. Five new isolates of CMV were isolated from naturally infected Hippeastrum hybridum, Dahlia pinnata, Hemerocallis fulva, Acorus calamus and Typhonium trilobatum plants, all exhibiting severe leaf mosaic symptoms. For molecular identification and sequence analyses, the complete coat protein (CP) gene of these isolates was amplified by RT-PCR. The resulting amplicons were cloned and sequenced and isolates were designated as HH (KP698590), DP (JF682239), HF (KP698589), AC (KP698588) and TT (JX570732). For study of genetic diversity among these isolates, the sequence data were analysed by BLASTn, multiple alignment and generating phylogenetic trees along with the respective sequences of other CMV isolates available in GenBank Database were done. The isolates under study showed 82-99% sequence diversity among them at nucleotide and amino acid levels; however they showed close relationships with CMV isolates of subgroup IB. In alignment analysis of amino acid sequences of HH and AC isolates, we have found fifteen and twelve unique substitutions, compared to HF, DP and TT isolates, suggesting the cause of high genetic diversity.

  5. Segment 2 based characterization of a novel Indian Bluetongue virus isolate

    Directory of Open Access Journals (Sweden)

    Gaya Prasad

    2013-10-01

    Full Text Available Aim: The study was conducted to characterize and serotype the novel isolate of bluetongue virus (BTV isolated from India. Materials and Methods: The BTV isolate was propagated in BHK-21 cell line. Nucleic acid (dsRNA was extracted using Trizol method and cDNA was prepared using a process called reverse transcription. The cDNA was subjected to group specific PCR using ns1 gene specific primer to confirm the isolate as BTV. The type specific PCR was conducted to confirm the serotype of the virus using vp2 gene specific primers for all the BTV serotype including BTV10. The vp2 gene specific PCR amplicon was sequenced and in-silico restriction enzyme analysis and phylogenetic analysis was conducted. Results: Group specific PCR using ns1 gene specific primers showed a single 274bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The type specific PCR using BTV10 vp2 gene specific primer showed a single amplicon of 647bp. Remaining BTV serotype specific primers didn't show any amplification. The vp2 gene PCR amplicon was sequenced. The in-silico restriction enzyme analysis of vp2 gene of Indian BTV10 isolate along with other isolates from GenBank database using HindIII, XhoII and ApoI showed a common pattern between Indian and USA isolates. Similarly, phylogenetic analyses using vp2 gene nucleotide as well as deduced amino acid sequence of Indian BTV10 isolate and global isolates showed that Indian and most of the USA isolates placed in a single clad. Conclusion: A novel BTV isolate was isolated and confirmed as BTV serotype 10. Upon molecular analysis Indian BTV10 isolate was found closer to that of USA isolates than other global isolates. [Vet World 2013; 6(5.000: 244-248

  6. Molecular characterization and phylogenetic analysis of human parainfluenza virus type 3 isolated from Saudi Arabia.

    Science.gov (United States)

    Almajhdi, Fahad N; Alshaman, Mohamed S; Amer, Haitham M

    2012-08-01

    Human parainfluenza virus 3 (HPIV-3) is a leading cause of respiratory disease in children worldwide. Previous sequence analyses of the entire virus genome, among different HPIV-3 strains, demonstrated that HN is the most variable gene. There is a dearth of data on HPIV-3 strains circulating in Saudi Arabia. In this report, HPIV-3 was screened in nasopharyngeal aspirates collected from hospitalized children with acute respiratory disease during two successive seasons (2007/08 and 2008/09) using nested RT-PCR. Out of 73 samples collected during 2007/08, seven (9.59%) were positive; while 3 out of 107 samples collected during 2008/09 (2.8%) were positive. Virus isolation in cell culture was successful using HEp2, but not Vero cells. The identity of the isolated viruses was confirmed using immunofluorescence and neutralization assays. To elucidate the genetic characteristics and phylogeny of Saudi HPIV-3 strains, the complete HN gene sequence of two selected Saudi strains was analyzed in comparison to 20 strains isolated by others from different countries worldwide. Both strains showed the highest degree of sequence homology with Indian strains, followed by Chinese and most Japanese strains. Phylogenetic analysis confirmed that these strains fell into a distinct Asian lineage. This study is the first in Saudi Arabia to recover HPIV-3 isolates of confirmed identity, and to generate sequence data that may help in understanding virus diversity and evolution.

  7. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presenc...

  8. Phylogenetic analysis of surface proteins of novel H1N1 virus isolated from 2009 pandemic.

    Science.gov (United States)

    Danishuddin, Mohd; Khan, Shahper N; Khan, Asad U

    2009-09-30

    Swine Influenza Virus (H1N1) is a known causative agent of swine flu. Transmission of Swine Influenza Virus form pig to human is not a common event and may not always cause human influenza. The 2009 outbreak by subtype H1N1 in humans is due to transfer of Swine Influenza Virus from pig to human. Thus to analyze the origin of this novel virus we compared two surface proteins (HA and NA) with influenza viruses of swine, avian and humans isolates recovered from 1918 to 2008 outbreaks. Phylogenetic analyses of hemagglutinin gene from 2009 pandemic found to be clustered with swine influenza virus (H1N2) circulated in U.S.A during the 1999-2004 outbreaks. Whereas, neuraminidase gene was clustered with H1N1 strains isolated from Europe and Asia during 1992-2007 outbreaks. This study concludes that the new H1N1 strain appeared in 2009 outbreak with high pathogenicity to human was originated as result of re-assortment (exchange of gene). Moreover, our data also suggest that the virus will remain sensitive to the pre-existing therapeutic strategies.

  9. Receptor binding properties of human and animal H1 influenza virus isolates.

    Science.gov (United States)

    Rogers, G N; D'Souza, B L

    1989-11-01

    It has been previously reported that several human H1 influenza viruses isolated prior to 1956, in contrast to human H3 isolates which are quite specific for SA alpha 2,6Gal sequences, apparently recognize both SA alpha 2,3Gal and SA alpha 2,6Gal sequences (Rogers, G.N., and Paulson, J.C., Virology 127, 361-373, 1983). In this report human H1 isolates representative of two epidemic periods, from 1934 to 1957 and from 1977 to 1986, and H1 influenza isolated from pigs, ducks, and turkeys were compared for their ability to utilize sialyloligosaccharide structures containing terminal SA alpha 2,3Gal or SA alpha 2,6Gal sequences as receptor determinants. Five of the eight human isolates from the first epidemic period recognize both SA alpha 2,3Gal and SA alpha 2,6Gal linkages, in agreement with our previous results. Of the remaining three strains, all isolated towards the end of the first epidemic, two appear to prefer SA alpha 2,6Gal sequences while the third preferentially binds SA alpha 2,3Gal sequences. In contrast to the early isolates, 11 of 13 human strains isolated during the second epidemic period preferentially bind SA alpha 2,6Gal containing oligosaccharides. On the basis of changes in receptor binding associated with continued passage in the laboratory for some of these later strains, it seems likely that human H1 isolates preferentially bind SA alpha 2,6Gal sequences in nature, and that acquisition of SA alpha 2,3Gal-binding is associated with laboratory passage. Influenza H1 viruses isolated from pigs were predominantly SA alpha 2,6Gal-specific while those isolated from ducks were primarily SA alpha 2,3Gal-specific. Thus, as has been previously reported for H3 influenza isolates, receptor specificity for influenza H1 viruses appears to be influenced by the species from which they were isolated, human isolates binding preferentially to SA alpha 2,6Gal-containing oligosaccharides while those isolated from ducks prefer SA alpha 2,3Gal

  10. Complete Genome Sequence of Zika Virus Isolated in Mexico, 2016

    Science.gov (United States)

    Peña-Alonso, Rocío; Mendieta-Condado, Edgar; Garcés-Ayala, Fabiola; González-Durán, Elizabeth; Escobar-Escamilla, Noé; Vázquez-Pichardo, Mauricio; Torres-Rodríguez, María de la Luz; Núñez-León, Alma; Torres-Longoria, Belem; López-Martínez, Irma; Ruíz-Matus, Cuitláhuac; Kuri-Morales, Pablo

    2016-01-01

    Zika virus belongs to the genus Flavivirus, and its spread remains an international public health emergency. In this report, we describe the obtainment and molecular characterization of a complete viral genome through the direct metagenomic analysis from saliva from an autochthonous transmission case in Mexico. PMID:27491989

  11. Diversity of Papaya ringspot virus isolates in Puerto Rico

    Science.gov (United States)

    Papaya ringspot virus (PRSV) devastates papaya production worldwide. In Puerto Rico, papaya fields can be completely infected with PRSV within a year of planting. Information about the diversity of the Puerto Rican PRSV population is relevant in order to establish a control strategy in the island. T...

  12. Complete Genome Sequence of Zika Virus Isolated in Mexico, 2016.

    Science.gov (United States)

    Díaz-Quiñonez, José Alberto; Peña-Alonso, Rocío; Mendieta-Condado, Edgar; Garcés-Ayala, Fabiola; González-Durán, Elizabeth; Escobar-Escamilla, Noé; Vázquez-Pichardo, Mauricio; Torres-Rodríguez, María de la Luz; Núñez-León, Alma; Torres-Longoria, Belem; López-Martínez, Irma; Ruíz-Matus, Cuitláhuac; Kuri-Morales, Pablo; Ramírez-González, José Ernesto

    2016-08-04

    Zika virus belongs to the genus Flavivirus, and its spread remains an international public health emergency. In this report, we describe the obtainment and molecular characterization of a complete viral genome through the direct metagenomic analysis from saliva from an autochthonous transmission case in Mexico.

  13. Prevalence of HIV infection in seronegative high-risk individuals examined by virus isolation and PCR

    DEFF Research Database (Denmark)

    Nielsen, C; Teglbjærg, Lars Stubbe; Pedersen, C

    1991-01-01

    HIV seronegative individuals with high-risk behavior were tested for HIV infection by sensitive virus isolation techniques using T4 lymphocytes and monocyte/macrophages, and by detection of proviral DNA using PCR with three different sets of nested primers. No evidence of HIV infection was found...... among the 31 seronegative high-risk subjects, either by virus isolation of by PCR (97.5% confidence limits, 0-11). Our results indicate that ongoing HIV infection in seronegative persons at high risk of infection is a rare event....

  14. Isolation of saffold virus type 2 in green monkey kidney cells.

    Science.gov (United States)

    Blomqvist, Soile; Lappalainen, Maija; Paananen, Anja; Ylipaasto, Petri; Roivainen, Merja

    2012-09-01

    Saffold viruses (SAFV) have been discovered recently and they are classified into Theilovirus species in genus Cardiovirus in the Picornaviridae family. SAFV, especially those belonging to the genotype 2, have been difficult to propagate in laboratory cell lines. This study describes the successful isolation of an efficiently growing SAFV-2 strain directly from a stool specimen by standard virological methods. The availability of SAFV isolates that can be propagated to high titers is crucial to the future studies on pathogenesis and epidemiology of these novel human viruses. Copyright © 2012 Wiley Periodicals, Inc.

  15. Fish viruses: isolation of an icosahedral cytoplasmic deoxyribovirus from sheatfish (Silurus glanis).

    Science.gov (United States)

    Ahne, W; Schlotfeldt, H J; Thomsen, I

    1989-07-01

    An icosahedral cytoplasmic deoxyvirus has been isolated from moribund sheatfish (Silurus glanis) fry of a commercial warm water recirculation aquaculture unit with cumulative mortalities of up to 100%. The agent replicated in BF-2 and in FHM cells at 20-30 degrees C producing cytoplasmatic inclusion bodies followed by lysis of the cells. The DNA containing virus proved to be labile to chloroform. Infected BF-2 cells revealed hexagonal particles in the cytoplasm measuring about 125-135 nm in diameter. The virus consisted of a central electron-dense core and a electron-translucent zone. The isolate shares characteristics with the Iridoviridae.

  16. Genetic characterization of the influenza A pandemic (H1N1 2009 virus isolates from India.

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    Varsha A Potdar

    Full Text Available BACKGROUND: The Influenza A pandemic H1N1 2009 (H1N1pdm virus appeared in India in May 2009 and thereafter outbreaks with considerable morbidity and mortality have been reported from many parts of the country. Continuous monitoring of the genetic makeup of the virus is essential to understand its evolution within the country in relation to global diversification and to track the mutations that may affect the behavior of the virus. METHODS: H1N1pdm viruses were isolated from both recovered and fatal cases representing major cities and sequenced. Phylogenetic analyses of six concatenated whole genomes and the hemagglutinin (HA gene of seven more isolates from May-September 2009 was performed with reference to 685 whole genomes of global isolates available as of November 24, 2009. Molecular characterization of all the 8 segments was carried out for known pathogenic markers. RESULTS: The first isolate of May 2009 belonged to clade 5. Although clade 7 was the dominant H1N1pdm lineage in India, both clades 6 and 7 were found to be co-circulating. The neuraminidase of all the Indian isolates possessed H275, the marker for sensitivity to the neuraminidase inhibitor Oseltamivir. Some of the mutations in HA are at or in the vicinity of antigenic sites and may therefore be of possible antigenic significance. Among these a D222G mutation in the HA receptor binding domain was found in two of the eight Indian isolates obtained from fatal cases. CONCLUSIONS: The majority of the 13 Indian isolates grouped in the globally most widely circulating H1N1pdm clade 7. Further, correlations of the mutations specific to clade 7 Indian isolates to viral fitness and adaptability in the country remains to be understood. The D222G mutation in HA from isolates of fatal cases needs to be studied for pathogenicity.

  17. [Genetic characterisation of Powassan virus (POWV) isolated from Haemophysalis longicornis ticks in Primorye and two strains of Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus): Alma-Arasan virus (AAV) isolated from Ixodes persulcatus ticks in Kazakhstan and Malyshevo virus isolated from Aedes vexans nipponii mosquitoes in Khabarovsk kray].

    Science.gov (United States)

    L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Deriabin, P G; Gitel'man, A K; Botikov, A G; Aristova, V A

    2014-01-01

    The complete genomes of the three tick-borne flaviviruses (genus Flavivirus, fam. Bunyaviridae) were sequenced: Povassan virus (POWV, strain LEIV-3070Prm, isolated from Haemophysalis logicornis in Primorsky Krai, Russia in 1977), Alma-Arasan virus (AAV, strain LEIV-1380Kaz, isolated from Ixodes persulcatus ticks in Kazakhstan in 1977) and Malyshevo virus (isolated from a pool of Aedes vexans nipponii mosquitoes, in the Khabarovsk Krai, Russia in 1978). It is shown that AAV and Malyshevo virus are the strains of Tick-borne encephalitis virus (TBEV) and belong to Sibirian and Far-Eastern genotypes, respectively (GenBank ID: AAV KJ744033; strain Malyshevo KJ744034). Phylogenetically AAV is closest related (94,6% nt and 98,3% aa identity) to TBEV strains, isolated in Sibiria (Vasilchenko, Aino, Chita-653, Irkutsk-12). Malyshevo virus is closest related (96,4% nt and 98,3% nt identity) to strains of TBEV, isolated in Far Eastern part of Russia (1230, Spassk-72, Primorye-89). POWV LEIV-3070Prm has 99.7% identity with the prototype strain POWV LB, isolated in Canada and 99.5% of isolates with Far-Eastern strains of POWV (Spassk-9 and Nadezdinsk-1991).

  18. Molecular characterization of infectious bronchitis viruses isolated from broiler chicken farms in Iran, 2014-2015.

    Science.gov (United States)

    Najafi, Hamideh; Langeroudi, Arash Ghalyanchi; Hashemzadeh, Masoud; Karimi, Vahid; Madadgar, Omid; Ghafouri, Seyed Ali; Maghsoudlo, Hossein; Farahani, Reza Khaltabadi

    2016-01-01

    Infectious bronchitis (IB) is a viral avian disease with economic importance in the world, including Iran. S1 gene sequencing has been used for molecular epidemiological studies and genotypic characterization of infectious bronchitis virus (IBV). A total of 118 IBV isolates were obtained from tissue samples from chickens with clinically suspected IB from Iranian broiler farms (eight provinces, 200 samples). The isolates were confirmed by real-time polymerase chain reaction (PCR) and characterized by sequencing the spike glycoprotein gene. The isolates formed six distinct phylogenetic groups (IS/1494/06 [Var2] like, 4/91-like, IS/720-like, QX-like, IR-1 and Mass-like) that were related to variants isolated in the region. The most frequently detected viruses were of the Var2-like (IS/1494/06-like) genotype, with an overall prevalence of 34 %. Twenty-one percent of the isolates formed a cluster together with the 4/91 IBV type, 10 % were of the QX genotype, and 8 % were of the IS/720 genotype. In addition, 4 % and 3 % of the isolates belonged to the Massachusetts and IR-1 genotype, respectively. For the first time, we have isolated and characterized IBV variants from broiler farms in different provinces of Iran. This study demonstrates a constant evolution of IBV in Iran, demonstrating the need for continuous monitoring and development of new vaccines based on indigenous viruses.

  19. Molecular analysis of hepatitis B virus isolates in Mexico: Predominant circulation of hepatitis B virus genotype H

    Institute of Scientific and Technical Information of China (English)

    Cosme Alvarado-Esquivel; Erwin Sablon; Carlos Jesús Conde-González; Luis Juárez-Figueroa; Lilia Ruiz-Maya; Sergio Aguilar-Benavides

    2006-01-01

    AIM:To determine the genotypes in Mexican hepatitis B virus (HBV) isolates and characterize their precore and core promoter mutations.METHODS: Forty-nine HBV isolates of Mexico obtained from sera of 15 hepatitis patients, 6 hemodialysis patients, 20 men seeking HIV testing, and 8 AIDS patients were analyzed. HBV isolates were amplified by PCR,and genotyped by line probe assay (INNO-LiPA HBV Genotyping; INNOGENETICS N V, Ghent, Belgium).HBV genotype confirmation was performed by DNA sequencing part of the sAg region. Precore and core promoter mutation characterization was performed by line probe assay (INNO-LiPA HBV PreCore; INNOGENETICS N.V.; Ghent, Belgium).RESULTS: Overall, HBV genotype H was found in 37(75.5%) out of the 49 isolates studied. HBV genotypes G, A, and D were found in 5 (10.2%), 4 (8.2%), and 3(6.1%) isolates, respectively. HBV genotype H was predominant in isolates from hemodialysis patients (100%),hepatitis patients (80%), and men seeking HIV testing (75%), and accounted for half of infections in AIDS patients (50%). Six (12.2%) out of the 49 HBV isolates showed both wild type and mutant populations at precore codon 28. These mixed wild type and precore murant populations were observed in one HBV genotype A isolate and in all HBV genotype G isolates. A dual variant core promoter mutation was observed in 1 (2%) of the isolates, which was genotype H.CONCLUSION: HBV genotype H is highly predominant in HBV isolates of Mexico followed by genotypes G, A and D. A low frequency of precore and core promoter mutations is observed in HBV Mexican isolates.

  20. Genomic analysis of highly virulent Georgia 2007/1 isolate of African swine fever virus.

    Science.gov (United States)

    Chapman, David A G; Darby, Alistair C; Da Silva, Melissa; Upton, Chris; Radford, Alan D; Dixon, Linda K

    2011-04-01

    African swine fever is widespread in Africa but has occasionally been introduced into other continents. In June 2007, African swine fever was isolated in the Caucasus Region of the Republic of Georgia and subsequently in neighboring countries (Armenia, Azerbaijan, and 9 states of the Russian Federation). Previous data for sequencing of 3 genes indicated that the Georgia 2007/1 isolate is closely related to isolates of genotype II, which has been identified in Mozambique, Madagascar, and Zambia. We report the complete genomic coding sequence of the Georgia 2007/1 isolate and comparison with other isolates. A genome sequence of 189,344 bp encoding 166 open reading frames (ORFs) was obtained. Phylogeny based on concatenated sequences of 125 conserved ORFs showed that this isolate clustered most closely with the Mkuzi 1979 isolate. Some ORFs clustered differently, suggesting that recombination may have occurred. Results provide a baseline for monitoring genomic changes in this virus.

  1. Genomic Characterizations of a Newcastle Disease Virus Isolated from Ducks in Live Bird Markets in China

    Science.gov (United States)

    Zhang, Yi; Zheng, Dongxia; Zhao, Yunling; Castellan, David; Liu, Hualei; Wang, Zhiliang

    2016-01-01

    One class I Newcastle disease virus (NDV), designated as duck/Guangxi/1261/2015 (GX1261), was isolated from asymptomatic ducks in live bird markets (LBM) from southern China during the national active surveillance for NDVs in 2015. The complete genome length of GX1261 isolate was 15,198 nucleotides with the gene order of 3’-NP-P-M-F-HN-L-5’. The motif at the cleavage site of F protein was 112ERQER/L117, which was typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on the complete F gene revealed that the isolate was clustered into sub-genotype 1c in class I, and showed a high level of similarity with the strains isolated from waterfowl in the United States of America. This is the first report of this kind of virus in the mainland of China. These results demonstrated that GX1261-like viruses might exist in asymptomatic waterfowl, and remain undetected or unidentified. Thus, more investigation needs to be done in order to identify the source of the virus. This study revealed the genetic and phylogenetic characteristics of GX1261 isolate and could help us to better understand the epidemiological context of class I NDV in China. PMID:27391305

  2. Genomic Characterizations of a Newcastle Disease Virus Isolated from Ducks in Live Bird Markets in China.

    Science.gov (United States)

    Wang, Jingjing; Lv, Yan; Zhang, Yi; Zheng, Dongxia; Zhao, Yunling; Castellan, David; Liu, Hualei; Wang, Zhiliang

    2016-01-01

    One class I Newcastle disease virus (NDV), designated as duck/Guangxi/1261/2015 (GX1261), was isolated from asymptomatic ducks in live bird markets (LBM) from southern China during the national active surveillance for NDVs in 2015. The complete genome length of GX1261 isolate was 15,198 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. The motif at the cleavage site of F protein was 112ERQER/L117, which was typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on the complete F gene revealed that the isolate was clustered into sub-genotype 1c in class I, and showed a high level of similarity with the strains isolated from waterfowl in the United States of America. This is the first report of this kind of virus in the mainland of China. These results demonstrated that GX1261-like viruses might exist in asymptomatic waterfowl, and remain undetected or unidentified. Thus, more investigation needs to be done in order to identify the source of the virus. This study revealed the genetic and phylogenetic characteristics of GX1261 isolate and could help us to better understand the epidemiological context of class I NDV in China.

  3. Genomic Characterizations of a Newcastle Disease Virus Isolated from Ducks in Live Bird Markets in China.

    Directory of Open Access Journals (Sweden)

    Jingjing Wang

    Full Text Available One class I Newcastle disease virus (NDV, designated as duck/Guangxi/1261/2015 (GX1261, was isolated from asymptomatic ducks in live bird markets (LBM from southern China during the national active surveillance for NDVs in 2015. The complete genome length of GX1261 isolate was 15,198 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. The motif at the cleavage site of F protein was 112ERQER/L117, which was typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on the complete F gene revealed that the isolate was clustered into sub-genotype 1c in class I, and showed a high level of similarity with the strains isolated from waterfowl in the United States of America. This is the first report of this kind of virus in the mainland of China. These results demonstrated that GX1261-like viruses might exist in asymptomatic waterfowl, and remain undetected or unidentified. Thus, more investigation needs to be done in order to identify the source of the virus. This study revealed the genetic and phylogenetic characteristics of GX1261 isolate and could help us to better understand the epidemiological context of class I NDV in China.

  4. Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American genogroup M.

    Science.gov (United States)

    Enzmann, P J; Kurath, G; Fichtner, D; Bergmann, S M

    2005-09-23

    Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe.

  5. Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene.

    Science.gov (United States)

    Nishizawa, T; Kinoshita, S; Kim, W S; Higashi, S; Yoshimizu, M

    2006-08-30

    Infectious hematopoietic necrosis virus (IHNV), a member of the genus Novirhabdovirus, causes a highly lethal disease of salmonid fish. In the present study, G gene nucleotide sequences of 9 Japanese IHNV isolates obtained from 1971 to 1996 were analyzed to evaluate the genetic diversity and compared with IHNV isolates from North America and Europe. A radial phylogenetic tree revealed 5 major clusters including 3 genogroups (U, M and L) for North American isolates and 1 genogroup for European isolates. Five Japanese isolates from 1971 to 1982 appeared in the cluster for genogroup U, while the remaining Japanese isolates from 1980 to 1996 formed a new genogroup, JRt (Japanese rainbow trout). Maximum nucleotide diversity among the Japanese isolates was 4.5%, which was greater than that within the North American isolates (3.6%), and the degree of nucleotide diversity within Japanese isolates was increased by inclusion of the genogroup JRt isolates. It was concluded that Japanese isolates shared a common source with the genogroup U of the North American isolates and that there were large divergences between Japanese isolates before and after the 1980s.

  6. An avian leukosis virus subgroup J isolate with a Rous sarcoma virus-like 5'-LTR shows enhanced replication capability.

    Science.gov (United States)

    Gao, Yanni; Guan, Xiaolu; Liu, Yongzhen; Li, Xiaofei; Yun, Bingling; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Wang, Xiaomei; Gao, Yulong

    2015-01-01

    Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5'-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5'-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5'-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5'-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.

  7. Isolation of porcine reproductive and respiratory syndrome (PRRS) virus in a Danish swine herd and experimental infection of pregnant gilts with the virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Nielsen, Jens; Bille-Hansen, Vivi

    1994-01-01

    The first case of porcine reproductive and respiratory syndrome (PRRS) in Denmark was diagnosed in March 1992 by the detection of specific antibodies against PRRS virus in serum samples originating from sows in a herd located on the island of Als. Subsequently, PRRS virus was isolated from a 200......-sow farrow-to-finish herd with clinical signs consistent with PRRS. The virus was isolated by inoculation of pleural fluid from a stillborn piglet onto porcine pulmonary alveolar macrophages. The isolate was identified as PRRS virus by staining with a specific antiserum. By electron microscopy...... infection was demonstrated by the re-isolation of PRRS virus from approximately 45% of the piglets from the experimentally infected gilts. However, the experimental infection produced no significant reproductive disorders or other clinical signs. At autopsy, histopathological examination revealed slight...

  8. Isolation and characterization of bovine parainfluenza virus type 3 from water buffaloes (Bubalus bubalis in Argentina

    Directory of Open Access Journals (Sweden)

    Maidana Silvina S

    2012-06-01

    Full Text Available Abstract Background Parainfluenza virus type 3 (PIV3 was isolated from dairy buffaloes (Bubalus bubalis naturally affected with respiratory and reproductive clinical conditions. Results Examination of nasal and vaginal swabs collected from 12 diseased buffaloes led to the isolation of three paramyxovirus isolates from two animals. Antigenic, morphological and biological characteristics of these three isolates were essentially similar to those of members of the Paramyxoviridae family. Antigenic analysis by direct immunofluorescence and cross neutralization test placed these isolates together with bovine parainfluenza virus type 3 (BPIV3. Nucleotide and amino acid phylogenetic analysis of partial matrix gene sequences of the buffalo isolates and six field BPIV3 isolates from bovines in Argentina were studied. Buffalo isolates were similar to genotype B (BPIV3b while the six BPIV3 isolates were similar to genotypes A (BPIV3a and C (BPIV3c. Conclusions This is the first characterization of BPIV3 in water buffalo. According to the samples analyzed, in Argentina, the genotype B was found in buffalo and the genotypes A and C were found in cattle.

  9. Sequence diversity and virulence in Zea mays of Maize streak virus isolates.

    Science.gov (United States)

    Martin, D P; Willment, J A; Billharz, R; Velders, R; Odhiambo, B; Njuguna, J; James, D; Rybicki, E P

    2001-09-30

    Full genomic sequences were determined for 12 Maize streak virus (MSV) isolates obtained from Zea mays and wild grass species. These and 10 other publicly available full-length sequences were used to classify a total of 66 additional MSV isolates that had been characterized by PCR-restriction fragment length polymorphism and/or partial nucleotide sequence analysis. A description is given of the host and geographical distribution of the MSV strain and subtype groupings identified. The relationship between the genotypes of 21 fully sequenced virus isolates and their virulence in differentially MSV-resistant Z. mays genotypes was examined. Within the only MSV strain grouping that produced severe symptoms in maize, highly virulent and widely distributed genotypes were identified that are likely to pose the most serious threat to maize production in Africa. Evidence is presented that certain of the isolates investigated may be the products of either intra- or interspecific recombination.

  10. Use of ultrafiltration to isolate viruses from seawater which are pathogens of marine phytoplankton.

    Science.gov (United States)

    Suttle, C A; Chan, A M; Cottrell, M T

    1991-03-01

    Viruses may be major structuring elements of phytoplankton communities and hence important regulators of nutrient and energy fluxes in aquatic environments. In order to ascertain whether viruses are potentially important in dictating phytoplankton community structure, it is essential to determine the extent to which representative phytoplankton taxa are susceptible to viral infection. We used a spiral ultrafiltration cartridge (30,000-molecular-weight cutoff) to concentrate viruses from seawater at efficiencies approaching 100%. Natural virus communities were concentrated from stations in the Gulf of Mexico, a barrier island pass, and a hypersaline lagoon (Laguna Madre) and added to cultures of potential phytoplankton hosts. By following changes in in vivo fluorescence over time, it was possible to isolate several viruses that were pathogens to a variety of marine phytoplankton, including a prasinophyte (Micromonas pusilla), a pennate diatom (likely a Navicula sp.), a centric diatom (of unknown taxa), and a chroococcoid cyanobacterium (a Synechococcus sp.). As well, we observed changes in fluorescence in cultures of a cryptophyte (a Rhodomonas sp.) and a chlorophyte (Nannochloropsis oculata) which were consistent with the presence of viral pathogens. Although pathogens were isolated from all stations, all the pathogens were not isolated from every station. Filterability studies on the viruses infecting M. pusilla and the Navicula sp. showed that the viruses were consistently infective after filtration through polycarbonate and glass-fiber filters but were affected by most other filter types. Establishment of phytoplankton-pathogen systems will be important in elucidating the effect that viruses have on primary producers in aquatic systems.

  11. Isolation and characterization of bovine plasma prekallikrein (Fletcher factor).

    Science.gov (United States)

    Heimark, R L; Davie, E W

    1979-12-11

    Prekallikrein (Fletcher factor) has been purified from bovine plasma approximately 25 000-fold with an overall yield of 14%. Purification steps included ammonium sulfate fractionation and column chromatography on heparin-agarose, DEAE-Sephadex, CM-Sephadex, benzamidine-agarose, and arginine methyl ester-agarose. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Bovine plasma prekallikrein is a glycoprotein with a molecular weight of 82 000 as determined by sedimentation equilibrium centrifugation. It contains 12.9% carbohydrate, including 6.2% hexose, 4.5% N-acetylglucosamine, and 2.2% N-acetylneuraminic acid. Prekallikrein is a single polypeptide chain with an amino-terminal sequence of Gly-Cys-Leu-Thr-Gln-Leu-Tyr-His-Asn-Ile-Phe-Phe-Arg-Gly-Gly. This sequence is homologous to the amino-terminal sequence of human factor XI (plasma thromboplastin antecedent). Both prekallikrein and kallikrein require kaolin to correct Fletcher factor deficient plasma. Kallikrein, however, has a specific activity 3.5 times greater than prekallikrein. Prekallikrein does not correct plasma deficient in factor XII (Hageman factor), factor XI, or high molecular weight kininogen (Fitzgerald factor).

  12. Characterization of H3N6 avian influenza virus isolated from a wild white pelican in Zambia.

    Science.gov (United States)

    Simulundu, Edgar; Mweene, Aaron S; Tomabechi, Daisuke; Hang'ombe, Bernard M; Ishii, Akihiro; Suzuki, Yuka; Nakamura, Ichiro; Sawa, Hirofumi; Sugimoto, Chihiro; Ito, Kimihito; Kida, Hiroshi; Saiwana, Lewis; Takada, Ayato

    2009-01-01

    We characterized an influenza virus isolated from a great white pelican in Zambia. Phylogenetic analysis showed that all of its gene segments belonged to the Eurasian lineage and that they appear to have evolved in distinct geographical regions in Europe, Asia, and Africa, suggesting reassortment of virus genes maintained in wild aquatic birds whose flyways overlap across these continents. It is notable that this virus might possess some genes of the same origin as those of highly pathogenic H7 and H5 viruses isolated in Eurasia. The present study underscores the need for continued monitoring of avian influenza viruses in Eurasia and Africa.

  13. Characterization of a New Tomato Spotted Wilt Virus Isolates Found in Hippeastrum hybridum (Hort. Plants in Poland

    Directory of Open Access Journals (Sweden)

    Berniak Hanna

    2016-06-01

    Full Text Available Two Tomato spotted wilt virus (TSWV isolates H1 and H2 found in Hippeastrum hybridum plants were characterized based on biological, serological, and molecular properties. Virus isolates showed differences in symptom expression – H1 isolate displayed severe necrotic spots and patterns, whereas mild mosaic symptoms were observed on H2-infected H. hybridum plants. Both TSWV isolates showed comparable reactivity with TSWV-specific antibodies and they induced similar symptoms on herbaceous indicator plants, but some differences between these isolates were detected at the nucleotide sequence level of genomic S and M ssRNAs segment fragments. The nucleotide sequences encoding nucleocapsid (N and nonstructural (NSs and NSm proteins showed 98.2%, 97.5%, and 96.5% identity, respectively. Phylogenetic analysis of N and NSs sequences conducted for tested isolates and 31 TSWV isolates included for comparison revealed that H1 and H2 isolates fell into the same cluster and they were grouped together with isolates found previously in different vegetables, ornamentals, and weeds. When NSm ORF was analyzed, the tested isolates formed a separate cluster: H1 isolate showed the highest affinity with TSWV isolates infecting chrysanthemum and pepper plants, whereas H2 isolate was most closely related to other virus isolates found in sweet pepper and tomatoes. These results indicate that both isolates were reassortants between different virus isolates, and represented two novel genetic patterns of TSWV.

  14. Genomic characterization of recent chicken anemia virus isolates in China

    Science.gov (United States)

    Chicken infectious anemiavirus (CIAV) causes diseases in young chickens, which include increased pathogenicity of secondary infectious agents, generalized lymphoid depletion, and immune-repression. In the present study, we have identified 22 CIAV strains isolated from several commercial chicken farm...

  15. Escherichia coli and virus isolated from ''sticky kits''

    DEFF Research Database (Denmark)

    Jørgensen, M.; Scheutz, F.; Strandbygaard, Bertel

    1996-01-01

    A total of 121 Escherichia coli strains isolated from 3-week-old mink kits were serotyped and examined for virulence factors. 56 strains were isolated from healthy kits while 65 were from ''sticky kits''. Among these, 34 different serotypes were detected. No difference in serotypes or the presenc...... of virulence factors could be detected between healthy and diseased kits. By electron microscopy of faecal samples corona-, rota-, and calicivirus were demonstrated among healthy as well as diseased kits....

  16. Characterisation of bovine viral diarrhoea virus (BVDV) isolates from an outbreak with haemorrhagic enteritis and severe pneumonia.

    Science.gov (United States)

    Yeşilbağ, Kadir; Förster, Christine; Ozyiğit, M Ozgür; Alpay, Gizem; Tuncer, Pelin; Thiel, Heinz-Jürgen; König, Matthias

    2014-02-21

    During 2007 a disease outbreak occurred in cattle in the Marmara region of western Turkey characterised by severe pneumonia and haemorrhagic enteritis in calves. Cases from three farms at different locations were examined and bovine viral diarrhoea virus (BVDV) isolated in all cases. Phylogenetic characterisation of the virus isolates allocated them in a new cluster tentatively named as BVDV-1r.

  17. Arrested relaxation in an isolated molecular ultracold plasma

    Science.gov (United States)

    Haenel, R.; Schulz-Weiling, M.; Sous, J.; Sadeghi, H.; Aghigh, M.; Melo, L.; Keller, J. S.; Grant, E. R.

    2017-08-01

    Spontaneous avalanche to plasma splits the core of an ellipsoidal Rydberg gas of nitric oxide. Ambipolar expansion first quenches the electron temperature of this core plasma. Then, long-range, resonant charge transfer from ballistic ions to frozen Rydberg molecules in the wings of the ellipsoid quenches the ion-Rydberg-molecule relative velocity distribution. This sequence of steps gives rise to a remarkable mechanics of self-assembly, in which the kinetic energy of initially formed hot electrons and ions drives an observed separation of plasma volumes. These dynamics adiabatically sequester energy in a reservoir of mass transport, starting a process that anneals separating volumes to form an apparent glass of strongly coupled ions and electrons. Short-time electron spectroscopy provides experimental evidence for complete ionization. The long lifetime of this system, particularly its stability with respect to recombination and neutral dissociation, suggests that this transformation affords a robust state of arrested relaxation, far from thermal equilibrium.

  18. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

    OpenAIRE

    Wang Nan; Sun Changjiang; Wang Quankai; Du Rui; Wang Shijie; Gao Yugang; Zhang Pengju; Zhang Lianxue

    2011-01-01

    Abstract Background Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical...

  19. Ilheus virus isolation in the Pantanal, west-central Brazil.

    Science.gov (United States)

    Pauvolid-Corrêa, Alex; Kenney, Joan L; Couto-Lima, Dinair; Campos, Zilca M S; Schatzmayr, Hermann G; Nogueira, Rita M R; Brault, Aaron C; Komar, Nicholas

    2013-01-01

    The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolândia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal.

  20. Analysis of Arbovirus Isolates from Australia Identifies Novel Bunyaviruses Including a Mapputta Group Virus from Western Australia That Links Gan Gan and Maprik Viruses.

    Science.gov (United States)

    Briese, Thomas; Williams, David T; Kapoor, Vishal; Diviney, Sinead M; Certoma, Andrea; Wang, Jianning; Johansen, Cheryl A; Chowdhary, Rashmi; Mackenzie, John S; Lipkin, W Ian

    2016-01-01

    The Mapputta group comprises antigenically related viruses indigenous to Australia and Papua New Guinea that are included in the family Bunyaviridae but not currently assigned to a specific genus. We determined and analyzed the genome sequences of five Australian viruses isolated from mosquitoes collected during routine arbovirus surveillance in Western Australia (K10441, SW27571, K13190, and K42904) and New South Wales (12005). Based on matching sequences of all three genome segments to prototype MRM3630 of Trubanaman virus (TRUV), NB6057 of Gan Gan virus (GGV), and MK7532 of Maprik virus (MPKV), isolates K13190 and SW27571 were identified as TRUV, 12005 as GGV, and K42904 as a Mapputta group virus from Western Australia linking GGV and MPKV. The results confirmed serum neutralization data that had linked SW27571 to TRUV. The fifth virus, K10441 from Willare, was most closely related to Batai orthobunyavirus, presumably representing an Australian variant of the virus. Phylogenetic analysis also confirmed the close relationship of our TRUV and GGV isolates to two other recently described Australian viruses, Murrumbidgee virus and Salt Ash virus, respectively. Our findings indicate that TRUV has a wide circulation throughout the Australian continent, demonstrating for the first time its presence in Western Australia. Similarly, the presence of a virus related to GGV, which had been linked to human disease and previously known only from the Australian southeast, was demonstrated in Western Australia. Finally, a Batai virus isolate was identified in Western Australia. The expanding availability of genomic sequence for novel Australian bunyavirus variants supports the identification of suitably conserved or diverse primer-binding target regions to establish group-wide as well as virus-specific nucleic acid tests in support of specific diagnostic and surveillance efforts throughout Australasia.

  1. Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity with meq gene polymorphism.

    Science.gov (United States)

    Renz, Katrin G; Cooke, Julie; Clarke, Nadeene; Cheetham, Brian F; Hussain, Zahid; Fakhrul Islam, A F M; Tannock, Gregory A; Walkden-Brown, Stephen W

    2012-01-01

    We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with meq gene polymorphism. Unvaccinated and herpesvirus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94% of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDV1 vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The meq gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the meq sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four prolines (PPPP repeats) in the meq gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.

  2. Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry.

    Science.gov (United States)

    Nagarajan, S; Rajukumar, K; Tosh, C; Ramaswamy, V; Purohit, K; Saxena, G; Behera, P; Pattnaik, B; Pradhan, H K; Dubey, S C

    2009-01-01

    A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.

  3. Genomic characterization of influenza A (H7N9) viruses isolated in Shenzhen, Southern China, during the second epidemic wave.

    Science.gov (United States)

    Fang, Shisong; Wang, Xin; Dong, Fangyuan; Jin, Tao; Liu, Guang; Lu, Xing; Peng, Bo; Wu, Weihua; Liu, Hui; Kong, Dongfeng; Tang, Xiujuan; Qin, Yanmin; Mei, Shujiang; Xie, Xu; He, Jianfan; Ma, Hanwu; Zhang, Renli; Cheng, Jinquan

    2016-08-01

    There were three epidemic waves of human infection with avian influenza A (H7N9) virus in 2013-2014. While many analyses of the genomic origin, evolution, and molecular characteristics of the influenza A (H7N9) virus have been performed using sequences from the first epidemic wave, genomic characterization of the virus from the second epidemic wave has been comparatively less reported. In this study, an in-depth analysis was performed with respect to the genomic characteristics of 11 H7N9 virus strains isolated from confirmed cases and four H7N9 virus strains isolated from environmental samples in Shenzhen during the second epidemic wave. Phylogenetic analysis demonstrated that six internal segments of the influenza A (H7N9) virus isolated from confirmed cases and environmental samples in Shenzhen were clustered into two different clades and that the origin of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen was different from that of viruses isolated during the first wave. In addition, H9N2 viruses, which were prevalent in southern China, played an important role in the reassortment of the influenza A (H7N9) virus isolated in Shenzhen. HA-R47K and -T122A, PB2-V139I, PB1-I397M, and NS1-T216P were the signature amino acids of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen. We found that the HA, NA, M, and PA genes of the A(H7N9) viruses underwent positive selection in the human population. Therefore, enhanced surveillance should be carried out to determine the origin and mode of transmission of the novel influenza A (H7N9) virus and to facilitate the formulation of effective policies for prevention and containment of a human infection epidemics.

  4. Isolation of mixed subtypes of influenza A virus from a bald eagle (Haliaeetus leucocephalus).

    Science.gov (United States)

    Goyal, Sagar M; Jindal, Naresh; Chander, Yogesh; Ramakrishnan, Muthanan A; Redig, Patrick T; Sreevatsan, Srinand

    2010-07-28

    From April 2007 to March 2008, cloacal swabs were obtained from 246 casualty raptors recovered by various wildlife rehabilitation centers in the United States. The swabs were placed in a virus transport medium and transported to the laboratory on ice packs. At the laboratory, the samples were pooled with each pool consisting of five samples. All pools (n = 50) were screened for the presence of avian influenza virus (AIV) using a real time reverse transcription-polymerase chain reaction (rRT-PCR); one of the pools was found positive. All five samples in this pool were tested individually by rRT-PCR; one sample from a bald eagle was found positive. This sample was inoculated in embryonated chicken eggs for virus isolation and a hemagglutinating virus was isolated. Complete genome sequencing of the isolate revealed a mixed infection with H1N1 and H2N1 subtypes. Further analysis revealed that the PB1-F2 gene sequence of H1N1 virus had the N66S virulence-associated substitution. Further studies on ecology and epidemiology of AIV in raptors are needed to help understand their role in the maintenance and evolution of AIV.

  5. Molecular characterization of banana bunchy top virus isolate from Sri Lanka and its genetic relationship with other isolates.

    Science.gov (United States)

    Wickramaarachchi, W A R T; Shankarappa, K S; Rangaswamy, K T; Maruthi, M N; Rajapakse, R G A S; Ghosh, Saptarshi

    2016-06-01

    Bunchy top disease of banana caused by Banana bunchy top virus (BBTV, genus Babuvirus family Nanoviridae) is one of the most important constraints in production of banana in the different parts of the world. Six genomic DNA components of BBTV isolate from Kandy, Sri Lanka (BBTV-K) were amplified by polymerase chain reaction (PCR) with specific primers using total DNA extracted from banana tissues showing typical symptoms of bunchy top disease. The amplicons were of expected size of 1.0-1.1 kb, which were cloned and sequenced. Analysis of sequence data revealed the presence of six DNA components; DNA-R, DNA-U3, DNA-S, DNA-N, DNA-M and DNA-C for Sri Lanka isolate. Comparisons of sequence data of DNA components followed by the phylogenetic analysis, grouped Sri Lanka-(Kandy) isolate in the Pacific Indian Oceans (PIO) group. Sri Lanka-(Kandy) isolate of BBTV is classified a new member of PIO group based on analysis of six components of the virus.

  6. The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses.

    Science.gov (United States)

    Richards, R Souza; Adams, I P; Kreuze, J F; De Souza, J; Cuellar, W; Dullemans, A M; Van Der Vlugt, R A A; Glover, R; Hany, U; Dickinson, M; Boonham, N

    2014-04-01

    The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.

  7. Avian H11 influenza virus isolated from domestic poultry in a Colombian live animal market.

    Science.gov (United States)

    Jiménez-Bluhm, Pedro; Karlsson, Erik A; Ciuoderis, Karl A; Cortez, Valerie; Marvin, Shauna A; Hamilton-West, Christopher; Schultz-Cherry, Stacey; Osorio, Jorge E

    2016-12-07

    Live animal markets (LAMs) are an essential source of food and trade in Latin American countries; however, they can also serve as 'hotbeds' for the emergence and potential spillover of avian influenza viruses (AIV). Despite extensive knowledge of AIV in Asian LAMs, little is known about the prevalence South American LAMs. To fill this gap in knowledge, active surveillance was carried out at the major LAM in Medellin, Colombia between February and September 2015. During this period, overall prevalence in the market was 2.67% and a North American origin H11N2 AIV most similar to a virus isolated from Chilean shorebirds asymptomatically spread through multiple bird species in the market resulting in 17.0% positivity at peak of infection. Phenotypically, the H11 viruses displayed no known molecular markers associated with increased virulence in birds or mammals, had α2,3-sialic acid binding preference, and caused minimal replication in vitro and little morbidity in vivo. However, the Colombian H11N2 virus replicated and transmitted effectively in chickens explaining the spread throughout the market. Genetic similarity to H11 viruses isolated from North and South American shorebirds suggest that the LAM occurrence may have resulted from a wild bird to domestic poultry spillover event. The ability to spread in domestic poultry as well as potential for human infection by H11 viruses highlight the need for enhanced AIV surveillance in South America in both avian species and humans.

  8. Avian H11 influenza virus isolated from domestic poultry in a Colombian live animal market

    Science.gov (United States)

    Jiménez-Bluhm, Pedro; Karlsson, Erik A; Ciuoderis, Karl A; Cortez, Valerie; Marvin, Shauna A; Hamilton-West, Christopher; Schultz-Cherry, Stacey; Osorio, Jorge E

    2016-01-01

    Live animal markets (LAMs) are an essential source of food and trade in Latin American countries; however, they can also serve as ‘hotbeds' for the emergence and potential spillover of avian influenza viruses (AIV). Despite extensive knowledge of AIV in Asian LAMs, little is known about the prevalence South American LAMs. To fill this gap in knowledge, active surveillance was carried out at the major LAM in Medellin, Colombia between February and September 2015. During this period, overall prevalence in the market was 2.67% and a North American origin H11N2 AIV most similar to a virus isolated from Chilean shorebirds asymptomatically spread through multiple bird species in the market resulting in 17.0% positivity at peak of infection. Phenotypically, the H11 viruses displayed no known molecular markers associated with increased virulence in birds or mammals, had α2,3-sialic acid binding preference, and caused minimal replication in vitro and little morbidity in vivo. However, the Colombian H11N2 virus replicated and transmitted effectively in chickens explaining the spread throughout the market. Genetic similarity to H11 viruses isolated from North and South American shorebirds suggest that the LAM occurrence may have resulted from a wild bird to domestic poultry spillover event. The ability to spread in domestic poultry as well as potential for human infection by H11 viruses highlight the need for enhanced AIV surveillance in South America in both avian species and humans. PMID:27924808

  9. Identification and isolation of Genotype-I Japanese Encephalitis virus from encephalitis patients

    Directory of Open Access Journals (Sweden)

    Gao Xiaoyan

    2010-11-01

    Full Text Available Abstract Historically, Japanese Encephalitis virus (JEV genotype III (GIII has been responsible for human diseases. In recent years, JEV genotype I (GI has been isolated from mosquitoes collected in numerous countries, but has not been isolated from patients with encephalitis. In this study, we report recovery of JEV GI live virus and identification of JEV GI RNA from cerebrospinal fluid (CSF of encephalitis patients in JE endemic areas of China. Whole-genome sequencing and molecular phylogenetic analysis of the JEV isolate from the CSF samples was performed. The isolate in this study is highly similar to other JEV GI strains which isolated from mosquitoes at both the nucleotide and deduced amino acid levels. Phylogenetic analysis based on the genomic sequence showed that the isolate belongs to JEV GI, which is consistent with the phylogenetic analysis based on the pre-membrane (PrM and Glycoprotein genes. As a conclusion, this is the first time to isolate JEV GI strain from CSF samples of encephalitis patients, so continuous survey and evaluate the infectivity and pathogenecity of JEV GI strains are necessary, especially for the JEV GI strains from encephalitis patients. With respect to the latter, because all current JEV vaccines (live and inactivated are derived from JEV GIII strains, future studies should be aimed at investigating and monitoring cross-protection of the human JEV GI isolates against widely used JEV vaccines.

  10. Genome Sequence of Bluetongue virus Serotype 17 Isolated in Brazil in 2014.

    Science.gov (United States)

    Matos, Ana Carolina Diniz; Rosa, Júlio César Câmara; Nomikou, Kyriaki; Guimarães, Lorena Lima Barbosa; Costa, Érica Azevedo; Guedes, Maria Isabel Maldonado Coelho; Driemeier, David; Lobato, Zélia Inês Portela; Mertens, Peter Paul Clement

    2016-10-27

    The complete genome sequence of Bluetongue virus (BTV) serotype 17 strain 17/BRA/2014/73, isolated from a sheep in Brazil in 2014, is reported here. All segments clustered with western topotype strains and indicated reassortment events with other BTV from the Americas. The strain 17/BRA/2014/73 represents a novel reference strain for BTV-17 from South America.

  11. Complete Genome Sequence of a South Korean Isolate of Habenaria mosaic virus.

    Science.gov (United States)

    Igori, Davaajargal; Lim, Seungmo; Zhao, Fumei; Baek, Dasom; Moon, Jae Sun

    2016-09-08

    Habenaria mosaic virus (HaMV), a member of the genus Potyvirus in the family Potyviridae, was first discovered from Habenaria radiata in Japan. The complete genomic sequence of a South Korean isolate (PA1) of HaMV infecting Plantago asiatica L. was determined with high-throughput RNA sequencing.

  12. Alstroemeria-infecting cucumber mosaic virus isolates contain additional sequences in the RNA 3 segment.

    NARCIS (Netherlands)

    Chen, Y.K.; Prins, M.W.; Derks, A.F.L.M.; Langeveld, S.A.; Goldbach, R.W.

    2002-01-01

    The coat protein (CP) genes and flanking regions of three alstroemeria-infecting cucumber mosaic virus isolates (CMV-ALS), denoted ALS-LBO, ALS-IPO, and ALS-NAK, were cloned and their nucleotide sequence determined and compared at both nucleic acid and deduced protein level with the published sequen

  13. Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus

    NARCIS (Netherlands)

    Komatsu, Ken; Yamashita, Kazuo; Sugawara, Kota; Verbeek, Martin; Fujita, Naoko; Hanada, Kaoru; Uehara-Ichiki, Tamaki; Fuji, Shin Ichi

    2017-01-01

    Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and t

  14. Complete genome sequence of genotype VI Newcastle disease viruses isolated from pigeons in Pakistan

    Science.gov (United States)

    Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion prot...

  15. Molecular characterization and phylogenetic study of Newcastle disease virus isolates from recent outbreaks in eastern Uganda

    DEFF Research Database (Denmark)

    Otim, Maxwell O.; Christensen, Henrik; Jørgensen, Poul Henrik;

    2004-01-01

    Newcastle disease virus isolates from chickens in eastern Uganda in 2001 were found to be velogenic by fusion protein cleavage site sequence analysis and biological characterization; the intracerebral pathogenicity index was 1.8. Analysis of their hemagglutinin-neuraminidase protein gene sequences...

  16. Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates

    DEFF Research Database (Denmark)

    Johansson, Tove; Einer-Jensen, Katja; Batts, William

    2009-01-01

    Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13/98 11...

  17. Genome sequences of nine vesicular stomatitis virus isolates from South America

    Science.gov (United States)

    We report nine full-genome sequences of vesicular stomatitis virus obtrained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species, clade III, while two...

  18. Alstroemeria-infecting cucumber mosaic virus isolates contain additional sequences in the RNA 3 segment.

    NARCIS (Netherlands)

    Chen, Y.K.; Prins, M.W.; Derks, A.F.L.M.; Langeveld, S.A.; Goldbach, R.W.

    2002-01-01

    The coat protein (CP) genes and flanking regions of three alstroemeria-infecting cucumber mosaic virus isolates (CMV-ALS), denoted ALS-LBO, ALS-IPO, and ALS-NAK, were cloned and their nucleotide sequence determined and compared at both nucleic acid and deduced protein level with the published

  19. First complete genome sequence of an emerging cucumber green mottle mosaic virus isolate in North America

    Science.gov (United States)

    The complete genome sequence (6,423 nt) of an emerging Cucumber green mottle mosaic virus (CGMMV) isolate on cucumber in North America was determined through deep sequencing of sRNA and rapid amplification of cDNA ends. It shares 99% nucleotide sequence identity to the Asian genotype, but only 90% t...

  20. Complete Genome Sequence of a Newcastle Disease Virus Isolate from an Outbreak in Central India

    Science.gov (United States)

    Gogoi, Polakshee; Morla, Sudhir; Kaore, Megha; Kurkure, Nitin Vasantrao

    2015-01-01

    The complete genome sequence of a Newcastle disease virus (NDV) strain NDV/Chicken/Nagpur/01/12 was isolated from vaccinated chicken farms in India during outbreaks in 2012. The genome is 15,192 nucleotides in length and is classified as genotype VII in class II. PMID:25593257

  1. First Complete Genome Sequences of Zika Virus Isolated from Febrile Patient Sera in Ecuador

    Science.gov (United States)

    Márquez, S.; Carrera, J.; Pullan, S. T.; Lewandowski, K.; Paz, V.; Loman, N.; Quick, J.; Bonsall, D.; Powell, R.; Thézé, J.; Pybus, O. G.; Klenerman, P.; Eisenberg, J.; Coloma, J.; Carroll, M. W.; Trueba, G.

    2017-01-01

    ABSTRACT Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, EcEs062_16 and EcEs089_16, isolated from the sera of febrile patients in Esmeraldas City, in the northern coastal province of Esmeraldas, Ecuador, in April 2016. These are the first complete ZIKV genomes to be reported from Ecuador. PMID:28232448

  2. In vitro and in vivo isolation and characterization of Duvenhage virus

    NARCIS (Netherlands)

    P. Koraka (Penelope); B.E.E. Martina (Byron); J.M. Roose (Jouke M.); P.P.A.M. van Thiel (Pieter); G. van Amerongen (Geert); T. Kuiken (Thijs); A.D.M.E. Osterhaus (Albert)

    2012-01-01

    textabstractA fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization o

  3. Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

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    M.P. Miagostovich

    2002-08-01

    Full Text Available The genetic characterization of dengue virus type 3 (DEN-3 strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550 of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures.

  4. Selection of tomato plants resistant to a local Polish isolate of tomato spotted wilt virus (TSWV).

    Science.gov (United States)

    Czech, Andrzej S; Szklarczyk, Marek; Gajewski, Zbigniew; Zukowska, Ewa; Michalik, Barbara; Kobyłko, Tadeusz; Strzałka, Kazimierz

    2003-01-01

    We found that the Sw-5 gene confers resistance to one of the Polish isolates of tomato spotted wilt virus (TSWV). A series of tomato breeding accessions was analysed along with standards of resistance and susceptibility to TSWV. The presence of the Sw-5 gene was determined using the available PCR marker. Subsequently plants from these accessions were grown in the presence of the TSWV isolate from Poland. Some of them developed severe symptoms of the TSWV disease. Expression of the virus proteins was also assayed in tissues of the investigated plants. We found general agreement between either lack or presence of the disease symptoms, virus proteins and resistance gene. Some observed discrepancies of these data are also discussed. Our results indicate that marker-assisted selection can be used for breeding of the TSWV-resistant tomato in Poland.

  5. Emergence of a new arbovirus disease in Brazil. III. Isolation of Rocio virus from Psorophora Ferox (Humboldt, 1819).

    Science.gov (United States)

    de Souza Lopes, O; de Abreu Sacchetta, L; Francy, D B; Jakob, W L; Calisher, C H

    1981-02-01

    A newly described flavivirus was responsible for a large encephalitis epidemic in São Paulo State, Brazil. The etiologic agent, Rocio virus, was isolated from human patients and sentinel mice. The natural history of the virus is unknown although presumed to be arthropod-borne. Rocio virus was isolated from a single pool containing 19 Psorophora ferox of 47 pools (283 specimens) of this species tested. The positive pool contained 16 deplete, 2 gravid, and 2 engorged mosquitoes. No isolations were made from 2183 pools of other species. The positive pool was collected during the year of the epidemic at the same approximate time and place where vertebrate isolations were made.

  6. Isolation and genetic characteristics of human genotype 1 Japanese encephalitis virus, China, 2009.

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    Jiu-Song Zhang

    Full Text Available BACKGROUND: Several studies have shown that the predominant genotype of Chinese Japanese encephalitis virus (JEV is evolving from genotype 3 to genotype 1. However, in recent years, almost all genotype 1 isolates were from mosquitoes, and genotype 1 has been less associated with human disease than genotype 3. This study reports the isolation of human genotype 1 JEV and its genetic characteristics to provide additional insights into human JE pathogens that are currently circulating in China. METHODS AND RESULTS: In 2009, 31 cerebrospinal fluid samples were collected from patients living in Yunnan and Shanxi provinces and were used to inoculate Aedes albopictus C6/36 cells for virus isolation. The JEV strains were identified using immunofluorescent assays and the reverse transcription-polymerase chain reaction. Phylogenetic analyses based on the partial capsid/pre-membrane and full envelope (E sequences were performed using Clustalx 1.8 software. Three JEV isolates were obtained from a 4-year-old girl and a 2-year-old boy living in Yunnan and an 82-year-old woman in Shanxi. The boy had been immunized with one dose of JE live attenuated vaccine. New isolates were grouped into genotype 1. Amino acid sequence for the viral E protein indicated 95% to 100% identity with each other and with other JEV strains. When compared with a consensus sequence of E protein, two amino acid substitutions were found: Ser(E-123-Asn in the two Yunnan isolates and Lys(E-166-Arg in the Shanxi isolate. CONCLUSIONS: Our findings indicate that the genotype 1 of JEV is causing human infections in China. Our observation of a previously vaccinated boy developing JE from genotype 1 virus infection also calls for more detailed studies, both in vitro and in vivo neutralization tests as well as active surveillance, to examine the possibility of a lack of complete protection conferred by the live attenuated JE vaccine against genotype 1 virus.

  7. Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru.

    Science.gov (United States)

    Vasilakis, Nikos; Castro-Llanos, Fanny; Widen, Steven G; Aguilar, Patricia V; Guzman, Hilda; Guevara, Carolina; Fernandez, Roberto; Auguste, Albert J; Wood, Thomas G; Popov, Vsevolod; Mundal, Kirk; Ghedin, Elodie; Kochel, Tadeusz J; Holmes, Edward C; Walker, Peter J; Tesh, Robert B

    2014-04-01

    Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.

  8. Biological and Molecular Characterization of Cucumber mosaic virus Subgroup II Isolate Causing Severe Mosaic in Cucumber.

    Science.gov (United States)

    Kumari, Reenu; Bhardwaj, Pooja; Singh, Lakhmir; Zaidi, Aijaz A; Hallan, Vipin

    2013-06-01

    Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricultural and ornamental crops. Earlier reports showed the prevalence of CMV subgroup I isolates in India. However, some recent reports point towards increasing incidence of subgroup II isolates in the country. The complete genome of a CMV isolate causing severe mosaic in cucumber was characterized and its phylogenetic analysis with other 21 CMV isolates reported worldwide clustered it with subgroup II strains. The genome comprised of RNA 1 (3,379 nucleotides), RNA 2 (3,038 nucleotides) and RNA 3 (2,206 nucleotides). The isolate showed highest homology with subgroup II isolates: 95.1-98.7, 87.7-98.0, and 85.4-97.1 % within RNA1, RNA2, and RNA3, respectively. RNA1 and RNA2 were closely related to the Japanese isolate while RNA3 clustered with an American isolate. Host range studies revealed that isolate showed severe mosaic symptoms on Nicotiana spp. and Cucumis spp. The isolate induced leaf deformation and mild filiform type symptoms in tomato. To best of our knowledge this is the first report of complete genome of CMV subgroup II isolate from India.

  9. R5 to X4 coreceptor switch of human immunodeficiency virus type 1 B' and B'/C recombinant subtype isolates in China

    Institute of Scientific and Technical Information of China (English)

    GUO Yan-fang; ZHANG Xiao-yan; RUAN Yu-hua; ZHANG Yao-xin; SHAO Yi-ming; MA Li-ying; YUAN Lin; WANG Shu-hua; SUN Jian-ping; XU Wei-si; Xu Jian-qing; XING Hui; HONG Kun-xue

    2007-01-01

    @@ The chemokine receptors CCR5 and CXCR4 play an important role as coreceptors for human immunodeficiency virus type 1 (HIV-1) entring into cells.HIV-1 isolates can be distinguished by the chemokine coreceptors. Nonsyncytium inducing (NSI), macrophage tropic viruses utilizing CCR5, are called R5 viruses;syncytium inducing (SI) isolates use CXCR4 and known as X4 viruses.

  10. A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk

    NARCIS (Netherlands)

    Kramps, J.A.; Maanen, van C.; Wetering, van de G.; Stienstra, G.; Quak, S.; Brinkhof, J.; Ronsholt, L.; Nylin, B.

    1997-01-01

    To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-struct

  11. Complete genome sequence of a J subgroup avian leukosis virus isolated from local commercial broilers.

    Science.gov (United States)

    Li, Hongxin; Xue, Chunyi; Ji, Jun; Chang, Shuang; Shang, Huiqin; Zhang, Lingjun; Ma, Jingyun; Bi, Yingzuo; Xie, Qingmei

    2012-11-01

    Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5'-GGCACCTCC-3') for c-myb was identified in the GDKP1202 genome. These findings will provide additional insights into the molecular characteristics in the genomes and pathogenicity of ALV-J.

  12. Genotypic characteristics of bovine viral diarrhea virus 2 strains isolated in northern Italy.

    Science.gov (United States)

    Giangaspero, M; Harasawa, R; Zecconi, A; Luzzago, C

    2001-09-01

    Two strains of Bovine viral diarrhea virus 2 (BVDV-2) were isolated from calves in northern Italy. Variations in the 5'-untranslated region (UTR) of the genome were studied by primary structure alignment and neighbor-joining method based phylogenetic tree analyses and by palindromic nucleotide substitutions at the three variable loci in the 5'-UTR. Genetic analysis indicated their appurtenance to genovar BVDV-2a. Nucleotide sequence at the 5'-UTR of strain BS-95-II, one of the Italian isolates from healthy calves, showed 98% homology to that of the Japanese isolate OY89, a cytopathic strain derived from cattle with mucosal disease.

  13. Epornitic of avian pox in common buzzards (Buteo buteo): virus isolation and molecular biological characterization.

    Science.gov (United States)

    Rampin, T; Pisoni, G; Manarolla, G; Gallazzi, D; Sironi, G

    2007-04-01

    Six common buzzards from a bird rescue centre showed wart-like lesions on their toes. The lesions consisted of multiple crusty and proliferative nodules surrounded by skin swelling. Histologically, epithelial cell hypertrophy and hyperplasia with ballooning degeneration and large intracytoplasmic inclusion bodies consistent with avipoxvirus infection were seen. The virus was isolated in embryonated chicken eggs. Positive chorioallantoic membranes and samples of skin lesions were submitted for polymerase chain reaction. Molecular characterization based on the 4b core protein indicates a 100% homology of the isolated poxvirus with avian poxviruses belonging to subclade A2. However, analysis of fpv139 locus does not reveal similarities of the isolate with other avian poxviruses.

  14. Comparison of the pathogenicity of Nipah virus isolates from Bangladesh and Malaysia in the Syrian hamster.

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    Blair L DeBuysscher

    Full Text Available Nipah virus is a zoonotic pathogen that causes severe disease in humans. The mechanisms of pathogenesis are not well described. The first Nipah virus outbreak occurred in Malaysia, where human disease had a strong neurological component. Subsequent outbreaks have occurred in Bangladesh and India and transmission and disease processes in these outbreaks appear to be different from those of the Malaysian outbreak. Until this point, virtually all Nipah virus studies in vitro and in vivo, including vaccine and pathogenesis studies, have utilized a virus isolate from the original Malaysian outbreak (NiV-M. To investigate potential differences between NiV-M and a Nipah virus isolate from Bangladesh (NiV-B, we compared NiV-M and NiV-B infection in vitro and in vivo. In hamster kidney cells, NiV-M-infection resulted in extensive syncytia formation and cytopathic effects, whereas NiV-B-infection resulted in little to no morphological changes. In vivo, NiV-M-infected Syrian hamsters had accelerated virus replication, pathology and death when compared to NiV-B-infected animals. NiV-M infection also resulted in the activation of host immune response genes at an earlier time point. Pathogenicity was not only a result of direct effects of virus replication, but likely also had an immunopathogenic component. The differences observed between NiV-M and NiV-B pathogeneis in hamsters may relate to differences observed in human cases. Characterization of the hamster model for NiV-B infection allows for further research of the strain of Nipah virus responsible for the more recent outbreaks in humans. This model can be used to study NiV-B pathogenesis, transmission, and countermeasures that could be used to control outbreaks.

  15. Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia.

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    Simon Delgado

    2008-04-01

    Full Text Available A small focus of hemorrhagic fever (HF cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá. RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.

  16. Virus movements on the plasma membrane support infection and transmission between cells.

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    Christoph J Burckhardt

    2009-11-01

    Full Text Available How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus-host interactions upstream of infectious entry offer new perspectives for anti-viral interference.

  17. Phylogenetic analysis of Chinese sheeppox and goatpox virus isolates

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    Zhou Tao

    2012-01-01

    Full Text Available Abstract Background Sheeppox virus (SPPV and goatpox virus (GTPV, members of the Capripoxvirus genus of the Poxviridae family are causative agents of sheep pox and goat pox respectively, which are important contagious diseases and endemic in central and northern Africa, the Middle and Far East, and the Indian sub-continent. Both sheep pox and goat pox can cause wool and hide damage, and reduce the production of mutton and milk, which may result in significant economic losses and threaten the stockbreeding. In this study, three SPPVs and two GTPVs were collected from China in 2009 and 2011. We described the sequence features and phylogenetic analysis of the P32 gene, GPCR gene and RPO30 gene of the SPPVs and GTPVs to reveal their genetic relatedness. Results Sequence and phylogenetic analysis showed that there was a close relationship among SPPV/GanS/2/2011/China, SPPV/GanS/1/2011/China and SPPV/NingX/2009/China. They were clustered on the same SPPV clade. GTPV/HuB/2009/China and GS-V1 belonged to the GTPV lineage. GS-V1 was closely related to other GTPV vaccine strains. GTPV/HuB/2009/China and GS-V1 were clustered with GTPVs from China and some southern Asian countries. Conclusion This study may expand the datum for spread trend research of Chinese SPPVs and GTPVs, meanwhile provide theoretical references to improve the preventive and control strategy.

  18. First Report of Cucumber mosaic virus Isolated from Wild Vigna angularis var. nipponensis in Korea

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    Mi-Kyeong Kim

    2014-06-01

    Full Text Available A viral disease causing severe mosaic, necrotic, and yellow symptoms on Vigna angularis var. nipponensis was prevalent around Suwon area in Korea. The causal virus was characterized as Cucumber mosaic virus (CMV on the basis of biological and nucleotide sequence properties of RNAs 1, 2 and 3 and named as CMV-wVa. CMV-wVa isolate caused mosaic symptoms on indicator plants, Nicotiana tabacum cv. Xanthi-nc, Petunia hybrida, and Cucumis sativus. Strikingly, CMV-wVa induced severe mosaic and malformation on Cucurbita pepo, and Solanum lycopersicum. Moreover, it caused necrotic or mosaic symptoms on V. angularis and V. radiate of Fabaceae. Symptoms of necrotic local or pin point were observed on inoculated leaves of V. unguiculata, Vicia fava, Pisum sativum and Phaseolus vulgaris. However, CMV-wVa isolate failed to infect in Glycine max cvs. ‘Sorok’, ‘Sodam’ and ‘Somyeong’. To assess genetic variation between CMV-wVa and the other known CMV isolates, phylogenetic analysis using 16 complete nucleotide sequences of CMV RNA1, RNA2, and RNA3 including CMV-wVa was performed. CMV-wVa was more closely related to CMV isolates belonging to CMV subgroup I showing about 85.1–100% nucleotide sequences identity to those of subgroup I isolates. This is the first report of CMV as the causal virus infecting wild Vigna angularis var. nipponensis in Korea.

  19. First Report of Cucumber mosaic virus Isolated from Wild Vigna angularis var. nipponensis in Korea.

    Science.gov (United States)

    Kim, Mi-Kyeong; Jeong, Rae-Dong; Kwak, Hae-Ryun; Lee, Su-Heon; Kim, Jeong-Soo; Kim, Kook-Hyung; Cha, Byeongjin; Choi, Hong-Soo

    2014-06-01

    A viral disease causing severe mosaic, necrotic, and yellow symptoms on Vigna angularis var. nipponensis was prevalent around Suwon area in Korea. The causal virus was characterized as Cucumber mosaic virus (CMV) on the basis of biological and nucleotide sequence properties of RNAs 1, 2 and 3 and named as CMV-wVa. CMV-wVa isolate caused mosaic symptoms on indicator plants, Nicotiana tabacum cv. Xanthi-nc, Petunia hybrida, and Cucumis sativus. Strikingly, CMV-wVa induced severe mosaic and malformation on Cucurbita pepo, and Solanum lycopersicum. Moreover, it caused necrotic or mosaic symptoms on V. angularis and V. radiate of Fabaceae. Symptoms of necrotic local or pin point were observed on inoculated leaves of V. unguiculata, Vicia fava, Pisum sativum and Phaseolus vulgaris. However, CMV-wVa isolate failed to infect in Glycine max cvs. 'Sorok', 'Sodam' and 'Somyeong'. To assess genetic variation between CMV-wVa and the other known CMV isolates, phylogenetic analysis using 16 complete nucleotide sequences of CMV RNA1, RNA2, and RNA3 including CMV-wVa was performed. CMV-wVa was more closely related to CMV isolates belonging to CMV subgroup I showing about 85.1-100% nucleotide sequences identity to those of subgroup I isolates. This is the first report of CMV as the causal virus infecting wild Vigna angularis var. nipponensis in Korea.

  20. Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

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    FAVORETTO Silvana Regina

    2002-01-01

    Full Text Available Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog, 3 (Desmodus rotundus, 4 (Tadarida brasiliensis, 5 (vampire bat from Venezuela, 6 (Lasiurus cinereus and Lab (reacted to all used antibodies. Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus. These findings may be related to the existence of multiple independent transmission cycles, involving different bat species.

  1. Genotypic and pathotypic characterization of Newcastle disease virus isolated from racing pigeons in China.

    Science.gov (United States)

    Liu, Mengda; Qu, Yajin; Wang, Fangkun; Liu, Sidang; Sun, Honglei

    2015-07-01

    A Newcastle disease virus (NDV) isolated from an outbreak in racing pigeons in China was characterized in this study. Complete gene of the NDV isolate was sequenced and phylogenetic analysis. Pathogenicity experiment was carried out in pigeons, chickens, and ducks. Phylogenetic analysis revealed that the strain clustered with the Class II viruses, has highly phylogenetically similar to NDV strains isolated from pigeons in China, but was distant from the viruses prevalence in chickens and vaccine strains used in China. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the virulent motif (112)RRQKRF(117) at the cleavage site, but it caused no appearance disease in chickens and ducks. However, the isolate had virulence in pigeons, resulting in severe nervous signs and highly mortality. Pigeons were considered as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the NDV infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks.

  2. Genomic characterization of three bovine viral diarrhea virus isolates from cattle.

    Science.gov (United States)

    Cai, Dongjie; Song, Quanjiang; Wang, Jiufeng; Zhu, Yaohong

    2016-12-01

    Three strains of the bovine viral diarrhea virus (BVDV) were isolated from cattle in Beijing, China. To investigate their genomic features, we sequenced and characterized the complete genome of each of the isolates. Each of the three virus genomes is about 12,220 bp in length, containing a 5' untranslated region (UTR), one open reading frame (ORF) encoding a 3897-amino-acid polypeptide, and a 3' UTR. The nucleotide sequence of the three isolates were 99.0 % identical to each and other shared nucleotide sequence identities of 73.4 % to 98.3 % with other BVDV-1 strains, about 70.0 % with BVDV-2 strains, about 67.0 % with BVDV-3, and less than 67.0 % with other pestiviruses. Phylogenetic analysis of the full-length genome, 3' UTR, and the N(pro) gene demonstrated that the three viruses were BVDV-1 isolates. This is the first report of complete genome sequences of BVDV 1d isolates from China and might have implications for vaccine development.

  3. Variation in the Coat Protein Gene of Papaya ringspot Virus Isolates from Multiple Locations of China

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The potyvirus Papaya ringspot virus (PRSV) is an important pathogen of papaya that causes severe losses in economic crops for papaya production globally. The coat protein (CP) genes of five PRSV isolates originating from different locations in China were cloned and sequenced. The CP-coding region varied in size from 864-873 nucleotides, encoding proteins of 288-291 amino acids. The five Chinese isolates of PRSV have been characterized as papaya-infecting (PRSV-P). The CP sequences of the Chinese isolates were compared with those of previously published PRSV isolates originating from different countries at amino acid levels. A number of KE repeat boxes in the N terminus of the PRSV-CP were found in all Chinese isolates. The phylogenetic branching pattern revealed that there was certain extended grouping between geographic locations, and the Asian type probably represents the oldest population of PRSV. The information of CP genes will be useful in designing and developing durable virus resistant-PRSV transgenic papaya in China. Meanwhile broad-spectrum-virus resistant, strongly resistant-PRSV and good safe papaya lines are required.

  4. Differential cytopathogenesis of respiratory syncytial virus prototypic and clinical isolates in primary pediatric bronchial epithelial cells

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    Coyle Peter V

    2011-01-01

    Full Text Available Abstract Background Human respiratory syncytial virus (RSV causes severe respiratory disease in infants. Airway epithelial cells are the principle targets of RSV infection. However, the mechanisms by which it causes disease are poorly understood. Most RSV pathogenesis data are derived using laboratory-adapted prototypic strains. We hypothesized that such strains may be poorly representative of recent clinical isolates in terms of virus/host interactions in primary human bronchial epithelial cells (PBECs. Methods To address this hypothesis, we isolated three RSV strains from infants hospitalized with bronchiolitis and compared them with the prototypic RSV A2 in terms of cytopathology, virus growth kinetics and chemokine secretion in infected PBEC monolayers. Results RSV A2 rapidly obliterated the PBECs, whereas the clinical isolates caused much less cytopathology. Concomitantly, RSV A2 also grew faster and to higher titers in PBECs. Furthermore, dramatically increased secretion of IP-10 and RANTES was evident following A2 infection compared with the clinical isolates. Conclusions The prototypic RSV strain A2 is poorly representative of recent clinical isolates in terms of cytopathogenicity, viral growth kinetics and pro-inflammatory responses induced following infection of PBEC monolayers. Thus, the choice of RSV strain may have important implications for future RSV pathogenesis studies.

  5. Nucleotide sequences of two Korean isolates of Cucumber green mottle mosaic virus.

    Science.gov (United States)

    Kim, Sang-Min; Lee, Jung-Myung; Yim, Kyu-Ock; Oh, Man-Ho; Park, Jin-Woo; Kim, Kook-Hyung

    2003-12-31

    The nucleotide sequences of the genomic RNAs of Cucumber green mottle mosaic virus Korean watermelon isolate (CGMMV-KW) and Korean oriental melon isolate (CGMMV-KOM) were determined and compared to the sequences of other tobamoviruses including CGMMV strains W and SH. Each CGMMV isolate had a genome of 6,424 nucleotides. Each also had 60 and 176 nucleotides of 5' and 3' untranslated regions (UTRs), respectively, and four open reading frames (ORF1-4). ORFs 1 to 4 encode proteins of 129, 186, 29, and 17.4 kDa, respectively. The nucleotide and deduced amino acid sequences of CGMMV-KOM and CGMMV-KW were more than 98.3% identical. When compared to other CGMMV strains in a phylogenetic analysis they were found to form a distinct virus clade, and were more distantly related to other tobamoviruses (23.5-56.7% identity).

  6. Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

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    Ana Luiza Pamplona Mosimann

    2011-05-01

    Full Text Available Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection.

  7. Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India.

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    Morla, Sudhir; Deka, Pankaj; Kumar, Sachin

    2016-04-01

    Infectious bursal disease virus (IBDV) is a highly infectious disease of young chicken that predominantly affects the immune system. In the present study, we are reporting first comprehensive study of IBDV outbreaks from the Northeastern part of India. Northeast India shares a porous border with four different countries; and as a rule any outbreak in the neighboring countries substantially affects the poultry population in the adjoining states. Nucleotide sequence analysis of the VP2 gene of the IBDV isolates from the Northeastern part of India suggested the extreme virulent nature of the virus. The virulent marker amino acids (A222, I242, Q253, I256 and S299) in the hypervariable region of the Northeastern isolates were found identical with the reported very virulent strains of IBDV. A unique insertion of I/L294V was recorded in all the isolates of the Northeastern India. The study will be useful in understanding the circulating pathotypes of IBDV in India.

  8. Partial sequencing and phylogenetic analysis of Soybean mosaic virus isolated in Ukraine

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    Polischuk V. P.

    2011-12-01

    Full Text Available The aim of the present study is to compare the biological and molecular properties of Ukrainian soybean mosaic virus (SMV isolates with those of known strains or isolates from other countries, and to trace their possible origin. The methods of mechanical inoculation, reverse transcription polymerase chain reaction, DNA sequencing and phylogenetic analysis have been used. Results. Five SMV isolates have been collected and biologically purified from breeding plots in Vinnitsa region of Ukraine. It has been found that all these isolates show the same reaction patterns when infecting 11 differential soybean cultivars. Phylogenetic analysis of sequences of the coat protein coding region and P1 coding region revealed strong genetic relationships between representative Ukrainian (UA1Gr and SMV-VA2 isolates which together were sorted in one clade with G2 strain. The investigation of sequence identity showed that different genomic regions of SMV were under different evolutionary constraints. Conclusions. SMV, isolated in Ukraine for the first time, belongs to the G2 strain group that is widespread in North America. The SMV isolates obtained in this work may be employed in the Ukrainian national breeding programs to create soybean with durable virus resistance.

  9. Lassa Virus Isolates from Mali and the Ivory Coast Represent an Emerging Fifth Lineage

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    John Tyler Manning

    2015-10-01

    Full Text Available Previous imported cases of Lassa fever into the United Kingdom from the Ivory Coast and Mali, as well as the detection of Lassa virus among the Mastomys natalensis population within Mali has led to the suggestion that the endemic area for Lassa fever is expanding. Initial phylogenetic analyses arrange isolates from Mali and the Ivory Coast separately from the classical lineage IV isolates taken from Sierra Leone, Guinea, and Liberia. The availability of full genome sequences continues to increase, allowing for a more complete phylogenetic comparison of the isolates from Mali and the Ivory Coast to the other existing isolates. In this study, we utilized a Bayesian approach to infer the demographic histories of each Lassa virus isolate for which the full sequence was available. Our results indicate that the isolates from Mali and the Ivory Coast group separately from the isolates of lineage IV, comprising a distinct fifth lineage. The split between lineages IV and V is estimated to have occurred around 200-300 years ago, which coincides with the colonial period of West Africa.

  10. Molecular detection, epidemiology, and genetic characterization of novel European field isolates of equine infectious anemia virus.

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    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.

  11. Molecular characterization of Dasheen mosaic virus isolates infecting edible aroids in India.

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    Babu, B; Hegde, V

    2014-01-01

    Dasheen mosaic virus (DsMV) infecting three major edible aroids namely Amorphophallus paeoniifolius, Colocasia esculenta, and Xanthosoma sagittifolium cultivated in India was characterized. Infected plants showing typical DsMV symptoms were subjected to reverse transcription-polymerase chain reaction, and an amplification of a 963 bp fragment which encoded the coat protein (CP) gene was obtained. BLAST analysis of the cloned DNA amplicon revealed the identity of the virus to be that of DsMV. Sequence identity matrix of the nucleotide sequences among the three isolates showed that the DsMV isolate infecting A. paeoniifolius and C. esculenta shared an identity as high as 93%, while the DsMV isolate from X. sagittifolium shared an identity of only 73% and 76% with the DsMV isolates from A. paeoniifolius and C. esculenta, respectively. Comparative analysis of the coat protein of the three DsMV isolates showed the presence of DVG motif (A. paeoniifolius and C. esculenta) and DTG motif in X. sagittifolium and several varying potential threonine and asparagine rich N-glycosylation motifs. Single amino acid substitution of the several conserved motifs occurs in all the three DsMV isolates. This is the first characterization of DsMV isolates infecting A. paeoniifolius, C. esculenta, and X. sagittifolium plants in India.

  12. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

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    Wang Nan

    2011-02-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. Results we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE, indirect immunoperoxidase test (IPX and electron microscopy(EM. The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV and 3 strains of border disease virus(BDV in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. Conclusion To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

  13. Molecular characterization of cucumber mosaic virus isolates infecting tomato in Hamedan and Tehran provinces of Iran.

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    Safaeizadeh, M; Saidi, A; Palukaitis, P

    2015-06-01

    Here we identified four isolates, MS, 3H, 50A, and 2K of cucumber mosaic virus (CMV) infecting tomato, on the basis of their non-coding intergenic region and a part of the coat protein (CP) sequence in the CMV genomic RNA3. The sequences from the four isolates were compared with other previously characterized isolates of CMV isolated from different plant species across the globe. Sequence comparisons revealed that the two CMV isolates from Hamedan province (MS and 3H) had the highest sequence identity with CMV-G10 (98%), which was previously reported as a severe Hellenic tomato isolate of CMV, while the CMV isolates from Tehran province, including CMV-2K (isolated from Karaj region) and CMV-50A (isolated from Varamin region), had the highest sequence identity with that of CMV-ALF (99%). Phylogenetic analysis of the nucleotide sequences showed that CMV-MS and CMV-3H belong to group IB, while CMV-2K and CMV-50A belong to group IA. This is the first report on the molecular characterization of novel isolates of CMV infecting tomato plants in Iran.

  14. Comparison of multiple genes of spring viremia of carp viruses isolated in the United States.

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    Warg, Janet V; Dikkeboom, Audrey L; Goodwin, Andrew E; Snekvik, Kevin; Whitney, John

    2007-08-01

    Five spring viremia of carp viruses (SVCV), Rhabdovirus carpio, were isolated in the United States (US) between 2002 and 2004. Single tube reverse transcription-polymerase chain reaction (RT-PCR) was used to generate overlapping cDNA fragments from the US isolates of SVCV. Multiple pairs of specific primers were designed to amplify a portion of the phosphoprotein gene, the matrix gene, and the glycoprotein gene of SVCV genogroup Id (corresponding to nucleotides 2174-4942 of GenBank accession NC_002803). Sequences were proofread and aligned to generate a consensus sequence for each isolate. Phylogenetic analysis of the 2705 nucleotide consensus sequence revealed that all five US isolates belong to SVCV genogroup Ia, Asian origin isolates, and a PCR primer binding site unique to SVCV genogroup Ia was identified.

  15. Diagnosis and sequence analysis of avian leukosis virus subgroup J isolated from Chinese Partridge Shank chickens.

    Science.gov (United States)

    Dong, Xuan; Zhao, Peng; Li, Weihua; Chang, Shuang; Li, Jianliang; Li, Yang; Ju, Sidi; Sun, Peng; Meng, Fanfeng; Liu, Juan; Cui, Zhizhong

    2015-04-01

    The diagnosis of avian leukosis virus subgroup J (ALV-J) infection in Chinese Partridge Shank chickens was confirmed by necropsy, histopathological examinations, antibody tests, viral isolation, immunofluorescence assays, and sequence analysis. Myelocytoma, myeloma, and fibrosarcoma were simultaneously found in Partridge Shank flock with ALV-J infection. Sequence analysis of the env genes of ALV-J demonstrated that both gp85 and gp37 were highly homologous among the three strains from local chickens of those among ALV-J strains isolated from white meat-type chickens. The phylogenetic trees indicated that the three strains isolated in this study were closely related to reference strains isolated in so-called Chinese yellow chickens and some strains isolated from white meat-type chickens, both from the USA and China. The observed ALV-J infection was the first report on Partridge Shank chickens, and myelocytoma, myeloma, and fibrosarcoma were found at the same time in this batch of local chickens.

  16. Biological and molecular characterization of Brazilian isolates of Zucchini yellow mosaic virus

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    David Marques de Almeida Spadotti

    2015-02-01

    Full Text Available Zucchini yellow mosaic virus (ZYMV causes substantial economic losses in cucurbit crops. Although ZYMV has been present in Brazil for more than 20 years, there is little information about the biological and molecular characteristics of the isolates found in the country. This study aimed to characterize the experimental hosts, pathotypes and genetic diversity of a collection of eleven Brazilian ZYMV isolates within the coat protein gene. For biological analysis, plant species from Amaranthaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Pedaliaceae were mechanically inoculated and pathotypes were identified based on the reaction of a resistant Cucumis melo, accession PI414723. All of the cucurbit species/varieties and Sesamum indicum were systemically infected with all isolates. The nucleotide sequence variability of the coat protein gene ranged from 82 % to 99 % compared to the corresponding sequences of ZYMV isolates from different geographical locations. No recombination event was detected in the coat protein gene of the isolates.

  17. Hepatitis delta virus:Making the point from virus isolation up to 2014

    Institute of Scientific and Technical Information of China (English)

    Raffaella; Romeo; Riccardo; Perbellini

    2015-01-01

    Chronic infection with hepatitis delta virus(HDV) has lately regained clinical importance because of the recent evidence of increasing prevalence in several European countries,due to immigration from highly endemic areas. HDV requires the mandatory presence ofhepatitis B virus(HBV) for propagation to hepatocytes. It is transmitted by the same routes of HBV and it can be acquired either by co-infection(simultaneous transmission of the two viruses) or super-infection(acquisition of HDV by an already chronic carrier of HBV). As a consequence,every HBV carrier is potentially at risk for HDV superinfection. Since the clinical course of superinfection can be severe,early diagnosis of HDV infection is necessary.

  18. Differences between the glycosylation patterns of haptoglobin isolated from skin scales and plasma of psoriatic patients.

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    Bernardetta Maresca

    Full Text Available Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt, a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.

  19. Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

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    Abdel-Ghany Ahmad E

    2010-04-01

    Full Text Available Abstract Background The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences. Methods Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE. Reverse transcriptase polymerase chain reaction (RT-PCR and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI assay on 105 donkey serum samples. Results We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate. Conclusions These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas.

  20. Characterization of Brugmansia mosaic virus Isolated from Brugmansia spp. in Korea

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    Chung Youl Park

    2014-12-01

    Full Text Available In May 2013, an angel’s trumpet leaves showing mosaic and malformation symptoms were collected from Suwon city, Gyeonggi-do. An analysis of the collected sample by transmission electron microscopy observation showed filamentous rod particles of 720-800 nm in length. On the basis of the these observations, we performed PCR against three reported Potyviruses (Brugmansia mosaic virus, Colombian datura virus and Brugmansia suaveolens mottle virus, and the sample was positive for BruMV. Pathogenicity and host range test of BruMV was determined by mechanical inoculation. Solanaceae (tobacco, tomato and eggplant and Amaranthaceae (ground cherry appeared typical virus symptoms. To determine coat protein of this virus, we designed specific primer pairs, and performed PCR amplification, cloning, and sequencing. Phylogenetic analysis showed that BruMV-SW was most closely related to BruMV isolate SK. Comparison of the BruMV-SW coat protein nucleotide sequences showed 92% to 99% identities to the other BruMV isolates.

  1. Exosome-associated hepatitis C virus in cell cultures and patient plasma.

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    Liu, Ziqing; Zhang, Xiugen; Yu, Qigui; He, Johnny J

    2014-12-12

    Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

  2. SPF rabbits infected with rabbit hepatitis E virus isolate experimentally showing the chronicity of hepatitis.

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    Han, Jian; Lei, Yaxin; Liu, Lin; Liu, Peng; Xia, Junke; Zhang, Yulin; Zeng, Hang; Wang, Lin; Wang, Ling; Zhuang, Hui

    2014-01-01

    This study focused on investigating the pathogenesis seen in specific-pathogen-free (SPF) rabbits following infection with a homologous rabbit HEV isolate (CHN-BJ-rb14) and comparing it to that seen following infection with a heterologous swine genotype 4 HEV isolate (CHN-XJ-SW13). Three of the four animals inoculated with the homologous rabbit HEV became infected, exhibiting an intermittent viremia, obvious fluctuations of liver function biomarkers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and persistent fecal virus shedding throughout the nine month study. In addition, liver histopathology showed both chronic inflammation and some degree of fibrosis. Both positive and negative-stranded HEV RNA and HEV antigen expression were detected in liver, brain, stomach, duodenum and kidney from the necropsied rabbits. Inflammation of extrahepatic tissue (duodenum and kidney) was also observed. Three of the four rabbits inoculated with the heterologous genotype 4 swine HEV also became infected, showing similar levels of anti-HEV antibody to that generated following infection with the homologous virus isolate. The duration of both viremia and fecal shedding of virus was however shorter following infection with the heterologous virus and there was no significant elevation of liver function biomarkers. These results suggest that rabbit HEV infection may cause more severe hepatitis and prolong the course of the disease, with a possible chronic trend of hepatitis in SPF rabbits.

  3. Field Trials of CpGV Virus Isolates Overcoming Resistance to CpGV-M

    Institute of Scientific and Technical Information of China (English)

    M. Berling; J. -B. Rey; S. -J. Ondet; Y. Tallot; O. Soubabère; A. Bonhomme; B. Sauphanor; M. Lopez-Ferber

    2009-01-01

    The Cydia pomonella granulovirus (CpGV) has been used for many years as biological agent for codling moth control in apple orchards. Resistance to the Mexican strain of CpGV was detected in orchards in Germany, France and Italy. A laboratory insect colony was started from insects collected in a French resistant orchard. It was named RGV. Various virus isolates were identified as active against this resistant insect colony. Field tests were carried out in 2007 to test if the two virus isolates CpGV-I12 and NPP-R1 were effective in the field. Although these virus isolates were not able to reduce insect caused fruit damages, they significantly reduced the overwintering insect populations. NPP-R1 was subjected to eight passages on RGV larvae (NPP-R1.8) that improved its biological activity on RGV larvae. 2008 field trials were set up to test this improved virus strain, compared to CpGV-I12 and Madex plus active on RGV. These tests confirmed the ability to control both in susceptible and resistant insect populations.

  4. Nucleotide and amino acid sequences of a coat protein of an Ukrainian isolate of Potato virus Y: comparison with homologous sequences of other isolates and phylogenetic analysis

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    Budzanivska I. G.

    2014-03-01

    Full Text Available Aim. Identification of the widespread Ukrainian isolate(s of PVY (Potato virus Y in different potato cultivars and subsequent phylogenetic analysis of detected PVY isolates based on NA and AA sequences of coat protein. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. PVY has been identified serologically in potato cultivars of Ukrainian selection. In this work we have optimized a method for total RNA extraction from potato samples and offered a sensitive and specific PCR-based test system of own design for diagnostics of the Ukrainian PVY isolates. Part of the CP gene of the Ukrainian PVY isolate has been sequenced and analyzed phylogenetically. It is demonstrated that the Ukrainian isolate of Potato virus Y (CP gene has a higher percentage of homology with the recombinant isolates (strains of this pathogen (approx. 98.8– 99.8 % of homology for both nucleotide and translated amino acid sequences of the CP gene. The Ukrainian isolate of PVY is positioned in the separate cluster together with the isolates found in Syria, Japan and Iran; these isolates possibly have common origin. The Ukrainian PVY isolate is confirmed to be recombinant. Conclusions. This work underlines the need and provides the means for accurate monitoring of Potato virus Y in the agroecosystems of Ukraine. Most importantly, the phylogenetic analysis demonstrated the recombinant nature of this PVY isolate which has been attributed to the strain group O, subclade N:O.

  5. Phylogenetic and pathogenic analyses of avian influenza A H5N1 viruses isolated from poultry in Vietnam.

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    Dongming Zhao

    Full Text Available Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature.

  6. Development, characterization and application of monoclonal antibodies against Brazilian Dengue virus isolates.

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    Camila Zanluca

    Full Text Available Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV detection through the production and characterization of 22 monoclonal antibodies (mAbs against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3 and dengue serotype-specific (DENV-2 or -3. Additionally, some mAbs cross-reacted with yellow fever virus (YFV, West Nile virus (WNV and Saint Louis encephalitis virus (SLEV. None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV. Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.

  7. Isolation of avian influenza H5N1 virus from vaccinated commercial layer flock in Egypt

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    El-Zoghby Elham F

    2012-11-01

    Full Text Available Abstract Background Uninterrupted transmission of highly pathogenic avian influenza virus (HPAIV H5N1 of clade 2.2.1 in Egypt since 2006 resulted in establishment of two main genetic clusters. The 2.2.1/C group where all recent human and majority of backyard origin viruses clustered together, meanwhile the majority of viruses derived from vaccinated poultry in commercial farms grouped in 2.2.1.1 clade. Findings In the present investigation, an HPAIV H5N1 was isolated from twenty weeks old layers chickens that were vaccinated with a homologous H5N1 vaccine at 1, 7 and 16 weeks old. At twenty weeks of age, birds showed cyanosis of comb and wattle, decrease in egg production and up to 27% mortality. Examined serum samples showed low antibody titer in HI test (Log2 3.2± 4.2. The hemagglutinin (HA and neuraminidase (NA genes of the isolated virus were closely related to viruses in 2.2.1/C group isolated from poultry in live bird market (LBM and backyards or from infected people. Conspicuous mutations in the HA and NA genes including a deletion within the receptor binding domain in the HA globular head region were observed. Conclusions Despite repeated vaccination of layer chickens using a homologous H5N1 vaccine, infection with HPAIV H5N1 resulted in significant morbidity and mortality. In endemic countries like Egypt, rigorous control measures including enforcement of biosecurity, culling of infected birds and constant update of vaccine virus strains are highly required to prevent circulation of HPAIV H5N1 between backyard birds, commercial poultry, LBM and humans.

  8. Newcastle Disease Viruses Causing Recent Outbreaks Worldwide Show Unexpectedly High Genetic Similarity to Historical Virulent Isolates from the 1940s.

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    Dimitrov, Kiril M; Lee, Dong-Hun; Williams-Coplin, Dawn; Olivier, Timothy L; Miller, Patti J; Afonso, Claudio L

    2016-05-01

    Virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), a devastating disease of poultry and wild birds. Phylogenetic analyses clearly distinguish historical isolates (obtained prior to 1960) from currently circulating viruses of class II genotypes V, VI, VII, and XII through XVIII. Here, partial and complete genomic sequences of recent virulent isolates of genotypes II and IX from China, Egypt, and India were found to be nearly identical to those of historical viruses isolated in the 1940s. Phylogenetic analysis, nucleotide distances, and rates of change demonstrate that these recent isolates have not evolved significantly from the most closely related ancestors from the 1940s. The low rates of change for these virulent viruses (7.05 × 10(-5) and 2.05 × 10(-5) per year, respectively) and the minimal genetic distances existing between these and historical viruses (0.3 to 1.2%) of the same genotypes indicate an unnatural origin. As with any other RNA virus, Newcastle disease virus is expected to evolve naturally; thus, these findings suggest that some recent field isolates should be excluded from evolutionary studies. Furthermore, phylogenetic analyses show that these recent virulent isolates are more closely related to virulent strains isolated during the 1940s, which have been and continue to be used in laboratory and experimental challenge studies. Since the preservation of viable viruses in the environment for over 6 decades is highly unlikely, it is possible that the source of some of the recent virulent viruses isolated from poultry and wild birds might be laboratory viruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Types of variation in DNA-A among isolates of East African cassava mosaic virus from Kenya, Malawi and Tanzania.

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    Zhou, X; Robinson, D J; Harrison, B D

    1998-11-01

    Complete nucleotide sequences of the DNA-A-like molecules of three East African cassava mosaic virus (EACMV) isolates from Kenya (-K, 2801 nt) and Malawi (-MH and -MK, both 2804 nt) were determined. These sequences were compared with that published for a Tanzanian isolate (-T, 2801 nt) and the partial sequence of a third Malawian isolate. Intergenic region sequences of all isolates, and deduced amino acid sequences of their AC1 (Rep) proteins, each formed a tightly related cluster that was distinct from the comparable components of other begomoviruses. Other complementary-sense genes (AC2, AC3, AC4) differed between EACMV isolates in a way consistent with the accumulation of point mutations. In contrast, virus-sense genes (CP, AV2) of isolates -MH and -MK differed (substantially for AV2) from those of other EACMV isolates but somewhat resembled those of tomato yellow leaf curl virus-Israel, suggesting they had been acquired by recombination with an unidentified begomovirus.

  10. Prevalence and genetic diversity of fig mosaic virus isolates infecting fig tree in Iran.

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    Danesh-Amuz, S; Rakhshandehroo, F; Rezaee, S

    2014-01-01

    Commercial and outdoor fig orchards in four Iranian provinces were surveyed for the incidence of fig mosaic virus (FMV), fig leaf mottle associated virus 2 (FLMaV-2) and fig mild mottle associated virus (FMMaV) from March 2011 to October 2012. A total of 350 asymptomatic and symptomatic fig samples were collected and tested by dot-immunobinding assay (DIBA) for the fig mosaic disease (FMD) using a polyclonal antiserum. According to DIBA results, FMD was present in 73% of the collected symptomatic samples from all visited regions. Samples with positive reactions in DIBA were then analyzed by RT-PCR using with specific primers. PCR results showed that about 14.8% of the FMD-positive samples from three inspected provinces are infected with at least one virus. FMV was the most widely spread virus (14%) followed by FLMaV-2 (1.5%), whereas FMMaV was not found. Phylogenetic analysis of the glycoprotein nucleotide and amino acid sequences of known FMV isolates showed two independent groups with high bootstrap values, with all Iranian isolates distinctly clustered in group I, subgroup IA beside those reported in Turkey. Nucleotide diversity was high within but low between different selected geographic regions and except for Europe, nucleotide distance within geographic regions was low. Statistical analyses indicated a correlation between the genetic structure of the FMV isolates and the geographical origin of isolation. Our analyses suggested that the FMV population is in a state of increase following a bottleneck or founder event in Iran.

  11. MOLECULAR EPIDEMIOLOGY FEATURES OF HEPATITIS C VIRUS ISOLATES FROM DIFFERENT REGIONS OF THE REPUBLIC SAKHA (YAKUTIA

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    A. V. Semenov

    2015-01-01

    Full Text Available According to WHO data about 3% of population are infected by hepatitic C virus (HCV worldwide. Chronic hepatitis C is the leading cause of cirrhosis and hepatocellular carcinoma, thus it becoming one of the global public health problems. Clinical manifestations are varied and depend mainly of the virus biological properties and its interaction with the host immune system. Determination of virus genotype and subtype is important for a better understanding of the epidemiological and virological features of the disease. The prevalence genotypes hepatitis C virus is varies in different geographical regions of the world. The data about HCV genotypes distribution in some Russian Federation regions are very limited, especially about HCV genotypes prevalence in Siberia, Far East and some rural regions. One of such regions is Yakutia. In our study we identified genetic variants of HCV in chronic hepatitis C patients with moderate and high viral load from Yakutia by direct sequencing of HCV RNA NS5B region. Based on phylogenetic analysis we found the prevalent genotype 1 (88.3%, than genotype 2 (6.7% and 3 (3.2% among HCV patients with moderate and high viral load. Our results on the prevalence of subtype 1b are consistent with the data on the connection between this subtype with high levels of viremia, greater duration and severity of liver disease, as well as the development of chronic hepatits C in patients infected by HCV subtype 1b, compared with those infected with other subtypes of hepatitis virus C. The similarity of some Yakutian isolates with isolates from the United States, Brazil and Ireland was found. We discuss HCV subtype 2a isolates identified origin from isolates found in China. First in the territory of the Russian Federation HCV subtype 3g was identified, presumably imported from South Asia. Interconnected use of molecular, virological, demographic and epidemiological methods and information to monitor the infections will contribute to

  12. Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells

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    A. Sasikalaveni

    2015-05-01

    Full Text Available Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA cells, which enable quicker isolation of street rabies virus with minimum passages. Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

  13. Plasma membrane phosphatidylinositol 4,5 bisphosphate is required for internalization of foot-and-mouth disease virus and vesicular stomatitis virus.

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    Angela Vázquez-Calvo

    Full Text Available Phosphatidylinositol-4,5-bisphosphate, PI(4,5P(2, is a phospholipid which plays important roles in clathrin-mediated endocytosis. To investigate the possible role of this lipid on viral entry, two viruses important for animal health were selected: the enveloped vesicular stomatitis virus (VSV - which uses a well characterized clathrin mediated endocytic route - and two different variants of the non-enveloped foot-and-mouth disease virus (FMDV with distinct receptor specificities. The expression of a dominant negative dynamin, a PI(4,5P(2 effector protein, inhibited the internalization and infection of VSV and both FMDV isolates. Depletion of PI(4,5P(2 from plasma membrane using ionomycin or an inducible system, and inhibition of its de novo synthesis with 1-butanol revealed that VSV as well as FMDV C-S8c1, which uses integrins as receptor, displayed a high dependence on PI(4,5P(2 for internalization. Expression of a kinase dead mutant (KD of phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K-Iα, an enzyme responsible for PI(4,5P(2 synthesis that regulates clathrin-dependent endocytosis, also impaired entry and infection of VSV and FMDV C-S8c1. Interestingly FMDV MARLS variant that uses receptors other than integrins for cell entry was less sensitive to PI(4,5P(2 depletion, and was not inhibited by the expression of the KD PIP5K-Iα mutant suggesting the involvement of endocytic routes other than the clathrin-mediated on its entry. These results highlight the role of PI(4,5P(2 and PIP5K-Iα on clathrin-mediated viral entry.

  14. Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

    Indian Academy of Sciences (India)

    A Srivastava; S K Raj

    2008-06-01

    Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

  15. Full Genome Sequence of Egg Drop Syndrome Virus Strain FJ12025 Isolated from Muscovy Duckling.

    Science.gov (United States)

    Fu, Guanghua; Chen, Hongmei; Huang, Yu; Cheng, Longfei; Fu, Qiuling; Shi, Shaohua; Wan, Chunhe; Chen, Cuiteng; Lin, Jiansheng

    2013-08-22

    Egg drop syndrome virus (EDSV) strain FJ12025 was isolated from a 9-day-old Muscovy duckling. The results of the sequence showed that the genome of strain FJ12025 is 33,213 bp in length, with a G+C content of 43.03%. When comparing the genome sequence of strain FJ12025 to that of laying duck original strain AV-127, we found 50 single-nucleotide polymorphisms (SNPs) between the two viral genome sequences. A genomic sequence comparison of FJ12025 and AV-127 will help to understand the phenotypic differences between the two viruses.

  16. Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

    Science.gov (United States)

    Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

    2014-07-01

    Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.

  17. Molecular analysis of bovine viral diarrhoea virus isolates from South Africa

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    N. Kabongo

    2003-11-01

    Full Text Available The presence of bovine viral diarrhoea virus in South Africa has been confirmed by several serological surveys. However, little is known about its biological properties. Twenty five isolates obtained by isolation in tissue culture and detected by means of the antigen capture ELISA from clinically sick cattle and from foetal calf serum in South Africa were characterized on the basis of analysis of the 5' non-translated (NTR region of the genome. A reverse-transcription polymerase chain reaction (RT-PCR was used to amplify specific sequences from the 5'NTR of the genome. The oligonucleotide primers corresponding to positions 105-125 and 399-378, respectively, in the sequence of BVDV strain NADL were used to generate the PCR products. Both strands were sequenced directly with these primers and fluorescence-labelled dideoxynucleotides in an automated nucleic acid sequencer. Reference strains of pestiviruses [(BVDV type I, BVDV type II, border disease virus (BDV and hog cholera virus (HCV] and isolates from a previous investigation on BVDV in southern Africa were included for comparative purposes. All the BVDV strains obtained during this study belong to subgroups of BVDV genotype I. No association could be demonstrated between the geographic origin of the isolates. A number of isolates formed another branch separate from the existing branches Ia, Ib and Ic. These findings suggest that extensive genetic diversity can be found within BVDV type I isolates from southern Africa. Isolates that group with the classical BVDV type I strains, particularly of American origin, coexist with variants that appear to represent a local genetic pool and or variants evolving from the classical strains.

  18. Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil

    Science.gov (United States)

    Drumond, Betania Paiva; da Silva Fagundes, Luiz Gustavo; Rocha, Raissa Prado; Fumagalli, Marcilio Jorge; Araki, Carlos Shigueru; Colombo, Tatiana Elisa; Nogueira, Mauricio Lacerda; Castilho, Thiago Elias; da Silveira, Nelson José Freitas; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-01-01

    Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1–4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population. PMID:26887252

  19. Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil.

    Science.gov (United States)

    Drumond, Betania Paiva; Fagundes, Luiz Gustavo da Silva; Rocha, Raissa Prado; Fumagalli, Marcilio Jorge; Araki, Carlos Shigueru; Colombo, Tatiana Elisa; Nogueira, Mauricio Lacerda; Castilho, Thiago Elias; da Silveira, Nelson José Freitas; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-01-01

    Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1-4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population.

  20. Phylogenetic characterization of virulent Newcastle disease viruses isolated during outbreaks in northwestern Iran in 2010.

    Science.gov (United States)

    Ahmadi, Elham; Pourbakhsh, Seyed Ali; Ahmadi, Malahat; Mardani, Karim; Talebi, Alireza

    2016-11-01

    The northwest of Iran shares long borders with three neighboring countries; therefore, it is considered one of the main entry portals of Newcastle disease virus (NDV) into the country. Ten virulent NDVs were recovered from 19 poultry farms of various prefectures in northwestern Iran during Newcastle disease outbreaks in 2010. The isolates were genotypically analyzed using an F-gene-specific reverse transcription polymerase chain reaction (RT-PCR) assay. The amplified F gene (nucleotides 189-1666) sequences of the NDV isolates were compared phylogenetically with those of previously published strains in GenBank. All of the NDV isolates belonged to genotype VIIb and were closely related to some isolates from Iran, Russia, and Sweden. Therefore, it can be postulated that these isolates evolved from previously reported strains. The velogenic viruses carried the motif (112)R-R-Q-K-R/F(117) at the F0 cleavage site and a unique substitution of (190)L→F which had never been reported in any NDV genotype VIIb isolate. They shared high sequence similarity with each other but were distinct from current NDV vaccines and NDV strains reported from other countries. This information is fundamental for improving the efficacy of controlling strategies and vaccine development for NDV.

  1. Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs.

    Science.gov (United States)

    Stevens, James; Chen, Li-Mei; Carney, Paul J; Garten, Rebecca; Foust, Angie; Le, Jianhua; Pokorny, Barbara A; Manojkumar, Ramanunninair; Silverman, Jeanmarie; Devis, Rene; Rhea, Karen; Xu, Xiyan; Bucher, Doris J; Paulson, James C; Paulson, James; Cox, Nancy J; Klimov, Alexander; Donis, Ruben O

    2010-08-01

    Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of alpha2-6 and alpha2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to alpha2-6- and alpha2-3-type receptors but retained substantial binding to specific O- and N-linked alpha2-3 glycans, including alpha2-3GalNAc and fucosylated alpha2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.

  2. Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

    Science.gov (United States)

    Shin, Dongyoung; Richards, Stephanie L; Alto, Barry W; Bettinardi, David J; Smartt, Chelsea T

    2013-01-01

    Dengue virus (DENV) is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL), including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1) and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR) to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW), FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI) and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

  3. Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

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    Dongyoung Shin

    Full Text Available Dengue virus (DENV is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL, including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1 and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW, FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

  4. Characterization of pigeon paramyxoviruses (Newcastle disease virus isolated in South Africa from 2001 to 2006

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    C. Abolnik

    2008-08-01

    Full Text Available Pigeon paramyxovirus type 1 (PPMV-1, a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced in to South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbil(l Bucorvus leadbeateri which becamea cutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had lCPl and MDT values characteristic of PPMV-1s trains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

  5. Pinhal Virus, a New Arenavirus Isolated from Calomys tener in Brazil.

    Science.gov (United States)

    Bisordi, Ivani; Levis, Silvana; Maeda, Adriana Y; Suzuki, Akemi; Nagasse-Sugahara, Teresa K; de Souza, Renato P; Pereira, Luiz E; Garcia, Jorge B; Cerroni, Matheus de P; de A e Silva, Franko; dos Santos, Cecília L S; da Fonseca, Benedito A L

    2015-11-01

    Arenavirus Sabiá was originally isolated from a fatal human infection in Brazil, and after the occurrence of the second fatal human case in São Paulo state, epidemiologic and virologic studies were performed in the area where the patient lived, aiming at the identification of the Sabiá natural rodent reservoir. A broadly cross-reactive enzyme-linked immunosorbent assay (ELISA) was used to screen for antibody-positive samples. Antibodies to arenavirus were detected in two of the 55 samples of Calomys tener, and from these results, samples of rodents were analyzed by a broad RT-PCR assay. RT-PCR amplification detected arenavirus sequences in five of the 55 C. tener samples, and sequencing showed that this virus is a distinct form of Sabiá virus. Thus, we describe here the evidence for the circulation of a new arenavirus in Brazil (proposed name Pinhal virus) and its genetic characterization compared to other arenaviruses. This study also suggests C. tener as a probable rodent reservoir for this virus and associates this new virus with the lineage C of New World arenaviruses. Although we have defined some characteristics of this virus, so far, there is no evidence of its involvement in human disease.

  6. Isolation and characterization of influenza C virus inhibitor in rat serum.

    Science.gov (United States)

    Kitame, F; Nakamura, K; Saito, A; Sinohara, H; Homma, M

    1985-10-01

    Two hemagglutination inhibitors for influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 22, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the hemagglutination by influenza C virus strains but did not affect the hemagglutination by influenza A and B virus strains. Hemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with influenza C virus at 37 degrees C but not with the other types of influenza virus, indicating that the inhibitors are specific for influenza C virus. The inhibitory activity was also destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is cleavable by the former neuraminidase but not by the latter is essential for the hemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.

  7. Phylogenetic characterization and virulence of two Newcastle disease viruses isolated from wild birds in China.

    Science.gov (United States)

    Liu, Haijin; Zhang, Peng; Wu, Pengpeng; Chen, Shengli; Mu, Guohui; Duan, Xuji; Hao, Huafang; Du, Enqi; Wang, Xinglong; Yang, Zengqi

    2013-12-01

    Wild birds are considered as a natural reservoir of Newcastle disease virus (NDV). However, there is no information about genotype IX NDV from wild birds, especially from Columbiformes. In this study, two genotype IX NDV viruses were isolated from wild birds. One was from Eurasian Blackbird, while the other was from Spotted-necked dove. After purification by plaque technique, complete genomes of both viruses were sequenced. Phylogenetic analysis of partial fusion (F) gene and complete genome indicated both strains belonged to genotype IX. Based on intracerebral pathogenicity index (ICPI), the virus from Eurasian Blackbird was velogenic virus, while the strain from Spotted-necked dove was lentogenic virus. However, both strains showed one of velogenic cleavage sites. In addition, the strain from Eurasian Blackbird showed greater replication ability and generated larger fusion foci in vitro than that of strain from Spotted-necked dove. Comparing all the corresponding protein sequences of both strains, there were only 9 different amino acid residues between them. Furthermore, after analysis of these differences, the information about lentogenic NDV with multi-basic cleavage site was presented.

  8. Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope.

    Science.gov (United States)

    Muller, Vanessa Danielle; Soares, Ricardo Oliveira; dos Santos, Nilton Nascimento; Trabuco, Amanda Cristina; Cintra, Adelia Cristina; Figueiredo, Luiz Tadeu; Caliri, Antonio; Sampaio, Suely Vilela; Aquino, Victor Hugo

    2014-01-01

    The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.

  9. Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope.

    Directory of Open Access Journals (Sweden)

    Vanessa Danielle Muller

    Full Text Available The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.

  10. Genotype diversity of H9N2 viruses isolated from wild birds and chickens in Hunan Province, China.

    Science.gov (United States)

    Wang, Ba; Liu, Zhihua; Chen, Quanjiao; Gao, Zhimin; Fang, Fang; Chang, Haiyan; Chen, Jianjun; Xu, Bing; Chen, Ze

    2014-01-01

    Three H9N2 avian influenza viruses were isolated from the Dongting Lake wetland, among which one was from fresh egret feces, the other two were from chicken cloacal swabs in poultry markets. Phylogenetic analyses suggested that eight genes of the egret-derived H9N2 virus might come from Korean-like or American-like lineages. The two poultry-derived H9N2 viruses were reassortants between the CK/BJ/94-like and G1-like viruses. Except the PB1 genes (90.6%), the nucleotide sequence of other internal genes of the two viruses exhibited high homology (>95%). In addition, they also exhibited high homology (96-98.3%) with some genes of the H7N9 virus that caused an epidemic in China in 2013. Nucleotide sequence of the poultry-derived and egret-derived H9N2 viruses shared low homology. Infection studies showed that the egret-derived H9N2 virus was non-pathogenic to both mice and chickens, and the virus was unable to infect chickens even through 8 passages continuously in the lung. On the other hand, the chickens infected by poultry-derived viruses showed obvious clinical symptoms and even died; the infected mice showed no noticeable clinical symptoms and weight loss, but viruses could be detected in their lungs. In conclusion, for the egret-derived H9N2 virus, it would take a long adaptation process to achieve cross-species transmission in poultry and mammals. H9N2 viruses isolated at different times from the same host species in the same geographical region presented different evolutionary status, and virus isolated from different hosts in the same geographical region exhibited genetic diversity. Therefore, it is important to continue the H9N2 virus surveillance for understanding their evolutionary trends so as to provide guidance for disease control and prevention.

  11. ISOLATION OF VIRUS IN PRODUCTS OF CONCEPTION IN SPONTANEOUS ABORTION

    Directory of Open Access Journals (Sweden)

    KAMYAB S.D. AZARI S.D. NATEGH R

    1980-08-01

    Full Text Available Two hundred pa t ient s o f t he l ow socioe conomic g roup wi th an aborti on o f the f i r s t t r i mest e r were stud 1ed . The s ter i l e produc ts o f concept ion were c ul t ured on Gr e e n Monke y Kidney ti s sue cul t ure f or v i r a l de t e c - tion. Four po s i t i ve Herpe s virus, two positive Rubella"nv~ru s and t hree pos i tive unidentifie d Entero v irus l ike Vlrus were obtained The sera of 192 of these patients were titrated for Rubella antibody. The sera of a control group of 150 normal pregnant women were titrated for Rubella antibody and 8.8 per cent of these patients had a negative titre and 21.3 per cent , 1 had a tltre over 40

  12. Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates.

    Science.gov (United States)

    Johansson, Tove; Einer-Jensen, Katja; Batts, William; Ahrens, Peter; Björkblom, Carina; Kurath, Gael; Björklund, Harry; Lorenzen, Niels

    2009-11-09

    Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13/98 11621, DE-DF 4/99-8/99, AU-9695338 and RU-FR1 were sequenced and compared with IHNV isolates from the North American genogroups U, M and L. In phylogenetic studies the N gene of the Italian, French, German and Austrian isolates clustered in the M genogroup, though in a different subgroup than the isolates from the USA. Analyses of the partial G gene of these European isolates clustered them in the M genogroup close to the root while the Russian isolate clustered in the U genogroup. The European isolates together with US-WRAC and US-Col-80 were also tested in an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) against the N protein. MAbs 136-1 and 136-3 reacted equally at all concentrations with the isolates tested, indicating that these antibodies identify a common epitope. MAb 34D3 separated the M and L genogroup isolates from the U genogroup isolate. MAb 1DW14D divided the European isolates into 2 groups. MAb 1DW14D reacted more strongly with DE-DF 13/98 11621 and RU-FR1 than with IT-217A, FR-32/87, DE-DF 4/99-8/99 and AU-9695338. In the phylogenetic studies, the Italian, French, German and Austrian isolates clustered in the M genogroup, whereas in the serological studies using MAbs, the European M genogroup isolates could not be placed in the same specific group. These results indicate that genotypic and serotypic classification do not correlate.

  13. Plasma Epstein-Barr virus DNA as a biomarker for nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    KC Allen Chan

    2014-01-01

    Nasopharyngeal carcinoma (NPC) is common in southern China and Southeast Asia. Epstein-Barr virus (EBV) infection is an important etiology for NPC, and EBV genome can be detected in almost all tumor tissues of NPC in this region. Plasma EBV DNA, when quantitatively analyzed using real-time polymerase chain reaction (PCR), has been developed as a biomarker for NPC. In this review, the different clinical applications of plasma EBV DNA in the management of NPC, including screening, monitoring, and prognostication, are discussed. In addition, the biological issues of circulating EBV DNA, including the molecular nature and clearance kinetics, are also explored.

  14. Molecular and biological characterization of Potato mop-top virus (PMTV, Pomovirus) isolates from potato-growing regions in Colombia

    DEFF Research Database (Denmark)

    Gil, José; Adams, Ian; Boonham, Neil

    2016-01-01

    samples were taken from the main potato-producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons......Potato mop-top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil...

  15. A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods

    Directory of Open Access Journals (Sweden)

    Abraham A

    2009-01-01

    Full Text Available Background: Typing of Herpes simplex virus (HSV isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF assays and polymerase chain reaction (PCR. This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb based IF test. Study design : This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42 of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42 were HSV-2, and two (4.8% were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30, whereas the cost per MAb IF test is about Rs 1,500 (USD 35 including all overheads (reagents, instruments, personnel time, and consumables. Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers is now more widespread than fluorescence microscopes in a country like India.

  16. Exosome-associated hepatitis C virus in cell cultures and patient plasma

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziqing [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Zhang, Xiugen [Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Yu, Qigui [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); He, Johnny J., E-mail: johnny.he@unthsc.edu [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2014-12-12

    Highlights: • HCV occurs in both exosome-free and exosome-associated forms. • Exosome-associated HCV is infectious and resistant to neutralizing antibodies. • More exosome-associated HCV than exosome-free HCV is present in patient plasma. - Abstract: Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

  17. High-throughput isolation of giant viruses in liquid medium using automated flow cytometry and fluorescence staining.

    Directory of Open Access Journals (Sweden)

    Jacques Yaacoub Bou Khalil

    2016-01-01

    Full Text Available The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than ten strains of previously known species of giant viruses and 7 new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.

  18. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

    Science.gov (United States)

    Khalil, Jacques Y. B.; Robert, Stephane; Reteno, Dorine G.; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  19. A survey of fish viruses isolated from wild marine fishes from the coastal waters of southern Korea.

    Science.gov (United States)

    Kim, Wi-Sik; Choi, Shin-Young; Kim, Do-Hyung; Oh, Myung-Joo

    2013-11-01

    A survey was conducted to investigate viral infection in 253 wild marine fishes harvested in the southern coastal area of Korea from 2010 to 2012. The fish that were captured by local anglers were randomly bought and sampled for virus examination. The samples were tested for presence of virus by virus isolation with FHM, FSP, and BF-2 cells and molecular methods (polymerase chain reaction and sequencing). Of the 253 fish sampled, 9 fish were infected with virus. Aquabirnaviruses (ABVs), Viral hemorrhagic septicemia virus (VHSV), and Red seabream iridovirus (RSIV) were detected in 7, 1, and 1 fish, respectively. Molecular phylogenies demonstrated the detected viruses (ABV, VHSV, and RSIV) were more closely related to viruses reported of the same type from Korea and Japan than from other countries, suggesting these viruses may be indigenous to Korean and Japanese coastal waters.

  20. Ecologic observations of Venezuelan encephalitis virus in vertebrates and isolations of Nepuyo and Patois viruses from sentinel hamsters at Pacific and Atlantic habitats in Guatemala, 1968-1980.

    Science.gov (United States)

    Scherer, W F; Dickerman, R W; Cupp, E W; Ordonez, J V

    1985-07-01

    La Avellana and Puerto Barrios, two enzootic foci of Venezuelan encephalitis (VE) virus on the Pacific and Caribbean lowlands (respectively) of Guatemala have been studied over a 13-year period. Data from sentinel hamsters and guinea pigs and wild and domestic vertebrates are reported. VE virus strains were isolated from hamsters each period they were exposed during the rainy seasons 1968-1980 and at the end of the dry season 1974. Rates of isolation of VE virus ranged from 0.2%-5.7% hamster/days/exposure. All strains tested were free of epizootic virions. Although virus was isolated from sentinel guinea pigs, their deaths were not attributable to infection with VE virus. Antibody titers in 26 of 28 terrestrial mammals bled at La Avellana in 1971 were higher to enzootic than to epizootic VE strains. Thirty-seven percent of 109 residents of Puerto Barrios had antibody to VE virus. In 13 of 20 tested, antibodies were engendered by the enzootic strain. Nepuyo and Patois viruses were isolated from sentinel hamsters at both La Avellana and Puerto Barrios.

  1. Complete genome sequences of two biologically distinct isolates of Asparagus virus 1.

    Science.gov (United States)

    Blockus, S; Lesker, T; Maiss, E

    2015-02-01

    The complete genome sequences of two asparagus virus 1 (AV-1) isolates differing in their ability to cause systemic infection in Nicotiana benthamiana were determined. Their genomes had 9,741 nucleotides excluding the 3'-terminal poly(A) tail, encoded a polyprotein of 3,112 amino acids, and shared 99.6 % nucleotide sequence identity. They differed at 37 nucleotide and 15 amino acid sequence positions (99.5 % identity) scattered over the polyprotein. The closest relatives of AV-1 in amino acid sequence identity were plum pox virus (54 %) and turnip mosaic virus (53 %), corroborating the classification of AV-1 as a member of a distinct species in the genus Potyvirus.

  2. Isolation and characterization of influenza C virus inhibitors in rat serum.

    Science.gov (United States)

    Kitame, F; Nakamura, K; Saito, A; Sinohara, H; Homma, M

    1985-09-01

    Two inhibitors against haemagglutination by influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 2, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the haemagglutination by influenza C virus strains but did not affect the haemagglutination by influenza A and B virus strains. Haemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is excised by the former neuraminidase but not by the latter is essential for the haemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.

  3. Cymbidium chlorotic mosaic virus, a new sobemovirus isolated from a spring orchid (Cymbidium goeringii) in Japan.

    Science.gov (United States)

    Kondo, Hideki; Takemoto, Shogo; Maruyama, Kazuyuki; Chiba, Sotaro; Andika, Ida Bagus; Suzuki, Nobuhiro

    2015-08-01

    Cymbidium chlorotic mosaic virus (CyCMV), isolated from a spring orchid (Cymbidium goeringii), was characterized molecularly. CyCMV isometric virions comprise a single, positive-strand RNA genome of 4,083 nucleotides and 30-kDa coat protein. The virus genome contains five overlapping open reading frames with a genomic organization similar to that of sobemoviruses. BLAST searches and phylogenetic analysis revealed that CyCMV is most closely related to papaya lethal yellowing virus, a proposed dicot-infecting sobemovirus (58.8 % nucleotide sequence identity), but has a relatively distant relationship to monocot-infecting sobemoviruses, with only modest sequence identities. This suggests that CyCMV is a new monocot-infecting member of the floating genus Sobemovirus.

  4. Development of a reverse genetics system to generate recombinant Marburg virus derived from a bat isolate.

    Science.gov (United States)

    Albariño, César G; Uebelhoer, Luke S; Vincent, Joel P; Khristova, Marina L; Chakrabarti, Ayan K; McElroy, Anita; Nichol, Stuart T; Towner, Jonathan S

    2013-11-01

    Recent investigations have shown the Egyptian fruit bat (Rousettus aegyptiacus) to be a natural reservoir for marburgviruses. To better understand the life cycle of these viruses in the natural host, a new reverse genetics system was developed for the reliable rescue of a Marburg virus (MARV) originally isolated directly from a R. aegyptiacus bat (371Bat). To develop this system, the exact terminal sequences were first determined by 5' and 3' RACE, followed by the cloning of viral proteins NP, VP35, VP30 and L into expression plasmids. Novel conditions were then developed to efficiently replicate virus mini-genomes followed by the construction of full-length genomic clones from which recombinant wild type and GFP-containing MARVs were rescued. Surprisingly, when these recombinant MARVs were propagated in primary human macrophages, a dramatic difference was found in their ability to grow and to elicit anti-viral cytokine responses.

  5. [Sequencing and analysis of the complete genome of a rabies virus isolate from Sika deer].

    Science.gov (United States)

    Zhao, Yun-Jiao; Guo, Li; Huang, Ying; Zhang, Li-Shi; Qian, Ai-Dong

    2008-05-01

    One DRV strain was isolated from Sika Deer brain and sequenced. Nine overlapped gene fragments were amplified by RT-PCR through 3'-RACE and 5'-RACE method, and the complete DRV genome sequence was assembled. The length of the complete genome is 11863bp. The DRV genome organization was similar to other rabies viruses which were composed of five genes and the initiation sites and termination sites were highly conservative. There were mutated amino acids in important antigen sites of nucleoprotein and glycoprotein. The nucleotide and amino acid homologies of gene N, P, M, G, L in strains with completed genomie sequencing were compared. Compared with N gene sequence of other typical rabies viruses, a phylogenetic tree was established . These results indicated that DRV belonged to gene type 1. The highest homology compared with Chinese vaccine strain 3aG was 94%, and the lowest was 71% compared with WCBV. These findings provided theoretical reference for further research in rabies virus.

  6. Characterization of a new isolate of pepper chlorotic spot virus from Yunnan province, China.

    Science.gov (United States)

    Zheng, Kuanyu; Chen, Tsung-Chi; Yeh, Shyi-Dong; Rahman, Md Siddiqur; Su, Xiaoxia; Wu, Kuo; Li, Ting; Zhang, Zhongkai; Dong, Jiahong

    2017-05-19

    A new isolate, 14YV733, of pepper chlorotic spot virus (PCSV) from chili peppers in Yunnan province of China was identified. The typical tospoviral particles of 80-120 nm in diameter were observed by electron microscopy. The virus caused systemic symptoms in several solanaceous plants and the Brassica rapa L. Chinensis group with mechanical inoculation. The sap from infected leaves reacted positively to a rabbit antibody to the N protein of watermelon silver mottle virus (WSMoV) in immunoblotting. The S, M, and L RNAs of PCSV-14YV733 are 3310 nts, 4711 nts, and 8913 nts long, respectively. This is the first report of complete sequences of PCSV in mainland China. Phylogenetic analysis of all tospoviral proteins indicated that PCSV-14YV733 is closely related to members of the WSMoV serogroup.

  7. First genome analysis and molecular characterization of Chickpea chlorotic dwarf virus Egyptian isolate infecting squash.

    Science.gov (United States)

    Fahmy, Inas Farouk; Taha, Omnia; El-Ashry, Abdel Nasser

    2015-06-01

    This study aims to identifying and characterizing some molecular properties of geminiviruses co-infection in squash field crop cultivated in Egypt. Squash crops observed to be heavily infected with several insect vectors, also severe chlorosis and stunting was observed. Electron microscopic analysis has revealed geminate capsid particles which indicate the infection of Geminiviruses, especially SqLCV which represent an economic problem to squash filed crop in Egypt. We have investigated possible mixed infections with different plant viruses associated with chlorotic stunt diseases and or other genus groups of geminiviruses. The main objective of this study is to investigate the recombination events, possible recombinants and variants among these genera in the same family differing in vector transmission. This is the first report of the molecular characterization, phylogenetic analysis and putative recombination events of the full length genome of the Chickpea Chlorotic Dwarf Mastrevirus in Egypt. And the first report of co-infection with another begomovirus infecting squash plants. A full length clone of both viruses were isolated and characterized at the molecular level. The complete nucleotide sequence of DNA-A was determined (2,572 bp) and submitted to the genbank under accession no. KF692356. The isolate from Egypt has about 97.8 % homology with the Chickpea chlorotic dwarf virus (CpCDV) isolate from Syria DNA-A isolate FR687959, a 83.2 % homology with the Sudan isolate AM933134 and a 82.7 % homology with Pakistan isolate FR687960. To best of our knowledge this is the first report of complete genome of CpCDV that infect squash plants in Egypt and worldwide.

  8. Typing of feline calicivirus isolates from different clinical groups by virus neutralisation tests.

    Science.gov (United States)

    Dawson, S; McArdle, F; Bennett, M; Carter, M; Milton, I P; Turner, P; Meanger, J; Gaskell, R M

    1993-07-03

    One hundred and thirteen isolates of feline calicivirus originating from seven different clinical groups were typed by virus neutralisation tests using eight different cat antisera. The clinical groups comprised 'healthy' cats, cases of acute oral/respiratory disease, chronic stomatitis, acute febrile lameness syndrome, vaccine reactions (clinical disease seen within 21 days of vaccination) and vaccine breakdowns (clinical disease seen more than 21 days after but within one year of vaccination). Isolates from the vaccine reaction cases were grouped into those associated with acute oral/respiratory disease alone and those associated with the lameness syndrome, and the latter group was further subdivided according to the vaccine used. Two groups appeared significantly different from others with some of the antisera. Thus the lameness vaccine reaction isolates associated with vaccine B were significantly different from the isolates from all the other clinical groups, including other lameness isolates, with a number of the antisera. In addition, the chronic stomatitis isolates were significantly different from those from the 'healthy' and the acute oral/respiratory disease groups with one or two of the antisera. Eighty-five to 88 per cent of the isolates were neutralised by antisera raised against F9 or F9-like vaccine strains at a dilution of 1 in 2. Twenty antibody units of such antisera neutralised 42 to 80 per cent of the isolates. A bivalent antiserum raised against a vaccine F9 strain and field strain LS015 neutralised 96 per cent of the isolates at a dilution of 1 in 2, and 20 antibody units neutralised 68 per cent of isolates. Antisera to field strain F65 neutralised all the remaining isolates at a dilution of 1 in 2 and 44 per cent of the remaining isolates at a dilution of 20 antibody units. Therefore, strains LS015 and F65 may be of use in the production of a polyvalent feline calicivirus vaccine, together with the widely used strain F9.

  9. 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.

  10. Emergence of simian virus 40 variants during serial passage of plaque isolates.

    Science.gov (United States)

    Norkin, L C; Tirrell, S M

    1982-01-01

    Three serial passage series of simian virus 40 (SV40) in CV-1 cells were initiated by infection directly from the same wild-type plaque isolate, three series were initiated by infection with another plaque isolate, and two series were initiated with each of two other plaque isolates. Aberrant SV40 genomes were not detected in any of the passage series until after the fifty undiluted passage, and each series generated a different array of variant genomes. The results show that the variants were not present in the original plaque isolates but, instead, were randomly generated during subsequent high-input multiplicity passages. Although many of the aberrant viral genomes in each passage series contained reiterations of the SV40 origin of replication and some also contained host cell sequences, there was no indication that SV40 is predisposed toward generating any particular variant. Images PMID:6283180

  11. Cloning of full genome sequence of hepatitis E virus of Shanghai swine isolate using RACE method

    Directory of Open Access Journals (Sweden)

    Mou Jing

    2007-10-01

    Full Text Available Abstract Genotype 4 hepatitis E virus (HEV was reportedly transmitted freely between humans and swine in eastern China. The full-length genomic sequence of Shanghai swine isolate (SH-SW-zs1 recovered from feces sample of a pig which was infected with HEV RNA positive swine serum was determined using RT-PCR and RACE (Rapid Amplification of cDNA Ends methods. The full genome of the SH-SW-zs1 isolate was 7265 nucleotides in length and phylogenetic analysis indicated that this isolate belonged to genotype 4. Comparison of the 3' UTR sequence with the corresponding regions of other 38 HEV strains from different region revealed that the Shanghai swine isolate is 21–49 bp longer than the other stains.

  12. Emergence of simian virus 40 variants during serial passage of plaque isolates.

    Science.gov (United States)

    Norkin, L C; Tirrell, S M

    1982-05-01

    Three serial passage series of simian virus 40 (SV40) in CV-1 cells were initiated by infection directly from the same wild-type plaque isolate, three series were initiated by infection with another plaque isolate, and two series were initiated with each of two other plaque isolates. Aberrant SV40 genomes were not detected in any of the passage series until after the fifty undiluted passage, and each series generated a different array of variant genomes. The results show that the variants were not present in the original plaque isolates but, instead, were randomly generated during subsequent high-input multiplicity passages. Although many of the aberrant viral genomes in each passage series contained reiterations of the SV40 origin of replication and some also contained host cell sequences, there was no indication that SV40 is predisposed toward generating any particular variant.

  13. Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions

    DEFF Research Database (Denmark)

    Mansfield, K.L.; Racloz, V.; McElhinney, L.M.

    2006-01-01

    We report a Molecular epidemiological study of rabies in Arctic Countries by comparing a panel of novel Greenland isolates to a larger cohort of viral sequences from both Arctic and Baltic regions. Rabies Virus isolates originating from wildlife (Arctic/red foxes, raccoon-dogs and reindeer), from...

  14. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A1

    Science.gov (United States)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen; Folch-Puy, Emma; Foronjy, Robert; Jalili, Roxana; Jendresen, Christian Bille; Kimura, Masashi; Kraft, Edward; Lindemose, Søren; Lu, Jin; McLain, Teri; Nutt, Leta; Ramon-Garcia, Santiago; Smith, Joseph; Spivak, Aaron; Wang, Michael L.; Zanic, Marija; Lin, Sue-Hwa

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic-bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers. PMID:18765283

  15. Isolation of Toscana virus from the cerebrospinal fluid of a man with meningitis in Marseille, France, 2010.

    Science.gov (United States)

    Nougairede, Antoine; Bichaud, Laurence; Thiberville, Simon-Djamel; Ninove, Laetitia; Zandotti, Christine; de Lamballerie, Xavier; Brouqui, Philippe; Charrel, Remi N

    2013-09-01

    Toscana virus (TOSV; Bunyaviridae, Phlebovirus) is an emerging arthropod-borne virus transmitted by phlebotomine sandflies. TOSV is a frequent cause of central nervous system infection during the warm season in several countries bordering the Mediterranean Sea. Here, we report a case of TOSV aseptic meningitis diagnosed in 2012 in Marseille, France. The virus strain was recovered in cell culture from the cerebrospinal fluid. New-generation sequencing based on Ion Torrent technology was used to determine its complete genome sequence. Phylogenetic analysis based on the partial L segment revealed that this isolate belongs to the lineage B together with other French, Spanish, and Moroccan strains. Although several cases of TOSV meningitis are reported in the literature, few of them are diagnosed by RT-PCR combined with virus isolation and further sequence characterization. This case report supports that virus isolation should be attempted whenever possible because this remains the gold standard technique for diagnosis of arthropod-borne viral infections.

  16. Genotyping and phylogenetic analysis of bovine viral diarrhea virus (BVDV) isolates in Kosovo.

    Science.gov (United States)

    Goga, Izedin; Berxholi, Kristaq; Hulaj, Beqe; Sylejmani, Driton; Yakobson, Boris; Stram, Yehuda

    2014-01-01

    Three serum samples positive in Antigen ELISA BVDV have been tested to characterise genetic diversity of bovine viral diarrhea virus (BVDV) in Kosovo. Samples were obtained in 2011 from heifers and were amplified by reverse transcription-polymerase chain reaction, sequenced and analysed by computer-assisted phylogenetic analysis. Amplified products and nucleotide sequence showed that all 3 isolates belonged to BVDV 1 genotype and 1b sub genotype. These results enrich the extant knowledge of BVDV and represent the first documented data about Kosovo BVDV isolates.

  17. Occurrence and characterization of plum pox virus strain D isolates from European Russia and Crimea.

    Science.gov (United States)

    Chirkov, Sergei; Ivanov, Peter; Sheveleva, Anna; Kudryavtseva, Anna; Prikhodko, Yuri; Mitrofanova, Irina

    2016-02-01

    Numerous plum pox virus (PPV) strain D isolates have been found in geographically distant regions of European Russia and the Crimean peninsula on different stone fruit hosts. Phylogenetic analysis of their partial and complete genomes suggests multiple introductions of PPV-D into Russia. Distinct natural isolates from Prunus tomentosa were found to bear unique amino acid substitutions in the N-terminus of the coat protein (CP) that may contribute to the adaptation of PPV-D to this host. Serological analysis using the PPV-D-specific monoclonal antibody 4DG5 provided further evidence that mutations at positions 58 and 59 of the CP are crucial for antibody binding.

  18. Fish viruses: transmission and pathogenicity of an icosahedral cytoplasmic deoxyribovirus isolated from sheatfish (Silurus glanis).

    Science.gov (United States)

    Ahne, W; Ogawa, M; Schlotfeldt, H J

    1990-05-01

    An iridovirus-like agent which was isolated from sheatfish fry showed to be of high virulence to those fish. 100% of sheatfish fry infected by water bath died within 8 days. The agent could be transmitted by infected fish causing 100% mortality in 11 days. Moribund fry showed spiralic swimming and exhibited hemorrhages in the skin. The infection studies demonstrate that the isolated virus was responsible for a sudden epizootic in 1988 with 100% mortality occurring in a warm water recirculating aquaculture unit in Northwest Germany, regularly controlled by the State Fish Epidemics Control Service of Lower Saxony and Fish Health Service.

  19. Short communication. Genotyping and phylogenetic analysis of bovine viral diarrhea virus (BVDV isolates in Kosovo

    Directory of Open Access Journals (Sweden)

    Izedin Goga

    2014-03-01

    Full Text Available Three serum samples positive in Antigen ELISA BVDV have been tested to characterise genetic diversity of bovine viral diarrhea virus (BVDV in Kosovo. Samples were obtained in 2011 from heifers and were amplified by reverse transcription-polymerase chain reaction, sequenced and analysed by computer-assisted phylogenetic analysis. Amplified products and nucleotide sequence showed that all 3 isolates belonged to BVDV 1 genotype and 1b sub genotype. These results enrich the extant knowledge of BVDV and represent the first documented data about Kosovo BVDV isolates.

  20. Tissue distribution of virus replication in cats experimentally infected with distinct feline calicivirus isolates.

    Science.gov (United States)

    Truyen, U; Geissler, K; Hirschberger, J

    1999-09-01

    Four specific pathogen-free (SPF) cats were each inoculated with one of two genetically and antigenically well characterized feline caliciviruses originally isolated from cats with acute respiratory disease (FCV-KS100/2), or with chronic stomatitis (FCV-KS20). Two cats of each group were euthanized at day 10 post infection and two cats at day 28. No clear differences between the clinical disease induced by the two isolates could be observed, and no apparent differences in the tissue spectrum were seen between day 10 and 28. No persistent virus shedding was observed over the 4-week period of this experiment.

  1. Biological and immunological studies of five Brazilian rabies virus isolates

    Directory of Open Access Journals (Sweden)

    Fernanda Bernardi

    2005-08-01

    Full Text Available Estudou-se em camundongos aspectos do comportamento biológico de amostras brasileiras de virus rábico isoladas de cão, bovino, eqüino, morcegos hematófago e insetívoro. Observou-se transmissão oral em camundongos alimentados com cérebros infectados de morcego insetívoro (8,82%, cão (8,57% e eqüino (3,03%. O período de incubação médio para todas as amostras foi de 6 dias após a inoculação intracerebral, com sintomas variando, desde hiperexitabilidade (amostra canina, paralisia progressiva principalmente de membros posteriores e maior duração do curso clínico até a morte (eqüino e morte repentina, sem sintomas aparentes (morcego insetívoro. Pela imunoistoquímica detectou-se produção de IFN nos cérebros dos camundongos inoculados com amostra de bovino e morcego insetívoro, TNF e iNOS nos animais infectados com amostra de cão, bovino e morcego insetívoro e reação astrocitária com aumento da expressão de GFAP em todas as cinco amostras. A eficácia de 2 vacinas comerciais inativadas, uma nacional e outra importada, para a proteção contra a infecção experimental em camundongos foi avaliada através dos testes NIH e CDC, usando as amostras de campo para o desafio. Não houve diferença significativa entre o desempenho das vacinas, quando comparadas para um mesmo teste de potência e amostra de desafio sugerindo que não há necessidade de se produzir vacinas com amostras isoladas de campo.

  2. Genetic Diversity of a Natural Population of Apple stem pitting virus Isolated from Apple in Korea

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    Ju Yeon Yoon

    2014-06-01

    Full Text Available Apple stem pitting virus (ASPV, of the Foveavirus genus in the family Betaflexiviridae, is one of the most common viruses of apple and pear trees. To examine variability of the coat protein (CP gene from ASPV, eight isolates originating from 251 apple trees, which were collected from 22 apple orchards located in intensive apple growing areas of the North Gyeongsang and North Jeolla Provinces in Korea, were sequenced and compared. The nucleotide sequence identity of the CP gene of eight ASPV isolates ranged from 77.0 to 97.0%, while the amino acid sequence identity ranged from 87.7 to 98.5%. The N-terminal region of the viral CP gene was highly variable, whereas the C-terminal region was conserved. Genetic algorithm recombination detection (GARD and single breakpoint recombination (SBP analyses identified base substitutions between eight ASPV isolates at positions 54 and 57 and position 771, respectively. GABranch analysis was used to determine whether the eight isolates evolved due to positive selection. All values in the GABranch analysis showed a ratio of substitution rates at non-synonymous and synonymous sites (dNS/dS below 1, suggestive of strong negative selection forces during ASPV CP history. Although negative selection dominated CP evolution in the eight ASPV isolates, SLAC and FEL tests identified four possible positive selection sites at codons 10, 22, 102, and 158. This is the first study of the ASPV genome in Korea.

  3. Genetic Diversity of a Natural Population of Apple stem pitting virus Isolated from Apple in Korea.

    Science.gov (United States)

    Yoon, Ju Yeon; Joa, Jae Ho; Choi, Kyung San; Do, Ki Seck; Lim, Han Cheol; Chung, Bong Nam

    2014-06-01

    Apple stem pitting virus (ASPV), of the Foveavirus genus in the family Betaflexiviridae, is one of the most common viruses of apple and pear trees. To examine variability of the coat protein (CP) gene from ASPV, eight isolates originating from 251 apple trees, which were collected from 22 apple orchards located in intensive apple growing areas of the North Gyeongsang and North Jeolla Provinces in Korea, were sequenced and compared. The nucleotide sequence identity of the CP gene of eight ASPV isolates ranged from 77.0 to 97.0%, while the amino acid sequence identity ranged from 87.7 to 98.5%. The N-terminal region of the viral CP gene was highly variable, whereas the C-terminal region was conserved. Genetic algorithm recombination detection (GARD) and single breakpoint recombination (SBP) analyses identified base substitutions between eight ASPV isolates at positions 54 and 57 and position 771, respectively. GABranch analysis was used to determine whether the eight isolates evolved due to positive selection. All values in the GABranch analysis showed a ratio of substitution rates at non-synonymous and synonymous sites (dNS/dS) below 1, suggestive of strong negative selection forces during ASPV CP history. Although negative selection dominated CP evolution in the eight ASPV isolates, SLAC and FEL tests identified four possible positive selection sites at codons 10, 22, 102, and 158. This is the first study of the ASPV genome in Korea.

  4. Screening, isolation and optimization of anti-white spot syndrome virus drug derived from terrestrial plants

    Institute of Scientific and Technical Information of China (English)

    Upasana Ghosh; Somnath Chakraborty; Thangavel Balasubramanian; Punyabrata Das

    2014-01-01

    Objective: To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model.Methods:Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug.Results: Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection.Conclusions:The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug.

  5. Phylogenetic study on the 5'-untranslated region of bovine viral diarrhoea virus isolates from Iran

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    Majid Esmaelizad

    2014-09-01

    Full Text Available Bovine viral diarrhoea virus is a pathogen of bovids associated with reproduction system, causing in infected animals a range of ailments, from abortion to congenital defects. In this article, the nucleotide structure of the 5'-untranslated region (5-UTR from 7 Iranian bovine diarrhoea virus (BVDV isolates was characterized and subjected to comparative analysis against a panel of BVDV isolates from different sources. To this end, a 288 bp-long stretch of the internal ribosome entry site was amplified by RT-PCR. The PCR products subsequently cloned into PTZ57T vector and sequenced using T7 promoter primers. This resulted in detection of 3 new point mutations G→A and G→T in 2 isolates. When these findings were phylogenetically assessed, all the examined Iranian isolates were deemed to belong to the type1 of BVDV. Besides, 2 subtypes were identified among these isolates. In group A, a high level of similarity (99.2% between Iranian isolates with a cytopathic Australian strain of BVDV-1c was detected; while in group B, the 4 Iranian isolates proved to be very similar to NADL-like BVDV-1a strains. We believe that the surprisingly high level of similarity between group A Iranian isolates and their corresponding Australian strain is likely to be an indication of a shared common ancestor. If correct, the most likely explanation of this observation is the introduction of such strains from Australia to Iran, possibly through exportation of infected live animals or animal productions (e.g. semen and meat at some points in the past. Nevertheless, this hypothesis remains to be proved as further epidemiological work at genomic level is required to understand population of BVDV in Iran.

  6. Isolation and characterization of tick-borne encephalitis virus from Ixodes persulcatus in Mongolia in 2012.

    Science.gov (United States)

    Muto, Memi; Bazartseren, Boldbaatar; Tsevel, Bazartseren; Dashzevge, Erdenechimeg; Yoshii, Kentaro; Kariwa, Hiroaki

    2015-07-01

    Tick-borne encephalitis virus (TBEV) is a zoonotic virus belonging to the genus Flavivirus, in the family Flaviviridae. The virus, which is endemic in Europe and northern parts of Asia, causes severe encephalitis. Tick-borne encephalitis (TBE) has been reported in Mongolia since the 1980s, but details about the biological characteristics of the endemic virus are lacking. In this study, 680 ticks (Ixodes persulcatus) were collected in Selenge aimag, northern Mongolia, in 2012. Nine Mongolian TBEV strains were isolated from tick homogenates. A sequence analysis of the envelope protein gene revealed that all isolates belonged to the Siberian subtype of TBEV. Two strains showed similar growth properties in cultured cells, but their virulence in mice differed. Whole genome sequencing revealed only thirteen amino acid differences between these Mongolian TBEV strains. Our results suggest that these naturally occurring amino acid mutations affected the pathogenicity of Mongolian TBEV. Our results may be an important platform for monitoring TBEV to evaluate the epidemiological risk in TBE endemic areas of Mongolia.

  7. Laboratory diagnosis of contagious ecthyma: comparison of different PCR protocols with virus isolation in cell culture.

    Science.gov (United States)

    Kottaridi, Christine; Nomikou, Kiki; Lelli, Rossella; Markoulatos, Panayotis; Mangana, Olga

    2006-06-01

    A new polymerase chain reaction (PCR) assay for rapid diagnosis of contagious ecthyma was designed and applied to 21 clinical samples from Greece. This assay, which detects a highly conserved gene from the parapox genome, was evaluated for its sensitivity and specificity in order to be considered as a useful diagnostic tool. A comparative study with two published PCR protocols one using primers PPP1-PPP3, PPP1-PPP4 which targets putative virion envelope gene B2L and the other using VIR1-VIR2 primers which amplifies ORF virus interferon resistant (VIR) gene, as well as cell culture virus neutralization assay was carried out. All samples tested were amplified successfully with the PCR protocol established in the laboratory. The combination of primers PPP1-PPP3 and PPP1-PPP4 in a semi-nested PCR gave a positive result in 20 of 21 samples while primers VIR1-VIR2 failed to amplify successfully 7 of 21 samples. The diagnostic value of parapox viral DNA amplification was also compared with the results of virus isolation by cell culture and was positive in three samples that the virus isolation was obtained.

  8. Isolation and diagnosis of Chikungunya virus causing outbreaks in Andhra Pradesh, India

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    C.V.M. Naresh Kumar

    2013-01-01

    Full Text Available Background: Chikungunya fever has recently re-emerged in India with a high morbidity. However, the prevalence of chikungunya fever in India has been underreported due to non-availability of specialized kits to confirm the disease in most of the laboratories. Methods: Nine hundred and fifty six serum samples were collected from subjects presenting with a short febrile illness from various places in Chittoor district, Andhra Pradesh, between January to October 2009 and were screened for Chikungunya Virus (CHIKV infection. Virus isolation, reverse transcriptase - polymerase chain reaction (RT-PCR and immunoglobulin M (IgM rapid strip method were employed for the identification of the causative agent. Results: Chikungunya Virus (CHIKV infection was confirmed in 520 (68.1% patients by RT-PCR. Seventy seven (40.1% patients showed the presence of anti-CHIKV IgM antibodies while 12 (6.3% patients showed the presence of both anti-CHIKV IgM and immunoglobulin G (IgG antibodies respectively. The isolation of CHIKV was successful from five patients. Conclusions: The re-emergence and persistence of CHIKV in Andhra Pradesh suggests the need for continuous monitoring and identification of the pathogen and thereby prevention of the spread of the virus to other parts of the country.

  9. Tobacco curly shoot virus iso-lated in Yunnan is a distinct species of Begomovirus

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observation showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in complementary-sense DNA. Comparisons with total DNA-A, intergenic region and deduced amino acid sequences of individual ORFs showed that Y1 is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is proposed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Pakistan (98% amino acid sequence identity).

  10. Complete genome sequence analysis of two Citrus tatter leaf virus (CTLV) isolates from China

    Institute of Scientific and Technical Information of China (English)

    SONG Zhen; LI Zhong-an; LIU Ke-hong; ZHOU Chang-yong

    2015-01-01

    In order to understand molecular characterization of Citrus tatter leaf virus (CTLV) isolated from China, ful-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome ampliifcation by RT-PCR. The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6 497 nucleotides in length and shared 79.9–91.0%and 78.8–98.0%nucleotide sequence identity, respectively, with other Apple stem grooving virus (ASGV) or CTLV strains available in GenBank. Unexpectedly, CTLV-MTH showed the highest nucleo-tide sequence identity (91%) with an apple isolate of ASGV, fol owed by 86.5%with ASGV-HH and 85.7%with ASGV-CHN. Furthermore, CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree, suggesting it has a closer relationship to ASGV than to CTLV. Therefore, it can be concluded roughly that CTLV may be not a distinct strains of ASGV. We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.

  11. Isolation and pathogenic analysis of virulent Marek's disease virus field strain in China.

    Science.gov (United States)

    Cui, Ning; Su, Shuai; Sun, Peng; Zhang, Yankun; Han, Ni; Cui, Zhizhong

    2016-07-01

    Marek's disease (MD) has become increasingly common in China, resulting in considerable economic loss. The etiological agent is unclear. In this study, we isolated a field MD virus (MDV) strain, designated SX1301, from CVI988/Rispens-vaccinated chickens with tumors. Co-infection of avian leukosis virus, reticuloendotheliosis virus, and chicken infectious anemia virus was excluded by polymerase chain reaction, enzyme-linked immunosorbant assay, DNA blotting hybridization, and indirect immunofluorescence assay. As with most strains isolated in China, SX1301 had the same amino acid mutation of meq protein at positions 77(E), 80(Y), and 115(A) Animal experimental results showed development of lethal MD in 57% and MD tumor in 23% of the specific pathogen-free chickens inoculated with SX1301, with tumors mainly distributed in spleen, liver, and kidney. CVI988/Rispens protected 83% of chickens upon challenge with SX1301, with a mortality rate and tumor incidence of 10% and 7%, respectively. These results implicated SX1301 as a virulent MDV strain, with commercial MDV vaccine CVI988/Rispens unable to confer adequate protection against SX1301. There have been no reports of very virulent (vv) plus MDV in China, but frequently occurring virulent MDV may account for the repeated outbreaks of MD. Vaccines with greater efficacy are needed to protect against MDV.

  12. Complete Genome Sequence and Phylogenetic Relatedness of Hepatitis B Virus Isolates in Papua, Indonesia▿ †

    OpenAIRE

    Utsumi, Takako; Lusida, Maria Inge; Yano, Yoshihiko; Nugrahaputra, Victor Eka; Amin, Mochamad; Juniastuti,; Soetjipto; Hayashi, Yoshitake; Hotta, Hak

    2009-01-01

    Each hepatitis B virus (HBV) genotype and subgenotype is associated with a particular geographic distribution, ethnicity, and anthropological history. Our previous study showed the novel HBV subgenotypes C6 (HBV/C6) and D6 (HBV/D6), based on the S gene sequences of isolates in Papua, Indonesia. The present study investigated the complete genome sequence of 22 strains from Papua and subjected them to molecular evolutionary analysis. A phylogenetic analysis revealed that 9 out of 22 strains wer...

  13. Characterization of West Nile Viruses Isolated from Captive American Flamingoes (Phoenicopterus ruber) in Medellin, Colombia

    OpenAIRE

    Jorge E Osorio; Ciuoderis, Karl A; Lopera, Juan G.; Piedrahita, Leidy D.; Murphy, Darby; Levasseur, James; Carrillo, Lina; Ocampo, Martha C.; Hofmeister, Erik

    2012-01-01

    Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequen...

  14. Genetic structure and molecular variability of Cucumber mosaic virus isolates in the United States.

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    Shahideh Nouri

    Full Text Available Cucumber mosaic virus (CMV has a worldwide distribution and the widest host range of any known plant virus. From 2000 to 2012, epidemics of CMV severely affected the production of snap bean (Phaseulos vulgaris L. in the Midwest and Northeastern United States. Virus diversity leading to emergence of new strains is often considered a significant factor in virus epidemics. In addition to epidemics, new disease phenotypes arising from genetic exchanges or mutation can compromise effectiveness of plant disease management strategies. Here, we captured a snapshot of genetic variation of 32 CMV isolates collected from different regions of the U.S including new field as well as historic isolates. Nucleotide diversity (π was low for U.S. CMV isolates. Sequence and phylogenetic analyses revealed that CMV subgroup I is predominant in the US and further showed that the CMV population is a mixture of subgroups IA and IB. Furthermore, phylogenetic analysis suggests likely reassortment between subgroups IA and IB within five CMV isolates. Based on phylogenetic and computational analysis, recombination between subgroups I and II as well as IA and IB in RNA 3 was detected. This is the first report of recombination between CMV subgroups I and II. Neutrality tests illustrated that negative selection was the major force operating upon the CMV genome, although some positively selected sites were detected for all encoded proteins. Together, these data suggest that different regions of the CMV genome are under different evolutionary constraints. These results also delineate composition of the CMV population in the US, and further suggest that recombination and reassortment among strain subgroups does occur but at a low frequency, and point towards CMV genomic regions that differ in types of selection pressure.

  15. Molecular and antigenic characteristics of Newcastle disease virus isolates from domestic ducks in China.

    Science.gov (United States)

    Wu, Wei; Liu, Huairan; Zhang, Tingting; Han, Zongxi; Jiang, Yanyu; Xu, Qianqian; Shao, Yuhao; Li, Huixin; Kong, Xiangang; Chen, Hongyan; Liu, Shengwang

    2015-06-01

    Newcastle disease (ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus (NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl, such as ducks and geese, are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decade. In this study, 18 NDV strains isolated from domestic ducks in southern and eastern China, between 2005 and 2013, were genetically and phylogenetically characterized. The complete genomes of these strains were sequenced, and they exhibited genome sizes of 15,186 nucleotides (nt), 15,192 nt, and 15,198 nt, which follow the "rule of six" that is required for the replication of NDV strains. Based on the cleavage site of the F protein and pathogenicity tests in chickens, 17 of our NDV isolates were categorized as lentogenic viruses, and one was characterized as a velogenic virus. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks; GD1, AH224, and AH209 belong to genotypes VIId, Ib, and II of class II NDVs, respectively, and the remaining 15 isolates belong to genotype 1b of class I NDVs. Cross-reactive hemagglutination inhibition tests demonstrated that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts. These results suggest that though those NDV isolates were from duck, they still don't form a phylogenetic group because they came from the same species; however, they may play an important role in promoting the evolution of NDVs.

  16. Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

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    L.C. Kreutz

    2000-12-01

    Full Text Available Three Brazilian isolates of bovine viral diarrhea virus (BVDV, antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs. Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11, were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid and 1:100 (hybridoma culture supernatant in IFA and immunoperoxidase (IPX staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

  17. Isolation of dual-tropic murine leukemia virus from radiation-induced thymoma in RF mouse

    Energy Technology Data Exchange (ETDEWEB)

    Okumoto, M.; Nishikawa, R.; Takamori, Y.; Iwai, Y.; Iwai, M. (Radiation Center of Osaka Prefecture, Sakai (Japan))

    1981-03-01

    Two different murine leukemia viruses (MuLV) were isolated from radiation-induced thymoma in RF mouse by co-cultivation method with mink lung cells. One is ecotropic MuLV, which replicates efficiently in NIH Swiss mouse embryo (NIH ME) and SC-1 cells and was able to form XC plaques. The other was dualtropic MuLV, which replicates in both mink lung cells and NIH ME cells but was unable to form XC plaques.

  18. Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer

    Directory of Open Access Journals (Sweden)

    Chang-Sook Hong

    2016-03-01

    Full Text Available Objective: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. Methods: Plasma specimens (1 mL of patients with acute myeloid leukaemia (AML or a head and neck squamous cell carcinoma (HNSCC were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC. Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. Results: Exosomes eluting in fractions #3–5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003 than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004, and HNSCC exosomes suppressed activation (p<0.01 and proliferation of activated T lymphocytes (p<0.03. Conclusions: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications.

  19. The isolation of plasma membrane from protoplasts of soybean suspension cultures.

    Science.gov (United States)

    Galbraith, D W; Northcote, D H

    1977-04-01

    A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

  20. A cytoplasmic polyhedrosis virus isolated from the pine processionary caterpillar, Thaumetopoea pityocampa.

    Science.gov (United States)

    Ince, Ikbal Agah; Demir, Ismail; Demirbag, Zihni; Nalcacioglu, Remziye

    2007-04-01

    A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 microm in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae.

  1. A complete sequence and comparative analysis of a SARS-associated virus (Isolate BJ01)

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-associated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs. We report the complete genome sequence and comparative analysis of an isolate (BJ01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 2 ORFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS- associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymousmutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host. Two amino acid changes have been detected in the M protein, which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).

  2. Newcastle disease viruses causing recent outbreaks worldwide show unexpectedly high genetic similarity with historical virulent isolates from the 1940s

    Science.gov (United States)

    Virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), a devastating disease of poultry and wild birds. Phylogenetic analyses clearly distinguish historical isolates (obtained prior to 1960) from currently circulating viruses of class II genotypes V, VI, VII, and XII throug...

  3. Excretion of bovine herpesvirus 1 in semen is detected much longer by PCR than by virus isolation

    NARCIS (Netherlands)

    Engelenburg, van F.A.C.; Schie, van F.W.; Rijsewijk, F.A.M.; Oirschot, van J.T.

    1995-01-01

    To compare the sensitivities of PCR and virus isolation and to examine the course of virus excretion in semen, we intrapreputially inoculated eight bulls with bovine herpesvirus 1 (BHV1) and used two bulls as sentinels. From these bulls, we collected a large panel of semen samples during 65 days pos

  4. Infectivity analysis of a blackgram isolate of Mungbean yellow mosaic virus and genetic assortment with MYMIV in selective hosts.

    Science.gov (United States)

    Haq, Q M I; Rouhibakhsh, A; Ali, Arif; Malathi, V G

    2011-06-01

    Yellow mosaic disease in grain legumes in Indian subcontinent is caused by two important virus species viz. Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV), belonging to the genus Begomovirus of the family Geminiviridae. The genomic components of a begomovirus causing yellow mosaic disease in blackgram in southern India were cloned and sequenced. Nucleotide sequence comparison of DNA A component shows the virus isolate to be a variant of Mungbean yellow mosaic virus:-(MYMV-[IN:Vam:05]). However, DNA B component of the present virus isolate has greater similarity (92%) to Mungbean yellow mosaic India virus. Agroinoculations of the viral clones produced typical yellow mosaic symptoms in blackgram and mungbean, severe leaf curl and stunting in French bean, similar to blackgram isolate of MYMIV. Blackgram isolates of both the virus species were only mildly infectious on cowpea, produced atypical leaf curl symptoms and not yellow or golden mosaic. In agroinoculations done by exchanging genomic components, symptom expression was seen only in French bean. In cowpea, blackgram and mungbean there was no visible symptoms though viral DNA could be detected by PCR.

  5. Full-Genome Sequence of a Reassortant H1N1 Swine Influenza Virus Isolated from Pigs in Italy.

    Science.gov (United States)

    Chiapponi, Chiara; Baioni, Laura; Luppi, Andrea; Moreno, Ana; Castellan, Alberto; Foni, Emanuela

    2013-10-03

    In this study, the full-genome sequence of a novel reassortant H1N1 swine influenza virus (SIV) is reported. The isolate has a hemagglutinin (HA) gene of the pandemic H1N1 influenza virus, but it carries the seven genome segments of the avian-origin H1N1 SIV currently circulating in European pig farms.

  6. Blueberry fruit drop associated virus: A new member of the family Caulimoviridae isolated from blueberry exhibiting fruit drop symptoms

    Science.gov (United States)

    This study describes the nucleotide sequence and genome organization of a new DNA virus isolated from ‘Bluecrop’ blueberry plants named Blueberry fruit drop associated virus (BFDaV). Infected bushes lose nearly 100% of their fruit prior to harvest, and in springtime young leaves and flowers develop ...

  7. Biochemical isolation and purification of ovulation-inducing factor (OIF in seminal plasma of llamas

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    Pierson Roger A

    2011-02-01

    Full Text Available Abstract Background The objective of the present study was to isolate and purify the protein fraction(s of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate. Methods Semen collected from male llamas by artificial vagina was centrifuged and the seminal plasma was harvested and stored frozen. Seminal plasma was thawed and loaded onto a Type 1 macro-prep ceramic hydroxylapatite column and elution was carried out using a lineal gradient with 350 mM sodium phosphate. Three protein fractions were identified clearly (Fractions A, B, and C, where a prominent protein band with a mass of 14 kDa was identified in Fraction C. Fraction C was loaded into a sephacryl gel filtration column for further purification using fast protein liquid chromatography (FPLC. Isocratic elution resulted in 2 distinct protein fractions (Fractions C1 and C2. An in vivo bioassay (n = 10 to 11 llamas per group was used to determine the ovarian effect of each fraction involving treatment with saline (negative control, whole seminal plasma (positive control, or seminal plasma Fractions A, B or C2. Ultrasonography was done to detect ovulation and CL formation, and blood samples were taken to measure plasma progesterone and LH concentrations. Results Ovulation and CL formation was detected in 0/10, 10/11, 0/10, 2/11, and 10/11 llamas treated with saline, whole seminal plasma, Fractions A, B and C2 respectively (P Conclusion Ovulation-inducing factor was isolated from llama seminal plasma as a 14 kDa protein molecule that elicits a preovulatory LH surge followed by ovulation and CL formation in llamas, suggesting an endocrine effect at the level of the hypothalamus (release of GnRH or the pituitary (gonadotrophs.

  8. Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

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    Cisneros Alejandro

    2009-12-01

    Full Text Available Abstract Background Dengue (DEN is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating. Results To study whether there are isolates from Oaxaca having recombination, we obtained the sequence of 6 different isolates of DENV-2 Asian/American genotype from the outbreak 2005-6, one clone of the C(91-prM-E-NS1(2400 structural genes, and 10 clones of the E gene from the isolate MEX_OAX_1656_05. Evidence of recombination was found by using different methods along with two softwares: RDP3 and GARD. The Oaxaca MEX_OAX_1656_05 and MEX_OAX_1038_05 isolates sequenced in this study were recombinant viruses that incorporate the genome sequence from the Cosmopolitan genotype. Furthermore, the clone of the E gene namely MEX_OAX_165607_05 from this study was also recombinant, incorporating genome sequence from the American genotype. Conclusions This is the first report of recombination in DENV-2 in Mexico. Given such a recombinant activity new genomic combinations were produced, this could play a significant role in the DENV evolution and must be considered as a potentially important mechanism generating genetic variation in this virus with serious implications for

  9. Detection of hepatitis C virus core antigen for early diagnosis of hepatitis C virus infection in plasma donor in China

    Institute of Scientific and Technical Information of China (English)

    He-Qiu Zhang; Shao-Bo Li; Guo-Hua Wang; Kun Chen; Xiao-Guo Song; Xiao-Yan Feng

    2007-01-01

    AIM: To evaluate the efficacy of a new hepatitis C virus (HCV) core antigen assay developed in China.METHODS: After the determination of HCV infection, 49 serial samples were selected from 11 regular plasma donors in 5 different plasma stations. To compare the performance of HCV core antigen detection and HCV PCR, these samples were genotyped, and each specimen was analyzed by ELISA for the detection of HCV core antigen and by qualitative HCV PCR.RESULTS: Among all of the sequential samples, the original 13 specimens were HCV RNA-negative, and 36 samples were HCV RNA-positive. Twenty-seven samples (75%) were HCV core antigen-positive from these HCV RNA-positive specimens. Conversely, 27 samples (93.1%) were found HCV RNA-positive in HCV core antigen-positive samples. Intervals between HCV RNA and HCV core antigen-positive, as well as between HCV core antigen-positive and HCV antibody-positive were 36.0 and 32.8 d, respectively.CONCLUSION: This HCV core antigen assay, developed in China, is able to detect much of anti-HCV-negative, HCV RNA-positive preseroconversion window period (PWP) plasma donations.

  10. A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma.

    Science.gov (United States)

    Grant, Ryan; Ansa-Addo, Ephraim; Stratton, Dan; Antwi-Baffour, Samuel; Jorfi, Samireh; Kholia, Sharad; Krige, Lizelle; Lange, Sigrun; Inal, Jameel

    2011-08-31

    The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 μm in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 μm pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 μm) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 μM) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects.

  11. Lassa virus isolates from Mali and the Ivory Coast represent an emerging fifth lineage.

    Science.gov (United States)

    Manning, John T; Forrester, Naomi; Paessler, Slobodan

    2015-01-01

    Previous imported cases of Lassa fever (LF) into the United Kingdom from the Ivory Coast and Mali, as well as the detection of Lassa virus (LASV) among the Mastomys natalensis population within Mali has led to the suggestion that the endemic area for LF is expanding. Initial phylogenetic analyses arrange isolates from Mali and the Ivory Coast separately from the classical lineage IV isolates taken from Sierra Leone, Guinea, and Liberia. The availability of full genome sequences continues to increase, allowing for a more complete phylogenetic comparison of the isolates from Mali and the Ivory Coast to the other existing isolates. In this study, we utilized a Bayesian approach to infer the demographic histories of each LASV isolate for which the full sequence was available. Our results indicate that the isolates from Mali and the Ivory Coast group separately from the isolates of lineage IV, comprising a distinct fifth lineage. The split between lineages IV and V is estimated to have occurred around 200-300 years ago, which coincides with the colonial period of West Africa.

  12. First isolation of dengue virus from Lao PDR in a Chinese traveler

    Science.gov (United States)

    2013-01-01

    Background Epidemic dengue activity has been demonstrated in several southern regions of China, but not in Yunnan province, which borders countries in Southeast Asia where dengue is endemic. Many dengue cases imported from Southeast Asia to Yunnan have been reported, but dengue virus (DENV) has not been isolated from any patients. This study is the first to report the isolation of DENV from a Chinese traveler returning to Yunnan from Lao PDR. Findings A serum sample was collected from a patient presenting with a febrile illness who returned from Lao PDR in 2009 and was used to inoculate Aedes albopictus C6/36 cells for viral isolation. The viral isolate was identified using reverse transcription-polymerase chain reaction, and phylogenetic analyses based on the full E sequence were performed using Clustalx 1.8 software. The analyses detected DENV genome, and thus, a DENV isolate was obtained from the patient’s serum sample. The new DENV isolate was grouped into genotype Asia 1, serotype 2. The viral E protein shared the greatest nucleotide sequence identity (99.6%) with the D2/Thailand/0606aTw strain isolated from Thailand in 2006 and demonstrated 94.3% to 100% identity with the predicted amino acid sequence of other DENV 2 strains. Conclusions Our findings indicate that DENV serotype 2 is circulating in Lao PDR, and surveillance of patients suspected of infection with dengue should be conducted not only by a serological test but also by pathogenic detection methods. PMID:23497045

  13. Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

    Science.gov (United States)

    Trucco, Verónica; de Breuil, Soledad; Bejerman, Nicolás; Lenardon, Sergio; Giolitti, Fabián

    2014-06-01

    The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

  14. High-throughput blood cell focusing and plasma isolation using spiral inertial microfluidic devices.

    Science.gov (United States)

    Xiang, Nan; Ni, Zhonghua

    2015-12-01

    Herein, we explored the blood cell focusing and plasma isolation using a spiral inertial microfluidic device. First, the flow-rate and concentration effects on the migration dynamics of blood cells were systematically investigated to uncover the focusing mechanisms and steric crowding effects of cells in Dean-coupled inertial flows. A novel phenomenon that the focusing status of discoid red blood cells (RBCs) changes according to the channel height was discovered. These experimental data may provide valuable insights for the high-throughput processing of blood samples using inertial microfluidics. On the basis of the improved understandings on blood cell focusing, efficient isolation of plasma from whole blood with a 20-fold dilution was achieved at a throughput up to 700 μl/min. The purity of the isolated blood plasma was close to 100 %, and the plasma yield was calculated to be 38.5 %. As compared with previously-reported devices, our spiral inertial microfluidic device provides a balanced overall performance, and has overriding advantages in terms of processing throughput and operating efficiency.

  15. Characterization of low pathogenicity avian influenza viruses isolated from wild birds in Mongolia 2005 through 2007

    Directory of Open Access Journals (Sweden)

    Sodnomdarjaa Ruuragchaa

    2009-11-01

    Full Text Available Abstract Background Since the emergence of H5N1 high pathogenicity (HP avian influenza virus (AIV in Asia, numerous efforts worldwide have focused on elucidating the relative roles of wild birds and domestic poultry movement in virus dissemination. In accordance with this a surveillance program for AIV in wild birds was conducted in Mongolia from 2005-2007. An important feature of Mongolia is that there is little domestic poultry production in the country, therefore AIV detection in wild birds would not likely be from spill-over from domestic poultry. Results During 2005-2007 2,139 specimens representing 4,077 individual birds of 45 species were tested for AIV by real time RT-PCR (rRT-PCR and/or virus isolation. Bird age and health status were recorded. Ninety rRT-PCR AIV positive samples representing 89 individual birds of 19 species including 9 low pathogenicity (LP AIVs were isolated from 6 species. A Bar-headed goose (Anser indicus, a Whooper swan (Cygnus cygnus and 2 Ruddy shelducks (Tadorna ferruginea were positive for H12N3 LP AIV. H16N3 and H13N6 viruses were isolated from Black-headed gulls (Larus ridibundus. A Red-crested pochard (Rhodonessa rufina and 2 Mongolian gulls (Larus vagae mongolicus were positive for H3N6 and H16N6 LP AIV, respectively. Full genomes of each virus isolate were sequenced and analyzed phylogenetically and were most closely related to recent European and Asian wild bird lineage AIVs and individual genes loosely grouped by year. Reassortment occurred within and among different years and subtypes. Conclusion Detection and/or isolation of AIV infection in numerous wild bird species, including 2 which have not been previously described as hosts, reinforces the wide host range of AIV within avian species. Reassortment complexity within the genomes indicate the introduction of new AIV strains into wild bird populations annually, however there is enough over-lap of infection for reassortment to occur. Further work is

  16. Propagation of Asian isolates of canine distemper virus (CDV in hamster cell lines

    Directory of Open Access Journals (Sweden)

    Yamaguchi Ryoji

    2009-10-01

    Full Text Available Abstract Backgrounds The aim of this study was to confirm the propagation of various canine distemper viruses (CDV in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. Methods The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains and by observing the development of cytopathic effect (CPE in infected cultures of hamster cell lines. Results Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. Conclusion The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

  17. Isolation and characterization of a variant porcine epidemic diarrhea virus in China

    Directory of Open Access Journals (Sweden)

    Pan Yongfei

    2012-09-01

    Full Text Available Abstract An outbreak of diarrhea in pigs started in Guangdong, South China in January 2011. Cases were characterized by watery diarrhea, dehydration and vomiting, with 80–100% morbidity and 50–90% mortality in suckling piglets. The causative agent of the diarrhea was ultimately identified as porcine epidemic diarrhea virus (PEDV. In this study, we isolated a PEDV strain designated CHGD-01 from piglet intestines using Vero cell cultures, and its specific cytopathic effects were confirmed in susceptible cells by direct immunofluorescence testing and electron microscopy. The complete genome of CHGD-01 was shown to be 28,035 nucleotides in length, with a similar structure to that of PEDV reference strains. Phylogenetic analyses based on the whole genome revealed that CHGD-01 shared nucleotide sequence identities of 98.2–98.4% with two other Chinese isolates reported in the same year, thus constituting a new cluster. Amino acid sequence analysis based on individual virus genes indicated a close relationship between the spike protein gene of CHGD-01 and the field strain KNU0802 in Korea. Its ORF3 and nucleoprotein genes, however, were divergent from all other sequenced PEDV isolate clusters and therefore formed a new group, suggesting a new variant PEDV isolate in China. Further studies will be required to determine the immunogenicity and pathogenicity of this new variant.

  18. Molecular Typing of Field Isolates from two outbreaks of Infectious Bursal Disease Virus from Pakistan

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    Q. M. Khan and M. J. Arshed

    Full Text Available A reverse transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR/RFLP technique was used for the identification and characterization of Pakistani field isolates of infectious bursal disease virus (IBDV. A total of 8 bursa samples were collected from two outbreaks during September and October 2003 from Tehsil Sumandri, Dist. Faisalabad with 40-50% mortality in commercially reared broiler chicken flocks experiencing signs typical of infectious bursal disease (IBD. Four samples were found to contain IBDV genome by One Step RTPCR using VP2 gene specific primers. The assay amplified a 743 bp fragment from 701-1444 nucleotides. RT-PCR product was further subjected to restriction digestion using MboI and MvaI restriction enzymes. A third enzyme SspI was used to identify the very virulent phenotype. The RFLP profile was found similar for all four isolates with MvaI enzyme but different for one isolate when digested with MboI. All three MvaI-positive viruses were further found positive for SspI digestion and yielded RFLP profile similar to vvIBDV in Europe whereas one isolate was SspI negative and had a RFLP profile similar to classic IBDV strains. The clinical history of high mortality and SspI restriction enzyme positivity revealed that vvIBDV strains exist in Pakistan. [Vet. World 2011; 4(7.000: 297-300

  19. Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan

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    N.A. Elmardi

    2016-06-01

    Full Text Available A reverse transcription-polymerase chain reaction (RT-PCR was used to amplify 1412 bp of the fusion protein gene (F gene of four Newcastle disease virus (NDV isolates; two velogenic (TY-1/90 and DIK-90 and two lentogenic isolates (Dongla 88/1 and GD.S.1. Following sequencing, nucleotide sequences were annotated and 894 bp were compared phylogenetically with those from strains previously reported in the Sudan and the virus strains published on the GenBank. It could be demonstrated that TY-1/90 and DIK-90 strains belong to the genotype VI of NDV and are in close genetic relationship to sub- genotype VIb. TY-1/90 and DIK-90 strains were observed to be genetically unrelated to the earlier Sudanese isolates of 1970/80s and the late of 2000s suggesting a different origin. The close genetic relationship to the European and African pigeon paramyxovirus type 1 (PPMV-1 suggests a common ancestor. Dongola, GD.S.1 strains were classified into genotype II that comprises non-pathogenic lentogenic NDV strains. The present genetic classification of NDV isolates of the Sudan provides valuable information on genotypes of NDV. Further molecular epidemiological investigations of the recent outbreaks of Newcastle disease in the Sudan are needed in order to improve the efficiency of control strategies and vaccine development.

  20. Identification of a new Newcastle disease virus isolate from Indonesia represents an ancestral lineage of class II genotype XIII.

    Science.gov (United States)

    Forrester, Naomi L; Widen, Steve G; Wood, Thomas G; Travassos da Rosa, Amelia P; Ksiazek, Thomas G; Vasilakis, Nikos; Tesh, Robert B

    2013-08-01

    An unknown virus was isolated from a mosquito pool collected in Jakarta during routine surveillance in 1979. Analysis of the sample using the Illumina platform resulted in the identification of a Newcastle disease virus (NDV) isolate. The sequence of the isolate indicated that it is an ancestral lineage of class II, genotype XIII. The source of the isolate is unusual, as newcastle disease virus is not believed to be vector-borne, although this mosquito pool was processed in a laboratory also handling samples for avian influenza surveillance and it is possible that this resulted in cross-contamination. This NDV isolate is still ancestral to most extant genotype XIII strains and provides a useful insight into historic NDV evolution.

  1. Complete genome sequence of an avian leukosis virus isolate associated with hemangioma and myeloid leukosis in egg-type and meat-type chickens

    Science.gov (United States)

    A new virus isolate was separated from a commercial egg-type flock of chickens in China and was determined as subgroup J avian leukosis virus (ALV-J). ALV-J is known to cause myeloid leukosis. But this new isolate of viruses causes both hemangioma and myeloid leukosis in chickens. Hemangioma is an a...

  2. Molecular characterization of quail bronchitis virus isolated from bobwhite quail in Minnesota.

    Science.gov (United States)

    Singh, Azad; Bekele, Aschalew Z; Patnayak, Devi P; Jindal, Naresh; Porter, Robert E; Mor, Sunil K; Goyal, Sagar M

    2016-12-01

    From 2008 to 2012, 4 separate cases of quail bronchitis virus infection were seen in bobwhite quail (Colinus virginianus) raised in Minnesota. The quail chicks ranged in age from 5 d to 8 wk and suffered from respiratory distress and elevated mortality. On necropsy, gross lesions consisted of mucus in trachea, congested lungs, caseous air sacculitis, accumulation of chalky white urates on internal organs, necrotic foci in liver, and enlarged spleen. Histologic examination revealed fibrinoheterophilic rhinitis, heterophilic bronchitis, heterophilic tracheitis, and interstitial pneumonia in addition to deciliation, desquamation, and necrosis of bronchial respiratory epithelium. Karyomegaly with basophilic intranuclear inclusions was also seen in affected epithelium. Severe epicarditis, pericarditis, myocarditis, multifocal necrotizing hepatitis, and splenitis were additional pathological findings. Quail bronchitis virus (QBV) was isolated from all four samples when inoculated in specific-pathogen-free (SPF) embryonated chicken eggs. The virus was confirmed by electron microscopy and polymerase chain reaction using fowl adenovirus (FAdV) hexon gene-specific primers. Nucleotide sequences of the four isolates showed 99.0% identity with CELO strain of fowl adenovirus A. Nine nucleotide substitutions were observed; 3 of these were nonsynonymous (A281G, C314T and G565C), leading to changes in deduced amino acid sequences (S94G, T105M and A189P, respectively). Based on partial sequence of the hexon gene, QBV isolates of this study clustered closely with fowl adenovirus A and were different from FAdV groups B through E and from adenoviruses of goose, duck, turkey, and pigeon. Further studies are indicated to determine the impact of nonsynonymous substitutions on host specific pathogenicity of these viruses. © 2016 Poultry Science Association Inc.

  3. Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

    Science.gov (United States)

    Aravind, S; Kamble, Nitin M; Gaikwad, Satish S; Shukla, Sanjeev Kumar; Dey, Sohini; Mohan, C Madhan

    2015-01-01

    Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis.

  4. Complete nucleotide sequence analysis of Cymbidium mosaic virus Indian isolate: further evidence for natural recombination among potexviruses

    Indian Academy of Sciences (India)

    Ang Rinzing Sherpa; Vipin Hallan; Promila Pathak; Aijaz Asghar Zaidi

    2007-06-01

    The complete nucleotide sequence of an Indian strain of Cymbidium mosaic virus (CymMV) was determined and compared with other potexviruses. Phylogenetic analyses on the basis of RNA-dependent RNA polymerase (RdRp), triple gene block protein and coat protein (CP) amino acid sequences revealed that CymMV is closely related to the Narcissus mosaic virus (NMV), Scallion virus X (SVX), Pepino mosaic virus (PepMV) and Potato aucuba mosaic virus (PAMV). Different sets of primers were used for the amplification of different regions of the genome through RT-PCR and the amplified genes were cloned in a suitable vector. The full genome of the Indian isolate of CymMV from Phaius tankervilliae shares 96–97% similarity with isolates reported from other countries. It was found that the CP gene of CymMV shares a high similarity with each other and other potexviruses. One of the Indian isolates seems to be a recombinant formed by the intermolecular recombination of two other CymMV isolates. The phylogenetic analyses, Recombination Detection Program (RDP2) analyses and sequence alignment survey provided evidence for the occurrence of a recombination between an Indian isolate (AM055720) as the major parent, and a Korean type-2 isolate (AF016914) as the minor parent. Recombination was also observed between a Singapore isolate (U62963) as the major parent, and a Taiwan CymMV (AY571289) as the minor parent.

  5. The spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma.

    Science.gov (United States)

    Gerber, Priscilla F; Xiao, Chao-Ting; Chen, Qi; Zhang, Jianqiang; Halbur, Patrick G; Opriessnig, Tanja

    2014-11-07

    Porcine epidemic diarrhea virus (PEDV) is considered an emergent pathogen associated with high economic losses in many pig rearing areas. Recently it has been suggested that PEDV could be transmitted to naïve pig populations through inclusion of spray-dried porcine plasma (SDPP) into the nursery diet which led to a ban of SDPP in several areas in North America and Europe. To determine the effect of spray-drying on PEDV infectivity, 3-week-old pigs were intragastrically inoculated with (1) raw porcine plasma spiked with PEDV (RAW-PEDV-CONTROL), (2) porcine plasma spiked with PEDV and then spray dried (SD-PEDV-CONTROL), (3) raw plasma from PEDV infected pigs (RAW-SICK), (4) spray-dried plasma from PEDV infected pigs (SD-SICK), or (5) spray-dried plasma from PEDV negative pigs (SD-NEG-CONTROL). For the spray-drying process, a tabletop spray-dryer with industry-like settings for inlet and outlet temperatures was used. In the RAW-PEDV-CONTROL group, PEDV RNA was present in feces at day post infection (dpi) 3 and the pigs seroconverted by dpi 14. In contrast, PEDV RNA in feces was not detected in any of the pigs in the other groups including the SD-PEDV-CONTROL group and none of the pigs had seroconverted by termination of the project at dpi 28. This work provides direct evidence that the experimental spray-drying process used in this study was effective in inactivating infectious PEDV in the plasma. Additionally, plasma collected from PEDV infected pigs at peak disease did not contain infectious PEDV. These findings suggest that the risk for PEDV transmission through commercially produced SDPP is minimal.

  6. Plasma EBV-DNA monitoring in Epstein-Barr virus-positive Hodgkin lymphoma patients.

    Science.gov (United States)

    Spacek, Martin; Hubacek, Petr; Markova, Jana; Zajac, Miroslav; Vernerova, Zdenka; Kamaradova, Katerina; Stuchly, Jan; Kozak, Tomas

    2011-01-01

    Epstein-Barr virus (EBV) is associated with approximately one-third of Hodgkin lymphoma (HL) cases. EBV-DNA is often present in the plasma and whole blood of EBV-associated HL patients. However, the significance of EBV-DNA monitoring is debated. In a cohort of 165 adult HL patients, EBV-DNA viral load was prospectively monitored both in the plasma and whole blood. Diagnostic tissue samples of all patients were histologically reviewed; in 72% nodular sclerosis was detected, 24% presented with mixed cellularity (MC), and 5% had other type of HL. Tissues from 150 patients were also analyzed for the presence of latent EBV infection using in situ hybridization for EBV-encoded RNA (EBER) and immunohistochemistry for latent membrane protein (LMP1). Using these methods, 29 (19%) patients were classified as EBV positive. Using real-time quantitative PCR, 22 (76%) of EBV-positive HL patients had detectable EBV-DNA in the plasma and 19 (66%) patients in whole blood prior to therapy. In the group of EBV-negative HL cases, three (2%) patients had detectable plasma EBV-DNA and 30 (25%) patients whole blood EBV-DNA before treatment. EBV-positive HL was significantly associated with EBV-DNA positivity both in the plasma and whole blood in pretreatment samples, increasing age and MC subtype. Serial analysis of plasma EBV-DNA showed that response to therapy was associated with decline in viral load. Moreover, significantly increased plasma EBV-DNA level recurred before disease relapse in one patient. Our results further suggest that the assessment of plasma EBV-DNA viral load might be of value for estimation of prognosis and follow-up of patients with EBV-positive HL.

  7. Isolation and characterization of Mayaro virus from a human in Acre, Brazil.

    Science.gov (United States)

    Terzian, Ana Carolina B; Auguste, Albert J; Vedovello, Danila; Ferreira, Marcelo U; da Silva-Nunes, Mônica; Sperança, Márcia A; Suzuki, Rodrigo B; Juncansen, Camila; Araújo, João P; Weaver, Scott C; Nogueira, Maurício L

    2015-02-01

    Mayaro virus (MAYV) is widely distributed throughout South America and is the etiologic agent of Mayaro fever, an acute febrile illness often presenting with arthralgic manifestations. The true incidence of MAYV infection is likely grossly underestimated because the symptomatic presentation is very similar to that of dengue fever and other acute febrile tropical diseases. We report the complete genome sequence of a MAYV isolate detected from an Acrelândia patient presenting with fever, chills, and sweating, but with no arthralgia. Results show that this isolate belongs to genotype D and is closely related to Bolivian strains. Our results suggest that the Acre/Mayaro strain is closely related to the progenitor of these Bolivian strains that were isolated between 2002 and 2006.

  8. Characterization of Pigeon Paramyxoviruses (Newcastle disease virus) Isolated in Kazakhstan in 2005

    Institute of Scientific and Technical Information of China (English)

    Andrey Bogoyavlenskiy; Vladimir Berezin; Alexey Prilipov; Eugeniy Usachev; Ilya Korotetskiy; Irina Zaitceva; Aydyn Kydyrmanov; Marat Sayatov

    2012-01-01

    Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene.Part of the amplified F protein DNA product (nucleotide sequence 47-422) and the deduced amino acid sequenceswere compared phylogenetically with those from strains previously reported in other geographic regions.Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype Ⅵ or 4bii.To our knowledge,this is the first reported Ⅵ isolates that possess the sequences of 112 GKRQKR116* F117 within the F0 protein.The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.

  9. Isolation and complete genome sequencing of Mimivirus bombay, a Giant Virus in sewage of Mumbai, India

    Directory of Open Access Journals (Sweden)

    Anirvan Chatterjee

    2016-09-01

    Full Text Available We report the isolation and complete genome sequencing of a new Mimiviridae family member, infecting Acanthamoeba castellanii, from sewage in Mumbai, India. The isolated virus has a particle size of about 435 nm and a 1,182,200-bp genome. A phylogeny based on the DNA polymerase sequence placed the isolate as a new member of the Mimiviridae family lineage A and was named as Mimivirus bombay. Extensive presence of Mimiviridae family members in different environmental niches, with remarkably similar genome size and genetic makeup, point towards an evolutionary advantage that needs to be further investigated. The complete genome sequence of Mimivirus bombay was deposited at GenBank/EMBL/DDBJ under the accession number KU761889.

  10. The complete genome sequence of a Crimean-Congo Hemorrhagic Fever virus isolated from an endemic region in Kosovo

    Science.gov (United States)

    Duh, Darja; Nichol, Stuart T; Khristova, Marina L; Saksida, Ana; Hafner-Bratkovič, Iva; Petrovec, Miroslav; Dedushaj, Iusuf; Ahmeti, Salih; Avšič-Županc, Tatjana

    2008-01-01

    The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF) endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti) isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt) S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01). This suggests that distinct virus strains can coexist in highly endemic areas. PMID:18197964

  11. The complete genome sequence of a Crimean-Congo Hemorrhagic Fever virus isolated from an endemic region in Kosovo

    Directory of Open Access Journals (Sweden)

    Dedushaj Iusuf

    2008-01-01

    Full Text Available Abstract The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01. This suggests that distinct virus strains can coexist in highly endemic areas.

  12. Experimental infection of rainbow trout Oncorhynchus mykiss with viral haemorrhagic septicaemia virus isolates from European marine and farmed fishes

    DEFF Research Database (Denmark)

    Skall, Helle Frank; Slierendrecht, W.J.; King, J.A.

    2004-01-01

    The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes in Europ......The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes...... in European waters (115 isolates), farmed turbot from Scotland and Ireland (2 isolates), and farmed rainbow trout (22 isolates). The isolates were tested by immersion and/or intraperitoneal injection either as pooled or single isolates. The isolates from wild marine fishes did not cause mortality by immersion...... while some of the isolates caused mortality when injected. All VHSV isolates from farmed rainbow trout caused significant mortality by immersion. Currently, pathogenicity trials are the only way to differentiate VHSV isolates from wild marine fishes and farmed rainbow trout. The 2 farmed turbot isolates...

  13. Suppression of human anti-inflammatory plasma cytokines IL-10 and IL-1RA with elevation of proinflammatory cytokine IFN-gamma during the isolation of the Antarctic winter

    Science.gov (United States)

    Shearer, William T.; Lee, Bang-Ning; Cron, Stanley G.; Rosenblatt, Howard M.; Smith, E. O'Brian; Lugg, Desmond J.; Nickolls, Peter M.; Sharp, Robert M.; Rollings, Karl; Reuben, James M.

    2002-01-01

    Cellular immune function has been shown to be decreased and latent virus shedding to be increased in human beings isolated during the Antarctic winter, a model used for assessing some effects of space flight. However, the balance of proinflammatory (IFN-gamma) and anti-inflammatory (IL-10 and IL-1RA) cytokines has not previously been evaluated. We therefore sought to determine whether isolation during the Antarctic winter would alter the proinflammatory and anti-inflammatory cytokine balance. Cytokine levels were measured with ELISA in monthly plasma samples from January through September 1999 in 21 study subjects in the Antarctic and 7 control subjects on Macquarie Island. There was a significant time-dependent increase in plasma IFN-gamma (P =.039) as well as decreases in IL-10 (P =.042) and IL-1RA (P =.053) in the study subjects compared with the control subjects. The study subjects also had significantly increased plasma IFN-gamma levels (P IL-10 and IL-1RA levels (P < or =.036) at individual time points of isolation. Isolation of human beings in the Antarctic appears to shift the plasma cytokine balance toward a proinflammatory profile. These observations are consistent with T-cell activation that might be due to activation of latent viruses, and they could hold importance for determining the risks of space flight.

  14. Host Reaction of Watermelon mosaic virus Isolates Infecting Melon from Different Geographical Origins in Xinjiang of China

    Directory of Open Access Journals (Sweden)

    Dong WANG

    2017-01-01

    Full Text Available Watermelon mosaic virus (WMV is one of the major viruses infecting cucurbit crops worldwide. Although WMV is very common worldwide, little is known about the biological traits of WMV isolates from China. Hence, this study aimed to characterize 11 WMV isolates infecting melon from different geographical origins in Xinjiang based on experimental hosts. Sap inoculation of the 11 WMV isolates onto a range of 13 plant species revealed some differences compared to the WMV isolates collected from other countries. Our results showed that, overall, there were no obvious correlations of host responses to inoculation with WMV isolates from different geographical origins. However, isolate JS-1 caused mild mosaic on Cucurbita moschata, whereas the remaining 10 isolates were asymptomatic on this plant species. Moreover, in Datura stramonium, isolate TYG-1 induced mosaic, whereas the remaining 10 isolates did not infect this species. All isolates infected systemically Cucurbita pepo and Cucumis melo plants, causing severe symptoms. All isolates did not induce any symptoms on Cucumis sativus, but the virus could be detected using RT-PCR. Additionally, all isolates infected systemically Nicotiana tabacum plants, causing mild mosaics. Chenopodium amaranticolor and Chenopodium quinoa reacted to all isolates by chlorotic local lesions in the inoculated leaves, and the virus was detected in the inoculated leaves using RT-PCR. In addition, the attempts to transmit the isolates to Luffa cylindrica, Vicia faba, Phaseolus vulgaris, Vigna unguiculata or Pisum sativum failed as confirmed by negative RT-PCR. Our results would be useful for understanding the biological variability of WMV.

  15. Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa.

    Science.gov (United States)

    Kimbi, Gerald C; Kramvis, Anna; Kew, Michael C

    2004-05-01

    Phylogenetic analysis of hepatitis B virus (HBV) has led to its classification into eight genotypes, A to H. The dominant genotype in South Africa is genotype A, which consists of two subgenotypes, A1 and A2. Subgenotype A1 (previously subgroup A') predominates over subgenotype A2 (previously subgroup A minus A'). The complete genome of HBV isolated from 18 asymptomatic carriers of the virus and five acute hepatitis B patients was amplified; the resulting amplicons were cloned and sequenced. All acute hepatitis isolates belonged to subgenotype A1 and had no distinguishing mutations relative to the isolates from asymptomatic carriers, which had a distribution of ten subgenotype A1, two subgenotype A2 and six genotype D. The presence of the previously described amino acid residues that distinguish subgenotype A1 (subgroup A') from the remainder of genotype A in the S and polymerase genes was confirmed. Moreover, the large number of subgenotype A1 isolates sequenced allowed identification in the other open reading frames of additional nucleotide and amino acid changes that are characteristic of subgenotype A1. In particular, nucleotide mutations at positions 1809-1812 that alter the Kozak sequence of the precore/core open reading frame, and A(1888) in the precore region, were found exclusively in subgenotype A1 isolates. Unique sequence alterations of the transcriptional regulatory elements were also found in subgenotype A1 isolates. The mean nucleotide divergence of subgenotype A1 was greater than that of subgenotype A2, suggesting that this subgenotype has been endemic for a longer time in the South African black population than had subgenotype A2.

  16. Intersubtype Reassortments of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Quail.

    Science.gov (United States)

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Hung, Vu-Khac; Nguyen, Duc Tan; Kim, Wonyong

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) viruses are considered a threat to national animal industries, causing production losses and high mortality in domestic poultry. In recent years, quail has become a popular terrestrial poultry species raised for production of meat and eggs in Asia. In this study, to better understand the roles of quail in H5N1 viral evolution, two H5N1-positive samples, designated A/quail/Vietnam/CVVI-49/2010 (CVVI-49/2010) and A/quail/Vietnam/CVVI-50/2014 (CVVI-50/2014), were isolated from quail during H5N1 outbreaks in Vietnam, and their whole genome were analyzed. The phylogenetic analysis reveals new evolutionary variation in the worldwide H5N1 viruses. The quail HA genes were clustered into clades 1.1.1 (CVVI-49/2010) and clade 2.3.2.1c (CVVI-50/2014), which may have evolved from viruses circulating from chickens and/or ducks in Cambodia, mainland of China, Taiwan, Indonesia, and South Korea in recent years. Interestingly, the M2 gene of the CVVI-49/2010 strain contained amino acid substitutions at position 26L-I and 31S-N that are related to amantadine-resistance. In particular, the CVVI-50/2014 strain revealed evidence of multiple intersubtype reassortment events between virus clades 2.3.2.1c, 2.3.2.1b, and 2.3.2.1a. Data from this study supports the possible role of quail as an important intermediate host in avian influenza virus evolution. Therefore, additional surveillance is needed to monitor these HPAI viruses both serologically and virologically in quail.

  17. Phylogenetic Analysis of Citrus tristeza virus Isolates of Wild Type Citrus in China

    Institute of Scientific and Technical Information of China (English)

    YI Long; ZHOU Chang-yong

    2014-01-01

    The genetic variation and phylogenetic relationships of Citrus tristeza virus (CTV) isolates collected from Chinese wild type citrus were analyzed by comparing the sequences of nine genomic regions (p23, p20, p13, p18, p25, p27, POL, HEL and k17) with the CTV isolates of cultivated citrus from different countries. The results showed that the divergence pattern of genomic RNA of the CTV isolates from wild type citrus was similar to that of other isolates from cultivated citrus, the 3´ proximal region was relatively conserved, and the 5´ proximal region had greater variability. The nine genomic regions of CTV isolates analyzed were found to have been under purifying selection in the evolution process. Phylogenetic analysis showed that the eleven Chinese wild CTV isolates were located at different clades and did not relfect their geographical origins, suggesting genetic diversity among the Chinese wild CTV populations. These results will aid in the understanding of molecular evolution of the Chinese CTV populations.

  18. Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA

    DEFF Research Database (Denmark)

    Uttenthal, Åse; Storgaard, Torben; Oleksiewicz, M.B.

    2003-01-01

    We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been...... without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak....

  19. Plasma charging and electron-based reactions at the plasma-liquid interface of an isolated liquid droplet

    Science.gov (United States)

    Maguire, Paul; Mahony, Charles; Kelsey, Colin; Rutherford, David; Mariotti, Davide; Diver, Declan

    2016-09-01

    The study of plasma-liquid interactions opens up exciting new opportunities for applications but numerous investigative challenges remain. The use of isolated and stable spherical liquid microdroplets in a non-thermal equilibrium atmospheric pressure plasma offers a new platform for experimental and theoretical investigations. Since the droplet assumes floating potential, a high flux of electrons with low net energy ( thermal) becomes fixed and solvated within the first monolayers of the liquid leading to highly reactive and rapid chemical reactions. We observe such reactions, e.g. H2 O2 and metal nanoparticle formation, at rates that are much higher than reported elsewhere. Since the isolated droplet radius is greater than Debye lengths and mean free paths, we have an opportunity to directly compare, for the first time, long-standing collisional probe theories in this important regime. We measure a lower bound average charge of >1E5 electrons on a 13um droplet. Simulations of unipolar corona charging for this size predict 1E3 electrons. A Comsol-based drift-diffusion model is currently under development and so far experiment and theory match within 1 order of magnitude but improvements in measurement technique are in progress. Funding from EPSRC acknowledged (Grants EP/K006088/1 and EP/K006142/1).

  20. Sinu virus, a novel and divergent orthomyxovirus related to members of the genus Thogotovirus isolated from mosquitoes in Colombia.

    Science.gov (United States)

    Contreras-Gutiérrez, María Angélica; Nunes, Marcio R T; Guzman, Hilda; Uribe, Sandra; Gallego Gómez, Juan Carlos; Suaza Vasco, Juan David; Cardoso, Jedson F; Popov, Vsevolod L; Widen, Steven G; Wood, Thomas G; Vasilakis, Nikos; Tesh, Robert B

    2017-01-15

    The genome and structural organization of a novel insect-specific orthomyxovirus, designated Sinu virus, is described. Sinu virus (SINUV) was isolated in cultures of C6/36 cells from a pool of mosquitoes collected in northwestern Colombia. The virus has six negative-sense ssRNA segments. Genetic analysis of each segment demonstrated the presence of six distinct ORFs encoding the following genes: PB2 (Segment 1), PB1, (Segment 2), PA protein (Segment 3), envelope GP gene (Segment 4), the NP (Segment 5), and M-like gene (Segment 6). Phylogenetically, SINUV appears to be most closed related to viruses in the genus Thogotovirus.

  1. The Development of Pathogenicity of Avian Influenza Virus Isolated from Indonesia

    Directory of Open Access Journals (Sweden)

    Michael Haryadi Wibowo

    2015-11-01

    Full Text Available Highly pathogenic avian infl uenza outbreak in Indonesia has been reported in various poultry due toH5N1 subtype. The presence of multiple basic amino acids within the cleavage site of HA glycoprotein hasbeen identifi ed to be associated with the pathogenicity of avian infl uenza virus. The study was retrospectivestudy which was designed to characterize the cleavage site and fusion site region of haemagglutinin gene ofAIV isolated from various poultry in 2003 to 2013. Isolation, Identifi cation and propagation were carried outto collect viral stock. For virus detection, reverse transcriptase PCR (RT-PCR method on H5 and N1 genefragment was performed. All of RT-PCR HA gene positive products were sequenced for further nucleotideanalysis and to determine the nucleotide composition at the targeted fragment. The results are all AIV isolateswere identifi ed as H5N1 subtype. The sequence analyses revealed some motives of basic amino acid motivethat were classifi ed as highly pathogenic avian infl uenza virus. Further analyses on fusion domain of all AIVisolated during the period 2003 to 2013 showed conserved amino acid.Keywords: avian infl uenza, haemagglutinin, cleavage site, basic amino acid, fusion site

  2. Isolation and characterization of ZH14 with antiviral activity against Tobacco mosaic virus.

    Science.gov (United States)

    Zhou, Wen-Wen; Zhang, Li-Xiang; Zhang, Bin; Wang, Fei; Liang, Zhi-Hong; Niu, Tian-Gui

    2008-06-01

    A large number of bacteria were isolated from plant samples and screened for antiviral activity against the Tobacco mosaic virus (TMV). The bacterium ZH14, which was isolated from Chinese Anxi oolong tea, secreted the antiviral substances, having 94.2% virus inhibition when the bacterial culture filtrate and TMV extract were mixed at a ratio of 1:1. The ZH14 strain is a gram-positive, spore-forming rod and has the ability to degrade ribonucleic acid. Based on its effectiveness on virus inhibition, ZH14 was selected for characterization and was identified as a strain of the Bacillus cereus group based on phenotypic tests and comparative analysis of its 16S rDNA sequence. At the same time, we determined the antiviral product of ZH14 as an extracellular protein with high molecular mass, having an optimum temperature of 15-60 degrees C and an optimum pH of 6-10. Hence, the ZH14 strain and its culture filtrate have potential application in controlling plant diseases caused by TMV.

  3. Potato virus X isolation and purification and coat protein gene cloning

    Directory of Open Access Journals (Sweden)

    L. Duplat

    2011-12-01

    Full Text Available In this work we describe the construction of potato virus X coat protein (PVX-CP gene clones from a Colombian isolate. Virus was isolated from field potato plants cultured at páramo de San Jorge. PVX particles were purified from Nicotiana tabacum leaves infected with a local lesion taken from Datura stramonium plants. The genomic RNA present in the virus particles consisted of a single species of about 6 Kb. cDNA synthesis representing genomic RNA was followed by Digoxigenin incorporation after priming with oligonucleotide 4DT/KPN. PCR amplification of CP gene sequence was made by using oligonucleotides OX6 and L/Kpn. An expected fragment of 870 bp, besides some 600-123 bp fragments, was obtained after PCR. The blunt-ended fragments were clonned into the PCR cloning pMOSblue plasmid. The insert size of the selected recombinant cDNA clones was determined by restriction analysis of the plasmidDNAwith Sma I and Hind III enzymes. Positive clones were also screened by direct colony PCR screening. The selected recombinant cDNA clones were stored in glycerol at .70 °C.

  4. Molecular characteristics and pathogenicity of an avian leukosis virus isolated from avian neurofibrosarcoma.

    Science.gov (United States)

    Ochi, Akihiro; Ochiai, Kenji; Nakamura, Sayuri; Kobara, Akiko; Sunden, Yuji; Umemura, Takashi

    2012-03-01

    Peripheral nerve sheath tumors (PNSTs) are rare in chickens and their etiology remains to be elucidated. In this study, a naturally occurring PNST in a Japanese native fowl (Gallus gallus domesticus) was pathologically examined and the strain of avian leukosis virus (ALV) isolated from the neoplasm was characterized by molecular biological analysis. The fowl presented with a firm subcutaneous mass in the neck. The mass, connected to the adjacent spinal cord (C9-14), was microscopically composed of highly cellular tissue of spindle cells arranged in interlacing bundles, streams, and palisading patterns with Verocay bodies and less cellular tissue with abundant collagen. Immunohistochemically, neoplastic cells were divided into two types: perineurial cells positive for vimentin, glucose transporter 1 (GLUT1), and claudin1; and Schwann cells positive for vimentin, occasionally positive for S-100 alpha/beta but negative for GLUT1. Based on these findings, a diagnosis of neurofibrosarcoma was made. The complete nucleotide sequence of an ALV strain, CTS_5371, isolated from the neoplasm was determined and phylogenetic analysis indicated that the strain was a novel recombinant virus from avian leukosis/sarcoma viruses previously reported. Additionally, experimental infection revealed that CTS_5371 induced the proliferation of Schwann cells and perineurial cells. These results suggest that this ALV strain has the ability to induce PNSTs in chickens.

  5. Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

    Directory of Open Access Journals (Sweden)

    María de los Angeles Ribas Antúnez

    2013-04-01

    Full Text Available Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV procedure using 24-well tissue culture plates with the mouse inoculation test (MIT, which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36% were positive at 24 h, 10 (40% were positive at 48 h and six (24% were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

  6. Isolation of Highly Pathogenic Avian Influenza H5N1 Virus from Saker Falcons (Falco cherrug in the Middle East

    Directory of Open Access Journals (Sweden)

    Henju Marjuki

    2009-01-01

    Full Text Available There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses. However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them.

  7. Inactivation of virus in solution by cold atmospheric pressure plasma: identification of chemical inactivation pathways

    Science.gov (United States)

    Aboubakr, Hamada A.; Gangal, Urvashi; Youssef, Mohammed M.; Goyal, Sagar M.; Bruggeman, Peter J.

    2016-05-01

    Cold atmospheric pressure plasma (CAP) inactivates bacteria and virus through in situ production of reactive oxygen and nitrogen species (RONS). While the bactericidal and virucidal efficiency of plasmas is well established, there is limited knowledge about the chemistry leading to the pathogen inactivation. This article describes a chemical analysis of the CAP reactive chemistry involved in the inactivation of feline calicivirus. We used a remote radio frequency CAP produced in varying gas mixtures leading to different plasma-induced chemistries. A study of the effects of selected scavengers complemented with positive control measurements of relevant RONS reveal two distinctive pathways based on singlet oxygen and peroxynitrous acid. The first mechanism is favored in the presence of oxygen and the second in the presence of air when a significant pH reduction is induced in the solution by the plasma. Additionally, smaller effects of the H2O2, O3 and \\text{NO}2- produced were also found. Identification of singlet oxygen-mediated 2-imidazolone/2-oxo-His (His  +14 Da)—an oxidative modification of His 262 comprising the capsid protein of feline calicivirus links the plasma induced singlet oxygen chemistry to viral inactivation.

  8. Plasma virus load evaluation in relation to disease progression in HIV-infected children.

    Science.gov (United States)

    Tetali, S; Bakshi, S; Than, S; Pahwa, S; Abrams, E; Romano, J; Pahwa, S G

    1998-05-01

    The objective of this study was to investigate the relationship of plasma HIV RNA load with survival and disease progression in HIV-infected children and to determine its correlation with cellular HIV DNA. Virus load (VL, HIV RNA copies/ml) was determined retrospectively by nucleic acid sequence-based amplification (NASBA) assay in 144 stored plasma samples between birth and 48 months in 50 children of whom 40 are alive (age range, 2-13 years). On the basis of clinical and immunologic status children were classified as rapid progressors (RPs), or nonrapid progressors (NRPs). Proviral HIV DNA quantitated by QC-PCR (quantitative competitive polymerase chain reaction) in 24 children was compared with plasma HIV RNA. At age 2 years (p or =750,000 copies/ml. Increasing mortality was observed with increasing plasma HIV RNA levels at ages 3-24 months and baseline VL of infants who died before age 24 months was significantly higher (p = 0.004) than baseline VL of those who survived beyond 24 months. Although baseline VL in infants classified as RPs was higher than that of NRPs, the difference was not statistically significant. Among surviving children 2-13 years of age, the baseline VL obtained at 80%. We conclude that high plasma HIV RNA in infancy is associated with increased mortality.

  9. Single strain isolation method for cell culture-adapted hepatitis C virus by end-point dilution and infection.

    Directory of Open Access Journals (Sweden)

    Nao Sugiyama

    Full Text Available The hepatitis C virus (HCV culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.

  10. Detection and some properties of cowpea mild mottle virus isolated from soybean in Iran.

    Science.gov (United States)

    Tavassoli, M; Shahraeen, N; Ghorbani, S

    2008-12-01

    During 2006-2007 growing seasons, survey were carried to identify a virus disease causing mosaic of soybean in the field in Southern region (Khozestan Province) of Iran. To detect the viral infection, diseased leaf samples showing mild mosaic and leaf malformation were collected from soybean fields in Dezful, located in Khozestan Province. Infected samples were carried to the lab in a proper condition on ice packages. TPIA and DAS-ELISA serological tests were applied to identify the viral agent. To investigate the host-range, several indicator plants were mechanically inoculated under green-house condition. Seed transmission of CPMMV was examined using the seeds obtained from infected plants. The virus isolate was not found to be seed-borne in Clark variety of soybean. Different steps of ultracentrifugation including sucrose density gradient (10-40%) were carried out in order to obtain partial purified virus. On the basis of biological, serological and EM results, CPMMV-Carla virus was identified in the infected soybean samples. This is the first report of CPMMV infection of soybean in Iran.

  11. Cross-protection or enhanced symptom display in greenhouse tomato co-infected with different Pepino mosaic virus isolates

    NARCIS (Netherlands)

    Hanssen, I.M.; Gutiérrez-Aguirre, I.; Paeleman, A.; Goen, K.; Wittemans, L.; Lievens, B.; Vanachter, A.C.R.C.; Ravnikar, M.; Thomma, B.P.H.J.

    2010-01-01

    The potential of three mild Pepino mosaic virus (PepMV) isolates, belonging to the CH2, EU and LP genotypes, to protect a tomato (Solanum lycopersicum) crop against an aggressive challenge isolate of the CH2 genotype was assessed in greenhouse trials and PepMV symptoms were rated at regular time poi

  12. Complete Genome Sequences of Two Chikungunya Viruses Isolated in the Central African Republic in the 1970s and 1980s

    Science.gov (United States)

    Desdouits, Marion; Nakouné, Emmanuel; Gessain, Antoine; Kazanji, Mirdad; Berthet, Nicolas

    2017-01-01

    ABSTRACT Some arboviruses threaten human global health with potentially explosive emergence. Analysis of whole-genome sequences of decades-old isolates might contribute to the understanding of the complex dynamics which drive their circulation and emergence. Here, we report the whole-genome sequences of two Chikungunya viruses isolated in the Central African Republic in the 1970s and 1980s. PMID:28254965

  13. Characterization and Sequencing of a Genotype XII Newcastle Disease Virus Isolated from a Peacock (Pavo cristatus) in Peru

    Science.gov (United States)

    Tataje-Lavanda, Luis; Figueroa, Aling; Segovia, Karen; Gonzalez, Rosa; Cribillero, Giovana; Montalvan, Angela; Fernández-Díaz, Manolo; Icochea, Eliana

    2015-01-01

    Here, we report the first complete sequence and biological characterization of a Newcastle disease virus (NDV) isolated from a peacock in South America (NDV/peacock/Peru/2011). This isolate, classified as genotype XII in class II, highlights the need for increased surveillance of noncommercial avian species. PMID:26227592

  14. Characterization of avian influenza virus isolates submitted to the National Centre for Foreign Animal Disease between 1997 and 2001.

    Science.gov (United States)

    Pasick, J; Weingartl, H; Clavijo, A; Riva, J; Kehler, H; Handel, K; Watkins, E; Hills, K

    2003-01-01

    The National Centre for Foreign Animal Disease (NCFAD) in Winnipeg, Manitoba, is the Canadian Food Inspection Agency's (CFIA) newest high biocontainment laboratory. One of the functions of the NCFAD is to serve as a national reference laboratory for avian influenza. Between 1997 and 2001, 15 avian influenza virus isolates were characterized. These isolates originated from domestic poultry, imported caged birds held in quarantine, and wild birds. Diagnostic specimens were submitted to the NCFAD by CFIA field veterinarians, provincial veterinary diagnostic laboratories, and veterinary colleges. Characterization of isolates included the determination of H and N subtypes: H1, H6, H7, and H10 subtypes were isolated from domestic poultry; H3, H4, and three H13 viruses were isolated from water fowl, and six H3 viruses were isolated from caged birds being held in import quarantine. Selected isolates were characterized with respect to their pathogenic potential by intravenous inoculation of 4-to-6-wk-old chickens. A molecular-based protocol was used to assess the pathogenicity of one H7 isolate. During this period, work was also carried out toward validating our molecular pathotyping protocol for avian influenza viruses with H5 and H7 hemagglutinin subtypes.

  15. Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates

    NARCIS (Netherlands)

    Souza, M.T.; Níckel, O.; Gonsalves, D.

    2005-01-01

    Line 63-1 is a 'Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R, plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA),

  16. Complete Genome Sequence of an American Avian Leukosis Virus Subgroup J Isolate That Causes Hemangiomas and Myeloid Leukosis

    OpenAIRE

    Malhotra, Sanandan; Justice, James; De Lee, Nathan; Li, Yingying; Zavala, Guillermo; Ruano, Miguel; Morgan, Robin; Beemon, Karen

    2015-01-01

    We report the complete genome sequence of avian leukosis virus subgroup J (ALV-J) isolate PDRC-59831, which causes myeloid leukosis and hemangiomas in chickens. This is an American ALV-J isolate, which was found in a 38-week-old broiler breeder chicken on a farm in Georgia in 2007.

  17. Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates

    NARCIS (Netherlands)

    Souza, M.T.; Níckel, O.; Gonsalves, D.

    2005-01-01

    Line 63-1 is a 'Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R, plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA), Thai

  18. Complete Genome Sequence of a Newcastle Disease Virus Isolated from a Rock Dove (Columba livia) in the Russian Federation.

    Science.gov (United States)

    Yurchenko, Kseniya S; Sivay, Mariya V; Glushchenko, Alexandra V; Alkhovsky, Sergey V; Shchetinin, Alexey M; Shchelkanov, Michail Y; Shestopalov, Alexander M

    2015-01-29

    We report here the complete genome sequence of a Newcastle disease virus (NDV) isolate, NDV/Altai/pigeon/770/2011, isolated from a rock dove in the Russian Federation. On the basis of phylogenetic analysis, this strain was clustered into genotype VIb class II.

  19. Cross-protection or enhanced symptom display in greenhouse tomato co-infected with different Pepino mosaic virus isolates

    NARCIS (Netherlands)

    Hanssen, I.M.; Gutiérrez-Aguirre, I.; Paeleman, A.; Goen, K.; Wittemans, L.; Lievens, B.; Vanachter, A.C.R.C.; Ravnikar, M.; Thomma, B.P.H.J.

    2010-01-01

    The potential of three mild Pepino mosaic virus (PepMV) isolates, belonging to the CH2, EU and LP genotypes, to protect a tomato (Solanum lycopersicum) crop against an aggressive challenge isolate of the CH2 genotype was assessed in greenhouse trials and PepMV symptoms were rated at regular time

  20. [Isolation and identification of avian leukosis virus-B from layer chickens infected with avian leukosis virus-J].

    Science.gov (United States)

    Liu, Gong-Zhen; Zhang, Hong-Hai; Liu, Qing; Qiu, Bo; Wang, Feng; Wang, Xiao-Wei; Chen, Hong-Bo; Cheng, Zi-Qiang

    2009-11-01

    Two strains of Avian leukosis virus subgroup B (ALV-B) were isolated for the first time in China Hy-line White on the cultured DF-1 cells which were inoculated tissue samples from by an ELISA assay, a histopathology examination and a PCR-based diagnosis. The results from the ELISA assay indicated that the positive rate of serum antibodies to ALV-B and ALV-J virus were 16.3% (15/92) and 13% (12/92), respectively. The histopathological examination indicated that two types of tumor cells existed at same focus in liver and spleen, which mainly were myelocytoma cells and lymphosarcoma cells. The PCR-based diagnosis were performed as follows: the cellular DNA was extracted from the inoculated DF-1 cells; the specific fragments of 1100 bp and 924 bp were obtained by a PCR system with the diagnostic primers of ALV-B and ALV-J; and the PCR results for ALV-A, MDV and REV were all negative. Then, the amplified fragments of the two ALV-B stains were partially sequenced and shown an identity of 92.8%,94.7% with the prototype strain of ALV-B (RSV Schmidt-ruppin B). The identities of two ALV-J strains with the prototype strain HPRS-103 at 96.9%, 91.5%; The identities of two ALV-J strains with the American prototype strain at 85.9%, 81.5%. Our study had shown that ALV-B was isolated for the first time from the ALV-J infected commercial layer flocks in China. It also indicated that the chance of genetic recombination among various subgroups of ALV was increased.

  1. Isolation of plasma from whole blood using a microfludic chip in a continuous cross-flow

    Institute of Scientific and Technical Information of China (English)

    CHEN Xing; CUI DaFu; ZHANG LuLu

    2009-01-01

    A novel microfluidic chip is developed for crossflow filtration plasma from the whole blood which is carried out in a continuous manner. This microfluidic chip was made of a silicon substrate sealed with a compound cover. The silicon substrate fabricated by micro-electro-mechanical system (MEMS)technology consisted of microposts array, microchannels and reservoirs. Then the silicon substrate was characterized by Scaning Electron Microscopy (SEM). The performance of the microfiuidic chip was valued by the experiments of plasma isolation. During more than one hour of continuous blood infusion through the chip, there were no problems of jamming or clogging, and the plasma selectivity of 97.78% was achieved. Due to the chip's simple structure and control mechanism with a continuous,real time operating manner, this microfluidic chip is easily expected to be integrated into micro total analytical system (uTAS) which will create a microanalysis system for point-of-care diagnostics.

  2. Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition.

    Science.gov (United States)

    Monneron, A; d'Alayer, J

    1978-04-01

    Thymocyte plasma and nuclear membranes obtained by the procedure described in the accompanying paper were analyzed for their biochemical composition. Plasma membranes were very rich in phospholipid, cholesterol, sialic aicd; they did not contain nucleic acids. In comparison, nuclear membranes had a lower phospholipid to protein ratio and contained much less sialic acid and cholesterol. 50% of the cellular cholesterol and of the membrane-bound sialic acid were found in the plasma membranes, 14% in the nuclear membranes. Live cells were labeled with 131I, and the acid-insoluble radioactivity was followed in the subfractions. A good correlation with the distribution and enrichment of plasma membrane market-enzymes was obtained. Label enrichment was about 50-fold in the two lightest of the three plasma membrane fractions. 60% of the label was contained in the plasma membranes, only 4% in the nuclear membranes. Cross-contamination of these two types of membranes was thus negligible. Sodium dodecyl sulfate-gel electrophoresis revealed three different patterns specific for, respectively, plasma membranes, the microsomal-mitochondrial fraction, and nuclear membranes. Each pattern was characterized by a set of proteins and glycoproteins, among which high molecular weight glycoproteins could be considered as marker-proteins of, respectively, 280,000, 260,000, and 230,000 daltons. 131I-labeling of live cells tagged with a very high specific activity three glycoproteins of mol wt 280,000, 200,000, and 135,000 daltons. Nuclear membranes prepared from labeled isolated nuclei had a set of labeled proteins completely different from plasma membranes.

  3. Evolutionary and Ecological Characterization of Mayaro Virus Strains Isolated during an Outbreak, Venezuela, 2010.

    Science.gov (United States)

    Auguste, Albert J; Liria, Jonathan; Forrester, Naomi L; Giambalvo, Dileyvic; Moncada, Maria; Long, Kanya C; Morón, Dulce; de Manzione, Nuris; Tesh, Robert B; Halsey, Eric S; Kochel, Tadeusz J; Hernandez, Rosa; Navarro, Juan-Carlos; Weaver, Scott C

    2015-10-01

    In 2010, an outbreak of febrile illness with arthralgic manifestations was detected at La Estación village, Portuguesa State, Venezuela. The etiologic agent was determined to be Mayaro virus (MAYV), a reemerging South American alphavirus. A total of 77 cases was reported and 19 were confirmed as seropositive. MAYV was isolated from acute-phase serum samples from 6 symptomatic patients. We sequenced 27 complete genomes representing the full spectrum of MAYV genetic diversity, which facilitated detection of a new genotype, designated N. Phylogenetic analysis of genomic sequences indicated that etiologic strains from Venezuela belong to genotype D. Results indicate that MAYV is highly conserved genetically, showing ≈17% nucleotide divergence across all 3 genotypes and 4% among genotype D strains in the most variable genes. Coalescent analyses suggested genotypes D and L diverged ≈150 years ago and genotype diverged N ≈250 years ago. This virus commonly infects persons residing near enzootic transmission foci because of anthropogenic incursions.

  4. Isolation and characterization of a temperature-sensitive mutant of avian myeloblastosis virus.

    Science.gov (United States)

    Moscovici, M G; Moscovici, C

    1983-01-01

    A temperature-sensitive (ts) mutant, GA 907/7, was isolated after mutagen treatment of avian myeloblastosis virus. When bone marrow cells or secondary yolk sac macrophages were infected with GA 907/7, the expression of transformation was greatly reduced at 41 degrees C. The results of temperature-shift experiments suggest that in GA 907/7 the putative v-myb gene product is functional only at 35.5 degrees C. Moreover, when ts-induced transformed cells were shifted to 41 degrees C, a partial morphological conversion to macrophage-like cells was obtained, while the majority of the cells underwent senescence and lysis. No leukemia was obtained when GA 907/7 was injected in 1-day-old chickens. Finally, a continuous cell line releasing genetically stable mutant virus was obtained after transformation of secondary yolk sac cells. Images PMID:6298790

  5. Different pattern of haemagglutinin immunoreactivity of equine influenza virus strains isolated in Poland

    Directory of Open Access Journals (Sweden)

    Kwaśnik Małgorzata

    2015-12-01

    Full Text Available The immunoreactivity of haemagglutinin (HA polypeptides of equine influenza virus was compared among the strains isolated in Poland, using H3 monoclonal antibody. A stronger signal in immunoblot reaction was observed for A/equi/Pulawy/2008 HA polypeptides compared to A/equi/Pulawy/2006, despite the fact that both strains are phylogenetically closely related and belong to Florida clade 2 of American lineage. The strongest signal, observed in the case of A/equi/Pulawy/2008, seemed to be connected with the presence of G135, I213, E379, and/or V530 instead of R135, M213, G379, and I530 present in A/equi/Pulawy/2006 HA sequence. This implies that point mutations within amino acid sequences of HA polypeptides of equine influenza virus may change their immunoreactivity even when they are not located within five basic antigenic sites.

  6. Complete Genomic Sequence of a Chinese Isolate of Duck Hepatitis Virus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The complete genomic sequence of Duck hepatitis virus 1 (DHV-1) ZJ-V isolate was sequenced and determined to be 7 691 nucleotides (nt) in length with a 5'-terminal un-translated region (UTR) of 626 nt and a 3'-terminal UTR of 315 nt (not including the poly(A) tail). One large open reading frame (ORF) was found within the genome (nt 627 to 7 373) coding for a polypeptide of 2 249amino acids. Our data also showed that the poly (A) tail of DHV-1 has at least 22 A's. Sequence comparison revealed significant homology (from 91.9% to 95.7%) between the protein sequences of the virus in the Picornaviridae family, its genome showed some unique characteristics. DHV-1 contains 3copies of the 2A gene and only 1 copy of the 3B gene, and its 3'-NCR is longer than those of other picornaviruses. Phylogenetic analysis to do sequence homology based on the VP1 protein sequences showed that the ZJ-V isolate shares high sequence homology with the reported DHV-1 isolates (from 92.9% to 99.2%), indicating that DHV-1 is genetically stable.

  7. Infectivity and complete nucleotide sequence of cucumber fruit mottle mosaic virus isolate Cm cDNA.

    Science.gov (United States)

    Rhee, Sun-Ju; Hong, Jin-Sung; Lee, Gung Pyo

    2014-07-01

    Three isolates of cucumber fruit mottle mosaic virus (CFMMV) were collected from melon, cucumber, and pumpkin plants in Korea. A full-length cDNA clone of CFMMV-Cm (melon isolate) was produced and evaluated for infectivity after T7 transcription in vitro (pT7CF-Cmflc). The complete CFMMV genome sequence of the infectious clone pT7CF-Cmflc was determined. The genome of CFMMV-Cm consisted of 6,571 nucleotides and shared high nucleotide sequence identity (98.8 %) with the Israel isolate of CFMMV. Based on the infectious clone pT7CF-Cmflc, a CaMV 35S-promoter driven cDNA clone (p35SCF-Cmflc) was subsequently constructed and sequenced. Mechanical inoculation with RNA transcripts of pT7CF-Cmflc and agro-inoculation with p35SCF-Cmflc resulted in systemic infection of cucumber and melon, producing symptoms similar to those produced by CFMMV-Cm. Progeny virus in infected plants was detected by RT-PCR, western blot assay, and transmission electron microscopy.

  8. [Identification of a new subgroup of avian leukosis virus isolated from Chinese indigenous chicken breeds].

    Science.gov (United States)

    Wang, Xin; Zhao, Peng; Cui, Zhi-Zhong

    2012-11-01

    In order to clarify Avian leukosis virus (ALV) characteristics from Chinese native chicken breeds, three ALV JS11C1, JS11C2 and JS11C3 were isolated from Chinese native breed "luhua" by inoculation of DF1 cell culture and detection of p27 antigen. Using PCR amplification of env gene, the amplified gp85 genes were analyzed and compared to all six chicken ALV subgroups reported. The gp85 genes of these three viruses were 1 005bp in length and encoded 335 amino acids, and the gp37 genes were 609bp and encoded 203 amino acids. The homology of gp85 among these three isolated strains was 91.9%-97.0%. Comparing to 18 stains of subgroup A, B, C, D, E published in GenBank, the homology was only in the range of 77.7%-84.6%, significantly lower than the gp85 homology observed within the common chicken subgroups A (88.2%-98.5%), B (91.6%-98.8%), and E (97.9%-99.4%). The gp85 homology compared with subgroup J was only 34.2%-36.5%. These results suggested that three isolated strains from Chinese native breed "luhua" belong to a new subgroup different from all six known subgroups from Chickens, and thus designated as subgroup K.

  9. Complete nucleotide sequence of a Spanish isolate of Parietaria mottle virus infecting tomato.

    Science.gov (United States)

    Galipienso, Luis; Rubio, Luis; López, Luis; Soler, Salvador; Aramburu, José

    2009-10-01

    The genome of a Spanish isolate of Parietaria mottle virus (PMoV) obtained from tomato (strain PMoV-T) was completely sequenced. Protein motifs conserved for RNA viruses were identified: the p1 protein contained a metyltransferase domain in its N-terminal half and a triphosphatase/ helicase domain in its C-terminal half, the p2 protein contained a RNA polymerase domain; the 3a protein contained a RNA-binding domain with α-helix and β-sheet secondary structures. In addition, stem-loop structures with potential capacity of protein interactions were predicted on the untranslated terminal regions. Comparison with the other sequenced PMoV isolate showed nucleotide identities of 93, 90, and 93% for genomic RNAs 1, 2 and 3, respectively, and amino acid identities ranging from 88 to 97% for the different proteins. A cytosine deletion was detected at position 1,366 of RNA 3, involving a start codon for the coat protein (CP) gene different from the other PMoV isolate, resulting in a CP 16 amino acids shorter. Comparison of synonymous and nonsynonymous mutations revealed different selective constraints along the genome.

  10. [Taxonomy of the Sokuluk virus (SOKV) (Flaviviridae, Flavivirus, Entebbe bat virus group) isolated from bats (Vespertilio pipistrellus Schreber, 1774), ticks (Argasidae Koch, 1844), and birds in Kyrgyzstan].

    Science.gov (United States)

    L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Shchetinin, A M; Deriabin, P G; Gitel'man, A K; Samokhvalov, E I; Botikov, A G

    2014-01-01

    Complete genome sequencing of the Sokuluk virus (SOKV) isolated in Kyrgyzstan from bats Vespertilio pipistrellus and their obligatory parasites--Argasidae Koch, 1844, ticks was carried out. SOKV was classified as attributed to the Flaviviridae family, Flavivirus genus. The maximum homology (71% for nucleotide and 79% for amino acid sequences) was detected with respect to the Entebbe bat virus (ENTV). ENTV and SOKV form a group joining to the yellow fever virus (YFV) within the limits of the mosquito flavivirus branch. Close relation of SOKV with bat covers and human housings permits to assume SOKV potentially patogenic to human health.

  11. Characteristics of a Lettuce mosaic virus Isolate Infecting Lettuce in Korea

    Directory of Open Access Journals (Sweden)

    Seungmo Lim

    2014-06-01

    Full Text Available Lettuce mosaic virus (LMV causes disease of plants in the family Asteraceae, especially lettuce crops. LMV isolates have previously been clustered in three main groups, LMV-Yar, LMV-Greek and LMVRoW. The first two groups, LMV-Yar and LMV-Greek, have similar characteristics such as no seed-borne transmission and non-resistance-breaking. The latter one, LMV-RoW, comprising a large percentage of the LMV isolates contains two large subgroups, LMV-Common and LMV-Most. To date, however, no Korean LMV isolate has been classified and characterized. In this study, LMV-Muju, the Korean LMV isolate, was isolated from lettuce showing pale green and mottle symptoms, and its complete genome sequence was determined. Classification method of LMV isolates based on nucleotide sequence divergence of the NIb-CP junction showed that LMV-Muju was categorized as LMV-Common. LMV-Muju was more similar to LMV-O (LMV-Common subgroup than to LMV-E (LMV-RoW group but not LMV-Common subgroup even in the amino acid domains of HC-Pro associated with pathogenicity, and in the CI and VPg regions related to ability to overcome resistance. Taken together, LMV-Muju belongs to the LMV-Common subgroup, and is expected to be a seed-borne, non-resistance-breaking isolate. According to our analysis, all other LMV isolates not previously assigned to a subgroup were also included in the LMV-RoW group.

  12. Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil

    Science.gov (United States)

    da Silva, Kely A. B.; de Castro, Marcia G.; Gerber, Alexandra L.; de Almeida, Luiz G. P.; Lourenço-de-Oliveira, Ricardo; Vasconcelos, Ana Tereza R.

    2016-01-01

    Background Zika virus (ZIKV) is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil. Methodology/Principal Findings Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak. Conclusions/Significance The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological

  13. Genetic diversity of bovine viral diarrhea virus 1: Italian isolates clustered in at least seven subgenotypes.

    Science.gov (United States)

    Giammarioli, Monica; Pellegrini, Claudia; Casciari, Cristina; Rossi, Elisabetta; De Mia, Gian Mario

    2008-11-01

    Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle. Two approved species are recognized, namely BVDV-1 and BVDV-2. To date, only 4 subgenotypes of BVDV-2 are known, and at least 11 distinct subgenotypes have been detected for BVDV-1. In a previous study, the genetic characteristics of 38 field isolates of BVDV from northern Italy were investigated, and all 38 isolates were classified as BVDV-1 and could be assigned to 5 different subgenotypes, namely BVDV-1b, BVDV-1d, BVDV-1e, BVDV-1h, and BVDV-1f. However, the circulation of BVDV-2 has been reported in Italy as well. The aim of the current study was to type 88 BVD viruses found throughout Italy. Genetic study was based on the 5'-UTR, supported by select comparison within the N(pro) coding region. Phylogenetic analysis showed that 5 isolates could be typed as BVDV-2a. The remaining 83 isolates were typed as BVDV-1 and were found to belong to 7 distinct subgenotypes, namely BVDV-1a (n = 8), BVDV-1b (n = 37), BVDV-1d (n = 3), BVDV-1e (n = 22), BVDV-1f (n = 4), BVDV-1g (n = 4), and BVDV-1h (n = 5). The majority of cattle farms in the current study were predominantly infected by BVDV-1b and BVDV-1e isolates, whereas the other BVDV subgenotypes occurred only sporadically. The results also provided evidence for circulation of additional subgenotypes BVDV-1a and BVDV-1g. The occurrence of BVDV-2 was also reconfirmed.

  14. Detection and phylogenetic analysis of peste des petits ruminants virus isolated from outbreaks in Punjab, Pakistan.

    Science.gov (United States)

    Munir, M; Zohari, S; Saeed, A; Khan, Q M; Abubakar, M; LeBlanc, N; Berg, M

    2012-02-01

    Peste des Petits Ruminants (PPR) is an important viral disease of small ruminants and is endemic in Pakistan. In the following study, samples from two outbreaks of PPR in goats have been subjected to laboratory investigations. The Peste des Petits Ruminants virus (PPRV) genome was detected using both conventional and real-time PCR. Genetic characterization of the local PPRV field isolates was conducted by sequencing 322 bp of the fusion (F) gene and 255 bp of the nucleoprotein (N) gene. The phylogenetic tree based on the F gene clustered samples from both outbreaks into lineage 4 along with other Asian isolates, specifically into subcluster 1 along with isolates from Middle East. Analysis of N gene revealed a different pattern. In this case, the Pakistani samples clustered with Chinese, Tajikistani and Iranian isolates, which probably represents the true geographical pattern of virus circulation. This is the first report presenting the phylogenetic tree based on N gene as well as performing a parallel comparison of the trees of F and N gene together from Pakistani isolates. The results of this study shed light on the PPRV population in Pakistan and emphasize the importance of using molecular methods to understand the epidemiology. Such understanding is essential in any efforts to control the number and impact of outbreaks that are occurring in endemic countries such as Pakistan, especially in the current scenario where OIE and FAO are eager to control and subsequently eradicate PPR from the globe, as has been achieved for Rinderpest. © 2011 Blackwell Verlag GmbH.

  15. Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil.

    Directory of Open Access Journals (Sweden)

    Myrna C Bonaldo

    2016-06-01

    Full Text Available Zika virus (ZIKV is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil.Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak.The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological relevance of this finding, regarding the contribution

  16. Genotypic lineages and restriction fragment length polymorphism of canine distemper virus isolates in Thailand.

    Science.gov (United States)

    Radtanakatikanon, Araya; Keawcharoen, Juthatip; Charoenvisal, Na Taya; Poovorawan, Yong; Prompetchara, Eakachai; Yamaguchi, Ryoji; Techangamsuwan, Somporn

    2013-09-27

    Canine distemper virus (CDV) is known to cause multisystemic disease in all families of terrestrial carnivores. Attenuated live vaccines have been used to control CDV in a variety of species for many decades, yet a number of CDV infections in vaccinated dogs are still observed. The aims of this study were to investigate the genetic diversity of CDV lineages based on phosphoprotein (P), hemagglutinin (H) and fusion protein (F) genes and to develop the restriction fragment length polymorphism (RFLP) technique for effective differentiation among individual wild-type and vaccine lineages in Thailand. Four commercial vaccine products, thirteen conjunctival swabs and various tissues from 9 necropsied dogs suspected of having CDV infections were included. Virus isolation was performed using Vero cell expressing canine signaling lymphocyte activation molecules (Vero-DST cells). Reverse-transcription polymerase chain reaction (RT-PCR) on 3 gene regions from the dog derived specimens and the vaccines were carried out, then RFLP analysis upon F-gene amplified fragments was developed. Nucleotide sequence and phylogenetic analysis were compared with other CDV lineages in Genbank. Phylogenetic relationships revealed that CDV field isolates were separated from the vaccine lineage and could be divided into two clusters; one of which belonged to the Asia-1 lineage and another, not related to any previous recognized lineages was proposed as 'Asia-4'. RFLP patterns demonstrating concordance with phylogenetic trees of the distemper virus allowed for differentiation between the Asia-1, Asia-4 and vaccine lineages. Thus, RFLP technique is able to effectively distinguish individual wild-type canine distemper virus from vaccine lineages in Thailand.

  17. Vaccine Potential of Two Previously Uncharacterized African Swine Fever Virus Isolates from Southern Africa and Heterologous Cross Protection of an Avirulent European Isolate.

    Science.gov (United States)

    Souto, R; Mutowembwa, P; van Heerden, J; Fosgate, G T; Heath, L; Vosloo, W

    2016-04-01

    African swine fever (ASF) is a mostly fatal viral infection of domestic pigs for which there is no vaccine available. The disease is endemic to most of sub-Saharan Africa, causes severe losses and threatens food security in large parts of the continent. Naturally occurring attenuated ASF viruses have been tested as vaccine candidates, but protection was variable depending on the challenge virus. In this study, the virulence of two African isolates, one from a tick vector and the other from an indigenous pig, was determined in domestic pigs to identify a potential vaccine strain for southern Africa. Neither isolate was suitable as the tick isolate was moderately virulent and the indigenous pig virus was highly virulent. The latter was subsequently used as heterologous challenge in pigs first vaccinated with a naturally attenuated isolate previously isolated in Portugal. Although a statistically significant reduction in death rate and virus load was observed compared with unvaccinated pigs post-challenge, all pigs succumbed to infection and died.

  18. Molecular phylogenetic and positive selection analysis of Japanese encephalitis virus strains isolated from pigs in China.

    Science.gov (United States)

    Liu, Wen-Jun; Zhu, Ming; Pei, Jing-Jing; Dong, Xiao-Ying; Liu, Wei; Zhao, Ming-Qiu; Wang, Jia-Ying; Gou, Hong-Chao; Luo, Yong-Wen; Chen, Jin-Ding

    2013-12-26

    Japanese encephalitis virus (JEV) is one of the most important virus which causes encephalitis. This disease is most prevalent in the south, southeast and the east region of Asia. In this study, two JEV strains, named JEV/SW/GD/01/2009 and JEV/SW/GZ/09/2004, were isolated from aborted fetuses and seminal fluid of pigs in China. To determine the characteristic of these virus isolates, the virulence of two newly JEV isolates was investigated, the result evidenced that the JEV/SW/GD/01/2009 did not kill mice, while the JEV/SW/GZ/09/2004 displayed neurovirulence with 0.925log10 p.f.u./LD50. Additionally, the full genome sequences of JEV were determined and compared with other known JEV strains. Results demonstrated that the genome of two JEV isolates was 10,976 nucleotides (nt) in length. As compared to the Chinese vaccine strain SA14-14-2, the JEV/SW/GD/01/2009 and the JEV/SW/GZ/09/2004 showed 99.7% and 97.5% identity at the nucleotide level, 99.6% and 96.7% identity at the amino acid level, respectively. Phylogenetic analysis, based on the full-length genome revealed that two JEV isolates were all clustered into genotype III compared to the reference strains. Furthermore, selection analyses revealed that dominant selective pressure acting on the JEV genome was purifying selection. Four sites under positive selection were identified: codon 521 (amino acid E-227), 2296 (amino acid NS4b-24), 3048 (amino acid NS5-521) and 3055 (amino acid NS5-528). Amino acid E-227 was proved to be related to neurovirulence. Taken together, the molecular epidemiology and functional of positively selected amino acid sites of two newly JEV isolates were fully understood, which might be helpful to predict possible changes in virulence. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. The first evidence of subgroup IB isolates of Cucumber mosaic virus in Ukraine

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    Shevchenko T. P.

    2015-02-01

    Full Text Available Aim. In current work, we proceeded with the strain attribution of Ukrainian isolates CMV based on the phylogenetic analysis of the partial sequences of the coat protein gene. Methods. ELISA, RT-PCR, DNA sequencing and phylogenetic analysis. Results. Cucumber mosaic virus (CMV is widespread among the variety of crops from the Cucurbitaceae and Solanaceae families in Ukraine. The symptomatic samples from different regions of Ukraine were collected and tested for the presence of CMV. The coat protein (CP gene of two isolates was amplified and sequenced. The partial nucleotide sequences of CP gene were determined and compared to those of other CMV strains belonging to the IA, IB and II subgroups. Comparison of the nucleotide sequences of Ukrainian isolates showed their similar identity percentages and close relationships with the subgroup IB strains from other countries. The highest nucleotide homology was shared with the strains ABI (Korea and SD (China. Conclusions. Based on the highest identities of the coat protein gene sequences and close phylogenetic relationships with the subgroup IB members of CMV, the Ukrainian isolates under study were identified as belonging to the subgroup IB. Our findings show for the first time an occurrence of the IB subgroup isolates of CMV in Ukraine.

  20. Association between IgM anti-herpes simplex virus and plasma amyloid-beta levels.

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    Catherine Féart

    Full Text Available OBJECTIVE: Herpes simplex virus (HSV reactivation has been identified as a possible risk factor for Alzheimer's disease (AD and plasma amyloid-beta (Aβ levels might be considered as possible biomarkers of the risk of AD. The aim of our study was to investigate the association between anti-HSV antibodies and plasma Aβ levels. METHODS: The study sample consisted of 1222 subjects (73.9 y in mean from the Three-City cohort. IgM and IgG anti-HSV antibodies were quantified using an ELISA kit, and plasma levels of Aβ(1-40 and Aβ(1-42 were measured using an xMAP-based assay technology. Cross-sectional analyses of the associations between anti-HSV antibodies and plasma Aβ levels were performed by multi-linear regression. RESULTS: After adjustment for study center, age, sex, education, and apolipoprotein E-e4 polymorphism, plasma Aβ(1-42 and Aβ(1-40 levels were specifically inversely associated with anti-HSV IgM levels (β = -20.7, P=0.001 and β = -92.4, P=0.007, respectively. In a sub-sample with information on CLU- and CR1-linked SNPs genotyping (n=754, additional adjustment for CR1 or CLU markers did not modify these associations (adjustment for CR1 rs6656401, β = -25.6, P=0.002 for Aβ(1-42 and β = -132.7, P=0.002 for Aβ(1-40; adjustment for CLU rs2279590, β = -25.6, P=0.002 for Aβ(1-42 and β = -134.8, P=0.002 for Aβ(1-40. No association between the plasma Aβ(1-42-to-Aβ(1-40 ratio and anti-HSV IgM or IgG were evidenced. CONCLUSION: High anti-HSV IgM levels, markers of HSV reactivation, are associated with lower plasma Aβ(1-40 and Aβ(1-42 levels, which suggest a possible involvement of the virus in the alterations of the APP processing and potentially in the pathogenesis of AD in human.

  1. Genetic analysis of ORF5 porcine reproductive and respiratory syndrome virus isolated in Vietnam.

    Science.gov (United States)

    Thuy, Nguyen Thi Dieu; Thu, Nguyen Thi Dieu; Son, Nguyen Giang; Ha, Le Thi Thu; Hung, Vo Khanh; Nguyen, Nguyen Thao; Khoa, Do Vo Anh

    2013-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens because it is highly infectious and causes economic losses due to decreased pig productivity. In this study, the 603 bp complete major envelope protein encoding gene (ORF5) of 32 field PRRSV isolates from Vietnam collected during 2008-2012 were sequenced and analyzed. Multiple nucleotide (nt) and deduced amino acid (aa) alignments of ORF5 were performed on the 32 isolates: the representative strains (European and North American genotypes), Chinese strains available in GenBank and vaccine strains licensed for use in Vietnam. The results showed 94.8-100.0% nt identity and 94.0-100% aa similarity among the 32 isolates. These isolates shared similarities with the prototype of the North American PRRSV strain (VR-2332; nt 87.8-89.3%, aa 87.5-90.0%), and Lelystat virus, the prototype of the European PRRSV strain (LV; nt 61.1-61.9%, aa 55.1-57.0%). There was greater similarity with QN07 (nt 96.5-98.5%, aa 96.0-99.0%) from the 2007 PRRS outbreak in QuangNam Province, CH-1a (nt 93.2-95.1%, 91.5-93.5%) isolated in China in 1995 and JXA1 (nt 96.5-98.6%, aa 95.0-98.0%), the highly pathogenic strain from China isolated in 2006. The Vietnamese isolates were more similar to JXA1-R (nt 96.5-98.6%, aa 95.0-98.0%), the strain used in Chinese vaccines, than to Ingelvac MLV/BSL-PS (nt 87.2-89.0%, aa 86.0-89.0%). Phylogenetic analysis showed that the 32 isolates were of the North American genotype and classified into sub-lineage 8.7. This sub-lineage contains highly pathogenic Chinese PRRSV strains. This study documents genetic variation in circulating PRRSV strains and could assist more effective use of PRRS vaccines in Vietnam.

  2. Characterization of a new dog isolate of canine distemper virus from China.

    Science.gov (United States)

    Qiao, J; Meng, Q; Chen, C; Xia, X; Cai, X; Ren, Y; Zhang, H

    2011-01-01

    Canine distemper virus (CDV) is a highly contagious pathogen of dogs. Vaccination is an effective way to protect dogs from CDV infection, but occasionally fails. In the present study, a wild type (wt) CDV, named XJ2, was isolated from a dead vaccinated dog. The hemagglutinin (H) gene of the XJ2 was amplified and analyzed for the molecular characteristics including N-glycosylation sites, phylogenesis, hydrophobicity and epitopes. The data indicated that XJ2 was a genetic variant strain of CDV. CDV-sero-negative dogs were inoculated intranasally with XJ2, developed severe clinical symptoms and died, suggesting high virulence.

  3. Evidence of different phylogenetic origins of two mexican sugarcane mosaic virus (scmv) isolates

    OpenAIRE

    Chaves Bedoya, Giovanni; Ortiz Rojas, Luz Yineth

    2012-01-01

    The molecular analysis of the Sugarcane mosaic virus (SCMV) for coat protein cistron reported in the public GenBank database, revealed the presence of 45 additional nucleotides coding for 15 amino acids in the N-terminal region of the coat protein sequence of the mexican isolate GU474635. BLAST analysis indicates this particular feature is also present in the coat protein sequence identified with the accession number D00949 reported in the USA in 1991. Phylogenetic analysis of 185 SCMV coat p...

  4. Hepatitis C virus infection influences the S-methadone metabolite plasma concentration.

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    Shiow-Ling Wu

    Full Text Available BACKGROUND AND OBJECTIVES: Heroin-dependent patients typically contract hepatitis C virus (HCV at a disproportionately high level due to needle exchange. The liver is the primary target organ of HCV infection and also the main organ responsible for drug metabolism. Methadone maintenance treatment (MMT is a major treatment regimen for opioid dependence. HCV infection may affect methadone metabolism but this has rarely been studied. In our current study, we aimed to test the hypothesis that HCV may influence the methadone dosage and its plasma metabolite concentrations in a MMT cohort from Taiwan. METHODS: A total of 366 MMT patients were recruited. The levels of plasma hepatitis B virus (HBV, HCV, human immunodeficiency virus (HIV antibodies (Ab, liver aspartate aminotransferase (AST and alanine aminotransferase (ALT, as well as methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP were measured along with the urine morphine concentration and amphetamine screening. RESULTS: Of the 352 subjects in our cohort with HCV test records, 95% were found to be positive for plasma anti-HCV antibody. The liver functional parameters of AST (Wilcoxon Rank-Sum test, P = 0.02 and ALT (Wilcoxon Rank-Sum test, P = 0.04, the plasma methadone concentrations (Wilcoxon Rank-Sum test, P = 0.043 and the R-enantiomer of methadone concentrations (Wilcoxon Rank-Sum test, P = 0.032 were significantly higher in the HCV antibody-positive subjects than in the HCV antibody-negative patients, but not the S-EDDP/methadone dose ratio. The HCV levels correlated with the methadone dose (β= 14.65 and 14.13; P = 0.029 and 0.03 and the S-EDDP/methadone dose ratio (β= -0.41 and -0.40; P = 0.00084 and 0.002 in both univariate and multivariate regression analyses. CONCLUSIONS: We conclude that HCV may influence the methadone dose and plasma S-EDDP/methadone dose ratio in MMT patients in this preliminary study.

  5. Mallard or chicken? Comparing the isolation of avian influenza A viruses in embryonated Mallard and chicken eggs

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    Josef D. Järhult

    2015-09-01

    Full Text Available Background: To date, the most efficient and robust method for isolating avian influenza A viruses (IAVs is using embryonated chicken eggs (ECEs. It is known that low-pathogenic avian IAVs undergo rapid genetic changes when introduced to poultry holdings, but the factors driving mutagenesis are not well understood. Despite this, there is limited data on the effects of the standard method of virus isolation of avian-derived viruses, that is, whether isolation in ECEs causes adaptive changes in avian IAVs. Eggs from a homologous species could potentially offer an isolation vessel less prone to induce adaptive changes. Methods: We performed eight serial passages of two avian IAVs isolated from fecal samples of wild Mallards in both ECEs and embryonated Mallard eggs, and hemagglutination assay titers and hemagglutinin sequences were compared. Results: There was no obvious difference in titers between ECEs and embryonated Mallard eggs. Sequence analyses of the isolates showed no apparent difference in the rate of introduction of amino acid substitutions in the hemagglutinin gene (three substitutions in total in embryonated Mallard eggs and two substitutions in ECEs. Conclusion: Embryonated Mallard eggs seem to be good isolation vessels for avian IAVs but carry some practical problems such as limited availability and short egg-laying season of Mallards. Our study finds isolation of Mallard-derived avian IAVs in ECEs non-inferior to isolation in embryonated Mallard eggs, but more research in the area may be warranted as this is a small-scale study.

  6. Genome Sequence Analysis of In Vitro and In Vivo Phenotypes of Bunyamwera and Ngari Virus Isolates from Northern Kenya

    Science.gov (United States)

    Odhiambo, Collins; Venter, Marietjie; Limbaso, Konongoi; Swanepoel, Robert; Sang, Rosemary

    2014-01-01

    Biological phenotypes of tri-segmented arboviruses display characteristics that map to mutation/s in the S, M or L segments of the genome. Plaque variants have been characterized for other viruses displaying varied phenotypes including attenuation in growth and/or pathogenesis. In order to characterize variants of Bunyamwera and Ngari viruses, we isolated individual plaque size variants; small plaque (SP) and large plaque (LP) and determined in vitro growth properties and in vivo pathogenesis in suckling mice. We performed gene sequencing to identify mutations that may be responsible for the observed phenotype. The LP generally replicated faster than the SP and the difference in growth rate was more pronounced in Bunyamwera virus isolates. Ngari virus isolates were more conserved with few point mutations compared to Bunyamwera virus isolates which displayed mutations in all three genome segments but majority were silent mutations. Contrary to expectation, the SP of Bunyamwera virus killed suckling mice significantly earlier than the LP. The LP attenuation may probably be due to a non-synonymous substitution (T858I) that mapped within the active site of the L protein. In this study, we identify natural mutations whose exact role in growth and pathogenesis need to be determined through site directed mutagenesis studies. PMID:25153316

  7. Genome sequence analysis of in vitro and in vivo phenotypes of Bunyamwera and Ngari virus isolates from northern Kenya.

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    Collins Odhiambo

    Full Text Available Biological phenotypes of tri-segmented arboviruses display characteristics that map to mutation/s in the S, M or L segments of the genome. Plaque variants have been characterized for other viruses displaying varied phenotypes including attenuation in growth and/or pathogenesis. In order to characterize variants of Bunyamwera and Ngari viruses, we isolated individual plaque size variants; small plaque (SP and large plaque (LP and determined in vitro growth properties and in vivo pathogenesis in suckling mice. We performed gene sequencing to identify mutations that may be responsible for the observed phenotype. The LP generally replicated faster than the SP and the difference in growth rate was more pronounced in Bunyamwera virus isolates. Ngari virus isolates were more conserved with few point mutations compared to Bunyamwera virus isolates which displayed mutations in all three genome segments but majority were silent mutations. Contrary to expectation, the SP of Bunyamwera virus killed suckling mice significantly earlier than the LP. The LP attenuation may probably be due to a non-synonymous substitution (T858I that mapped within the active site of the L protein. In this study, we identify natural mutations whose exact role in growth and pathogenesis need to be determined through site directed mutagenesis studies.

  8. Analysis of the complete sequences of two biologically distinct Zucchini yellow mosaic virus isolates further evidences the involvement of a single amino acid in the virus pathogenicity.

    Science.gov (United States)

    Nováková, S; Svoboda, J; Glasa, M

    2014-01-01

    The complete genome sequences of two Slovak Zucchini yellow mosaic virus isolates (ZYMV-H and ZYMV-SE04T) were determined. These isolates differ significantly in their pathogenicity, producing either severe or very mild symptoms on susceptible cucurbit hosts. The viral genome of both isolates consisted of 9593 nucleotides in size, and contained an open reading frame encoding a single polyprotein of 3080 amino acids. Despite their different biological properties, an extremely high nucleotide identity could be noted (99.8%), resulting in differences of only 5 aa, located in the HC-Pro, P3, and NIb, respectively. In silico analysis including 5 additional fully-sequenced and phylogenetically closely-related isolates known to induce different symptoms in cucurbits was performed. This suggested that the key single mutation responsible for virus pathogenicity is likely located in the N-terminal part of P3, adjacent to the PIPO.

  9. Hepatitis B virus subgenotype A1: evolutionary relationships between Brazilian, African and Asian isolates.

    Science.gov (United States)

    Lago, Bárbara V; Mello, Francisco C; Kramvis, Anna; Niel, Christian; Gomes, Selma A

    2014-01-01

    Brazil is a country of low hepatitis B virus (HBV) endemicity in which the genotype A of HBV (HBV/A) is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase), and one (Phe17) in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an 'Asian-American' clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300-400,000 captives forcibly removed from southeast Africa at the middle of the 19th century.

  10. Hepatitis B virus subgenotype A1: evolutionary relationships between Brazilian, African and Asian isolates.

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    Bárbara V Lago

    Full Text Available Brazil is a country of low hepatitis B virus (HBV endemicity in which the genotype A of HBV (HBV/A is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase, and one (Phe17 in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an 'Asian-American' clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300-400,000 captives forcibly removed from southeast Africa at the middle of the 19th century.

  11. In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection.

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    Judith Dalmau

    Full Text Available BACKGROUND: The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies. METHODOLOGY/PRINCIPAL FINDINGS: We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA. CONCLUSION: This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define

  12. [Detection and description of avian hepatitis E virus isolated in China--a review].

    Science.gov (United States)

    Zhao, Qin; Sun, Yani; Zhou, Enmin

    2012-03-04

    Avian hepatitis E virus (HEV), a member of Hepeviridae family, is genetically and antigenically related with human and swine HEV in the family. Since its discovery, avian HEV infection has been investigated in many countries from serology and molecular epidemiology studies. At present, five complete or near complete genomes of avian HEV isolates were reported in GenBank and were divided into three genotypes. The complete genome of avian HEV contains 3 ORFs of which ORF2 gene encodes capsid protein containing the primary epitopes of viral particles and is target gene for serodiagnostic antigen and vaccine candidate. Because avian HEV infection has significant impact on the poultry industry and potential zoonotic transmission, the researches on avian HEV have been given much attention. We here give a broad review of the research update on the aetiology, pathogenesis and the antigenicity of capsid protein of avian HEV based on identification of Chinese avian HEV isolate.

  13. Isolation and phylogenetic analysis of canine distemper virus among domestic dogs in Vietnam

    Science.gov (United States)

    NGUYEN, Dung Van; SUZUKI, Junko; MINAMI, Shohei; YONEMITSU, Kenzo; NAGATA, Nao; KUWATA, Ryusei; SHIMODA, Hiroshi; VU, Chien Kim; TRUONG, Thuy Quoc; MAEDA, Ken

    2016-01-01

    Canine distemper virus (CDV) is one of the most serious pathogens found in many species of carnivores, including domestic dogs. In this study, hemagglutinin (H) genes were detected in five domestic Vietnamese dogs with diarrhea, and two CDVs were successfully isolated from dogs positive for H genes. The complete genome of one isolate, CDV/dog/HCM/33/140816, was determined. Phylogenetic analysis showed that all Vietnamese CDVs belonged to the Asia-1 genotype. In addition, the H proteins of Vietnamese CDV strains were the most homologous to those of Chinese CDVs (98.4% to 99.3% identity). These results indicated that the Asia-1 genotype of CDV was the predominant genotype circulating among the domestic dog population in Vietnam and that transboundary transmission of CDV has occurred between Vietnam and China. PMID:27746406

  14. Cloning and Identification of S Gene from Chinese Isolate TH-98 of Transmissible Gastroenteritis Virus

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle (ST)cells. Two pairs of primers were designed according to the published sequence with Oligo 4. 1 and DNasis softwares. The products of RT-PCR were named Sa and Sb ,of 2. 3kb and 2. 1kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I sites of the same pUC18 plasmid.The recombinant designated pUC- S was verified and analyzed by corresponding restriction endonuclease (RE)and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid ,which was identified as S gene from Chinese isolate of TGEV.

  15. Sequencing and Phylogenetic Analysis of Potato virus Y Liaoning Isolate in China

    Institute of Scientific and Technical Information of China (English)

    WANG Fang; GAO Zheng-liang; AN Meng-nan; ZHOU Ben-guo; and WU Yuan-hua

    2013-01-01

    Complete genome sequence of Potato virus Y Liaoning isolate (PVY-LN) causing tobacco vein necrosis symptoms were isolated from Liaoning Province in China. Genome sequences of PVY-LN was 9 714 nucleotides in length, excluding the 3′-terminal poly (A) tail. PVY-LN encodes a single long open reading frame (ORF) of polyprotein that is predicted to be cleaved into ten mature proteins by three viral proteases. No recombination can be predicted in PVY-LN sequences compared with that of the other PVY strains using Recombination Detection Programe v. 4.16 (RDP4). Complete genome sequence comparison and phylogenetic analysis indicated that PVY-LN is closely related to PVY necrosis strain (PVYN).

  16. Serological and pathological studies of Newcastle disease viruses isolated from caged birds from Southeast Asia.

    Science.gov (United States)

    Kawamura, M; Nerome, K; Kodama, H; Izawa, H; Mikami, T

    1987-01-01

    Eleven isolates of Newcastle disease virus (NDV), from caged birds imported from or captured in Southeast Asia in 1979-80, were antigenically divided into five distinct groups. Most of them were distinguishable from more classical NDVs (vaccine B1 strain and Miyadera strain) on the basis of their reactivity to eight monoclonal antibodies against the HN molecule of NDV in hemagglutination-inhibition tests. However, when three representative isolates were evaluated for their biological properties and pathogenicity against 1-day-old chickens, all three were found to be velogenic types that could induce serious symptoms of Newcastle disease and which eventually killed all of the chickens, regardless of the route of infection. There was not any significant correlation between their reactivity patterns with the monoclonal antibodies and their virulence.

  17. Isolation of EEE virus from Ochlerotatus taeniorhynchus and Culiseta melanura in coastal South Carolina.

    Science.gov (United States)

    Ortiz, Diana I; Wozniak, Arthur; Tolson, Marsha W; Turner, Pearl E; Vaughan, David R

    2003-03-01

    A 1-year arbovirus study was conducted at The Wedge Plantation located in coastal South Carolina to determine the occurrence and level of arbovirus activity in mosquito species inhabiting the site. Mosquito species composition and temporal abundance were also determined. A total of 45,051 mosquitoes representing 27 species in 9 genera was collected and identified during 130 trap-nights between August, 1997, and July, 1998. The most abundant species was Culex salinarius (n = 20,954) followed by Ochlerotatus taeniorhynchus (n = 12,185). Eastern equine encephalomyelitis virus (EEE) was isolated from 2 pools collected in August, 1997; one pool of Oc. taeniorhynchus (minimum infection rate [MIR] = 0.6/1,000) and a second of Culiseta melanura (MIR = 3.8/1,000). This report represents the first record of an EEE isolation from Oc. taeniorhynchus and Cs. melanura in South Carolina.

  18. A first note on Japanese encephalitis virus isolation from Culex quinquefasciatus Say in Northern West Bengal.

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    V. Thenmozhi

    2014-03-01

    Full Text Available Japanese encephalitis (JE is endemic in many parts of India including the state of West Bengal. In West Bengal, the first major outbreaks of JE occurred in the districts of Bankura and Burdwan in 1973. The Culex vishnui subgroup of mosquitoes has been implicated as major vectors of JE. However in India, JE virus (JEV has been isolated from 16 species of mosquitoes. During September 2011, JE cases were reported from four districts -Jalpaiguri, Darjeeling, Dinajpur and Cooch Behar of West Bengal (North. Adult mosquitoes were collected, identified, pooled and screened for JEV using antigen capture ELISA. Out of 279 mosquito pools tested, one pool of Cx. pseudovishnui and three pools of Cx. quinquefasciatus were found positive for JEV. The ELISA positive pools were further confirmed as JEV by insect bioassay (Toxo-IFA. Two pools of Cx. quinquefasciatus were confirmed as JEV. This represents the first report of JEV isolation from Cx. quinquefasciatus in West Bengal.

  19. Diversity of beet curly top Iran virus isolated from different hosts in Iran.

    Science.gov (United States)

    Gharouni Kardani, Sara; Heydarnejad, Jahangir; Zakiaghl, Mohammad; Mehrvar, Mohsen; Kraberger, Simona; Varsani, Arvind

    2013-06-01

    Beet curly top Iran virus (BCTIV) is a major pathogen of sugar beet in Iran. In order to study diversity of BCTIV, we sampled 68 plants in Iran during the summer of 2010 with curly top disease symptoms on beans (Phaseolus vulgaris), cowpeas (Vigna unguiculata), tomatoes (Solanum lycopersicum L.), sea beets (Beta vulgaris subsp. maritima), and sugar beets (Beta vulgaris). Plant samples showing leaf curling, yellowing, and/or swelling of veins on the lower leaf surfaces were collected from various fields in Khorasan Razavi, Northern Khorasan (north-eastern Iran), East Azarbayejan, West Azarbayejan (north-western Iran), and Fars (southern Iran) provinces. Using rolling circle amplification coupled with restriction digests, cloning, and Sanger sequencing, we determined the genomes of nine new BCTIV isolates from bean, cowpea, tomato, sea beet, and sugar beet in Iran. Our analysis reveals ~11 % diversity amongst BCTIV isolates and we detect evidence of recombination within these genomes.

  20. Molecular characterization of double-stranded RNA virus in Trichomonas vaginalis Egyptian isolates and its association with pathogenicity.

    Science.gov (United States)

    El-Gayar, Eman K; Mokhtar, Amira B; Hassan, Wael A

    2016-10-01

    Trichomoniasis is a common human sexually transmitted infection caused by Trichomonas vaginalis. The parasite can be infected with double-stranded RNA viruses (TVV). This viral infection may have important implications on trichomonal virulence and disease pathogenesis. This study aimed to determine the prevalence of T. vaginalis virus among isolates obtained from infected (symptomatic and asymptomatic) women in Ismailia City, Egypt, and to correlate the virus-infected isolates with the clinical manifestations of patients. In addition, the pathogenicity of TVV infected isolates on mice was also evaluated. T. vaginalis isolates were obtained from symptomatic and asymptomatic female patients followed by axenic cultivation in Diamond's TYM medium. The presence of T. vaginalis virus was determined from total extraction of nucleic acids (DNA-RNA) followed by reverse transcriptase-PCR. Representative samples were inoculated intraperitoneally in female albino/BALB mice to assess the pathogenicity of different isolates. A total of 110 women were examined; 40 (36.3 %) samples were positive for T. vaginalis infection. Of these 40 isolates, 8 (20 %) were infected by TVV. Five isolates contained TVV-2 virus species, and the remaining three isolates were infected withTVV-4 variant. A significant association was found between the presence of TVV and particular clinical manifestations of trichomoniasis. Experimental mice infection showed varying degrees of pathogenicity. This is the first report on T. vaginalis infection by TVV in Egypt. The strong association detected between TVV and particular clinical features of trichomoniasis and also the degree of pathogenicity in experimentally infected mice may indicate a possible clinical significance of TVV infection of T. vaginalis isolates.

  1. Partial biological and molecular characterization of a Cucumber mosaic virus isolate naturally infecting Cucumis melo in Iran.

    Science.gov (United States)

    Rasoulpour, Rasoul; Afsharifar, Alireza; Izadpanah, Keramat

    2016-06-01

    Melon seedlings showing systemic chlorotic spots and mosaic symptoms were collected in central part of Iran, and a virus was isolated from diseased plants by mechanical inoculation. The virus systemically infected the most inoculated test plants by inducing mosaic symptoms, while, in the members of Fabaceae family and Chenopodium quinoa induced local lesions. Agar gel diffusion test using a polyclonal antiserum against a squash Cucumber mosaic virus (CMV) isolate showed the presence of CMV in the mechanically inoculated plants (designated CMV-Me). The virus was purified by polyethylene glycol precipitation and differential centrifugation. A polyclonal antiserum was produced against the virus that reacted specifically with virus antigen in ELISA and agar gel diffusion tests. The virus was molecularly characterized by PCR amplification of the full length of the coat protein gene using cucumovirus genus specific primer pair CPTALL-3/CPTALL-5 and sequence analysis of the resulting product. No RNA satellite was detected using the primer pair CMVsat3H/sat5T7P. Phylogenetic analysis based on the coat protein amino acid sequences showed that CMV-Me belongs to Subgroup IB. These results may be helpful in melon breeding programs, focusing on plant resistance to plant viruses including CMV.

  2. High frequency of hepatitis E virus infection in swine from South Brazil and close similarity to human HEV isolates.

    Science.gov (United States)

    Passos-Castilho, Ana Maria; Granato, Celso Francisco Hernandes

    2017-01-03

    Hepatitis E virus is responsible for acute and chronic liver infections worldwide. Swine hepatitis E virus has been isolated in Brazil, and a probable zoonotic transmission has been described, although data are still scarce. The aim of this study was to investigate the frequency of hepatitis E virus infection in pigs from a small-scale farm in the rural area of Paraná State, South Brazil. Fecal samples were collected from 170 pigs and screened for hepatitis E virus RNA using a duplex real-time RT-PCR targeting a highly conserved 70nt long sequence within overlapping parts of ORF2 and ORF3 as well as a 113nt sequence of ORF2. Positive samples with high viral loads were subjected to direct sequencing and phylogenetic analysis. hepatitis E virus RNA was detected in 34 (20.0%) of the 170 pigs following positive results in at least one set of screening real-time RT-PCR primers and probes. The swine hepatitis E virus strains clustered with the genotype hepatitis E virus-3b reference sequences in the phylogenetic analysis and showed close similarity to human hepatitis E virus isolates previously reported in Brazil.

  3. Platelet aggregation responses and virus isolation from platelets in calves experimentally infected with type I or type II bovine viral diarrhea virus.

    Science.gov (United States)

    Walz, P H; Bell, T G; Grooms, D L; Kaiser, L; Maes, R K; Baker, J C

    2001-10-01

    Altered platelet function has been reported in calves experimentally infected with type II bovine viral diarrhea virus (BVDV). The purpose of the present study was to further evaluate the ability of BVDV isolates to alter platelet function and to examine for the presence of a virus-platelet interaction during BVDV infection. Colostrum-deprived Holstein calves were obtained immediately after birth, housed in isolation, and assigned to 1 of 4 groups (1 control and 3 treatment groups). Control calves (n = 4) were sham inoculated, while calves in the infected groups (n = 4 for each group) were inoculated by intranasal instillation with 10(7) TCID50 of either BVDV 890 (type II), BVDV 7937 (type II), or BVDV TGAN (type I). Whole blood was collected prior to inoculation (day 0) and on days 4, 6, 8, 10, and 12 after inoculation for platelet function testing by optical aggregometry by using adenosine diphosphate and platelet activating factor. The maximum percentage aggregation and the slope of the aggregation curve decreased over time in BVDV-infected calves; however, statistically significant differences (Freidman repeated measures ANOVA on ranks, P infected with the type II BVDV isolates. Bovine viral diarrhea virus was not isolated from control calves, but was isolated from all calves infected with both type II BVDV isolates from days 4 through 12 after inoculation. In calves infected with type I BVDV, virus was isolated from 1 of 4 calves on days 4 and 12 after inoculation and from all calves on days 6 and 8 after inoculation. Altered platelet function was observed in calves infected with both type II BVDV isolates, but was not observed in calves infected with type I BVDV. Altered platelet function may be important as a difference in virulence between type I and type II BVDV infection.

  4. Molecular Characterization of Banana streak virus Isolate from Musa Acuminata in China

    Institute of Scientific and Technical Information of China (English)

    Jun Zhuang; Jian-hua Wang; Xin Zhang; Zhi-xinLiu

    2011-01-01

    Banana streak virus (BSV),a member of genus Badnavirus,is a causal agent of banana streak disease throughout the world.The genetic diversity of BSVs from different regions of banana plantations has previously been investigated,but there are relatively few reports of the genetic characteristic of episomal (non-integrated)BSV genomes isolated from China.Here,the complete genome,a total of 7722bp (GenBank accession number DQ092436),of an isolate of Banana streak virus (BSV) on cultivar Cavendish (BSAcYNV) in Yunnan,China was determined.The genome organises in the typical manner of badnaviruses.The intergenic region of genomic DNA contains a large stem-loop,which may contribute to the ribosome shift into the following open reading frames (ORFs).The coding region of BSAcYNV consists of three overlapping ORFs,ORF 1 with a non-AUG start eodon and ORF2 encoding two small proteins are individually involved in viral movement and ORF3 encodes a polyprotein.Besides the complete genome,a defective genome lacking the whole RNA leader region and a majority of ORF1 and which encompasses 6525bp was also isolated and sequenced from this BSV DNA reservoir in infected banana plants.Sequence analyses showed that BSAcYNV has closest similarity in terms of genome organization and the coding assignments with an BSV isolate from Vientam (BSAcVNV).The corresponding coding regions shared identities of 88% and ~95% at nucleotide and amino acid levels,respectively.Phylogenetic analysis also indicated BSAcYNV shared the closest geographical evolutionary relationship to BSAcVNV among sequenced banana streak badnaviruses.

  5. Hepatitis B virus (HBV) variants fluctuate in paired plasma and peripheral blood mononuclear cells among patient cohorts during different chronic hepatitis B (CHB) disease phases.

    Science.gov (United States)

    Coffin, C S; Osiowy, C; Gao, S; Nishikawa, S; van der Meer, F; van Marle, G

    2015-04-01

    Hepatitis B virus is classically considered a hepatotropic virus but also infects peripheral blood mononuclear cells. Chronic hepatitis B has different disease phases modulated by host immunity. We compared HBV variability, drug resistance and immune escape mutations in the overlapping HBV polymerase/surface gene in plasma and peripheral blood mononuclear cells in different disease phases. Plasma and peripheral blood mononuclear cells were isolated from 22 treatment naïve patient cohorts (five inactive, six immune-active, nine HBeAg negative and two immune-tolerant). HBV was genotyped via line probe assay, hepatitis B surface antigen titres were determined by an in-house immunoassay, and HBV DNA was quantified by kinetic PCR. The HBV polymerase/surface region, including full genome in some, was PCR-amplified and cloned, and ~20 clones/sample were sequenced. The sequences were subjected to various mutational and phylogenetic analyses. Clonal sequencing showed that only three of 22 patients had identical HBV genotype profiles in both sites. In immune-active chronic hepatitis B, viral diversity in plasma was higher compared with peripheral blood mononuclear cells. Mutations at residues, in a minority of clones, associated with drug resistance, and/or immune escape were found in both compartments but were more common in plasma. Immune escape mutations were more often observed in the peripheral blood mono