Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

Science.gov (United States)

Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

2015-01-01

A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

Single-virus tracking approach to reveal the interaction of Dengue virus with autophagy during the early stage of infection

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Chu, Li-Wei; Huang, Yi-Lung; Lee, Jin-Hui; Huang, Long-Ying; Chen, Wei-Jun; Lin, Ya-Hsuan; Chen, Jyun-Yu; Xiang, Rui; Lee, Chau-Hwang; Ping, Yueh-Hsin

2014-01-01

Dengue virus (DENV) is one of the major infectious pathogens worldwide. DENV infection is a highly dynamic process. Currently, no antiviral drug is available for treating DENV-induced diseases since little is known regarding how the virus interacts with host cells during infection. Advanced molecular imaging technologies are powerful tools to understand the dynamics of intracellular interactions and molecular trafficking. This study exploited a single-virus particle tracking technology to address whether DENV interacts with autophagy machinery during the early stage of infection. Using confocal microscopy and three-dimensional image analysis, we showed that DENV triggered the formation of green fluorescence protein-fused microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta, and DENV-induced autophagosomes engulfed DENV particles within 15-min postinfection. Moreover, single-virus particle tracking revealed that both DENV particles and autophagosomes traveled together during the viral infection. Finally, in the presence of autophagy suppressor 3-methyladenine, the replication of DENV was inhibited and the location of DENV particles spread in cytoplasma. In contrast, the numbers of newly synthesized DENV were elevated and the co-localization of DENV particles and autophagosomes was detected while the cells were treated with autophagy inducer rapamycin. Taken together, we propose that DENV particles interact with autophagosomes at the early stage of viral infection, which promotes the replication of DENV.

Reconstructing an icosahedral virus from single-particle diffraction experiments

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Saldin, D. K.; Poon, H.-C.; Schwander, P.; Uddin, M.; Schmidt, M.

2011-08-01

The first experimental data from single-particle scattering experiments from free electron lasers (FELs) are now becoming available. The first such experiments are being performed on relatively large objects such as viruses, which produce relatively low-resolution, low-noise diffraction patterns in so-called ``diffract-and-destroy'' experiments. We describe a very simple test on the angular correlations of measured diffraction data to determine if the scattering is from an icosahedral particle. If this is confirmed, the efficient algorithm proposed can then combine diffraction data from multiple shots of particles in random unknown orientations to generate a full 3D image of the icosahedral particle. We demonstrate this with a simulation for the satellite tobacco necrosis virus (STNV), the atomic coordinates of whose asymmetric unit is given in Protein Data Bank entry 2BUK.

Near-Complete Genome Sequence of a Novel Single-Stranded RNA Virus Discovered in Indoor Air.

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Rosario, Karyna; Fierer, Noah; Breitbart, Mya

2018-03-22

Viral metagenomic analysis of heating, ventilation, and air conditioning (HVAC) filters recovered the near-complete genome sequence of a novel virus, named HVAC-associated R NA v irus 1 (HVAC-RV1). The HVAC-RV1 genome is most similar to those of picorna-like viruses identified in arthropods but encodes a small domain observed only in negative-sense single-stranded RNA viruses. Copyright © 2018 Rosario et al.

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

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Remy Froissart

2005-03-01

Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5 to 4 x 10(-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

Energy Technology Data Exchange (ETDEWEB)

Hanke, Leo; Knockenhauer, Kevin E.; Brewer, R. Camille; van Diest, Eline; Schmidt, Florian I.; Schwartz, Thomas U.; Ploegh, Hidde L. (Whitehead); (MIT)

2016-12-13

Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies.

IMPORTANCEInfluenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and

Ammonia disinfection of hatchery waste for elimination of single-stranded RNA viruses.

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Emmoth, Eva; Ottoson, Jakob; Albihn, Ann; Belák, Sándor; Vinnerås, Björn

2011-06-01

Hatchery waste, an animal by-product of the poultry industry, needs sanitation treatment before further use as fertilizer or as a substrate in biogas or composting plants, owing to the potential presence of opportunistic pathogens, including zoonotic viruses. Effective sanitation is also important in viral epizootic outbreaks and as a routine, ensuring high hygiene standards on farms. This study examined the use of ammonia at different concentrations and temperatures to disinfect hatchery waste. Inactivation kinetics of high-pathogenic avian influenza virus H7N1 and low-pathogenic avian influenza virus H5N3, as representatives of notifiable avian viral diseases, were determined in spiked hatchery waste. Bovine parainfluenza virus type 3, feline coronavirus, and feline calicivirus were used as models for other important avian pathogens, such as Newcastle disease virus, infectious bronchitis virus, and avian hepatitis E virus. Bacteriophage MS2 was also monitored as a stable indicator. Coronavirus was the most sensitive virus, with decimal reduction (D) values of 1.2 and 0.63 h after addition of 0.5% (wt/wt) ammonia at 14 and 25°C, respectively. Under similar conditions, high-pathogenic avian influenza H7N1 was the most resistant, with D values of 3.0 and 1.4 h. MS2 was more resistant than the viruses to all treatments and proved to be a suitable indicator of viral inactivation. The results indicate that ammonia treatment of hatchery waste is efficient in inactivating enveloped and naked single-stranded RNA viruses. Based on the D values and confidence intervals obtained, guidelines for treatment were proposed, and one was successfully validated at full scale at a hatchery, with MS2 added to hatchery waste.

Novel Single-Stranded DNA Virus Genomes Recovered from Chimpanzee Feces Sampled from the Mambilla Plateau in Nigeria

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Walters, Matthew; Bawuro, Musa; Christopher, Alfred; Knight, Alexander; Kraberger, Simona; Stainton, Daisy; Chapman, Hazel

2017-01-01

ABSTRACT Metagenomic approaches are rapidly expanding our knowledge of the diversity of viruses. In the fecal matter of Nigerian chimpanzees we recovered three gokushovirus genomes, one circular replication-associated protein encoding single-stranded DNA virus (CRESS), and a CRESS DNA molecule. PMID:28254982

Quantification of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV-UG) in single and mixed infected Cassava (Manihot esculenta Crantz) using quantitative PCR.

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Naseem, Saadia; Winter, Stephan

2016-01-01

The quantity of genomic DNA-A and DNA-B of African cassava mosaic virus (ACMV) and East African cassava mosaic virus Uganda (Uganda variant, EACMV-UG) was analysed using quantitative PCR to assess virus concentrations in plants from susceptible and tolerant cultivars. The concentrations of genome components in absolute and relative quantification experiments in single and mixed viral infections were determined. Virus concentration was much higher in symptomatic leaf tissues compared to non-symptomatic leaves and corresponded with the severity of disease symptoms. In general, higher titres were recorded for EACMV-UG Ca055 compared to ACMV DRC6. The quantitative assessment also showed that the distribution of both viruses in the moderately resistant cassava cv. TMS 30572 was not different from the highly susceptible cv. TME 117. Natural mixed infections with both viruses gave severe disease symptoms. Relative quantification of virus genomes in mixed infections showed higher concentrations of EACMV-UG DNA-A compared to ACMV DNA-A, but a marked reduction of EACMV-UG DNA-B. The higher concentrations of EACMV-UG DNA-B compared to EACMV DNA-A accumulation in single infections were consistent. Since DNA-B is implicated in virus cell-to-cell spread and systemic movement, the abundance of the EACMV-UG DNA-B may be an important factor driving cassava mosaic disease epidemic. Copyright © 2015 Elsevier B.V. All rights reserved.

Elucidation of hepatitis C virus transmission and early diversification by single genome sequencing.

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Li, Hui; Stoddard, Mark B; Wang, Shuyi; Blair, Lily M; Giorgi, Elena E; Parrish, Erica H; Learn, Gerald H; Hraber, Peter; Goepfert, Paul A; Saag, Michael S; Denny, Thomas N; Haynes, Barton F; Hahn, Beatrice H; Ribeiro, Ruy M; Perelson, Alan S; Korber, Bette T; Bhattacharya, Tanmoy; Shaw, George M

2012-01-01

A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures.

Toward unstained cytology and complete blood counts at the point of care (Conference Presentation)

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Zuluaga, Andres F.; Pierce, Mark C.; MacAulay, Calum E.

2017-02-01

Cytology tests, whether performed on body fluids, aspirates, or scrapings are commonly used to detect, diagnose, and monitor a wide variety of health conditions. Complete blood counts (CBCs) quantify the number of red and white blood cells in a blood volume, as well as the different types of white blood cells. There is a critical unmet need for an instrument that can perform CBCs at the point of care (POC), and there is currently no product in the US that can perform this test at the bedside. We have developed a system that is capable of tomographic images with sub-cellular resolution with consumer-grade broadband (LED) sources and CMOS detectors suitable for POC implementation of CBC tests. The systems consists of cascaded static Michelson and Sagnac interferometers that map phase (encoding depth) and a transverse spatial dimension onto a two-dimensional output plane. Our approach requires a 5 microliter sample, can be performed in 5 minutes or less, and does not require staining or other processing as it relies on intrinsic contrast. We will show results directly imaging and differentiating unstained blood cells using supercontinuum fiber lasers and LEDs as sources and CMOS cameras as sensors. We will also lay out the follow up steps needed, including image segmentation, analysis and classification, to verify performance and advance toward CBCs that can be performed bedside and do not require CLIA-certified laboratories.

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus.

Science.gov (United States)

Rogers, Stephanie M; Payton, Mark; Allen, Robert W; Melcher, Ulrich; Carver, Jesse; Fletcher, Jacqueline

2012-05-17

The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough

Penerapan Metode Diagnosis Cepat Virus Avian Influenza H5N1 dengan Metode Single Step Multiplex RT-PCR

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Aris Haryanto

2010-12-01

Full Text Available Avian influenza (AI virus is a segmented single stranded (ss RNA virus with negative polarity andbelong to the Orthomyxoviridae family. Diagnose of AI virus can be performed using conventional methodsbut it has low sensitivity and specificity. The objective of the research was to apply rapid, precise, andaccurate diagnostic method for AI virus and also to determine its type and subtype based on the SingleStep Multiplex Reverse Transcriptase-Polymerase Chain Reaction targeting M, H5, and N1 genes. In thismethod M, H5 and NI genes were simultaneously amplified in one PCR tube. The steps of this researchconsist of collecting viral RNAs from 10 different AI samples originated from Maros Disease InvestigationCenter during 2007. DNA Amplification was conducted by Simplex RT-PCR using M primer set. Then, bysingle step multiplex RT-PCR were conducted simultaneously using M, H5 and N1 primers set. The RTPCRproducts were then separated on 1.5% agarose gel, stained by ethidum bromide and visualized underUV transilluminator. Results showed that 8 of 10 RNA virus samples could be amplified by Simplex RTPCRfor M gene which generating a DNA fragment of 276 bp. Amplification using multiplex RT-PCRmethod showed two of 10 samples were AI positive using multiplex RT-PCR, three DNA fragments weregenerated consisting of 276 bp for M gene, 189 bp for H5 gene, and 131 bp for N1. In this study, rapid andeffective diagnosis method for AI virus can be conducted by using simultaneous Single Step Multiplex RTPCR.By this technique type and subtype of AI virus, can also be determined, especially H5N1.

Single-particle cryo-electron microscopy of Rift Valley fever virus

OpenAIRE

Sherman, Michael B.; Freiberg, Alexander N.; Holbrook, Michael R.; Watowich, Stanley J.

2009-01-01

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human veterinary pathogen causing acute hepatitis in ruminants and has the potential to Single-particle cryo-EM reconstruction of RVFV MP-12 hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on...

Long-Term Single-Dose Efficacy of a Vesicular Stomatitis Virus-Based Andes Virus Vaccine in Syrian Hamsters

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Joseph Prescott

2014-01-01

Full Text Available Andes virus (ANDV is highly pathogenic in humans and is the primary etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in South America. Case-fatality rates are as high as 50% and there are no approved vaccines or specific therapies for infection. Our laboratory has recently developed a replication-competent recombinant vesicular stomatitis virus (VSV-based vaccine that expressed the glycoproteins of Andes virus in place of the native VSV glycoprotein (G. This vaccine is highly efficacious in the Syrian hamster model of HCPS when given 28 days before challenge with ANDV, or when given around the time of challenge (peri-exposure, and even protects when administered post-exposure. Herein, we sought to test the durability of the immune response to a single dose of this vaccine in Syrian hamsters. This vaccine was efficacious in hamsters challenged intranasally with ANDV 6 months after vaccination (p = 0.025, but animals were not significantly protected following 1 year of vaccination (p = 0.090. The decrease in protection correlated with a reduction of measurable neutralizing antibody responses, and suggests that a more robust vaccination schedule might be required to provide long-term immunity.

A single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus.

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Jiao, Yue-Ying; Fu, Yuan-Hui; Yan, Yi-Fei; Hua, Ying; Ma, Yao; Zhang, Xiu-Juan; Song, Jing-Dong; Peng, Xiang-Lei; Huang, Jiaqiang; Hong, Tao; He, Jin-Sheng

2017-08-01

Human respiratory syncytial virus (RSV) is an important pediatric pathogen causing acute viral respiratory disease in infants and young children. However, no licensed vaccines are currently available. Virus-like particles (VLPs) may bring new hope to producing RSV VLP vaccine with high immunogenicity and safety. Here, we constructed the recombinants of matrix protein (M) and fusion glycoprotein (F) of RSV, respectively into a replication-deficient first-generation adenoviral vector (FGAd), which were used to co-infect Vero cells to assemble RSV VLPs successfully. The resulting VLPs showed similar immunoreactivity and function to RSV virion in vitro. Moreover, Th1 polarized response, and effective mucosal virus-neutralizing antibody and CD8 + T-cell responses were induced by a single intranasal (i.n.) administration of RSV VLPs rather than intramuscular (i.m.) inoculation, although the comparable RSV F-specific serum IgG and long-lasting RSV-specific neutralizing antibody were detected in the mice immunized by both routes. Upon RSV challenge, VLP-immunized mice showed increased viral clearance but decreased signs of enhanced lung pathology and fewer eosinophils compared to mice immunized with formalin-inactivated RSV (FI-RSV). In addition, a single i.n. RSV VLP vaccine has the capability to induce RSV-specific long-lasting neutralizing antibody responses observable up to 15 months. Our results demonstrate that the long-term and memory immune responses in mice against RSV were induced by a single i.n. administration of RSV VLP vaccine, suggesting a successful approach of RSV VLPs as an effective and safe mucosal vaccine against RSV infection, and an applicable and qualified platform of FGAd-infected Vero cells for VLP production. Copyright © 2017. Published by Elsevier B.V.

Etiology and Clinical Characteristics of Single and Multiple Respiratory Virus Infections Diagnosed in Croatian Children in Two Respiratory Seasons

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Sunčanica Ljubin-Sternak

2016-01-01

Full Text Available The aim of this study was to determine the causative agent of acute respiratory infection (ARI in hospitalized children, as well as investigate the characteristics of ARIs with single and multiple virus detection in two respiratory seasons. In 2010 and 2015, nasopharyngeal and pharyngeal swabs from a total of 134 children, admitted to the hospital due to ARI, were tested using multiplex PCR. Viral etiology was established in 81.3% of the patients. Coinfection with two viruses was diagnosed in 27.6% of the patients, and concurrent detection of three or more viruses was diagnosed in 12.8% of the patients. The most commonly diagnosed virus in both seasons combined was respiratory syncytial virus (RSV (28.6%, followed by parainfluenza viruses (PIVs types 1–3 (18.4%, rhinovirus (HRV (14.3%, human metapneumovirus (10.1%, adenovirus (AdV (7.1%, influenza viruses types A and B (4.8%, and coronaviruses (4.2%. In 2015, additional pathogens were investigated with the following detection rate: enterovirus (13.2%, bocavirus (HBoV (10.5%, PIV-4 (2.6%, and parechovirus (1.3%. There were no statistical differences between single and multiple virus infection regarding patients age, localization of infection, and severity of disease (P>0.05. AdV, HRV, HBoV, and PIVs were significantly more often detected in multiple virus infections compared to the other respiratory viruses (P<0.001.

Packaging signals in single-stranded RNA viruses: nature's alternative to a purely electrostatic assembly mechanism.

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Stockley, Peter G; Twarock, Reidun; Bakker, Saskia E; Barker, Amy M; Borodavka, Alexander; Dykeman, Eric; Ford, Robert J; Pearson, Arwen R; Phillips, Simon E V; Ranson, Neil A; Tuma, Roman

2013-03-01

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA-coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus

Science.gov (United States)

2012-01-01

Background The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. Method This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Result Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. Conclusion The molecular typing method presented is one tool that could be incorporated into the forensic

Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics.

Science.gov (United States)

Roux, Simon; Hawley, Alyse K; Torres Beltran, Monica; Scofield, Melanie; Schwientek, Patrick; Stepanauskas, Ramunas; Woyke, Tanja; Hallam, Steven J; Sullivan, Matthew B

2014-08-29

Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus-host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host's energy metabolism. Together, these results demonstrate closely coupled SUP05 virus-host co-evolutionary dynamics with the potential to modulate biogeochemical cycling in climate-critical and expanding OMZs.

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

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Hsu, Hung-Lun; Millet, Jean K.; Costello, Deirdre A.; Whittaker, Gary R.; Daniel, Susan

2016-01-01

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses. PMID:27752100

Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein.

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Guowei Wei

Full Text Available Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.

Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments.

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Paul J Wichgers Schreur

2016-08-01

Full Text Available The bunyavirus genome comprises a small (S, medium (M, and large (L RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH, the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process.

De novo Genome Assembly and Single Nucleotide Variations for Soybean Mosaic Virus Using Soybean Seed Transcriptome Data

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Yeonhwa Jo

2017-10-01

Full Text Available Soybean is the most important legume crop in the world. Several diseases in soybean lead to serious yield losses in major soybean-producing countries. Moreover, soybean can be infected by diverse viruses. Recently, we carried out a large-scale screening to identify viruses infecting soybean using available soybean transcriptome data. Of the screened transcriptomes, a soybean transcriptome for soybean seed development analysis contains several virus-associated sequences. In this study, we identified five viruses, including soybean mosaic virus (SMV, infecting soybean by de novo transcriptome assembly followed by blast search. We assembled a nearly complete consensus genome sequence of SMV China using transcriptome data. Based on phylogenetic analysis, the consensus genome sequence of SMV China was closely related to SMV isolates from South Korea. We examined single nucleotide variations (SNVs for SMVs in the soybean seed transcriptome revealing 780 SNVs, which were evenly distributed on the SMV genome. Four SNVs, C-U, U-C, A-G, and G-A, were frequently identified. This result demonstrated the quasispecies variation of the SMV genome. Taken together, this study carried out bioinformatics analyses to identify viruses using soybean transcriptome data. In addition, we demonstrated the application of soybean transcriptome data for virus genome assembly and SNV analysis.

Pleolipoviridae, a newly proposed family comprising archaeal pleomorphic viruses with single-stranded or double-stranded DNA genomes.

Science.gov (United States)

Pietilä, Maija K; Roine, Elina; Sencilo, Ana; Bamford, Dennis H; Oksanen, Hanna M

2016-01-01

Viruses infecting archaea show a variety of virion morphotypes, and they are currently classified into more than ten viral families or corresponding groups. A pleomorphic virus morphotype is very common among haloarchaeal viruses, and to date, several such viruses have been isolated. Here, we propose the classification of eight such viruses and formation of a new family, Pleolipoviridae (from the Greek pleo for more or many and lipos for lipid), containing three genera, Alpha-, Beta-, and Gammapleolipovirus. The proposal is currently under review by the International Committee on Taxonomy of Viruses (ICTV). The members of the proposed family Pleolipoviridae infect halophilic archaea and are nonlytic. They share structural and genomic features and differ from any other classified virus. The virion of pleolipoviruses is composed of a pleomorphic membrane vesicle enclosing the genome. All pleolipoviruses have two major structural protein species, internal membrane and spike proteins. Although the genomes of the pleolipoviruses are single- or double-stranded, linear or circular DNA molecules, they share the same genome organization and gene synteny and show significant similarity at the amino acid level. The canonical features common to all members of the proposed family Pleolipoviridae show that they are closely related and thus form a new viral family.

Single-Genome Sequencing of Hepatitis C Virus in Donor-Recipient Pairs Distinguishes Modes and Models of Virus Transmission and Early Diversification.

Science.gov (United States)

Li, Hui; Stoddard, Mark B; Wang, Shuyi; Giorgi, Elena E; Blair, Lily M; Learn, Gerald H; Hahn, Beatrice H; Alter, Harvey J; Busch, Michael P; Fierer, Daniel S; Ribeiro, Ruy M; Perelson, Alan S; Bhattacharya, Tanmoy; Shaw, George M

2016-01-01

Despite the recent development of highly effective anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and development of an effective vaccine. A precise molecular identification of transmitted/founder (T/F) HCV genomes that lead to productive clinical infection could play a critical role in vaccine research, as it has for HIV-1. However, the replication schema of these two RNA viruses differ substantially, as do viral responses to innate and adaptive host defenses. These differences raise questions as to the certainty of T/F HCV genome inferences, particularly in cases where multiple closely related sequence lineages have been observed. To clarify these issues and distinguish between competing models of early HCV diversification, we examined seven cases of acute HCV infection in humans and chimpanzees, including three examples of virus transmission between linked donors and recipients. Using single-genome sequencing (SGS) of plasma vRNA, we found that inferred T/F sequences in recipients were identical to viral sequences in their respective donors. Early in infection, HCV genomes generally evolved according to a simple model of random evolution where the coalescent corresponded to the T/F sequence. Closely related sequence lineages could be explained by high multiplicity infection from a donor whose viral sequences had undergone a pretransmission bottleneck due to treatment, immune selection, or recent infection. These findings validate SGS, together with mathematical modeling and phylogenetic analysis, as a novel strategy to infer T/F HCV genome sequences. Despite the recent development of highly effective, interferon-sparing anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and the development of an effective

Experimental Treatment of Ebola Virus Disease with TKM-130803: A Single-Arm Phase 2 Clinical Trial

DEFF Research Database (Denmark)

Dunning, Jake; Sahr, Foday; Rojek, Amanda

2016-01-01

BACKGROUND: TKM-130803, a small interfering RNA lipid nanoparticle product, has been developed for the treatment of Ebola virus disease (EVD), but its efficacy and safety in humans has not been evaluated. METHODS AND FINDINGS: In this single-arm phase 2 trial, adults with laboratory-confirmed EVD...... of admission. After 14 adults with EVD had received TKM-130803, the pre-specified futility boundary was reached, indicating a probability of survival to day 14 of ≤0.55, and enrolment was stopped. Pre-treatment geometric mean Ebola virus load in the 14 TKM-130803 recipients was 2.24 × 109 RNA copies/ml plasma...

Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

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Guus Koch

2013-04-01

Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

Viral coinfection is shaped by host ecology and virus-virus interactions across diverse microbial taxa and environments.

Science.gov (United States)

Díaz-Muñoz, Samuel L

2017-01-01

Infection of more than one virus in a host, coinfection, is common across taxa and environments. Viral coinfection can enable genetic exchange, alter the dynamics of infections, and change the course of viral evolution. Yet, a systematic test of the factors explaining variation in viral coinfection across different taxa and environments awaits completion. Here I employ three microbial data sets of virus-host interactions covering cross-infectivity, culture coinfection, and single-cell coinfection (total: 6,564 microbial hosts, 13,103 viruses) to provide a broad, comprehensive picture of the ecological and biological factors shaping viral coinfection. I found evidence that ecology and virus-virus interactions are recurrent factors shaping coinfection patterns. Host ecology was a consistent and strong predictor of coinfection across all three data sets: cross-infectivity, culture coinfection, and single-cell coinfection. Host phylogeny or taxonomy was a less consistent predictor, being weak or absent in the cross-infectivity and single-cell coinfection models, yet it was the strongest predictor in the culture coinfection model. Virus-virus interactions strongly affected coinfection. In the largest test of superinfection exclusion to date, prophage sequences reduced culture coinfection by other prophages, with a weaker effect on extrachromosomal virus coinfection. At the single-cell level, prophage sequences eliminated coinfection. Virus-virus interactions also increased culture coinfection with ssDNA-dsDNA coinfections >2× more likely than ssDNA-only coinfections. The presence of CRISPR spacers was associated with a ∼50% reduction in single-cell coinfection in a marine bacteria, despite the absence of exact spacer matches in any active infection. Collectively, these results suggest the environment bacteria inhabit and the interactions among surrounding viruses are two factors consistently shaping viral coinfection patterns. These findings highlight the role of

Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype

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Kimberly A. Dowd

2016-08-01

Full Text Available Recent epidemics of Zika virus (ZIKV have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas.

MERS-CoV: Middle East respiratory syndrome corona virus: Can radiology be of help? Initial single center experience

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Ahmed Hamimi

2016-03-01

Conclusions: MERS CoV virus may have a specific pattern in chest X-ray and CT developing a single or multiple opacities progressing into a widespread multifocal bilateral patches of ground glass opacities or confluent consolidation resembling organizing pneumonia.

Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

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David Metzgar

Full Text Available For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based or remarkably insensitive (antibody-based. Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A

Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

Science.gov (United States)

Metzgar, David; Myers, Christopher A; Russell, Kevin L; Faix, Dennis; Blair, Patrick J; Brown, Jason; Vo, Scott; Swayne, David E; Thomas, Colleen; Stenger, David A; Lin, Baochuan; Malanoski, Anthony P; Wang, Zheng; Blaney, Kate M; Long, Nina C; Schnur, Joel M; Saad, Magdi D; Borsuk, Lisa A; Lichanska, Agnieszka M; Lorence, Matthew C; Weslowski, Brian; Schafer, Klaus O; Tibbetts, Clark

2010-02-03

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence

A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease.

Science.gov (United States)

Alfson, Kendra J; Avena, Laura E; Delgado, Jenny; Beadles, Michael W; Patterson, Jean L; Carrion, Ricardo; Griffiths, Anthony

2018-01-01

Marburg virus (MARV) causes disease with high case fatality rates, and there are no approved vaccines or therapies. Licensing of MARV countermeasures will likely require approval via the FDA's Animal Efficacy Rule, which requires well-characterized animal models that recapitulate human disease. This includes selection of the virus used for exposure and ensuring that it retains the properties of the original isolate. The consequences of amplification of MARV for challenge studies are unknown. Here, we serially passaged and characterized MARV through 13 passes from the original isolate. Surprisingly, the viral genome was very stable, except for a single nucleotide change that resulted in an amino acid substitution in the hydrophobic region of the signal peptide of the glycoprotein (GP). The particle/PFU ratio also decreased following passages, suggesting a role for the amino acid in viral infectivity. To determine if amplification introduces a phenotype in an animal model, cynomolgus macaques were exposed to either 100 or 0.01 PFU of low- and high-passage-number MARV. All animals succumbed when exposed to 100 PFU of either passage 3 or 13 viruses, although animals exposed to the high-passage-number virus survived longer. However, none of the passage 13 MARV-exposed animals succumbed to 0.01-PFU exposure compared to 75% of passage 3-exposed animals. This is consistent with other filovirus studies that show some particles that are unable to yield a plaque in cell culture can cause lethal disease in vivo . These results have important consequences for the design of experiments that investigate MARV pathogenesis and that test the efficacy of MARV countermeasures. IMPORTANCE Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV

Characterization of rice black-streaked dwarf virus- and rice stripe virus-derived siRNAs in singly and doubly infected insect vector Laodelphax striatellus.

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Junmin Li

Full Text Available Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi which generates viral-derived small interfering RNAs (siRNAs. However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus was infected by Rice black-streaked dwarf virus (RBSDV (Reoviridae; Fijivirus, more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV, a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5'- and 3'-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.

A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

International Nuclear Information System (INIS)

Li Jianwei; Faber, Milosz; Papaneri, Amy; Faber, Marie-Luise; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard

2006-01-01

Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

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Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant

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Victoria M. Pearson

2016-10-01

Full Text Available Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (Circoviridae and Geminiviridae, and model organisms for genetics and evolution studies (Microviridae. Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such as Circoviridae, Geminiviridae, and Microviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.

Single endemic genotype of measles virus continuously circulating in China for at least 16 years.

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Yan Zhang

Full Text Available The incidence of measles in China from 1991 to 2008 was reviewed, and the nucleotide sequences from 1507 measles viruses (MeV isolated during 1993 to 2008 were phylogenetically analyzed. The results showed that measles epidemics peaked approximately every 3 to 5 years with the range of measles cases detected between 56,850 and 140,048 per year. The Chinese MeV strains represented three genotypes; 1501 H1, 1 H2 and 5 A. Genotype H1 was the predominant genotype throughout China continuously circulating for at least 16 years. Genotype H1 sequences could be divided into two distinct clusters, H1a and H1b. A 4.2% average nucleotide divergence was found between the H1a and H1b clusters, and the nucleotide sequence and predicted amino acid homologies of H1a viruses were 92.3%-100% and 84.7%-100%, H1b were 97.1%-100% and 95.3%-100%, respectively. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Cluster H1a and H1b viruses were co-circulating during 1993 to 2005, while no H1b viruses were detected after 2005 and the transmission of that cluster has presumably been interrupted. Analysis of the nucleotide and predicted amino acid changes in the N proteins of H1a and H1b viruses showed no evidence of selective pressure. This study investigated the genotype and cluster distribution of MeV in China over a 16-year period to establish a genetic baseline before MeV elimination in Western Pacific Region (WPR. Continuous and extensive MeV surveillance and the ability to quickly identify imported cases of measles will become more critical as measles elimination goals are achieved in China in the near future. This is the first report that a single endemic genotype of measles virus has been found to be continuously circulating in one country for at least 16 years.

Monitoring virus entry into living cells using DiD-labeled dengue virus particles

NARCIS (Netherlands)

Ayala Nunez, Vanesa; Wilschut, Jan; Smit, Jolanda M.

2011-01-01

A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular

Optical redox imaging of fixed unstained tissue slides to identify biomarkers for breast cancer diagnosis/prognosis: feasibility study

Science.gov (United States)

Xu, He N.; Tchou, Julia; Li, Yusheng; Feng, Min; Zhang, Paul; Quinn, William J.; Baur, Joseph A.; Li, Lin Z.

2018-02-01

We previously showed that optical redox imaging (ORI) of snap-frozen breast biopsies by the Chance redox scanner readily discriminates cancer from normal tissue. Moreover, indices of redox heterogeneity differentiate among tumor xenografts with different metastatic potential. These observations suggest that ORI of fluorescence of NADH and oxidized flavoproteins (Fp) may provide diagnostic/prognostic value for clinical applications. In this work, we investigate whether ORI of formalin-fixed-paraffin-embedded (FFPE) unstained clinical tissue slides of breast tumors is feasible and comparable to ORI of snap-frozen tumors. If ORI of FFPE is validated, it will enhance the versatility of ORI as a novel diagnostic/prognostic assay as FFPE samples are readily available. ORI of fixed tissue slides was performed using a fluorescence microscope equipped with a precision automated stage and appropriate optical filters. We developed a vignette correction algorithm to remove the tiling effect of stitched-images. The preliminary data from imaging fixed slides of breast tumor xenografts showed intratumor redox heterogeneity patterns similar to that of the frozen tissues imaged by the Chance redox scanner. From ORI of human breast tissue slides we identified certain redox differences among normal, ductal carcinoma in situ, and invasive carcinoma. We found paraformaldehyde fixation causes no change in NADH signals but enhances Fp signals of fresh muscle fibers. We also investigated the stability of the fluorescence microscope and reproducibility of tissue slide fluorescence signals. We plan to validate the diagnostic/prognostic value of ORI using clinically annotated breast cancer sample set from patients with long-term follow-up data.

Single Endemic Genotype of Measles Virus Continuously Circulating in China for at Least 16 Years

Science.gov (United States)

Wang, Huiling; Zhu, Zhen; Ji, Yixin; Liu, Chunyu; Zhang, Xiaojie; Sun, Liwei; Zhou, Jianhui; Lu, Peishan; Hu, Ying; Feng, Daxing; Zhang, Zhenying; Wang, Changyin; Fang, Xueqiang; Zheng, Huanying; Liu, Leng; Sun, Xiaodong; Tang, Wei; Wang, Yan; Liu, Yan; Gao, Hui; Tian, Hong; Ma, Jiangtao; Gu, Suyi; Wang, Shuang; Feng, Yan; Bo, Fang; Liu, Jianfeng; Si, Yuan; Zhou, Shujie; Ma, Yuyan; Wu, Shengwei; Zhou, Shunde; Li, Fangcai; Ding, Zhengrong; Yang, Zhaohui; Rota, Paul A.; Featherstone, David; Jee, Youngmee; Bellini, William J.; Xu, Wenbo

2012-01-01

The incidence of measles in China from 1991 to 2008 was reviewed, and the nucleotide sequences from 1507 measles viruses (MeV) isolated during 1993 to 2008 were phylogenetically analyzed. The results showed that measles epidemics peaked approximately every 3 to 5 years with the range of measles cases detected between 56,850 and 140,048 per year. The Chinese MeV strains represented three genotypes; 1501 H1, 1 H2 and 5 A. Genotype H1 was the predominant genotype throughout China continuously circulating for at least 16 years. Genotype H1 sequences could be divided into two distinct clusters, H1a and H1b. A 4.2% average nucleotide divergence was found between the H1a and H1b clusters, and the nucleotide sequence and predicted amino acid homologies of H1a viruses were 92.3%–100% and 84.7%–100%, H1b were 97.1%–100% and 95.3%–100%, respectively. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Cluster H1a and H1b viruses were co-circulating during 1993 to 2005, while no H1b viruses were detected after 2005 and the transmission of that cluster has presumably been interrupted. Analysis of the nucleotide and predicted amino acid changes in the N proteins of H1a and H1b viruses showed no evidence of selective pressure. This study investigated the genotype and cluster distribution of MeV in China over a 16-year period to establish a genetic baseline before MeV elimination in Western Pacific Region (WPR). Continuous and extensive MeV surveillance and the ability to quickly identify imported cases of measles will become more critical as measles elimination goals are achieved in China in the near future. This is the first report that a single endemic genotype of measles virus has been found to be continuously circulating in one country for at least 16 years. PMID:22532829

SARS – virus jumps species

Indian Academy of Sciences (India)

SARS – virus jumps species. Coronavirus reshuffles genes; Rotteir et al, Rotterdam showed the virus to jump from cats to mouse cells after single gene mutation ? Human disease due to virus jumping from wild or domestic animals; Present favourite animal - the cat; - edible or domestic.

Packaging signals in two single-stranded RNA viruses imply a conserved assembly mechanism and geometry of the packaged genome.

Science.gov (United States)

Dykeman, Eric C; Stockley, Peter G; Twarock, Reidun

2013-09-09

The current paradigm for assembly of single-stranded RNA viruses is based on a mechanism involving non-sequence-specific packaging of genomic RNA driven by electrostatic interactions. Recent experiments, however, provide compelling evidence for sequence specificity in this process both in vitro and in vivo. The existence of multiple RNA packaging signals (PSs) within viral genomes has been proposed, which facilitates assembly by binding coat proteins in such a way that they promote the protein-protein contacts needed to build the capsid. The binding energy from these interactions enables the confinement or compaction of the genomic RNAs. Identifying the nature of such PSs is crucial for a full understanding of assembly, which is an as yet untapped potential drug target for this important class of pathogens. Here, for two related bacterial viruses, we determine the sequences and locations of their PSs using Hamiltonian paths, a concept from graph theory, in combination with bioinformatics and structural studies. Their PSs have a common secondary structure motif but distinct consensus sequences and positions within the respective genomes. Despite these differences, the distributions of PSs in both viruses imply defined conformations for the packaged RNA genomes in contact with the protein shell in the capsid, consistent with a recent asymmetric structure determination of the MS2 virion. The PS distributions identified moreover imply a preferred, evolutionarily conserved assembly pathway with respect to the RNA sequence with potentially profound implications for other single-stranded RNA viruses known to have RNA PSs, including many animal and human pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

DEFF Research Database (Denmark)

Hakhverdyan, Mikhayil; Hägglund, Sara; Larsen, Lars Erik

2005-01-01

understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format......, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR......, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can...

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

Science.gov (United States)

Martínez Martínez, Joaquín; Poulton, Nicole J; Stepanauskas, Ramunas; Sieracki, Michael E; Wilson, William H

2011-01-01

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Novel Positive-Sense, Single-Stranded RNA (+ssRNA) Virus with Di-Cistronic Genome from Intestinal Content of Freshwater Carp (Cyprinus carpio)

Science.gov (United States)

Pankovics, Péter; Simmonds, Peter

2011-01-01

A novel positive-sense, single-stranded RNA (+ssRNA) virus (Halastavi árva RNA virus, HalV; JN000306) with di-cistronic genome organization was serendipitously identified in intestinal contents of freshwater carps (Cyprinus carpio) fished by line-fishing from fishpond “Lőrinte halastó” located in Veszprém County, Hungary. The complete nucleotide (nt) sequence of the genomic RNA is 9565 nt in length and contains two long - non-in-frame - open reading frames (ORFs), which are separated by an intergenic region. The ORF1 (replicase) is preceded by an untranslated sequence of 827 nt, while an untranslated region of 139 nt follows the ORF2 (capsid proteins). The deduced amino acid (aa) sequences of the ORFs showed only low (less than 32%) and partial similarity to the non-structural (2C-like helicase, 3C-like cystein protease and 3D-like RNA dependent RNA polymerase) and structural proteins (VP2/VP4/VP3) of virus families in Picornavirales especially to members of the viruses with dicistronic genome. Halastavi árva RNA virus is present in intestinal contents of omnivorous freshwater carps but the origin and the host species of this virus remains unknown. The unique viral sequence and the actual position indicate that Halastavi árva RNA virus seems to be the first member of a new di-cistronic ssRNA virus. Further studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature. PMID:22195010

Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

Science.gov (United States)

Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

1985-09-01

The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

Engineering Molecular Immunity Against Plant Viruses

KAUST Repository

Zaidi, Syed Shan-e-Ali; Tashkandi, Manal; Mahfouz, Magdy M.

2017-01-01

Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.

Engineering Molecular Immunity Against Plant Viruses

KAUST Repository

Zaidi, Syed Shan-e-Ali

2017-04-26

Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.

A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

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Miguel A Martín-Acebes

Full Text Available West Nile virus (WNV is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu on the highly basic internal capsid or core (C protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

Science.gov (United States)

Martín-Acebes, Miguel A; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

2013-01-01

West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

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Joaquín Martínez Martínez

Full Text Available Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Role of electrostatics in the assembly pathway of a single-stranded RNA virus.

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Garmann, Rees F; Comas-Garcia, Mauricio; Koay, Melissa S T; Cornelissen, Jeroen J L M; Knobler, Charles M; Gelbart, William M

2014-09-01

We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of

Additive interactions of unrelated viruses in mixed infections of cowpea.

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Imade Yolanda Nsa

2015-10-01

Full Text Available This study was carried out to determine the effects of single infections and co-infections of three unrelated viruses on three cowpea cultivars (one commercial cowpea cultivar White and 2 IITA lines; IT81D-985 and TVu76. The plants were inoculated with Cowpea aphid-borne mosaic virus (CABMV, genus Potyvirus, Cowpea mottle virus (CMeV, genus Carmovirus and Southern bean mosaic virus (SBMV, genus Sobemovirus singly and in mixture (double and triple at 10, 20 and 30 days after planting (DAP. The treated plants were assessed for susceptibility to the viruses, growth and yield. In all cases of infection, early inoculation resulted in higher disease severity compared with late infection. The virus treated cowpea plants were relatively shorter than buffer inoculated control plants except the IT81D-985 plants that were taller and produced more foliage. Single infections by CABMV, CMeV and SBMV led to a complete loss of seeds in the three cowpea cultivars at 10DAP; only cultivar White produced some seeds at 30DAP. Double and triple virus infections led to a total loss of seeds in all three cowpea cultivars. None of the virus infected IITA lines produced any seeds except IT81D-985 plants co-infected with CABMV and SBMV at 30DAP with a reduction of 80%. Overall, the commercial cultivar White was the least susceptible to the virus treatments and produced the most yield (flowers, pods and seeds. CABMV was the most aggressive of these viruses and early single inoculations with this virus resulted in the premature death of some of the seedlings. The presence of the Potyvirus, CABMV in the double virus infections did not appear to increase disease severity or yield loss. There was no strong evidence for synergistic interactions between the viruses in the double virus mixtures.

Single treatment with ethanol hand rub is ineffective against human rhinovirus--hand washing with soap and water removes the virus efficiently.

Science.gov (United States)

Savolainen-Kopra, Carita; Korpela, Terttu; Simonen-Tikka, Marja-Leena; Amiryousefi, Ali; Ziegler, Thedi; Roivainen, Merja; Hovi, Tapani

2012-03-01

Ethanol-containing hand rubs are used frequently as a substitute for hand washing with water and soap. However, not all viruses are inactivated by a short term rubbing with alcohol. The capacity of a single round of instructed and controlled hand cleaning with water and soap or ethanol-containing hand rub, respectively, was tested for removal of human rhinovirus administered onto the skin of healthy volunteers on the back of the hands. Hand washing with soap and water appeared to be much more efficient for removing rhinoviruses from skin than rubbing hands with an ethanol-containing disinfectant. After washing with soap and water the virus was detected in 3/9 (33.3%) test persons from the left hand and 1/9 (11.1%) cases from the right hand, whereas the virus was detected invariably by real-time RT-PCR from both hands after cleaning with alcohol hand rub (P-value soap can clean efficiently hands contaminated with the virus responsible for an extensive share of common cold episodes. Copyright © 2012 Wiley Periodicals, Inc.

Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

NARCIS (Netherlands)

van der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaarl, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain SI on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by

Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

NARCIS (Netherlands)

van der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaarl, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

2007-01-01

In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain SI on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by

A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2)

Science.gov (United States)

An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), an important factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcription of RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested ...

Detection of sweet potato virus C, sweet potato virus 2 and sweet potato feathery mottle virus in Portugal.

Science.gov (United States)

Varanda, Carla M R; Santos, Susana J; Oliveira, Mônica D M; Clara, Maria Ivone E; Félix, Maria Rosário F

2015-06-01

Field sweet potato plants showing virus-like symptoms, as stunting, leaf distortion, mosaic and chlorosis, were collected in southwest Portugal and tested for the presence of four potyviruses, sweet potato virus C (SPVC), sweet potato virus 2 (SPV2), sweet potato feathery mottle virus (SPFMV), sweet potato virus G (SPVG), and the crinivirus sweet potato chlorotic stunt virus (SPCSV). DsRNA fractions were extracted from symptomatic leaves and used as templates in single and multiplex RT-PCR assays using previously described specific primers for each analyzed virus. The amplified reaction products for SPVC, SPV2 and SPFMV were of expected size, and direct sequencing of PCR products revealed that they correspond to the coat protein gene (CP) and showed 98%, 99% and 99% identity, respectively, to those viruses. Comparison of the CP genomic and amino acid sequences of the Portuguese viral isolates recovered here with those of ten other sequences of isolates obtained in different countries retrieved from the GenBank showed very few differences. The application of the RT-PCR assays revealed for the first time the presence of SPVC and SPFMV in the sweet potato crop in Portugal, the absence of SPVG and SPCSV in tested plants, as well as the occurrence of triple virus infections under field conditions.

Exposure to hepatitis E virus, hepatitis A virus and Borrelia spp. infections in forest rangers from a single forest district in western Poland.

Science.gov (United States)

Bura, Maciej; Bukowska, Alicja; Michalak, Michał; Bura, Aleksandra; Nawrocki, Mariusz J; Karczewski, Marek; Mozer-Lisewska, Iwona

2018-03-13

Hepatitis E virus (HEV) infection is an emerging problem in developed countries. At least 2 zoonotic genotypes of the virus (HEV-3 and HEV-4) infect human beings. There are some data suggesting that forest rangers (FRs) can be at a higher risk of contact with HEV. The aim of this study was to assess the prevalence of HEV exposure markers in FRs from a single forest district in Greater Poland in relation to anti-HAV (hepatitis A virus) IgG, and anti-Borrelia spp. IgM and IgG antibodies. In total, 138 participants (48 FRs and 90 blood donors - BDs) were tested for anti-HEV IgM and IgG (EUROIMMUN Medizinische Labordiagnostika AG, Luebeck, Germany) and 96 individuals (48 FRs and 48 BDs) were tested for anti-HAV IgG (ARCHITECT immunoassays, Abbott Laboratories, Wiesbaden, Germany); anti-Borrelia IgM and IgG (EUROIMMUN kits) were assessed in FRs only. Anti-HEV markers were detected in 3 participants (2.2%; IgM in 1 FR, IgG in 2 BDs), less frequently than anti-HAV (16 out of 96 individuals, about 17%; FRs 19% vs BDs 15%) or anti-Borrelia antibodies (18 out of 48 individuals, 37.5%) (p < 0.0001 for both). Older study participants (≥45 years of age) were more frequently HAV-seropositive (29% vs 4% of the younger individuals; p = 0.0012). We failed to unequivocally prove HEV exposure in FRs. The HAV seroprevalence in this study paralleled the situation in the general population. Exposure to Borrelia spp. in FRs was common.

MVA-based H5N1 vaccine affords cross-clade protection in mice against influenza A/H5N1 viruses at low doses and after single immunization.

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Joost H C M Kreijtz

Full Text Available Human infections with highly pathogenic avian influenza viruses of the H5N1 subtype, frequently reported since 2003, result in high morbidity and mortality. It is feared that these viruses become pandemic, therefore the development of safe and effective vaccines is desirable. MVA-based H5N1 vaccines already proved to be effective when two immunizations with high doses were used. Dose-sparing strategies would increase the number of people that can be vaccinated when the amount of vaccine preparations that can be produced is limited. Furthermore, protective immunity is induced ideally after a single immunization. Therefore the minimal requirements for induction of protective immunity with a MVA-based H5N1 vaccine were assessed in mice. To this end, mice were vaccinated once or twice with descending doses of a recombinant MVA expressing the HA gene of influenza virus A/Vietnam/1194/04. The protective efficacy was determined after challenge infection with the homologous clade 1 virus and a heterologous virus derived from clade 2.1, A/Indonesia/5/05 by assessing weight loss, virus replication and histopathological changes. It was concluded that MVA-based vaccines allowed significant dose-sparing and afford cross-clade protection, also after a single immunization, which are favorable properties for an H5N1 vaccine candidate.

Single-particle fusion of influenza viruses reveals complex interactions with target membranes

Science.gov (United States)

van der Borg, Guus; Braddock, Scarlett; Blijleven, Jelle S.; van Oijen, Antoine M.; Roos, Wouter H.

2018-05-01

The first step in infection of influenza A virus is contact with the host cell membrane, with which it later fuses. The composition of the target bilayer exerts a complex influence on both fusion efficiency and time. Here, an in vitro, single-particle approach is used to study this effect. Using total internal reflection fluorescence (TIRF) microscopy and a microfluidic flow cell, the hemifusion of single virions is visualized. Hemifusion efficiency and kinetics are studied while altering target bilayer cholesterol content and sialic-acid donor. Cholesterol ratios tested were 0%, 10%, 20%, and 40%. Sialic-acid donors GD1a and GYPA were used. Both cholesterol ratio and sialic-acid donors proved to have a significant effect on hemifusion efficiency. Furthermore, comparison between GD1a and GYPA conditions shows that the cholesterol dependence of the hemifusion time is severely affected by the sialic-acid donor. Only GD1a shows a clear increasing trend in hemifusion efficiency and time with increasing cholesterol concentration of the target bilayer with maximum rates for GD1A and 40% cholesterol. Overall our results show that sialic acid donor and target bilayer composition should be carefully chosen, depending on the desired hemifusion time and efficiency in the experiment.

Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves.

Science.gov (United States)

Xue, Wenzhi; Ellis, John; Mattick, Debra; Smith, Linda; Brady, Ryan; Trigo, Emilio

2010-05-14

The immunogenicity of an intranasally-administered modified-live virus (MLV) vaccine in 3-8 day old calves was evaluated against bovine viral diarrhea virus (BVDV) types 1 and 2, infectious bovine rhinotracheitis (IBR) virus, parainfluenza-3 (PI-3) virus and bovine respiratory syncytial virus (BRSV). Calves were intranasally vaccinated with a single dose of a multivalent MLV vaccine and were challenged with one of the respective viruses three to four weeks post-vaccination in five separate studies. There was significant sparing of diseases in calves intranasally vaccinated with the MLV vaccine, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding, greater white blood cell and platelet counts, and less severe pulmonary lesions than control animals. This was the first MLV combination vaccine to demonstrate efficacy against BVDV types 1 and 2, IBR, PI-3 and BRSV in calves 3-8 days of age. Copyright 2010 Elsevier Ltd. All rights reserved.

Purification and crystallization of Kokobera virus helicase

International Nuclear Information System (INIS)

De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno; Forrester, Naomi L.; Gould, Ernest; Canard, Bruno; Mattevi, Andrea

2007-01-01

Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3 1 21 (or P3 2 21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å

Purification and crystallization of Kokobera virus helicase

Energy Technology Data Exchange (ETDEWEB)

De Colibus, Luigi; Speroni, Silvia [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy); Coutard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Forrester, Naomi L.; Gould, Ernest [Centre for Ecology and Hydrology (formerly Institute of Virology), Mansfield Road, Oxford OX1 3SR (United Kingdom); Canard, Bruno [Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 13288 Marseille CEDEX 09 (France); Mattevi, Andrea, E-mail: mattevi@ipvgen.unipv.it [Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia (Italy)

2007-03-01

Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.

Development of a novel method for simultaneous concentration of viruses and protozoa from a single water sample.

Science.gov (United States)

Haramoto, Eiji; Katayama, Hiroyuki; Asami, Mari; Akiba, Michihiro

2012-06-01

A novel method, electronegative membrane-vortex (EMV) method, was developed for simultaneous concentration of viruses and protozoa from a single water sample. Viruses and protozoa in a water sample were mixed with a cation solution and adsorbed on an electronegative membrane. Concentrated virus and protozoa samples were obtained as supernatant and pellet fractions, respectively, by vigorous vortex mixing of the membrane and centrifugation of the eluted material. The highest recovery efficiencies of model microbes from river water and tap water by this EMV method were obtained using a mixed cellulose ester membrane with a pore size of 0.45 μm (Millipore) as the electronegative membrane and MgCl(2) as the cation solution. The recovery was 27.7-86.5% for poliovirus, 25.7-68.3% for coliphage Qβ, 28.0-60.0% for Cryptosporidium oocysts, and 35.0-53.0% for Giardia cysts. The EMV method detected successfully indigenous viruses and protozoa in wastewater and river water samples from the Kofu basin, Japan, showing an overall positive rate of 100% (43/43) for human adenovirus, 79% (34/43) for norovirus GI, 65% (28/43) for norovirus GII, 23% (10/43) for Cryptosporidium oocysts, and 60% (26/43) for Giardia cysts. By direct DNA sequencing, a total of four genotypes (AI, AII, B, and G) of Giardia intestinalis were identified in the water samples, indicating that the river water was contaminated with feces from various mammals, including humans. Copyright © 2012 Elsevier B.V. All rights reserved.

A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

Directory of Open Access Journals (Sweden)

Juana M Sánchez-Puig

Full Text Available Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

Science.gov (United States)

Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

2013-01-01

Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

Zika Virus: An Emergent Neuropathological Agent

Science.gov (United States)

White, Martyn K.; Wollebo, Hassen S.; Beckham, J. David; Tyler, Kenneth L.; Khalili, Kamel

2016-01-01

The emergence of Zika virus in the Americas has followed a pattern that is familiar from earlier epidemics of other viruses, where a new disease is introduced into a human population and then spreads rapidly with important public health consequences. In the case of Zika virus, an accumulating body of recent evidence implicates the virus in the etiology of serious pathologies of the human nervous system, that is, the occurrence of microcephaly in neonates and Guillain–Barré syndrome in adults. Zika virus is an arbovirus (arthropod-borne virus) and a member of the family Flaviviridae, genus Flavivirus. Zika virions are enveloped and icosahedral, and contain a nonsegmented, single-stranded, positive-sense RNA genome, which encodes 3 structural and 7 nonstructural proteins that are expressed as a single polyprotein that undergoes cleavage. Zika genomic RNA replicates in the cytoplasm of infected host cells. Zika virus was first detected in 1947 in the blood of a febrile monkey in Uganda’s Zika Forest and in crushed suspensions of the Aedes mosquito, which is one of the vectors for Zika virus. The virus remained obscure, with a few human cases confined to Africa and Asia. There are two lineages of the Zika virus, African and Asian, with the Asian strain causing outbreaks in Micronesia in 2007 and French Polynesia in 2013–2014. From here, the virus spread to Brazil with the first report of autochthonous Zika transmission in the Americas in March 2015. The rapid advance of the virus in the Americas and its likely association with microcephaly and Guillain–Barré syndrome make Zika an urgent public health concern. PMID:27464346

Vesicular Stomatitis Virus Pseudotyped with Ebola Virus Glycoprotein Serves as a Highly Protective, Non-infectious Vaccine Against Ebola Virus Challenge

Science.gov (United States)

2016-07-01

Single-Injection Trivalent Filovirus 428 Vaccine: Proof of Concept Study in Outbred Guinea Pigs . J Infect Dis. 429 29. Murin, C. D., M. L. Fusco, Z...Jahrling, and J. F. Smith. 2000. Recombinant RNA replicons derived from attenuated 442 Venezuelan equine encephalitis virus protect guinea pigs and...platform, 65 including ease of production and characterization, absence of virus replication concerns and the 66 robust immune stimulatory activity

Single-photon emission computed tomography in human immunodeficiency virus encephalopathy: A preliminary report

International Nuclear Information System (INIS)

Masdeu, J.C.; Yudd, A.; Van Heertum, R.L.; Grundman, M.; Hriso, E.; O'Connell, R.A.; Luck, D.; Camli, U.; King, L.N.

1991-01-01

Depression or psychosis in a previously asymptomatic individual infected with the human immunodeficiency virus (HIV) may be psychogenic, related to brain involvement by the HIV or both. Although prognosis and treatment differ depending on etiology, computed tomography (CT) and magnetic resonance imaging (MRI) are usually unrevealing in early HIV encephalopathy and therefore cannot differentiate it from psychogenic conditions. Thirty of 32 patients (94%) with HIV encephalopathy had single-photon emission computed tomography (SPECT) findings that differed from the findings in 15 patients with non-HIV psychoses and 6 controls. SPECT showed multifocal cortical and subcortical areas of hypoperfusion. In 4 cases, cognitive improvement after 6-8 weeks of zidovudine (AZT) therapy was reflected in amelioration of SPECT findings. CT remained unchanged. SPECT may be a useful technique for the evaluation of HIV encephalopathy

Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

Directory of Open Access Journals (Sweden)

Parida Manmohan

2008-01-01

Full Text Available Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever and alphavirus (Chikungunya. The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation vis a vis conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only. Conclusion These findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.

Single-cycle immunodeficiency viruses provide strategies for uncoupling in vivo expression levels from viral replicative capacity and for mimicking live-attenuated SIV vaccines

International Nuclear Information System (INIS)

Kuate, Seraphin; Stahl-Hennig, Christiane; Haaft, Peter ten; Heeney, Jonathan; Ueberla, Klaus

2003-01-01

To reduce the risks associated with live-attenuated immunodeficiency virus vaccines, single-cycle immunodeficiency viruses (SCIVs) were developed by primer complementation and production of the vaccine in the absence of vif in a vif-independent cell line. After a single intravenous injection of SCIVs into rhesus monkeys, peak viral RNA levels of 10 3 to 10 4 copies/ml plasma were observed, indicating efficient expression of SCIV in the vaccinee. After booster immunizations with SCIVs, SIV-specific humoral and cellular immune responses were observed. Although the vaccine doses used in this pilot study could not protect vaccinees from subsequent intravenous challenge with pathogenic SIVmac239, our results demonstrate that the novel SCIV approach allows us to uncouple in vivo expression levels from the viral replicative capacity facilitating the analysis of the relationship between viral expression levels or viral genes and immune responses induced by SIV

Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

Science.gov (United States)

Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Shi, Linqing; Zhang, Renxia; Jiang, Chunlai; Kong, Wei; Wu, Yongge

2017-04-28

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

Science.gov (United States)

Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

2016-03-01

Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses.

Directory of Open Access Journals (Sweden)

Graeme E Price

2010-10-01

Full Text Available The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, "universal" vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd expressing the conserved influenza virus antigens matrix 2 and nucleoprotein.In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals.Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.

Evaluation of recombinant influenza virus-simian immunodeficiency virus vaccines in macaques.

Science.gov (United States)

Sexton, Amy; De Rose, Robert; Reece, Jeanette C; Alcantara, Sheilajen; Loh, Liyen; Moffat, Jessica M; Laurie, Karen; Hurt, Aeron; Doherty, Peter C; Turner, Stephen J; Kent, Stephen J; Stambas, John

2009-08-01

There is an urgent need for human immunodeficiency virus (HIV) vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express simian immunodeficiency virus (SIV) CD8 T-cell epitopes and evaluated following administration to the respiratory tracts of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions, although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T-cell responses to influenza virus proteins. SIV-specific CD8 T-cell responses bearing the mucosal homing marker beta7 integrin were induced by vaccination of naïve animals. Further, SIV-specific CD8 T-cell responses could be boosted by recombinant influenza virus-SIV vaccination of animals with already-established SIV infection. Sequential vaccination with influenza virus-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T-cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T-cell response, but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest that influenza virus-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit cytotoxic T-lymphocyte escape.

New Taastrup-Like virus, Rhabdoviridae, lethal to leafhoppers

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A new viral pathogen (‘Taastrup Virus’) of leafhoppers was discovered. The unclassified virus is a negative sense, single-stranded RNA virus which appears to be a new member of the order Mononegavirales in the family Rhabdoviridae, and thus far it is only the second report of a Taastrup-like virus m...

Zika Virus-Associated Cognitive Impairment in Adolescent, 2016.

Science.gov (United States)

Zucker, Jason; Neu, Natalie; Chiriboga, Claudia A; Hinton, Veronica J; Leonardo, Marc; Sheikh, Arif; Thakur, Kiran

2017-06-01

Incidence of neurologic manifestations associated with Zika virus infection has been increasing. In 2016, neuropsychological and cognitive changes developed in an adolescent after travel to a Zika virus-endemic area. Single-photon emission computed tomography and neuropsychological testing raised the possibility that Zika virus infection may lead to neuropsychiatric and cognitive symptoms.

Hepatitis E Virus and Related Viruses in Animals.

Science.gov (United States)

Thiry, D; Mauroy, A; Pavio, N; Purdy, M A; Rose, N; Thiry, E; de Oliveira-Filho, E F

2017-02-01

Hepatitis E is an acute human liver disease in healthy individuals which may eventually become chronic. It is caused by the hepatitis E virus (HEV) and can have a zoonotic origin. Nearly 57,000 people die yearly from hepatitis E-related conditions. The disease is endemic in both developing and developed countries with distinct epidemiologic profiles. In developing countries, the disease is associated with inadequate water treatment, while in developed countries, transmission is associated with animal contact and the ingestion of raw or uncooked meat, especially liver. All human HEV are grouped into at least four genotypes, while HEV or HEV-related viruses have been identified in an increasing number of domestic and wild animal species. Despite a high genetic diversity, only one single HEV serotype has been described to date for HEV genotypes 1-4. The discovery of new HEV or HEV-related viruses leads to a continuing increase in the number of genotypes. In addition, the genome organization of all these viruses is variable with overlapping open reading frames (ORF) and differences in the location of ORF3. In spite of the role of some domestic and wild animals as reservoir, the origin of HEV and HEV-related viruses in humans and animals is still unclear. This review discusses aspects of the detection, molecular virology, zoonotic transmission and origin of HEV and HEV-related viruses in the context of 'One Health' and establishes a link between the previous and the new taxonomy of this growing virus family. © 2015 Blackwell Verlag GmbH.

Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

OpenAIRE

Parida Manmohan; Shrivastava Ambuj; Santhosh SR; Dash Paban; Saxena Parag; Rao PV

2008-01-01

Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Jap...

RNA viruses in the sea.

Science.gov (United States)

Lang, Andrew S; Rise, Matthew L; Culley, Alexander I; Steward, Grieg F

2009-03-01

Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.

The Genetic and Molecular Studies of Hepatitis C Virus: A Review ...

African Journals Online (AJOL)

The role of Hepatitis viruses, particularly Hepatitis c virus (HCV) as human pathogen and their transmission have been of interest over the years. The virus is a small (55-65nm in size), included in Group IV, enveloped, positive sense, single stranded RNA virus, the family Flaviviridae, genus Hepacivirus, and Hepatitis c virus ...

ICTV Virus Taxonomy Profile: Rhabdoviridae.

Science.gov (United States)

Walker, Peter J; Blasdell, Kim R; Calisher, Charles H; Dietzgen, Ralf G; Kondo, Hideki; Kurath, Gael; Longdon, Ben; Stone, David M; Tesh, Robert B; Tordo, Noël; Vasilakis, Nikos; Whitfield, Anna E; Nbsp Ictv Report Consortium

2018-04-01

The family Rhabdoviridae comprises viruses with negative-sense (-) single-stranded RNA genomes of 10.8-16.1 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants and animals including mammals, birds, reptiles and fish, as well as arthropods which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish and agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Rhabdoviridae, which is available at www.ictv.global/report/rhabdoviridae.

Transmission and pathogenesis of vesicular stomatitis viruses

Science.gov (United States)

Vesicular Stomatitis (VS) is caused by the Vesicular Stomatitis Virus (VSV), a negative single stranded RNA arthropod-borne virus member of the Family Rhabdoviridae. The virion is composed of the host derived plasma membrane, the envelope, and an internal ribonucleoprotein core. The envelope contain...

Data for increase of Lymantria dispar male survival after topical application of single-stranded RING domain fragment of IAP-3 gene of its nuclear polyhedrosis virus

Science.gov (United States)

Oberemok, Volodymyr V.; Laikova, Kateryna V.; Zaitsev, Aleksei S.; Gushchin, Vladimir A.; Skorokhod, Oleksii A.

2016-01-01

This data article is related to the research article entitled “The RING for gypsy moth control: topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide” [1]. This article reports on significantly higher survival of gypsy moth Lymantria dispar male individuals in response to topical application of single-stranded DNA, based on RING (really interesting new gene) domain fragment of LdMNPV (L. dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene and acted as DNA insecticide. PMID:27054151

Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice.

Science.gov (United States)

Kim, Ha-Hyun; Yang, Dong-Kun; Nah, Jin-Ju; Song, Jae-Young; Cho, In-Soo

2017-06-24

Rabies is an important viral zoonosis that causes acute encephalitis and death in mammals. To date, several recombinant vaccines have been developed based on G protein, which is considered to be the main antigen, and these vaccines are used for rabies control in many countries. Most recombinant viruses expressing RABV G protein retain the G gene from attenuated RABV. Not enough is currently known about the protective effect against RABV of a combination of recombinant adenoviruses expressing the G and N proteins of pathogenic street RABV. We constructed a recombinant adenovirus (Ad-0910Gsped) expressing the signal peptide and ectodomain (sped) of G protein of the Korean street strain, and evaluated the immunological protection conferred by a single and combination of three kinds of recombinant adenoviruses (Ad-0910Gsped and Ad-0910G with or without Ad-0910 N) in mice. A combination of Ad-0910G and Ad-0910 N conferred improved immunity against intracranial challenge compared to single administration of Ad-0910G. The Ad-0910G virus, expressing the complete G protein, was more immunogenic than Ad-0910Gsped, which expressed a truncated G protein with the transmembrane and cytoplasmic domains removed. Additionally, oral vaccination using a combination of viruses led to complete protection. Our results suggest that this combination of viruses is a viable new intramuscular and oral vaccine candidate.

TH-EF-207A-06: High-Resolution Optical-CT/ECT Imaging of Unstained Mice Femur, Brain, Spleen, and Tumor

International Nuclear Information System (INIS)

Yoon, S; Dewhirst, M; Oldham, M; Boss, M; Birer, S

2016-01-01

Purpose: Optical transmission and emission computed tomography (optical-CT/ECT) provides high-resolution 3D attenuation and emission maps in unsectioned large (∼1cm 3 ) ex vivo tissue samples at a resolution of 12.9µm 3 per voxel. Here we apply optical-CT/ECT to investigate high-resolution structure and auto-fluorescence in a range of optically cleared mice organs, including, for the first time, mouse bone (femur), opening the potential for study of bone metastasis and bone-mediated immune response. Methods: Three BALBc mice containing 4T1 flank tumors were sacrificed to obtain spleen, brain, tumor, and femur. Tissues were washed in 4% PFA, fixed in EtOH solution (for 5, 10, 10, and 2 days respectively), and then optically cleared for 3 days in BABBs. The femur was also placed in 0.25M aqueous EDTA for 15–30 days to remove calcium. Optical-CT/ECT attenuation and emission maps at 633nm (the latter using 530nm excitation light) were obtained for all samples. Bi-telecentric optical-CT was compared side-by-side with conventional optical projection tomography (OPT) imaging to evaluate imaging capability of these two rival techniques. Results: Auto-fluorescence mapping of femurs reveals vasculatures and fluorescence heterogeneity. High signals (A.U.=10) are reported in the medullary cavity but not in the cortical bone (A.U.=1). The brain strongly and uniform auto-fluoresces (A.U.=5). Thick, optically dense organs such as the spleen and the tumor (0.12, 0.46OD/mm) are reconstructed at depth without significant loss of resolution, which we attribute to the bi-telecentric optics of optical-CT. The attenuation map of tumor reveals vasculature, attenuation heterogeneity, and possibly necrotic tissue. Conclusion: We demonstrate the feasibility of optical-CT/ECT imaging of un-sectioned mice bones (femurs) and spleen with high resolution. This result, and the characterization of unstained organs, are important steps enabling future studies involving optical-CT/ECT applied

The prevalence of transfusion-transmitted virus (TTV) infection in ...

African Journals Online (AJOL)

Transfusion-transmitted virus (TTV) is an unenveloped circular single-stranded DNA virus with a diameter of 30 to 32 nm that was first described in 1997 in Japan. TTV was detected in various populations without proven pathology, including blood donors and in patients with chronic hepatitis B virus (HBV) and hepatitis C ...

Additive interactions of unrelated viruses in mixed infections of cowpea (Vigna unguiculata L. Walp).

Science.gov (United States)

Nsa, Imade Y; Kareem, Kehinde T

2015-01-01

This study was carried out to determine the effects of single infections and co-infections of three unrelated viruses on three cowpea cultivars (one commercial cowpea cultivar "White" and 2 IITA lines; IT81D-985 and TVu 76). The plants were inoculated with Cowpea aphid-borne mosaic virus (CABMV), genus Potyvirus, Cowpea mottle virus (CMeV), genus Carmovirus and Southern bean mosaic virus (SBMV), genus Sobemovirus singly and in mixture (double and triple) at 10, 20, and 30 days after planting (DAP). The treated plants were assessed for susceptibility to the viruses, growth, and yield. In all cases of infection, early inoculation resulted in higher disease severity compared with late infection. The virus treated cowpea plants were relatively shorter than buffer inoculated control plants except the IT81D-985 plants that were taller and produced more foliage. Single infections by CABMV, CMeV, and SBMV led to a complete loss of seeds in the three cowpea cultivars at 10 DAP; only cultivar White produced some seeds at 30 DAP. Double and triple virus infections led to a total loss of seeds in all three cowpea cultivars. None of the virus infected IITA lines produced any seeds except IT81D-985 plants co-infected with CABMV and SBMV at 30 DAP with a reduction of 80%. Overall, the commercial cultivar "White" was the least susceptible to the virus treatments and produced the most yield (flowers, pods, and seeds). CABMV was the most aggressive of these viruses and early single inoculations with this virus resulted in the premature death of some of the seedlings. The presence of the Potyvirus, CABMV in the double virus infections did not appear to increase disease severity or yield loss. There was no strong evidence for synergistic interactions between the viruses in the double virus mixtures.

Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

International Nuclear Information System (INIS)

Bukreyev, Alexander; Marzi, Andrea; Feldmann, Friederike; Zhang Liqun; Yang Lijuan; Ward, Jerrold M.; Dorward, David W.; Pickles, Raymond J.; Murphy, Brian R.; Feldmann, Heinz; Collins, Peter L.

2009-01-01

We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV

Phomopsis longicolla RNA virus 1 - Novel virus at the edge of myco- and plant viruses.

Science.gov (United States)

Hrabáková, Lenka; Koloniuk, Igor; Petrzik, Karel

2017-06-01

The complete nucleotide sequence of a new RNA mycovirus in the KY isolate of Phomopsis longicolla Hobbs 1985 and its protoplasts subcultures p5, p9, and ME711 was discovered. The virus, provisionally named Phomopsis longicolla RNA virus 1 (PlRV1), was localized in mitochondria and was determined to have a genome 2822 nucleotides long. A single open reading frame could be translated in silico by both standard and mitochondrial genetic codes into a product featuring conservative domains for an RNA-dependent RNA polymerase (RdRp). The RdRp of PlRV1 has no counterpart among mycoviruses, but it is about 30% identical with the RdRp of plant ourmiaviruses. Recently, new mycoviruses related to plant ourmiaviruses and forming one clade with PlRV1 have been discovered. This separate clade could represent the crucial link between plant and fungal viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

International Nuclear Information System (INIS)

Zamansky, G.B.; Kleinman, L.F.; Black, P.H.; Kaplan, J.C.

1980-01-01

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40(SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The UV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical conditions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line. (orig.)

RECOVIR Software for Identifying Viruses

Science.gov (United States)

Chakravarty, Sugoto; Fox, George E.; Zhu, Dianhui

2013-01-01

Most single-stranded RNA (ssRNA) viruses mutate rapidly to generate a large number of strains with highly divergent capsid sequences. Determining the capsid residues or nucleotides that uniquely characterize these strains is critical in understanding the strain diversity of these viruses. RECOVIR (an acronym for "recognize viruses") software predicts the strains of some ssRNA viruses from their limited sequence data. Novel phylogenetic-tree-based databases of protein or nucleic acid residues that uniquely characterize these virus strains are created. Strains of input virus sequences (partial or complete) are predicted through residue-wise comparisons with the databases. RECOVIR uses unique characterizing residues to identify automatically strains of partial or complete capsid sequences of picorna and caliciviruses, two of the most highly diverse ssRNA virus families. Partition-wise comparisons of the database residues with the corresponding residues of more than 300 complete and partial sequences of these viruses resulted in correct strain identification for all of these sequences. This study shows the feasibility of creating databases of hitherto unknown residues uniquely characterizing the capsid sequences of two of the most highly divergent ssRNA virus families. These databases enable automated strain identification from partial or complete capsid sequences of these human and animal pathogens.

Unraveling the web of viroinformatics: computational tools and databases in virus research.

Science.gov (United States)

Sharma, Deepak; Priyadarshini, Pragya; Vrati, Sudhanshu

2015-02-01

The beginning of the second century of research in the field of virology (the first virus was discovered in 1898) was marked by its amalgamation with bioinformatics, resulting in the birth of a new domain--viroinformatics. The availability of more than 100 Web servers and databases embracing all or specific viruses (for example, dengue virus, influenza virus, hepatitis virus, human immunodeficiency virus [HIV], hemorrhagic fever virus [HFV], human papillomavirus [HPV], West Nile virus, etc.) as well as distinct applications (comparative/diversity analysis, viral recombination, small interfering RNA [siRNA]/short hairpin RNA [shRNA]/microRNA [miRNA] studies, RNA folding, protein-protein interaction, structural analysis, and phylotyping and genotyping) will definitely aid the development of effective drugs and vaccines. However, information about their access and utility is not available at any single source or on any single platform. Therefore, a compendium of various computational tools and resources dedicated specifically to virology is presented in this article. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Human Respiratory Syncytial Virus and Human Metapneumovirus

OpenAIRE

Luciana Helena Antoniassi da Silva; Fernando Rosado Spilki; Adriana Gut Lopes Riccetto; Emilio Elias Baracat; Clarice Weis Arns

2009-01-01

The human respiratory syncytial virus (hRSV) and the human metapneumovírus (hMPV) are main etiological agents of acute respiratory infections (ARI). The ARI is an important cause of childhood morbidity and mortality worldwide.  hRSV and hMPV are members of the Paramyxoviridae. They are enveloped, non-segmented viruses, with negative-sense single stranded genomes. Respiratory syncytial virus (hRSV) is the best characterized agent viral of this group, associated with respiratory diseases in...

Efficacy of single dose of a bivalent vaccine containing inactivated Newcastle disease virus and reassortant highly pathogenic avian influenza H5N1 virus against lethal HPAI and NDV infection in chickens.

Directory of Open Access Journals (Sweden)

Dong-Hun Lee

Full Text Available Highly pathogenic avian influenza (HPAI and Newcastle disease (ND are 2 devastating diseases of poultry, which cause great economic losses to the poultry industry. In the present study, we developed a bivalent vaccine containing antigens of inactivated ND and reassortant HPAI H5N1 viruses as a candidate poultry vaccine, and we evaluated its immunogenicity and protective efficacy in specific pathogen-free chickens. The 6:2 reassortant H5N1 vaccine strain containing the surface genes of the A/Chicken/Korea/ES/2003(H5N1 virus was successfully generated by reverse genetics. A polybasic cleavage site of the hemagglutinin segment was replaced by a monobasic cleavage site. We characterized the reverse genetics-derived reassortant HPAI H5N1 clade 2.5 vaccine strain by evaluating its growth kinetics in eggs, minimum effective dose in chickens, and cross-clade immunogenicity against HPAI clade 1 and 2. The bivalent vaccine was prepared by emulsifying inactivated ND (La Sota strain and reassortant HPAI viruses with Montanide ISA 70 adjuvant. A single immunization with this vaccine induced high levels of hemagglutination-inhibiting antibody titers and protected chickens against a lethal challenge with the wild-type HPAI and ND viruses. Our results demonstrate that the bivalent, inactivated vaccine developed in this study is a promising approach for the control of both HPAI H5N1 and ND viral infections.

Deep sequencing of H7N8 avian influenza viruses from surveillance zone supports H7N8 high pathogenicity avian influenza was limited to a single outbreak farm in Indiana during 2016.

Science.gov (United States)

Lee, Dong-Hun; Torchetti, Mia Kim; Killian, Mary Lea; Swayne, David E

2017-07-01

In mid-January 2016, an outbreak of H7N8 high-pathogenicity avian influenza virus (HPAIV) in commercial turkeys occurred in Indiana. Surveillance within the 10km control zone identified H7N8 low-pathogenicity avian influenza virus (LPAIV) in nine surrounding turkey flocks but no other HPAIV-affected premises. We sequenced four of the H7N8 HPAIV isolated from the single farm and nine LPAIV identified during control zone surveillance. Evaluation included phylogenetic network analysis indicating close relatedness across the HPAIV and LPAIV, and that the progenitor H7N8 LPAIV spread among the affected turkey farms in Indiana, followed by spontaneous mutation to HPAIV on a single premise through acquisition of three basic amino acids at the hemagglutinin cleavage site. Deep sequencing of the available viruses failed to identify subpopulations in either the HPAIV or LPAIV suggesting mutation to HPAIV likely occurred on a single farm and the HPAIV did not spread to epidemiologically linked LPAIV-affected farms. Published by Elsevier Inc.

Electrostatics and the assembly of an RNA virus

NARCIS (Netherlands)

Schoot, van der P.P.A.M.; Bruinsma, R.

2005-01-01

Electrostatic interactions play a central role in the assembly of single-stranded RNA viruses. Under physiological conditions of salinity and acidity, virus capsid assembly requires the presence of genomic material that is oppositely charged to the core proteins. In this paper we apply basic polymer

A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

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Rui eZhang

2014-07-01

Full Text Available Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10 of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1. A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A tail. The genome possesses two non-overlapping open reading frames (ORFs: a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5'-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1. Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1and FgV1.

METODE PENAPISAN CABAI (CAPSICUM ANNUUM L. UNTUK KETAHANAN TERHADAP CHILLI VEINAL MOTTLE VIRUS (Chi VMV DAN CUCUMBER MOSAIC VIRUS (CMV

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Latifah, Sri Hendrastuti Hidayat, dan Sriani Sujiprihati .

2011-11-01

Full Text Available Screening Method for Chilli Veinal Mottle Virus  (Chi VMV and Cucumber Mosaic Virus  (CMV Resistance in Chillipepper.  ChiVMV and CMV have been reported as the causal agents of main diseases in chillipepper in Indonesia and other Asian countries.  Mix infection of this two viruses was commonly occurred in the field, causing severe disease .  The use of resistance varieties has been proposed for dealing with the yield losses causing by  the viruses.  Breeding program is undergoing for development of chillipepper varieties resistant to ChiVMV and CMV.  Methodology for routine screening activity of chillipepper for resistance to both ChiVMV and CMV needs to be established. This research was conducted in Cikabayan Glass House and Plant Virology Laboratory, Plant Protection Department, Bogor Agricultural University from May 2006 to June 2007. Aim of the research was to develop screening method for simultaneous infection by the two viruses, ChiVMV and CMV.  Inoculation of ChiVMV and CMV was done by single inoculation or repetitive inoculation methods.  In both methods, ChiVMV and CMV were inoculated in different sequences, either ChiVMV or CMV first.  The result showed that incubation period was shorter when CMV was inoculated in advance both in single and repetitive inoculation method.  Mosaic, mottle and malformation type symptom was observed in infected plants. Based on disease incidence, infection of ChiVMV was higher compared to CMV in repetitive inoculation as well as in single inoculation.  Repetitive inoculation methods with virus sequence ChiVMV-CMV-ChiVMV-CMV  was selected for resistance evaluation of chillipepper genotypes.

Photodynamic inactivation of rubella virus enhances recombination with a latent virus of a baby hamster kidney cell line BHK21

International Nuclear Information System (INIS)

Yamamoto, Nobuto; Urade, Masahiro

1989-01-01

Rubella virus is very sensitive to photodynamic action. When tested with 1.2 x 10 -5 M toluidine blue and 8 W fluorescent lamp at a fluence of 11 W/m 2 , inactivation kinetics showed a linear single hit curve with a k value of 1.48 min -1 . Photodynamic inactivation of rubella virus greatly enhanced recombination with a latent virus (R-virus) of baby hamster kidney BHK21 cells. In contrast, no hybrids were detected in lysates of the cells infected with either UV-treated or untreated rubella virus. Therefore, hybrid viruses were readily detected only in lysates of BHK21 cells infected with photodynamically treated rubella virus. Photodynamic damage of rubella virus genomes generated a new hybrid type (hybrid type 3) in addition to a previously described type 2 hybrid (formerly designated as HPV-RV variant). Although both of these hybrid types carry the CF antigens of rubella virus, plaque forming ability of type 3 hybrid is neutralized neither by anti-rubella serum nor by anti-latent virus serum while type 2 hybrid is neutralized by anti-latent virus serum. (author)

Complete genome sequence of pronghorn virus, a pestivirus

Science.gov (United States)

The complete genome sequence of Pronghorn virus, a member of the Pestivirus genus of the Flaviviridae, was determined. The virus, originally isolated from a pronghorn antelope, had a genome of 12,287 nucleotides with a single open reading frame of 11,694 bases encoding 3898 amino acids....

Single-particle cryo-electron microscopy of Rift Valley fever virus

International Nuclear Information System (INIS)

Sherman, Michael B.; Freiberg, Alexander N.; Holbrook, Michael R.; Watowich, Stanley J.

2009-01-01

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human and veterinary pathogen causing acute hepatitis in ruminants and has the potential to cause hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T = 12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit vaccines.

Single-particle cryo-electron microscopy of Rift Valley fever virus.

Science.gov (United States)

Sherman, Michael B; Freiberg, Alexander N; Holbrook, Michael R; Watowich, Stanley J

2009-04-25

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human and veterinary pathogen causing acute hepatitis in ruminants and has the potential to cause hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T=12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit vaccines.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

Directory of Open Access Journals (Sweden)

Qin E-de

2010-06-01

Full Text Available Abstract A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009 influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

Single particle tracking and single molecule energy transfer

CERN Document Server

Bräuchle, Christoph; Michaelis, Jens

2009-01-01

Closing a gap in the literature, this handbook gathers all the information on single particle tracking and single molecule energy transfer. It covers all aspects of this hot and modern topic, from detecting virus entry to membrane diffusion, and from protein folding using spFRET to coupled dye systems, as well recent achievements in the field. Throughout, the first-class editors and top international authors present content of the highest quality, making this a must-have for physical chemists, spectroscopists, molecular physicists and biochemists.

Yeast for virus research

Science.gov (United States)

Zhao, Richard Yuqi

2017-01-01

Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

Presence and Distribution of Economically Important Potato Viruses in Montenegro

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Jelena Zindović

2011-01-01

Full Text Available The research was carried out, in the period 2002-2004 in order to determine the presence and distribution of potato viruses at 12 different locations and on 9 different potato varieties grown in Montenegro. The research included collecting of samples in seed potato crops and testing of six economically important potato viruses: Potato leaf roll virus (PLRV, Potato virus Y (PVY, Potato virus X (PVX, Potato virus S (PVS, Potato virus A (PVA i Potato virus M (PVM. Using the direct enzyme-linked immunosorbent assay (DAS-ELISA and commercial antisera specific for six potato viruses, it was found that PVY was the most frequent virus during the three-year research period. The second frequent virus was PVS, followed by PVA, PLRV, PVM and PVX. Single and mixed infections were detected, and the most prevalent were the single infections of PVY. Also, in the period 2002-2004, PVY had the highest distribution and the number of present viruses was different at different localities and on different potato varieties. Further investigations were related to detailed characterization of the most prevalent virus (PVY, which is at the same time economically the most important one. Serological characterization of PVY was performed utilizing DAS-ELISA kit with commercial monoclonal antibodies specific for detection of the three strain groups of PVY, and the two straingroups - necrotic (PVYN/PVYNTN and common (PVYO, were identified. Necrotic strains were prevalent in 2002 and 2004, while in 2003 PVYO was the most frequent strain in virus population. The presence of stipple streak strain (PVYC was not detected in any of the testedsamples.

A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses.

Directory of Open Access Journals (Sweden)

Jeffrey W Koehler

Full Text Available For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III based on the protein sequence and structure. For Rift Valley fever virus (RVFV, the glycoprotein Gc (Class II fusion protein mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus, Class II (Andes virus, or Class III (vesicular stomatitis virus fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

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George G Daaboul

Full Text Available Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm, while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm. Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

Detection of hepatitis A virus by hybridization with single-stranded RNA probes

International Nuclear Information System (INIS)

Xi, J.; Estes, M.K.; Metcalf, T.G.

1987-01-01

An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32 P-labeled ssRNA probes were at least eightfold more sensitive than the 32 P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32 P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted an semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay

Transcriptional mapping of rabies virus in vivo

International Nuclear Information System (INIS)

Flamand, A.; Delagneau, J.F.

1978-01-01

Synthesis of the proteins of rabies virus was studied in hamster cell infected with uv-irradiated virus. The uv target size of genes L, N, M 1 , and M 2 was measured during primary transcription. Except for N, the target size of the remaining genes was considerably larger than that of their physical sizes. The data fit the hypothesis that four genes occupy a single transcriptional unit and that transcription of rabies virus proceeds in the order N, M 1 , M 2 , and L

Hepatitis C Virus and Hepatocellular Carcinoma

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Masao Omata

2013-01-01

Full Text Available Hepatitis C virus (HCV, a hepatotropic virus, is a single stranded-positive RNA virus of ~9,600 nt. length belonging to the Flaviviridae family. HCV infection causes acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC. It has been reported that HCV-coding proteins interact with host-cell factors that are involved in cell cycle regulation, transcriptional regulation, cell proliferation and apoptosis. Severe inflammation and advanced liver fibrosis in the liver background are also associated with the incidence of HCV-related HCC. In this review, we discuss the mechanism of hepatocarcinogenesis in HCV-related liver diseases.

Construction and Testing of orfA +/- FIV Reporter Viruses

Directory of Open Access Journals (Sweden)

Eric M. Poeschla

2012-01-01

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

Field investigation of Foot and Mouth Disease (FMD) virus infection ...

African Journals Online (AJOL)

Prof. Ogunji

Foot and Mouth Disease Virus (FMDV) is a non-enveloped, single stranded RNA virus ... continents of Asia, Africa, and some regions in the South America. .... FCT = Federal Capital Territory; NE = North East, NC = North Central; NW =.

Immunogenicity of UV-inactivated measles virus

International Nuclear Information System (INIS)

Zahorska, R.; Mazur, N.; Korbecki, M.

1978-01-01

By means of the antigen extinction limit test it was shown that a triple dose vaccination of guinea pigs with UV-inactivated measles virus gave better results, than a single dose vaccination which was proved by the very low immunogenicity index. For both vaccination schemes (single and triple) the immune response was only sligthly influenced by a change of dose from 10 5 to 10 6 HadU 50 /ml or by the addition of aluminum adjuvant. In the antigen extinction limit test the antibody levels were determined by two methods (HIT and NT) the results of which were statistically equivalent. The UV-inactivated measles virus was also found to induce hemolysis-inhibiting antibodies. (orig.) [de

Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single-stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva

International Nuclear Information System (INIS)

Valles, Steven M.; Oi, David H.; Becnel, James J.; Wetterer, James K.; LaPolla, John S.; Firth, Andrew E.

2016-01-01

We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1 nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species. - Highlights: • A new positive-strand RNA virus was discovered in the ant, Nylanderia fulva. • The Nylanderia fulva virus 1 genome was comprised of 10,881 nucleotides. • NfV-1 was detected in larval, pupal, queen and worker ants, but not eggs. • Replication of NfV-1 appeared to be limited to the larval stage.

Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single-stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva

Energy Technology Data Exchange (ETDEWEB)

Valles, Steven M., E-mail: steven.valles@ars.usda.gov [Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608 (United States); Oi, David H.; Becnel, James J. [Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608 (United States); Wetterer, James K. [Wilkes Honors College, Florida Atlantic University, 5353 Parkside Drive, Jupiter, FL 33458 (United States); LaPolla, John S. [Department of Biological Sciences, Towson University, 8000 York Road, Towson, MD 21252 (United States); Firth, Andrew E. [Department of Pathology, University of Cambridge, Cambridge CB2 1QP (United Kingdom)

2016-09-15

We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1 nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species. - Highlights: • A new positive-strand RNA virus was discovered in the ant, Nylanderia fulva. • The Nylanderia fulva virus 1 genome was comprised of 10,881 nucleotides. • NfV-1 was detected in larval, pupal, queen and worker ants, but not eggs. • Replication of NfV-1 appeared to be limited to the larval stage.

Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge.

Science.gov (United States)

Erasmus, Jesse H; Seymour, Robert L; Kaelber, Jason T; Kim, Dal Y; Leal, Grace; Sherman, Michael B; Frolov, Ilya; Chiu, Wah; Weaver, Scott C; Nasar, Farooq

2018-02-15

Most alphaviruses are mosquito borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. Recently, a host-restricted, mosquito-borne alphavirus, Eilat virus (EILV), was described with an inability to infect vertebrate cells based on defective attachment and/or entry, as well as a lack of genomic RNA replication. We investigated the utilization of EILV recombinant technology as a vaccine platform against eastern (EEEV) and Venezuelan equine encephalitis viruses (VEEV), two important pathogens of humans and domesticated animals. EILV chimeras containing structural proteins of EEEV or VEEV were engineered and successfully rescued in Aedes albopictus cells. Cryo-electron microscopy reconstructions at 8 and 11 Å of EILV/VEEV and EILV/EEEV, respectively, showed virion and glycoprotein spike structures similar to those of VEEV-TC83 and other alphaviruses. The chimeras were unable to replicate in vertebrate cell lines or in brains of newborn mice when injected intracranially. Histopathologic examinations of the brain tissues showed no evidence of pathological lesions and were indistinguishable from those of mock-infected animals. A single-dose immunization of either monovalent or multivalent EILV chimera(s) generated neutralizing antibody responses and protected animals against lethal challenge 70 days later. Lastly, a single dose of monovalent EILV chimeras generated protective responses as early as day 1 postvaccination and partial or complete protection by day 6. These data demonstrate the safety, immunogenicity, and efficacy of novel insect-specific EILV-based chimeras as potential EEEV and VEEV vaccines. IMPORTANCE Mostly in the last decade, insect-specific viruses have been discovered in several arbovirus families. However, most of these viruses are not well studied and largely have been ignored. We explored the use of the mosquito-specific alphavirus EILV as an alphavirus vaccine

Interrogating Host-virus Interactions and Elemental Transfer Using NanoSIMS

Science.gov (United States)

Pasulka, A.; Thamatrakoln, K.; Poulos, B.; Bidle, K. D.; Sullivan, M. B.; Orphan, V. J.

2016-02-01

Marine viruses (bacteriophage and eukaryotic viruses) impact microbial food webs by influencing microbial community structure, carbon and nutrient flow, and serving as agents of gene transfer. While the collective impact of viral activity has become more apparent over the last decade, there is a growing need for single-cell and single-virus level measurements of the associated carbon and nitrogen transfer, which ultimately shape the biogeochemical impact of viruses in the upper ocean. Stable isotopes have been used extensively for understanding trophic relationships and elemental cycling in marine food webs. While single-cell isotope approaches such as nanoscale secondary ion mass spectrometry (nanoSIMS) have been more readily used to study trophic interactions between microorganisms, isotopic enrichment in viruses has not been described. Here we used nanoSIMS to quantify the transfer of stable isotopes (13C and 15N) from host to individual viral particles in two distinct unicellular algal-virus model systems. These model systems represent a eukaryotic phytoplankton (Emiliania huxleyi strain CCMP374) and its 200nm coccolithovirus (EhV207), as well as a cyanobacterial phytoplankton (Synechococcus WH8101) and its 80nm virus (Syn1). Host cells were grown on labeled media for multiple generations, subjected to viral infection, and then viruses were harvested after lysis. In both cases, nanoSIMS measurements were able to detect 13C and 15N in the resulting viral particles significantly above the background noise. The isotopic enrichment in the viral particles mirrored that of the host. Through use of these laboratory model systems, we quantified the sensitivity (ion counts), spatial resolution, and reproducibility, including sources of methodological and biological variability, in stable isotope incorporation into viral particles. Our findings suggest that nanoSIMS can be successfully employed to directly probe virus-host interactions at the resolution of individual

Molecular structures of viruses from Raman optical activity

DEFF Research Database (Denmark)

Blanch, Ewan W.; Hecht, Lutz; Syme, Christopher D.

2002-01-01

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chira......A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity...... (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation...... and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible....

Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus.

Science.gov (United States)

Crowther, R A; Berriman, J A; Curran, W L; Allan, G M; Todd, D

2003-12-01

Circoviruses are small, nonenveloped icosahedral animal viruses characterized by circular single-stranded DNA genomes. Their genomes are the smallest possessed by animal viruses. Infections with circoviruses, which can lead to economically important diseases, frequently result in virus-induced damage to lymphoid tissue and immunosuppression. Within the family Circoviridae, different genera are distinguished by differences in genomic organization. Thus, Chicken anemia virus is in the genus Gyrovirus, while porcine circoviruses and Beak and feather disease virus belong to the genus CIRCOVIRUS: Little is known about the structures of circoviruses. Accordingly, we investigated the structures of these three viruses with a view to determining whether they are related. Three-dimensional maps computed from electron micrographs showed that all three viruses have a T=1 organization with capsids formed from 60 subunits. Porcine circovirus type 2 and beak and feather disease virus show similar capsid structures with flat pentameric morphological units, whereas chicken anemia virus has stikingly different protruding pentagonal trumpet-shaped units. It thus appears that the structures of viruses in the same genus are related but that those of viruses in different genera are unrelated.

Temperature and development of Zika virus infection: an Indonesian case

Institute of Scientific and Technical Information of China (English)

Ramadhan Tosepu

2017-01-01

Distribution of Zika virus has spread throughout the world. WHO has announced that Zika virus disease is a global health problem. As a tropical country, Indonesia has the potential to spread Zika virus. This letter is to report that there was a single case of zika in Jambi province published by the Eijkman Institute for Molecular Biology. This case was analyzed with the temperature in the scene, which is considered as an appropriate factor to development of Zika virus.

Imaging and manipulation of single viruses by atomic force microscopy

NARCIS (Netherlands)

Baclayon, M.; Wuite, G. J. L.; Roos, W. H.

2010-01-01

The recent developments in virus research and the application of functional viral particles in nanotechnology and medicine rely on sophisticated imaging and manipulation techniques at nanometre resolution in liquid, air and vacuum. Atomic force microscopy (AFM) is a tool that combines these

Analysis of single-nucleotide polymorphisms in the APOBEC3H gene of domestic cats (Felis catus) and their association with the susceptibility to feline immunodeficiency virus and feline leukemia virus infections.

Science.gov (United States)

de Castro, Fernanda Luz; Junqueira, Dennis Maletich; de Medeiros, Rúbia Marília; da Silva, Tailene Rabello; Costenaro, Jamile Girardi; Knak, Marcus Braga; de Matos Almeida, Sabrina Esteves; Campos, Fabrício Souza; Roehe, Paulo Michel; Franco, Ana Cláudia

2014-10-01

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are widely distributed retroviruses that infect domestic cats (Felis catus). Restriction factors are proteins that have the ability to hamper retroviruses' replication and are part of the conserved mechanisms of anti-viral immunity of mammals. The APOBEC3 protein family is the most studied class of restriction factors; they are cytidine deaminases that generate hypermutations in provirus DNA during reverse transcription, thus causing hypermutations in the viral genome, hindering virus replication. One of the feline APOBEC3 genes, named APOBEC3H, encodes two proteins (APOBEC3H and APOBEC3CH). In other mammals, APOBEC3H single-nucleotide polymorphisms (SNPs) can alter the stability and cellular localization of the encoded protein, thus influencing its subcellular localization and reducing its anti-viral effect. In cats, the association of APOBEC3H SNPs with susceptibility to retroviral infections was not yet demonstrated. Therefore, this study aimed the investigation on the variability of APOBEC3H and the possible association with FIV/FeLV infections. DNA obtained from whole blood of fifty FIV- and/or FeLV-infected cats and fifty-nine FIV- and/or FeLV-uninfected cats were used as templates to amplify two different regions of the APOBEC3H, with subsequent sequencing and analysis. The first region was highly conserved among all samples, while in the second, six single-nucleotide variation points were identified. One of the SNPs, A65S (A65I), was significantly correlated with the susceptibility to FIV and/or FeLV infections. On the other hand, the haplotype analysis showed that the combination "GGGGCC" was positively correlated with the lack of FIV and/or FeLV infections. Our results indicate that, as previously shown in other mammals, variability of restriction factors may contribute to susceptibility of domestic cats to retroviral infections; however, these results should be confirmed by more

How hepatitis D virus can hinder the control of hepatitis B virus.

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Maria Xiridou

Full Text Available BACKGROUND: Hepatitis D (or hepatitis delta virus is a defective virus that relies on hepatitis B virus (HBV for transmission; infection with hepatitis D can occur only as coinfection with HBV or superinfection of an existing HBV infection. Because of the bond between the two viruses, control measures for HBV may have also affected the spread of hepatitis D, as evidenced by the decline of hepatitis D in recent years. Since the presence of hepatitis D is associated with suppressed HBV replication and possibly infectivity, it is reasonable to speculate that hepatitis D may facilitate the control of HBV. METHODOLOGY AND PRINCIPAL FINDINGS: We introduced a mathematical model for the transmission of HBV and hepatitis D, where individuals with dual HBV and hepatitis D infection transmit both viruses. We calculated the reproduction numbers of single HBV infections and dual HBV and hepatitis D infections and examined the endemic prevalences of the two viruses. The results show that hepatitis D virus modulates not only the severity of the HBV epidemic, but also the impact of interventions for HBV. Surprisingly we find that the presence of hepatitis D virus may hamper the eradication of HBV. Interventions that aim to reduce the basic reproduction number of HBV below one may not be sufficient to eradicate the virus, as control of HBV depends also on the reproduction numbers of dual infections. CONCLUSIONS AND SIGNIFICANCE: For populations where hepatitis D is endemic, plans for control programs ignoring the presence of hepatitis D may underestimate the HBV epidemic and produce overoptimistic results. The current HBV surveillance should be augmented with monitoring of hepatitis D, in order to improve accuracy of the monitoring and the efficacy of control measures.

A Glimpse of the genomic diversity of haloarchaeal tailed viruses

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Ana eSencilo

2014-03-01

Full Text Available Tailed viruses are the most common isolates infecting prokaryotic hosts residing hypersaline environments. Archaeal tailed viruses represent only a small portion of all characterized tailed viruses of prokaryotes. But even this small dataset revealed that archaeal tailed viruses have many similarities to their counterparts infecting bacteria, the bacteriophages. Shared functional homologues and similar genome organizations suggested that all microbial tailed viruses have common virion architectural and assembly principles. Recent structural studies have provided evidence justifying this thereby grouping archaeal and bacterial tailed viruses into a single lineage. Currently there are 17 haloarchaeal tailed viruses with entirely sequenced genomes. Nine viruses have at least one close relative among the 17 viruses and, according to the similarities, can be divided into three groups. Two other viruses share some homologues and therefore are distantly related, whereas the rest of the viruses are rather divergent (or singletons. Comparative genomics analysis of these viruses offers a glimpse into the genetic diversity and structure of haloarchaeal tailed virus communities.

Automated high resolution full-field spatial coherence tomography for quantitative phase imaging of human red blood cells

Science.gov (United States)

Singla, Neeru; Dubey, Kavita; Srivastava, Vishal; Ahmad, Azeem; Mehta, D. S.

2018-02-01

We developed an automated high-resolution full-field spatial coherence tomography (FF-SCT) microscope for quantitative phase imaging that is based on the spatial, rather than the temporal, coherence gating. The Red and Green color laser light was used for finding the quantitative phase images of unstained human red blood cells (RBCs). This study uses morphological parameters of unstained RBCs phase images to distinguish between normal and infected cells. We recorded the single interferogram by a FF-SCT microscope for red and green color wavelength and average the two phase images to further reduced the noise artifacts. In order to characterize anemia infected from normal cells different morphological features were extracted and these features were used to train machine learning ensemble model to classify RBCs with high accuracy.

ICTV virus taxonomy profile

NARCIS (Netherlands)

García, María Laura; Bó, Dal Elena; Graça, da John V.; Gago-Zachert, Selma; Hammond, John; Moreno, Pedro; Natsuaki, Tomohide; Pallás, Vicente; Navarro, Jose A.; Reyes, Carina A.; Luna, Gabriel Robles; Sasaya, Takahide; Tzanetakis, Ioannis E.; Vaira, Anna María; Verbeek, Martin; Lefkowitz, Elliot J.; Davison, Andrew J.; Siddell, Stuart G.; Simmonds, Peter; Adams, Michael J.; Smith, Donald B.; Orton, Richard J.; Sanfaçon, Hélène

2017-01-01

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour

Disabled infectious single cycle-herpes simplex virus (DISC-HSV) as a vector for immunogene therapy of cancer.

Science.gov (United States)

Rees, Robert C; McArdle, Stephanie; Mian, Shahid; Li, Geng; Ahmad, Murrium; Parkinson, Richard; Ali, Selman A

2002-02-01

Disabled infectious single cycle-herpes simplex viruses (DISC-HSV) have been shown to be safe for use in humans and may be considered efficacious as vectors for immunogene therapy in cancer. Preclinical studies show that DISC-HSV is an efficient delivery system for cytokine genes and antigens. DISC-HSV infects a high proportion of cells, resulting in rapid gene expression for at least 72 h. The DISC-HSV-mGM-CSF vector, when inoculated into tumors, induces tumor regression in a high percentage of animals, concomitant with establishing a cytotoxic T-cell response, which is MHC class I restricted and directed against peptides of known tumor antigens. The inherent properties of DISC-HSV makes it a suitable vector for consideration in human immunogene therapy trials.

A Single-Amino-Acid Substitution at Position 225 in Hemagglutinin Alters the Transmissibility of Eurasian Avian-Like H1N1 Swine Influenza Virus in Guinea Pigs.

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Wang, Zeng; Yang, Huanliang; Chen, Yan; Tao, Shiyu; Liu, Liling; Kong, Huihui; Ma, Shujie; Meng, Fei; Suzuki, Yasuo; Qiao, Chuanling; Chen, Hualan

2017-11-01

Efficient transmission from human to human is the prerequisite for an influenza virus to cause a pandemic; however, the molecular determinants of influenza virus transmission are still largely unknown. In this study, we explored the molecular basis for transmission of Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses by comparing two viruses that are genetically similar but differ in their transmissibility in guinea pigs: the A/swine/Guangxi/18/2011 virus (GX/18) is highly transmissible by respiratory droplet in guinea pigs, whereas the A/swine/Heilongjiang/27/2012 virus (HLJ/27) does not transmit in this animal model. We used reverse genetics to generate a series of reassortants and mutants in the GX/18 background and tested their transmissibility in guinea pigs. We found that a single-amino-acid substitution of glycine (G) for glutamic acid (E) at position 225 (E225G) in the HA1 protein completely abolished the respiratory droplet transmission of GX/18, whereas the substitution of E for G at the same position (G225E) in HA1 enabled HLJ/27 to transmit in guinea pigs. We investigated the underlying mechanism and found that viruses bearing 225E in HA1 replicated more rapidly than viruses bearing 225G due to differences in assembly and budding efficiencies. Our study indicates that the amino acid 225E in HA1 plays a key role in EAH1N1 swine influenza virus transmission and provides important information for evaluating the pandemic potential of field influenza virus strains. IMPORTANCE Efficient transmission among humans is a prerequisite for a novel influenza virus to cause a human pandemic. Transmissibility of influenza viruses is a polygenic trait, and understanding the genetic determinants for transmissibility will provide useful insights for evaluating the pandemic potential of influenza viruses in the field. Several amino acids in the hemagglutinin (HA) protein of influenza viruses have been shown to be important for transmissibility, usually by

Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

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George P Anderson

Full Text Available Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

Astrovirology: Viruses at Large in the Universe.

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Berliner, Aaron J; Mochizuki, Tomohiro; Stedman, Kenneth M

2018-02-01

Viruses are the most abundant biological entities on modern Earth. They are highly diverse both in structure and genomic sequence, play critical roles in evolution, strongly influence terran biogeochemistry, and are believed to have played important roles in the origin and evolution of life. However, there is yet very little focus on viruses in astrobiology. Viruses arguably have coexisted with cellular life-forms since the earliest stages of life, may have been directly involved therein, and have profoundly influenced cellular evolution. Viruses are the only entities on modern Earth to use either RNA or DNA in both single- and double-stranded forms for their genetic material and thus may provide a model for the putative RNA-protein world. With this review, we hope to inspire integration of virus research into astrobiology and also point out pressing unanswered questions in astrovirology, particularly regarding the detection of virus biosignatures and whether viruses could be spread extraterrestrially. We present basic virology principles, an inclusive definition of viruses, review current virology research pertinent to astrobiology, and propose ideas for future astrovirology research foci. Key Words: Astrobiology-Virology-Biosignatures-Origin of life-Roadmap. Astrobiology 18, 207-223.

Lanthanum-Based Metal-Organic Frameworks for Specific Detection of Sudan Virus RNA Conservative Sequences down to Single-Base Mismatch.

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Yang, Shui-Ping; Zhao, Wei; Hu, Pei-Pei; Wu, Ke-Yang; Jiang, Zhi-Hong; Bai, Li-Ping; Li, Min-Min; Chen, Jin-Xiang

2017-12-18

Reactions of La(NO 3 ) 3 ·6H 2 O with the polar, tritopic quaternized carboxylate ligands N-carboxymethyl-3,5-dicarboxylpyridinium bromide (H 3 CmdcpBr) and N-(4-carboxybenzyl)-3,5-dicarboxylpyridinium bromide (H 3 CbdcpBr) afford two water-stable metal-organic frameworks (MOFs) of {[La 4 (Cmdcp) 6 (H 2 O) 9 ]} n (1, 3D) and {[La 2 (Cbdcp) 3 (H 2 O) 10 ]} n (2, 2D). MOFs 1 and 2 absorb the carboxyfluorescein (FAM)-tagged probe DNA (P-DNA) and quench the fluorescence of FAM via a photoinduced electron transfer (PET) process. The nonemissive P-DNA@MOF hybrids thus formed in turn function as sensing platforms to distinguish conservative linear, single-stranded RNA sequences of Sudan virus with high selectivity and low detection limits of 112 and 67 pM, respectively (at a signal-to-noise ratio of 3). These hybrids also exhibit high specificity and discriminate down to single-base mismatch RNA sequences.

Kinetics of single and dual infection of calves with an Asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus 1

DEFF Research Database (Denmark)

Larskaa, Magdalena; Polak, Mirosław P.; Riitho, Victor

2012-01-01

. Co-infection with both viruses led to prolonged fever in comparison to singlestrain inoculated groups and simultaneous replication of concurrent viruses in blood and in the upper respiratory tract. Following the infections all the calves seroconverted against homologous strains. Atypical pestiviruses......) and an Asianatypicalbovinepestivirus (Th/04_KhonKaen) in naïve calves, in comparison to singleinfections. Milder clinical signs were observed in the animals infected with single Th/04_KhonKaen strain. Leukocytopenia and lymphocytopenia were observed in all infected groups at a similar level which correlated with the onset of viraemia...... pose a serious threat to livestock health and BVDV eradication, since they may have the potential to be widely spread in cattle populations without being detected and differentiated from other BVDV infections....

Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses.

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Ekström, Jens-Ola; Habayeb, Mazen S; Srivastava, Vaibhav; Kieselbach, Thomas; Wingsle, Gunnar; Hultmark, Dan

2011-09-01

The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales. Copyright © 2011 Elsevier B.V. All rights reserved.

A multiplex PCR for detection of six viruses in ducks.

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Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

2017-10-01

In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10 2 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains

Science.gov (United States)

Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

2014-09-01

Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear-cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

Interaction between single-dose Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus vaccines on dually infected pigs.

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Park, Su-Jin; Seo, Hwi Won; Park, Changhoon; Chae, Chanhee

2014-06-01

The objective of this study was to determine the effects of Mycoplasma hyopneumoniae and/or porcine reproductive and respiratory syndrome virus (PRRSV) vaccination on dually infected pigs. In total, 72 pigs were randomly divided into nine groups (eight pigs per group), as follows: five vaccinated and challenged groups, three non-vaccinated and challenged groups, and a negative control group. Single-dose vaccination against M. hyopneumoniae alone decreased the levels of PRRSV viremia and PRRSV-induced pulmonary lesions, whereas single-dose vaccination against PRRSV alone did not decrease nasal shedding of M. hyopneumoniae and mycoplasma-induced pulmonary lesions in the dually infected pigs. The M. hyopneumoniae challenge impaired the protective cell-mediated immunity induced by the PRRSV vaccine, whereas the PRRSV challenge did not impair the protective cell-mediated immunity induced by the M. hyopneumoniae vaccine. The present study provides swine practitioners and producers with efficient vaccination regimes; vaccination against M. hyopneumoniae is the first step in protecting pigs against co-infection with M. hyopneumoniae and PRRSV. Copyright © 2014 Elsevier Ltd. All rights reserved.

Varicella-zoster virus infections in immunocompromised patients - a single centre 6-years analysis

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Liese Johannes

2011-05-01

Full Text Available Abstract Background Infection with varicella-zoster virus (VZV contemporaneously with malignant disease or immunosuppression represents a particular challenge and requires individualized decisions and treatment. Although the increasing use of varicella-vaccines in the general population and rapid initiation of VZV-immunoglobulins and acyclovir in case of exposure has been beneficial for some patients, immunocompromised individuals are still at risk for unfavourable courses. Methods In this single center, 6-year analysis we review incidence, hospitalization and complication rates of VZV-infections in our center and compare them to published data. Furthermore, we report three instructive cases. Results Hospitalization rate of referred children with VZV-infections was 45%, among these 17% with malignancies and 9% under immunosuppressive therapy. Rate of complications was not elevated in these two high-risk cohorts, but one ALL-patient died due to VZV-related complications. We report one 4-year old boy with initial diagnosis of acute lymphoblastic leukemia who showed a rapidly fatal outcome of his simultaneous varicella-infection, one 1.8-year old boy with an identical situation but a mild course of his disease, and an 8.5-year old boy with a steroid-dependent nephrotic syndrome. This boy developed severe hepatic involvement during his varicella-infection but responded to immediate withdrawl of steroids and administration of acyclovir plus single-dose cidofovir after nonresponse to acyclovir after 48 h. Conclusion Our data show that patients with malignant diseases or immunosuppressive therapy should be hospitalized and treated immediately with antiviral agents. Despite these measures the course of VZV-infections can be highly variable in these patients. We discuss aids to individual decision-making for these difficult situations.

Virus diseases of peppers (Capsicum spp.) and their control.

Science.gov (United States)

Kenyon, Lawrence; Kumar, Sanjeet; Tsai, Wen-Shi; Hughes, Jacqueline d'A

2014-01-01

The number of virus species infecting pepper (Capsicum spp.) crops and their incidences has increased considerably over the past 30 years, particularly in tropical and subtropical pepper production systems. This is probably due to a combination of factors, including the expansion and intensification of pepper cultivation in these regions, the increased volume and speed of global trade of fresh produce (including peppers) carrying viruses and vectors to new locations, and perhaps climate change expanding the geographic range suitable for the viruses and vectors. With the increased incidences of diverse virus species comes increased incidences of coinfection with two or more virus species in the same plant. There is then greater chance of synergistic interactions between virus species, increasing symptom severity and weakening host resistance, as well as the opportunity for genetic recombination and component exchange and a possible increase in aggressiveness, virulence, and transmissibility. The main virus groups infecting peppers are transmitted by aphids, whiteflies, or thrips, and a feature of many populations of these vector groups is that they can develop resistance to some of the commonly used insecticides relatively quickly. This, coupled with the increasing concern over the impact of over- or misuse of insecticides on the environment, growers, and consumers, means that there should be less reliance on insecticides to control the vectors of viruses infecting pepper crops. To improve the durability of pepper crop protection measures, there should be a shift away from the broadscale use of insecticides and the use of single, major gene resistance to viruses. Instead, integrated and pragmatic virus control measures should be sought that combine (1) cultural practices that reduce sources of virus inoculum and decrease the rate of spread of viruliferous vectors into the pepper crop, (2) synthetic insecticides, which should be used judiciously and only when the

Viruses as Sole Causative Agents of Severe Acute Respiratory Tract Infections in Children.

Science.gov (United States)

Moesker, Fleur M; van Kampen, Jeroen J A; van Rossum, Annemarie M C; de Hoog, Matthijs; Koopmans, Marion P G; Osterhaus, Albert D M E; Fraaij, Pieter L A

2016-01-01

Respiratory syncytial virus (RSV) and influenza A viruses are known to cause severe acute respiratory tract infections (SARIs) in children. For other viruses like human rhinoviruses (HRVs) this is less well established. Viral or bacterial co-infections are often considered essential for severe manifestations of these virus infections. The study aims at identifying viruses that may cause SARI in children in the absence of viral and bacterial co-infections, at identifying disease characteristics associated with these single virus infections, and at identifying a possible correlation between viral loads and disease severities. Between April 2007 and March 2012, we identified children (acute respiratory tract infection (ARTI) (controls). Data were extracted from the clinical and laboratory databases of our tertiary care paediatric hospital. Patient specimens were tested for fifteen respiratory viruses with real-time reverse transcriptase PCR assays and we selected patients with a single virus infection only. Typical bacterial co-infections were considered unlikely to have contributed to the PICU or MC admission based on C-reactive protein-levels or bacteriological test results if performed. We identified 44 patients admitted to PICU with SARI and 40 patients admitted to MC with ARTI. Twelve viruses were associated with SARI, ten of which were also associated with ARTI in the absence of typical bacterial and viral co-infections, with RSV and HRV being the most frequent causes. Viral loads were not different between PICU-SARI patients and MC-ARTI patients. Both SARI and ARTI may be caused by single viral pathogens in previously healthy children as well as in children with a medical history. No relationship between viral load and disease severity was identified.

Viruses and Multiple Sclerosis

Science.gov (United States)

Virtanen, Jussi Oskari; Jacobson, Steve

2016-01-01

Multiple sclerosis (MS) is a heterogeneous disease that develops as an interplay between the immune system and environmental stimuli in genetically susceptible individuals. There is increasing evidence that viruses may play a role in MS pathogenesis acting as these environmental triggers. However, it is not known if any single virus is causal, or rather several viruses can act as triggers in disease development. Here, we review the association of different viruses to MS with an emphasis on two herpesviruses, Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6). These two agents have generated the most impact during recent years as possible co-factors in MS disease development. The strongest argument for association of EBV with MS comes from the link between symptomatic infectious mononucleosis and MS and from seroepidemiological studies. In contrast to EBV, HHV-6 has been found significantly more often in MS plaques than in MS normal appearing white matter or non-MS brains and HHV-6 re-activation has been reported during MS clinical relapses. In this review we also suggest new strategies, including the development of new infectious animal models of MS and antiviral MS clinical trials, to elucidate roles of different viruses in the pathogenesis of this disease. Furthermore, we introduce the idea of using unbiased sequence-independent pathogen discovery methodologies, such as next generation sequencing, to study MS brain tissue or body fluids for detection of known viral sequences or potential novel viral agents. PMID:22583435

Virus fitness differences observed between two naturally occurring isolates of Ebola virus Makona variant using a reverse genetics approach.

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Albariño, César G; Guerrero, Lisa Wiggleton; Chakrabarti, Ayan K; Kainulainen, Markus H; Whitmer, Shannon L M; Welch, Stephen R; Nichol, Stuart T

2016-09-01

During the large outbreak of Ebola virus disease that occurred in Western Africa from late 2013 to early 2016, several hundred Ebola virus (EBOV) genomes have been sequenced and the virus genetic drift analyzed. In a previous report, we described an efficient reverse genetics system designed to generate recombinant EBOV based on a Makona variant isolate obtained in 2014. Using this system, we characterized the replication and fitness of 2 isolates of the Makona variant. These virus isolates are nearly identical at the genetic level, but have single amino acid differences in the VP30 and L proteins. The potential effects of these differences were tested using minigenomes and recombinant viruses. The results obtained with this approach are consistent with the role of VP30 and L as components of the EBOV RNA replication machinery. Moreover, the 2 isolates exhibited clear fitness differences in competitive growth assays. Published by Elsevier Inc.

Preliminary X-ray data analysis of crystalline hibiscus chlorotic ringspot virus

International Nuclear Information System (INIS)

Cheng, Ao; Speir, Jeffrey A.; Yuan, Y. Adam; Johnson, John E.; Wong, Sek-Man

2009-01-01

Hibiscus chlorotic ringspot virus is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family. Authentic virus harvested from infected host kenaf leaves was purified and virus crystals were grown in multiple conditions. One of the crystals diffracted to 3.2 Å resolution and allowed the collection of a partial data set. Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3.2 Å resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 Å. Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV

Virus Incidence of Sweet Potato in Korea from 2011 to 2014.

Science.gov (United States)

Kim, Jaedeok; Yang, Jung Wook; Kwak, Hae-Ryun; Kim, Mi-Kyeong; Seo, Jang-Kyun; Chung, Mi-Nam; Lee, Hyeong-Un; Lee, Kyeong-Bo; Nam, Sang Sik; Kim, Chang-Seok; Lee, Gwan-Seok; Kim, Jeong-Soo; Lee, Sukchan; Choi, Hong-Soo

2017-10-01

A nationwide survey was performed to investigate the current incidence of viral diseases in Korean sweet potatoes for germplasm and growing fields from 2011 to 2014. A total of 83.8% of the germplasm in Korea was infected with viruses in 2011. Commercial cultivars that were used to supply growing fields were infected at a rate of 62.1% in 2012. Among surveyed viruses, the incidence of five Potyvirus species that infect sweet potato decreased between 2012 and 2013, and then increased again in 2014. Representatively, the incidence of Sweet potato feathery mottle virus (SPFMV) was 87.0% in 2012, 20.7% in 2013 and then increased to 35.3% in 2014. Unlike RNA viruses, DNA viruses were shown to decrease continuously. The incidence of Sweet potato leaf curl virus (SPLCV) was 5.5% in 2003, 59.5% in 2011, and 47.4% in 2012. It then decreased continuously year by year to 33.2% in 2013, and then 25.6% in 2014. While the infection rate of each virus species showed a tendency to decline, the virus infection status was more variable in 2013 and 2014. Nevertheless, the high rate of single infections and mixed infection combinations were more variable than the survey results from 2012. As shown in the results from 2013, the most prevalent virus infection was a single infection at 27.6%, with the highest rate of infection belonging to sweet potato symptomless virus-1 (SPSMV-1) (12.9%). Compared to 2013, infection combinations were more varied in 2014, with a total of 122 kinds of mixed infection.

Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection.

Science.gov (United States)

De Paulis, Milena; Gilio, Alfredo Elias; Ferraro, Alexandre Archanjo; Ferronato, Angela Esposito; do Sacramento, Patrícia Rossi; Botosso, Viviane Fongaro; Oliveira, Danielle Bruna Leal de; Marinheiro, Juliana Cristina; Hársi, Charlotte Marianna; Durigon, Edison Luiz; Vieira, Sandra Elisabete

2011-01-01

To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.

Biology of human respiratory syncytial virus: a review | Aliyu | Bayero ...

African Journals Online (AJOL)

Acute lower respiratory tract infection is one of the major causes of mortality and morbidity in young children worldwide. Respiratory syncytial virus (RSV) is the single most important viral cause of lower respiratory tract infection during infancy and early childhood worldwide. Respiratory syncytial virus belongs to the ...

Novel RNA viruses within plant parasitic cyst nematodes.

Science.gov (United States)

Ruark, Casey L; Gardner, Michael; Mitchum, Melissa G; Davis, Eric L; Sit, Tim L

2018-01-01

The study of invertebrate-and particularly nematode-viruses is emerging with the advancement of transcriptome sequencing. Five single-stranded RNA viruses have now been confirmed within the economically important soybean cyst nematode (SCN; Heterodera glycines). From previous research, we know these viruses to be widespread in greenhouse and field populations of SCN. Several of the SCN viruses were also confirmed within clover (H. trifolii) and beet (H. schachtii) cyst nematodes. In the presented study, we sequenced the transcriptomes of several inbred SCN populations and identified two previously undiscovered viral-like genomes. Both of these proposed viruses are negative-sense RNA viruses and have been named SCN nyami-like virus (NLV) and SCN bunya-like virus (BLV). Finally, we analyzed publicly available transcriptome data of two potato cyst nematode (PCN) species, Globodera pallida and G. rostochiensis. From these data, a third potential virus was discovered and called PCN picorna-like virus (PLV). PCN PLV is a positive-sense RNA virus, and to the best of our knowledge, is the first virus described within PCN. The presence of these novel viruses was confirmed via qRT-PCR, endpoint PCR, and Sanger sequencing with the exception of PCN PLV due to quarantine restrictions on the nematode host. While much work needs to be done to understand the biological and evolutionary significance of these viruses, they offer insight into nematode ecology and the possibility of novel nematode management strategies.

A single site for N-linked glycosylation in the envelope glycoprotein of feline immunodeficiency virus modulates the virus-receptor interaction

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Samman Ayman

2008-08-01

Full Text Available Abstract Feline immunodeficiency virus (FIV targets helper T cells by attachment of the envelope glycoprotein (Env to CD134, a subsequent interaction with CXCR4 then facilitating the process of viral entry. As the CXCR4 binding site is not exposed until CD134-binding has occurred then the virus is protected from neutralising antibodies targeting the CXCR4-binding site on Env. Prototypic FIV vaccines based on the FL4 strain of FIV contain a cell culture-adapted strain of FIV Petaluma, a CD134-independent strain of FIV that interacts directly with CXCR4. In addition to a characteristic increase in charge in the V3 loop homologue of FIVFL4, we identified two mutations in potential sites for N-linked glycosylation in the region of FIV Env analogous to the V1–V2 region of HIV and SIV Env, T271I and N342Y. When these mutations were introduced into the primary GL8 and CPG41 strains of FIV, the T271I mutation was found to alter the nature of the virus-CD134 interaction; primary viruses carrying the T271I mutation no longer required determinants in cysteine-rich domain (CRD 2 of CD134 for viral entry. The T271I mutation did not confer CD134-independent infection upon GL8 or CPG41, nor did it increase the affinity of the CXCR4 interaction, suggesting that the principal effect was targeted at reducing the complexity of the Env-CD134 interaction.

Production of a phage-displayed single chain variable fragment ...

African Journals Online (AJOL)

Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

Data mining cDNAs reveals three new single stranded RNA viruses in Nasonia (Hymenopetera:Pteromalidae)

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Hymenopteran viruses may provide insights into colony collapse disorder in honey bees and other insect species. Three novel small RNA viruses were discovered during the genomics effort for the beneficial parasitoid of flies in the genus Nasonia (Hymenoptera). Genomics provides a great deal of inform...

A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo.

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Takamatsu, Yuki; Morita, Kouichi; Hayasaka, Daisuke

2015-06-01

We identified a unique amino acid of NS2A113, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

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Kaku Yoshihiro

2012-09-01

Full Text Available Abstract Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1. Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs. Findings Human single-fold scFv libraries (Tomlinson I + J underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA. After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display

Ebola Virus and Marburg Virus

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Ebola virus and Marburg virus Overview Ebola virus and Marburg virus are related viruses that cause hemorrhagic fevers — illnesses marked by severe bleeding (hemorrhage), organ failure and, in many ...

Chikungunya virus fusion properties elucidated by single-particle and bulk approaches

NARCIS (Netherlands)

van Duijl-Richter, Mareike K. S.; Blijleven, Jelle S.; van Oijen, Antoine M.; Smit, Jolanda M.

Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the

Singled out?

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Waller, Frank

2004-03-01

The increasing use of single use medical devices is being driven by a growing awareness of iatrogenic (from the Greek; caused by the doctor) and nosocomial infections. Public health perceptions relating to transmissible spongiform encephalopathies, specifically variant Creutzfeldt-Jakob disease (vCJD), the Human Immunodeficiency Virus (HIV) and Hepatitis B are high on the political agenda and a matter of concern to healthcare professionals.

Mutants of alfalfa mosaic virus

International Nuclear Information System (INIS)

Roosien, J.

1983-01-01

In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

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Davidson, Irit; Raibshtein, I; Al-Touri, A

2013-06-01

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

Identification of a novel virus in pigs--Bungowannah virus: a possible new species of pestivirus.

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Kirkland, P D; Frost, M J; Finlaison, D S; King, K R; Ridpath, J F; Gu, X

2007-10-01

In 2003 an outbreak of sudden deaths occurred in 3-4-week-old piglets on a farm in New South Wales, Australia. There was a marked increase in the birth of stillborn foetuses. Pathological changes consisted of a multifocal non-suppurative myocarditis. A viral infection was suspected but a wide range of known agents were excluded. A modified sequence independent single primer amplification (SISPA) method was used to identify a novel virus associated with this outbreak. Conserved 5'UTR motifs, the presence of a putative N(pro) coding region and limited antigenic cross-reactivity with other members of the Pestivirus genus, support the placement of this virus in the Pestivirus genus. Phylogenetic analysis of the 5'UTR, N(pro) and E2 coding regions showed this virus to be the most divergent pestivirus identified to date.

Coinfection with Epstein–Barr Virus (EBV, Human Papilloma Virus (HPV and Polyoma BK Virus (BKPyV in Laryngeal, Oropharyngeal and Oral Cavity Cancer

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Bartłomiej Drop

2017-12-01

Full Text Available Most research providing evidence for the role of oncogenic viruses in head and neck squamous cell carcinoma (SCC development is focused on one type of virus without analyzing possible interactions between two or more types of viruses. The aim of this study was to analyse the prevalence of co-infection with human papillomavirus (HPV, Epstein–Barr virus (EBV and polyoma BK virus (BKPyV in oral, oropharyngeal and laryngeal squamous cell carcinomas in Polish patients. The correlations between viral infection, SCC, demographic parameters, evidence of metastases and grading were also investigated. Fresh-frozen tumour tissue samples were collected from 146 patients with laryngeal, oropharyngeal and oral cancer. After DNA extraction, the DNA of the studied viruses was detected using polymerase chain rection (PCR assay. Males (87.7% with a history of smoking (70.6% and alcohol abuse (59.6% prevailed in the studied group. Histological type G2 was recognized in 64.4% cases. The patients were most frequently diagnosed with T2 stage (36.3% and with N1 stage (45.8%. Infection with at least two viruses was detected in 56.2% of patients. In this group, co-infection with HPV/EBV was identified in 34.1% of cases, EBV/BKV in 23.2%, HPV/BKV in 22.0%, and HPV/EBV/BKV in 20.7%. No difference of multiple infection in different locations of cancer was observed. The prevalence of poorly differentiated tumours (G3 was more frequent in co-infection with all three viruses than EBV or BKV alone. A significant correlation was observed between tumour dimensions (T and lymph-node involvement (N in co-infected patients compared to single infection. Further studies are necessary to clarify whether co-infection plays an important role in the initiation and/or progression of oncogenic transformation of oral, oropharyngeal and laryngeal epithelial cells.

Coinfection with Epstein-Barr Virus (EBV), Human Papilloma Virus (HPV) and Polyoma BK Virus (BKPyV) in Laryngeal, Oropharyngeal and Oral Cavity Cancer.

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Drop, Bartłomiej; Strycharz-Dudziak, Małgorzata; Kliszczewska, Ewa; Polz-Dacewicz, Małgorzata

2017-12-19

Most research providing evidence for the role of oncogenic viruses in head and neck squamous cell carcinoma (SCC) development is focused on one type of virus without analyzing possible interactions between two or more types of viruses. The aim of this study was to analyse the prevalence of co-infection with human papillomavirus (HPV), Epstein-Barr virus (EBV) and polyoma BK virus (BKPyV) in oral, oropharyngeal and laryngeal squamous cell carcinomas in Polish patients. The correlations between viral infection, SCC, demographic parameters, evidence of metastases and grading were also investigated. Fresh-frozen tumour tissue samples were collected from 146 patients with laryngeal, oropharyngeal and oral cancer. After DNA extraction, the DNA of the studied viruses was detected using polymerase chain rection (PCR) assay. Males (87.7%) with a history of smoking (70.6%) and alcohol abuse (59.6%) prevailed in the studied group. Histological type G2 was recognized in 64.4% cases. The patients were most frequently diagnosed with T2 stage (36.3%) and with N1 stage (45.8%). Infection with at least two viruses was detected in 56.2% of patients. In this group, co-infection with HPV/EBV was identified in 34.1% of cases, EBV/BKV in 23.2%, HPV/BKV in 22.0%, and HPV/EBV/BKV in 20.7%. No difference of multiple infection in different locations of cancer was observed. The prevalence of poorly differentiated tumours (G3) was more frequent in co-infection with all three viruses than EBV or BKV alone. A significant correlation was observed between tumour dimensions (T) and lymph-node involvement (N) in co-infected patients compared to single infection. Further studies are necessary to clarify whether co-infection plays an important role in the initiation and/or progression of oncogenic transformation of oral, oropharyngeal and laryngeal epithelial cells.

Coinfection with Epstein–Barr Virus (EBV), Human Papilloma Virus (HPV) and Polyoma BK Virus (BKPyV) in Laryngeal, Oropharyngeal and Oral Cavity Cancer

Science.gov (United States)

Drop, Bartłomiej; Strycharz-Dudziak, Małgorzata; Kliszczewska, Ewa; Polz-Dacewicz, Małgorzata

2017-01-01

Most research providing evidence for the role of oncogenic viruses in head and neck squamous cell carcinoma (SCC) development is focused on one type of virus without analyzing possible interactions between two or more types of viruses. The aim of this study was to analyse the prevalence of co-infection with human papillomavirus (HPV), Epstein–Barr virus (EBV) and polyoma BK virus (BKPyV) in oral, oropharyngeal and laryngeal squamous cell carcinomas in Polish patients. The correlations between viral infection, SCC, demographic parameters, evidence of metastases and grading were also investigated. Fresh-frozen tumour tissue samples were collected from 146 patients with laryngeal, oropharyngeal and oral cancer. After DNA extraction, the DNA of the studied viruses was detected using polymerase chain rection (PCR) assay. Males (87.7%) with a history of smoking (70.6%) and alcohol abuse (59.6%) prevailed in the studied group. Histological type G2 was recognized in 64.4% cases. The patients were most frequently diagnosed with T2 stage (36.3%) and with N1 stage (45.8%). Infection with at least two viruses was detected in 56.2% of patients. In this group, co-infection with HPV/EBV was identified in 34.1% of cases, EBV/BKV in 23.2%, HPV/BKV in 22.0%, and HPV/EBV/BKV in 20.7%. No difference of multiple infection in different locations of cancer was observed. The prevalence of poorly differentiated tumours (G3) was more frequent in co-infection with all three viruses than EBV or BKV alone. A significant correlation was observed between tumour dimensions (T) and lymph-node involvement (N) in co-infected patients compared to single infection. Further studies are necessary to clarify whether co-infection plays an important role in the initiation and/or progression of oncogenic transformation of oral, oropharyngeal and laryngeal epithelial cells. PMID:29257122

The Mutational Robustness of Influenza A Virus.

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Elisa Visher

2016-08-01

Full Text Available A virus' mutational robustness is described in terms of the strength and distribution of the mutational fitness effects, or MFE. The distribution of MFE is central to many questions in evolutionary theory and is a key parameter in models of molecular evolution. Here we define the mutational fitness effects in influenza A virus by generating 128 viruses, each with a single nucleotide mutation. In contrast to mutational scanning approaches, this strategy allowed us to unambiguously assign fitness values to individual mutations. The presence of each desired mutation and the absence of additional mutations were verified by next generation sequencing of each stock. A mutation was considered lethal only after we failed to rescue virus in three independent transfections. We measured the fitness of each viable mutant relative to the wild type by quantitative RT-PCR following direct competition on A549 cells. We found that 31.6% of the mutations in the genome-wide dataset were lethal and that the lethal fraction did not differ appreciably between the HA- and NA-encoding segments and the rest of the genome. Of the viable mutants, the fitness mean and standard deviation were 0.80 and 0.22 in the genome-wide dataset and best modeled as a beta distribution. The fitness impact of mutation was marginally lower in the segments coding for HA and NA (0.88 ± 0.16 than in the other 6 segments (0.78 ± 0.24, and their respective beta distributions had slightly different shape parameters. The results for influenza A virus are remarkably similar to our own analysis of CirSeq-derived fitness values from poliovirus and previously published data from other small, single stranded DNA and RNA viruses. These data suggest that genome size, and not nucleic acid type or mode of replication, is the main determinant of viral mutational fitness effects.

Infection of Mosquito Cells (C6/36) by Dengue-2 Virus Interferes with Subsequent Infection by Yellow Fever Virus.

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Abrao, Emiliana Pereira; da Fonseca, Benedito Antônio Lopes

2016-02-01

Dengue is one of the most important diseases caused by arboviruses in the world. Yellow fever is another arthropod-borne disease of great importance to public health that is endemic to tropical regions of Africa and the Americas. Both yellow fever and dengue viruses are flaviviruses transmitted by Aedes aegypti mosquitoes, and then, it is reasonable to consider that in a given moment, mosquito cells could be coinfected by both viruses. Therefore, we decided to evaluate if sequential infections of dengue and yellow fever viruses (and vice-versa) in mosquito cells could affect the virus replication patterns. Using immunofluorescence and real-time PCR-based replication assays in Aedes albopictus C6/36 cells with single or sequential infections with both viruses, we demonstrated the occurrence of viral interference, also called superinfection exclusion, between these two viruses. Our results show that this interference pattern is particularly evident when cells were first infected with dengue virus and subsequently with yellow fever virus (YFV). Reduction in dengue virus replication, although to a lower extent, was also observed when C6/36 cells were initially infected with YFV followed by dengue virus infection. Although the importance that these findings have on nature is unknown, this study provides evidence, at the cellular level, of the occurrence of replication interference between dengue and yellow fever viruses and raises the question if superinfection exclusion could be a possible explanation, at least partially, for the reported lack of urban yellow fever occurrence in regions where a high level of dengue transmission occurs.

Isolation and characterization of subgenomic DNAs encapsidated in 'single' T = 1 isometric particles of Maize streak virus

International Nuclear Information System (INIS)

Casado, Carolina G.; Javier Ortiz, G.; Padron, Eric; Bean, Samantha J.; McKenna, Robert; Agbandje-McKenna, Mavis; Boulton, Margaret I.

2004-01-01

'Single' T = 1 isometric particles of Maize streak virus (MSV) have been isolated from infected maize leaves. Biochemical and genetic characterizations show that these particles contain subgenomic (sg) MSV DNA encapsidated by the MSV coat protein. The largest sg DNA is 1.56 kb, slightly larger than half genome size, although sg DNAs as small as 0.2 kb were also cloned. The sg DNAs are not infectious, and they do not appear to play a role in the pathogenicity of MSV. This is the first report of sg DNAs for MSV and, to our knowledge, the first time that encapsidated sg DNAs have been characterized at the sequence level for any geminivirus. These data will assist in our investigations into the role of genomic DNA in the formation of the unique geminate capsid architecture of the Geminiviridae

Zika virus: a new arboviral public health problem.

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Demir, Tulin; Kilic, Selcuk

2016-11-01

Zika virus (ZIKV) is a single-stranded RNA virus in the Flaviviridae family and transmitted to human through infected mosquitos (Aedes aegypti and Aedes albopictus). Virus is closely related with other flaviviruses; dengue virus, yellow fever virus, West Nile virus, and Japanese encephalitis virus phylogenetically. Due to the possible relationship between virus and clinical features including microcephaly, ventricule, and eye deformities, Guillain-Barre syndrome increases the interest on this virus gradually. Along with the vector-borne transmission, exposure via blood transfusion and sexual contact are further concerns. Since December 2015, CDC reported 440.000-1.300.000 possible cases in Brazil and as of 19 January 2016, El Salvador, Venezuela, Colombia, Brazil, Surinam, French Guana, Honduras, Mexico, and Panama are the countries with active epidemic. CDC recommends ZIKV screening for all pregnants including asymptomatic cases those living in the active epidemic areas. Recently, virus is detected in the USA and most European countries including UK, Netherlands, Denmark, Switzerland, and Italy as a travel-associated infection. Owing to the changing world with increased capabilities for transportation globally, this vector-borne infection represents a valuable marker for the ability of spreading of any infection from its original area that it was first seen. In this review, we summarized the up-to-date data and reports in terms of the importance of the ZIKV infection in the public health.

A virus-based single-enzyme nanoreactor

NARCIS (Netherlands)

Comellas Aragones, M.; Engelkamp, H.; Claessen, V.I.; Sommerdijk, N.A.J.M.; Rowan, A.E.; Christianen, P.C.M.; Maan, J.C.; Verduin, B.J.M.; Cornelissen, J.J.L.M.; Nolte, R.J.M.

2007-01-01

Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or

ACVP-05: Virus Genetic Analysis from Cell-Free Plasma, Virally Infected Cells or Tissues and Cultured Supernatant Via Single Genome Amplification and Direct Sequencing | Frederick National Laboratory for Cancer Research

Science.gov (United States)

The Viral Evolution Core within the AIDS and Cancer Virus Program will extract viral RNA/DNA from cell-free or cell-associated samples. Complementary (cDNA) will be generated as needed, and cDNA or DNA will be diluted to a single copy prior to nested

The condensation of water on adsorbed viruses.

Science.gov (United States)

Alonso, José María; Tatti, Francesco; Chuvilin, Andrey; Mam, Keriya; Ondarçuhu, Thierry; Bittner, Alexander M

2013-11-26

The wetting and dewetting behavior of biological nanostructures and to a greater degree single molecules is not well-known even though their contact with water is the basis for all biology. Here, we show that environmental electron microscopy (EM) can be applied as a means of imaging the condensation of water onto viruses. We captured the formation of submicrometer water droplets and filaments on single viral particles by environmental EM and by environmental transmission EM. The condensate structures are compatible with capillary condensation between adsorbed virus particles and with known droplet shapes on patterned surfaces. Our results confirm that such droplets exist down to condensation/evaporation cycle as expected from their stability in air and water. Moreover we developed procedures that overcome problems of beam damage and of resolving structures with a low atomic number.

Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication.

Science.gov (United States)

Kjær, Jonas; Belsham, Graham J

2018-04-15

Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed "ribosome skipping" or "StopGo." Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E 14 , S 15 , and N 16 within the 2A sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P 17 , G 18 , or P 19 , which displayed little or no "cleavage" activity in vitro , were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E 14 , S 15 , N 16 , and P 19 resulted in partial "cleavage" of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational "cleavage." Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at

Single-Domain Antibodies As Therapeutics against Human Viral Diseases

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Yanling Wu

2017-12-01

Full Text Available In full-size formats, monoclonal antibodies have been highly successful as therapeutics against cancer and immune diseases. However, their large size leads to inaccessibility of some epitopes and relatively high production costs. As an alternative, single-domain antibodies (sdAbs offer special advantages compared to full-size antibodies, including smaller size, larger number of accessible epitopes, relatively low production costs and improved robustness. Currently, sdAbs are being developed against a number of viruses, including human immunodeficiency virus-1 (HIV-1, influenza viruses, hepatitis C virus (HCV, respiratory syncytial virus (RSV, and enteric viruses. Although sdAbs are very potent inhibitors of viral infections, no sdAbs have been approved for clinical use against virial infection or any other diseases. In this review, we discuss the current state of research on sdAbs against viruses and their potential as therapeutics against human viral diseases.

Nucleotide sequence and taxonomy of Cycas necrotic stunt virus. Brief report.

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Han, S S; Karasev, A V; Ieki, H; Iwanami, T

2002-11-01

Cycas necrotic stunt virus (CNSV) is the only well-characterized virus from gymnosperm. cDNA segments corresponding to the bipartite genome RNAs (RNA1, RNA2) were synthesized and sequenced. Each RNA encoded a single polyprotein, flanked by the 5' and 3' non-coding regions (NCR) and followed by a poly (A) tail. The putative polyproteins encoded by RNA1 and RNA2 had sets of motifs, which were characteristic of viruses in the genus Nepovirus. The polyproteins showed higher sequence identities to Artichoke Italian latent virus, Grapevine chrome mosaic virus and Tomato black ring virus, all of which belong to subgroup b of the genus Nepovirus, than to other nepoviruses. Phylogenetic analysis of RNA dependent RNA polymerase and coat protein also showed closer relationships with these viruses than other viruses. The data obtained supported the taxonomical status of CNSV as a definitive member of the genus Nepovirus, subgroup b.

Human Respiratory Syncytial Virus and Human Metapneumovirus

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Luciana Helena Antoniassi da Silva

2009-08-01

Full Text Available The human respiratory syncytial virus (hRSV and the human metapneumovírus (hMPV are main etiological agents of acute respiratory infections (ARI. The ARI is an important cause of childhood morbidity and mortality worldwide.  hRSV and hMPV are members of the Paramyxoviridae. They are enveloped, non-segmented viruses, with negative-sense single stranded genomes. Respiratory syncytial virus (hRSV is the best characterized agent viral of this group, associated with respiratory diseases in lower respiratory tract. Recently, a new human pathogen belonging to the subfamily Pneumovirinae was identified, the human metapneumovirus (hMPV, which is structurally similar to the hRSV, in genomic organization, viral structure, antigenicity and clinical symptoms.  The subfamily Pneumovirinae contains two genera: genus Pneumovirus contains hRSV, the bovine (bRSV, as well as the ovine and caprine respiratory syncytial virus and pneumonia virus of mice, the second genus Metapneumovirus, consists of avian metapneumovirus (aMPV and human metapneumovirus (hMPV. In this work, we present a brief narrative review of the literature on important aspects of the biology, epidemiology and clinical manifestations of infections by two respiratory viruses.

Some points of the X-ray pattern of acute viral primary pneumonia caused by acute respiratory disease viruses

International Nuclear Information System (INIS)

Stoyanov, V.

1991-01-01

An analysis is made of the results of the X-ray studies as well as of the virological and serological tests in 225 out-patients consulted in the first days of their complaints. A predominance of the viral (70.2%) over the viral-bacterial primary pneumonia is established. The acute viral primary pneumonia are caused mostly by single influenza viruses and more rarely - by single respiratory viruses; in the cases of combined influenza viruses influenza-influenza viruses prevail over the influenza-respiratory ones. The morphological changes in pneumonia due to isolated single influenza viruses involve mostly the interstitium and are projected on X-ray as patchy and stripped densities. The inflamatory changes in pneumonia caused by combined influenza viruses affect both ihe interstitium and the broncho-alveolar substrate of the lungs; they are manifested in two roentgenologic forms: creeping (migrating) and fusing (confluent). In viral-bacterial pneumonia the changes affect mostly the lobe. The right lung and the lower parts of the both lungs are affected in most cases. 5 figs., 21 refs

T T virus in chronic hepatitis B, C patients

International Nuclear Information System (INIS)

Abd Rabo, L.A.

2007-01-01

In 1997, in Japan, a non-enveloped single stranded circular DNA virus was recovered from a patient. Who developed post transfusion hepatitis not related to any of the know hepatitis viruses . The virus owes its name (T T) virus to the initials of the patient in whom the virus was first identified . Although this acronym might also stand for transfusion - transmitted virus, however, this name would emphasize only one, and certainly not the most frequent mode of this virus transmission. The taxonomy of the virus is uncertain but it is believed now that it may belong to a new family called paracircoviridae. TTDNA has been detected in liver, bone marrow, lung, spleen, pancreas, kidney, lymph nodes, skeletal muscles and thyroid gland as well as in saliva, tear, stool, bile, throat swabs, breast milk and semen while could not be detected in urine and sweat. TTV infection is transmitted parenterally by feco-oral or droplet routes, or sexual intercourse. These properties of virus influence its high prevalence in general population whether intrauterine transmission of virus is possible remains uncertain. The aim of this work is viewing the prevalence of TTV infection, mode of transmission, pathogenicity, diagnosis and management among chronic hepatitis B and C patients and control group

Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus

DEFF Research Database (Denmark)

Martinez-Torrecuadrada, Jorge L.; Cortes, Elena; Vela, Carmen

1998-01-01

Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal...

Clinical Features and Outcome of Ebola Virus Disease in Pediatric Patients

DEFF Research Database (Denmark)

Damkjær, Mads; Rudolf, Frauke; Mishra, Sharmistha

2016-01-01

Clinical and outcome data on pediatric Ebola virus disease are limited. We report a case-series of 33 pediatric patients with Ebola virus disease in a single Ebola Treatment Center in 2014-2015. The case-fatality rate was 42%, with the majority of deaths occurring within 10 days of admission....

Overlooking the smallest matter: viruses impact biological invasions.

Science.gov (United States)

Faillace, Cara A; Lorusso, Nicholas S; Duffy, Siobain

2017-04-01

Parasites and pathogens have recently received considerable attention for their ability to affect biological invasions, however, researchers have largely overlooked the distinct role of viruses afforded by their unique ability to rapidly mutate and adapt to new hosts. With high mutation and genomic substitution rates, RNA and single-stranded DNA (ssDNA) viruses may be important constituents of invaded ecosystems, and could potentially behave quite differently from other pathogens. We review evidence suggesting that rapidly evolving viruses impact invasion dynamics in three key ways: (1) Rapidly evolving viruses may prevent exotic species from establishing self-sustaining populations. (2) Viruses can cause population collapses of exotic species in the introduced range. (3) Viruses can alter the consequences of biological invasions by causing population collapses and extinctions of native species. The ubiquity and frequent host shifting of viruses make their ability to influence invasion events likely. Eludicating the viral ecology of biological invasions will lead to an improved understanding of the causes and consequences of invasions, particularly as regards establishment success and changes to community structure that cannot be explained by direct interspecific interactions among native and exotic species. © 2017 John Wiley & Sons Ltd/CNRS.

ICTV virus taxonomy profile: Baculoviridae

Science.gov (United States)

The Baculoviridae is a family of large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. Virions consist of enveloped rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15 to 15 µm. The occlusion bodies consist of a matrix composed of a single viral...

Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses

Directory of Open Access Journals (Sweden)

Wahyu eWulan

2015-06-01

Full Text Available Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs.

The molecular epidemiology of respiratory viruses associated with asthma attacks: A single-center observational study in Japan.

Science.gov (United States)

Saraya, Takeshi; Kimura, Hirokazu; Kurai, Daisuke; Ishii, Haruyuki; Takizawa, Hajime

2017-10-01

Few reports have described the significance of viral respiratory infections (VRIs) in exacerbation of asthma in adult patients. The aim of this study was to elucidate the profiles of VRIs in adult patients with asthma along with their molecular epidemiology.A cross-sectional observational study was conducted at Kyorin University Hospital from August 2012 to May 2015. To identify respiratory pathogens in inpatients and outpatients suffering from asthma attacks, RT-PCR/sequencing/phylogenetic analysis methods were applied alongside conventional microbiological methods. Phylogenetic and pairwise distance analyses of 10 viruses were performed.A total of 106 asthma attack patients enrolled in this study in both inpatient (n = 49) and outpatient (n = 57) settings. The total 106 respiratory samples were obtained from nasopharyngeal swab (n = 68) or sputum (n = 38). Among these, patients with virus alone (n = 39), virus and bacterial (n = 5), and bacterial alone (n = 5) were identified. The ratio of virus-positive patients in inpatient or outpatient to the total cases were 31.1% (n = 33) and 10.4% (n = 11), respectively. The frequency of virus-positive patients was significantly higher in inpatients (75.3%, n = 33) than in outpatients (19.3%, n = 11). Major VRIs included human rhinovirus (HRV) (n = 24), human metapneumovirus (hMPV) (n = 9), influenza virus (Inf-V) (n = 8), and respiratory syncytial virus (RSV) (n = 3) infections with seasonal variations. HRV-A and HRV-C were the most commonly detected viruses, with wide genetic divergence on phylogenetic analysis.Asthmatic exacerbations in adults are highly associated with VRIs such as HRV-A or HRV-C, hMPV, RSV, and Inf-V infections with seasonal variations and genetic divergence, but similar frequencies of VRIs occurred in asthma attack patients throughout the seasons.

Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

International Nuclear Information System (INIS)

Wanitchang, Asawin; Wongthida, Phonphimon; Jongkaewwattana, Anan

2016-01-01

The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.

Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

Energy Technology Data Exchange (ETDEWEB)

Wanitchang, Asawin; Wongthida, Phonphimon; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

2016-11-15

The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.

Proteolytic processing of the primary translation products of cowpea mosaic virus RNAs

NARCIS (Netherlands)

Franssen, H.

1984-01-01

Cowpea mosaic virus (CPMV) is the type member of a group of plant viruses, the comoviruses, with a genome consisting of two single stranded RNA molecules separately encapsidated in icosahedral particles. A characteristic feature of the two genome RNAs is that they are both polyadenylated at their

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper.

Science.gov (United States)

Almási, Asztéria; Nemes, Katalin; Csömör, Zsófia; Tóbiás, István; Palkovics, László; Salánki, Katalin

2017-06-01

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.

Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of ebola virus.

Science.gov (United States)

Hartman, Amy L; Bird, Brian H; Towner, Jonathan S; Antoniadou, Zoi-Anna; Zaki, Sherif R; Nichol, Stuart T

2008-03-01

Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.

Microscale memory characteristics of virus-quantum dot hybrids

Science.gov (United States)

Portney, Nathaniel G.; Tseng, Ricky J.; Destito, Giuseppe; Strable, Erica; Yang, Yang; Manchester, Marianne; Finn, M. G.; Ozkan, Mihrimah

2007-05-01

An electrical multi stability effect was observed for a single layer device fabricated, comprising a hybrid virus-semiconducting quantum dot (CdSe /ZnS core/shell Qds) assembled onto icosahedral-mutant-virus template (CPMV-T184C). A substrate based bottom-up pathway was used to conjugate two different color emitting Qds for fluorescence visualization and to insert a charging/decharging factor. Pulsed wave measurements depicted distinct conductive states with repeatable and nonvolatile behavior as a functioning memory element.

Recombination in hepatitis C virus.

Science.gov (United States)

González-Candelas, Fernando; López-Labrador, F Xavier; Bracho, María Alma

2011-10-01

Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus.

Virus-Clip: a fast and memory-efficient viral integration site detection tool at single-base resolution with annotation capability.

Science.gov (United States)

Ho, Daniel W H; Sze, Karen M F; Ng, Irene O L

2015-08-28

Viral integration into the human genome upon infection is an important risk factor for various human malignancies. We developed viral integration site detection tool called Virus-Clip, which makes use of information extracted from soft-clipped sequencing reads to identify exact positions of human and virus breakpoints of integration events. With initial read alignment to virus reference genome and streamlined procedures, Virus-Clip delivers a simple, fast and memory-efficient solution to viral integration site detection. Moreover, it can also automatically annotate the integration events with the corresponding affected human genes. Virus-Clip has been verified using whole-transcriptome sequencing data and its detection was validated to have satisfactory sensitivity and specificity. Marked advancement in performance was detected, compared to existing tools. It is applicable to versatile types of data including whole-genome sequencing, whole-transcriptome sequencing, and targeted sequencing. Virus-Clip is available at http://web.hku.hk/~dwhho/Virus-Clip.zip.

Efficacy of Vesicular Stomatitis Virus-Ebola Virus Postexposure Treatment in Rhesus Macaques Infected With Ebola Virus Makona.

Science.gov (United States)

Marzi, Andrea; Hanley, Patrick W; Haddock, Elaine; Martellaro, Cynthia; Kobinger, Gary; Feldmann, Heinz

2016-10-15

The Ebola virus (EBOV) epidemic in West Africa increased the focus on vaccine development against this hemorrhagic fever-causing pathogen, and as a consequence human clinical trials for a few selected platforms were accelerated. One of these vaccines is vesicular stomatitis virus (VSV)-EBOV, also known as rVSV-ZEBOV, a fast-acting vaccine against EBOV and so far the only vaccine with reported efficacy against EBOV infections in humans in phase III clinical trials. In this study, we analyzed the potential of VSV-EBOV for postexposure treatment of rhesus macaques infected with EBOV-Makona. We treated groups of animals with 1 dose of VSV-EBOV either in a single injection at 1 or 24 hours after EBOV exposure or with 2 injections, half the dose at each time point; 1 control group received the same dose of the VSV-based Marburg virus vaccine at both time points; another group remained untreated. Although all untreated animals succumbed to EBOV infection, 33%-67% of the animals in each treatment group survived the infection, including the group treated with the VSV-based Marburg virus vaccine. This result suggests that protection from postexposure vaccination may be antigen unspecific and due rather to an early activation of the innate immune system. In conclusion, VSV-EBOV remains a potent and fast-acting prophylactic vaccine but demonstrates only limited efficacy in postexposure treatment. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

OCCURRENCE OF SMALL HOMOLOGOUS AND COMPLEMENTARY FRAGMENTS IN HUMAN VIRUS GENOMES AND THEIR POSSIBLE ROLE

Directory of Open Access Journals (Sweden)

E. P. Kharchenko

2017-01-01

Full Text Available With computer analysis occurrence of small homologous and complementary fragments (21 nucleotides in length has been studied in genomes of 14 human viruses causing most dangerous infections. The sample includes viruses with (+ and (– single stranded RNA and DNA-containing hepatitis A virus. Analysis of occurrence of homologous sequences has shown the existence two extreme situations. On the one hand, the same virus contains homologous sequences to almost all other viruses (for example, Ebola virus, severe acute respiratory syndrome-related coronavirus, and mumps virus, and numerous homologous sequences to the same other virus (especially in severe acute respiratory syndrome-related coronavirus to Dengue virus and in Ebola virus to poliovirus. On the other hand, there are rare occurrence and not numerous homologous sequences in genomes of other viruses (rubella virus, hepatitis A virus, and hepatitis B virus. Similar situation exists for occurrence of complementary sequences. Rubella virus, the genome of which has the high content of guanine and cytosine, has no complementary sequences to almost all other viruses. Most viruses have moderate level of occurrence for homologous and complementary sequences. Autocomplementary sequences are numerous in most viruses and one may suggest that the genome of single stranded RNA viruses has branched secondary structure. In addition to possible role in recombination among strains autocomplementary sequences could be regulators of translation rate of virus proteins and determine its optimal proportion in virion assembly with genome and mRNA folding. Occurrence of small homologous and complementary sequences in RNA- and DNA-containing viruses may be the result of multiple recombinations in the past and the present and determine their adaptation and variability. Recombination may take place in coinfection of human and/or common hosts. Inclusion of homologous and complementary sequences into genome could not

Physical mode of bacteria and virus coevolution

Science.gov (United States)

Han, Pu; Niestemski, Liang; Deem, Michael

2013-03-01

Single-cell hosts such as bacteria or archaea possess an adaptive, heritable immune system that protects them from viral invasion. This system, known as the CRISPR-Cas system, allows the host to recognize and incorporate short foreign DNA or RNA sequences from viruses or plasmids. The sequences form what are called ``spacers'' in the CRISPR. Spacers in the CRISPR loci provide a record of the host and predator coevolution history. We develop a physical model to study the dynamics of this coevolution due to immune pressure. Hosts and viruses reproduce, die, and evolve due to viral infection pressure, host immune pressure, and mutation. We will discuss the differing effects of point mutation and recombination on CRISPR evolution. We will also discuss the effect of different spacer deletion mechanisms. We will describe population structure of hosts and viruses, how spacer diversity depends on position within CRISPR, and match of the CRISPR spacers to the virus population.

Virus-Vectored Influenza Virus Vaccines

Science.gov (United States)

Tripp, Ralph A.; Tompkins, S. Mark

2014-01-01

Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. PMID:25105278

Human papilloma virus prevalence in laryngeal squamous cell carcinoma.

Science.gov (United States)

Gungor, A; Cincik, H; Baloglu, H; Cekin, E; Dogru, S; Dursun, E

2007-08-01

To determine the prevalence and type of human papilloma virus deoxyribonucleic acid (DNA) in cases of laryngeal squamous cell carcinoma. We analysed the prevalence of human papilloma virus infection in archived paraffin block specimens taken from 99 cases of laryngeal squamous cell carcinoma between 1990 and 2005, using polymerase chain reaction techniques. Biopsy specimens from five proven verrucous skin lesions were used as positive controls, and peripheral blood samples from five healthy volunteers were used as negative controls. Four test samples were found to have inadequate deoxyribonucleic acid purity and were therefore excluded from the study. Human papilloma virus deoxyribonucleic acid was detected in seven of 95 cases of laryngeal squamous cell carcinoma (7.36 per cent). Human papilloma virus genotyping revealed double human papilloma virus infection in three cases and single human papilloma virus infection in the remaining four cases. The human papilloma virus genotypes detected were 6, 11 and 16 (the latter detected in only one case). In our series, a very low human papilloma virus prevalence was found among laryngeal squamous cell carcinoma cases. The human papilloma virus genotypes detected were mostly 6 and/or 11, and 16 in only one case. To the best of our knowledge, this is the first report of human papilloma virus prevalence in laryngeal squamous cell carcinoma, based on polymerase chain reaction genotyping in a Turkish population.

Development of a broad-spectrum antiviral with activity against Ebola virus.

Science.gov (United States)

Aman, M Javad; Kinch, Michael S; Warfield, Kelly; Warren, Travis; Yunus, Abdul; Enterlein, Sven; Stavale, Eric; Wang, Peifang; Chang, Shaojing; Tang, Qingsong; Porter, Kevin; Goldblatt, Michael; Bavari, Sina

2009-09-01

We report herein the identification of a small molecule therapeutic, FGI-106, which displays potent and broad-spectrum inhibition of lethal viral hemorrhagic fevers pathogens, including Ebola, Rift Valley and Dengue Fever viruses, in cell-based assays. Using mouse models of Ebola virus, we further demonstrate that FGI-106 can protect animals from an otherwise lethal infection when used either in a prophylactic or therapeutic setting. A single treatment, administered 1 day after infection, is sufficient to protect animals from lethal Ebola virus challenge. Cell-based assays also identified inhibitory activity against divergent virus families, which supports a hypothesis that FGI-106 interferes with a common pathway utilized by different viruses. These findings suggest FGI-106 may provide an opportunity for targeting viral diseases.

Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants.

Science.gov (United States)

Klingbeil, Katharina; Lange, Elke; Blohm, Ulrike; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

2015-03-02

Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba. Copyright © 2015 Elsevier B.V. All rights reserved.

Single-dose and steady-state pharmacokinetics of tenofovir disoproxil fumarate in human immunodeficiency virus-infected children.

Science.gov (United States)

Hazra, Rohan; Balis, Frank M; Tullio, Antonella N; DeCarlo, Ellen; Worrell, Carol J; Steinberg, Seth M; Flaherty, John F; Yale, Kitty; Poblenz, Marianne; Kearney, Brian P; Zhong, Lijie; Coakley, Dion F; Blanche, Stephane; Bresson, Jean Louis; Zuckerman, Judith A; Zeichner, Steven L

2004-01-01

Tenofovir disoproxil fumarate (DF) is a potent nucleotide analog reverse transcriptase inhibitor approved for the treatment of human immunodeficiency virus (HIV)-infected adults. The single-dose and steady-state pharmacokinetics of tenofovir were evaluated following administration of tenofovir DF in treatment-experienced HIV-infected children requiring a change in antiretroviral therapy. Using increments of tenofovir DF 75-mg tablets, the target dose was 175 mg/m(2); the median administered dose was 208 mg/m(2). Single-dose pharmacokinetics were evaluated in 18 subjects, and the geometric mean area under the concentration-time curve from 0 h to infinity (AUC(0- infinity )) was 2,150 ng. h/ml and the geometric mean maximum concentration (C(max)) was 266 ng/ml. Subsequently, other antiretrovirals were added to each patient's regimen based upon treatment history and baseline viral resistance results. Steady-state pharmacokinetics were evaluated in 16 subjects at week 4. The steady-state, geometric mean AUC for the 24-h dosing interval was 2,920 ng. h/ml and was significantly higher than the AUC(0- infinity ) after the first dose (P = 0.0004). The geometric mean C(max) at steady state was 302 ng/ml. Tenofovir DF was generally very well tolerated. Steady-state tenofovir exposures in children receiving tenofovir DF-containing combination antiretroviral therapy approached values seen in HIV-infected adults (AUC, approximately 3,000 ng. h/ml; C(max), approximately 300 ng/ml) treated with tenofovir DF at 300 mg.

An overview on hepatitis C virus genotypes and its control | Nouroz ...

African Journals Online (AJOL)

Hepatitis C virus (HCV) is a blood borne, circular and positive single stranded virus with high spread rates. With the passage of time the frequency of HCV is increasing in different parts of the world. HCV is a major cause, which may end in liver cirrhosis and hepatocellular carcinoma. HCV has six main genotypes with many ...

Virus surveys of Capsicum spp. in the Republic of Benin reveal the prevalence of pepper vein yellows virus and the identification of a previously uncharacterised polerovirus species.

Science.gov (United States)

Afouda, Leonard; Kone, Daouda; Zinsou, Valerien; Dossou, Laurence; Kenyon, Lawrence; Winter, Stephan; Knierim, Dennis

2017-06-01

Surveys were conducted in 2014 and 2015 in Southern and Northern Benin, respectively, to identify the viruses infecting peppers (Capsicum spp.). The samples were screened by ELISA for cucumber mosaic virus (CMV), pepper veinal mottle virus (PVMV), potato virus Y (PVY) and tomato yellow leaf curl virus (TYLCV). A generic reverse transcription PCR (RT-PCR) was used to test for the presence of poleroviruses. ELISA tests confirmed the prevalence of all viruses, while the RT-PCR detected pepper vein yellows virus (PeVYV) which is reported for the first time in Benin. A further, divergent polerovirus isolate was detected from a single pepper sample originating from southern Benin. Screening of samples collected from solanaceous plants during virus surveys in Mali (conducted in 2009) also detected this divergent polerovirus isolate in two samples from African eggplants. The complete genome sequence was obtained from the Mali isolate using transcriptome sequencing and by conventional Sanger sequencing of overlapping RT-PCR products. Based on the sequence characteristics of this isolate we propose a new polerovirus species, African eggplant yellowing virus (AeYV).

Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infections.

Science.gov (United States)

Iversen, Patrick L; Warren, Travis K; Wells, Jay B; Garza, Nicole L; Mourich, Dan V; Welch, Lisa S; Panchal, Rekha G; Bavari, Sina

2012-11-06

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

A complex adenovirus vaccine against chikungunya virus provides complete protection against viraemia and arthritis

Science.gov (United States)

Wang, Danher; Suhrbier, Andreas; Penn-Nicholson, Adam; Woraratanadharm, Jan; Gardner, Joy; Luo, Min; Le, Thuy T.; Anraku, Itaru; Sakalian, Michael; Einfeld, David; Dong, John Y.

2011-01-01

Chikungunya virus, a mosquito-borne alphavirus, recently caused the largest epidemic ever seen for this virus. Chikungunya disease primarily manifests as a painful and debilitating arthralgia/arthritis, and no effective drug or vaccine is currently available. Here we describe a recombinant chikungunya virus vaccine comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus. A single immunisation with this vaccine consistently induced high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic. The vaccine also completely protected mice against viraemia and arthritic disease caused by both virus isolates. PMID:21320541

Epstein-Barr virus latent gene sequences as geographical markers of viral origin: unique EBNA3 gene signatures identify Japanese viruses as distinct members of the Asian virus family.

Science.gov (United States)

Sawada, Akihisa; Croom-Carter, Deborah; Kondo, Osamu; Yasui, Masahiro; Koyama-Sato, Maho; Inoue, Masami; Kawa, Keisei; Rickinson, Alan B; Tierney, Rosemary J

2011-05-01

Polymorphisms in Epstein-Barr virus (EBV) latent genes can identify virus strains from different human populations and individual strains within a population. An Asian EBV signature has been defined almost exclusively from Chinese viruses, with little information from other Asian countries. Here we sequenced polymorphic regions of the EBNA1, 2, 3A, 3B, 3C and LMP1 genes of 31 Japanese strains from control donors and EBV-associated T/NK-cell lymphoproliferative disease (T/NK-LPD) patients. Though identical to Chinese strains in their dominant EBNA1 and LMP1 alleles, Japanese viruses were subtly different at other loci. Thus, while Chinese viruses mainly fall into two families with strongly linked 'Wu' or 'Li' alleles at EBNA2 and EBNA3A/B/C, Japanese viruses all have the consensus Wu EBNA2 allele but fall into two families at EBNA3A/B/C. One family has variant Li-like sequences at EBNA3A and 3B and the consensus Li sequence at EBNA3C; the other family has variant Wu-like sequences at EBNA3A, variants of a low frequency Chinese allele 'Sp' at EBNA3B and a consensus Sp sequence at EBNA3C. Thus, EBNA3A/B/C allelotypes clearly distinguish Japanese from Chinese strains. Interestingly, most Japanese viruses also lack those immune-escape mutations in the HLA-A11 epitope-encoding region of EBNA3B that are so characteristic of viruses from the highly A11-positive Chinese population. Control donor-derived and T/NK-LPD-derived strains were similarly distributed across allelotypes and, by using allelic polymorphisms to track virus strains in patients pre- and post-haematopoietic stem-cell transplant, we show that a single strain can induce both T/NK-LPD and B-cell-lymphoproliferative disease in the same patient.

Experimental Treatment of Ebola Virus Disease with TKM-130803: A Single-Arm Phase 2 Clinical Trial.

Science.gov (United States)

Dunning, Jake; Sahr, Foday; Rojek, Amanda; Gannon, Fiona; Carson, Gail; Idriss, Baimba; Massaquoi, Thomas; Gandi, Regina; Joseph, Sebatu; Osman, Hassan K; Brooks, Timothy J G; Simpson, Andrew J H; Goodfellow, Ian; Thorne, Lucy; Arias, Armando; Merson, Laura; Castle, Lyndsey; Howell-Jones, Rebecca; Pardinaz-Solis, Raul; Hope-Gill, Benjamin; Ferri, Mauricio; Grove, Jennifer; Kowalski, Mark; Stepniewska, Kasia; Lang, Trudie; Whitehead, John; Olliaro, Piero; Samai, Mohammed; Horby, Peter W

2016-04-01

TKM-130803, a small interfering RNA lipid nanoparticle product, has been developed for the treatment of Ebola virus disease (EVD), but its efficacy and safety in humans has not been evaluated. In this single-arm phase 2 trial, adults with laboratory-confirmed EVD received 0.3 mg/kg of TKM-130803 by intravenous infusion once daily for up to 7 d. On days when trial enrolment capacity was reached, patients were enrolled into a concurrent observational cohort. The primary outcome was survival to day 14 after admission, excluding patients who died within 48 h of admission. After 14 adults with EVD had received TKM-130803, the pre-specified futility boundary was reached, indicating a probability of survival to day 14 of ≤0.55, and enrolment was stopped. Pre-treatment geometric mean Ebola virus load in the 14 TKM-130803 recipients was 2.24 × 109 RNA copies/ml plasma (95% CI 7.52 × 108, 6.66 × 109). Two of the TKM-130803 recipients died within 48 h of admission and were therefore excluded from the primary outcome analysis. Of the remaining 12 TKM-130803 recipients, nine died and three survived. The probability that a TKM-130803 recipient who survived for 48 h will subsequently survive to day 14 was estimated to be 0.27 (95% CI 0.06, 0.58). TKM-130803 infusions were well tolerated, with 56 doses administered and only one possible infusion-related reaction observed. Three patients were enrolled in the observational cohort, of whom two died. Administration of TKM-130803 at a dose of 0.3 mg/kg/d by intravenous infusion to adult patients with severe EVD was not shown to improve survival when compared to historic controls. Pan African Clinical Trials Registry PACTR201501000997429.

First Report of Hepatitis E Virus Infection in Sika Deer in China

OpenAIRE

Zhang, Xiao-Xuan; Qin, Si-Yuan; Zhang, Yuan; Meng, Qing-Feng; Jiang, Jing; Yang, Gui-Lian; Zhao, Quan; Zhu, Xing-Quan

2015-01-01

Hepatitis E virus (HEV), a single stranded RNA, nonenveloped virus, belongs to the genus Hepevirus, in the family of Hepeviridae. In this study, 46 (5.43%) out of the 847 serum samples from sika deer (Cervus nippon) were detected as seropositive with hepatitis E virus (HEV) by enzyme linked immunosorbent assay (ELISA). These samples were collected from Inner Mongolia and Jilin and Heilongjiang provinces in China, between October 2012 and October 2013. Seroprevalence of HEV infection in male a...

Single Amino Acid Insertion in Loop 4 Confers Amphotropic Murine Leukemia Virus Receptor Function upon Murine Pit1

DEFF Research Database (Denmark)

Lundorf, Mikkel D.; Pedersen, Finn Skou; O'Hara, Bryan

1998-01-01

Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch...

Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

Directory of Open Access Journals (Sweden)

Scott A Walper

Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

Screen-Printed All-Polymer Aptasensor for Impedance Based Detection of Influenza A Virus

DEFF Research Database (Denmark)

Kirkegaard, Julie; Rozlosnik, Noemi

2017-01-01

are made by CO2 laser cutting of Poly(methyl methacrylate) (PMMA) sheets. Influenza A virus specific aptamers are immobilized onto the electrodes by UV cross-linking. Impedance based measurements at a single frequency, measured over time, are used to detect the virus in a buffer solution....

Genetic diversity among pandemic 2009 influenza viruses isolated from a transmission chain

DEFF Research Database (Denmark)

Fordyce, Sarah Louise; Bragstad, Karoline; Pedersen, Svend Stenvang

2013-01-01

Influenza viruses such as swine-origin influenza A(H1N1) virus (A(H1N1)pdm09) generate genetic diversity due to the high error rate of their RNA polymerase, often resulting in mixed genotype populations (intra-host variants) within a single infection. This variation helps influenza to rapidly res...

Sour and duke cherry viruses in South-West Europe

Directory of Open Access Journals (Sweden)

Rodrigo PÉREZ-SÁNCHEZ

2017-05-01

Full Text Available This study investigated the phytosanitary status of sour and duke cherry genetic resources in the Iberian Peninsula, and the incidence and leaf symptoms induced by the Prunus necrotic ringspot virus (PNRSV, Prune dwarf virus (PDV and Apple chlorotic leaf spot virus (ACLSV. Young leaf samples were taken from 204 sour and duke cherry trees belonging to ten cultivars, and were assayed by DAS-ELISA. Samples positive for any of the three viruses were also tested by RT-PCR. To associate the leaf symptoms with virus presence, 50 mature leaves from each infected tree were visually inspected during the summer. The ELISA and RT-PCR results indicated that 63% of the cherry trees were infected by at least one of these viruses. PNRSV occurred in all cultivars sampled and presented the highest infection rate (46%, followed by PDV (31% and ACLSV (6%. Many trees, (60 to 100%, were asymptomatic while harbouring single and mixed virus infections. The leaf symptoms associated with the viruses included chlorotic and dark brown necrotic ringspots on secondary veins and interveinal regions, for PNRSV, generalized chlorosis around the midveins, for PDV, chlorotic and reddish necrotic ringspots, for ACLSV, and generalized interveinal chlorosis, for mixed PNRSV and PDVinfections.

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat.

Science.gov (United States)

Galipienso, L; Vives, M C; Moreno, P; Milne, R G; Navarro, L; Guerri, J

2001-01-01

Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 x 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 x 10(6)). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 x 10(6). The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5' co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear.

A comparative analysis of measles virus RNA by oligonucleotide fingerprinting

International Nuclear Information System (INIS)

Stephenson, J.R.; Meulen, V. ter

1982-01-01

Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T 1 oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin. (Author)

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

Directory of Open Access Journals (Sweden)

Yong Huang

Full Text Available Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus and PCV2 (DNA virus from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29% and TGEV (11.7% preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

Science.gov (United States)

Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

2015-01-01

Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

Science.gov (United States)

Huang, Yong; Xing, Na; Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

2015-01-01

Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

Annulate lamellae in phloem cells of virus-infected Sonchus plants.

Science.gov (United States)

Steinkamp, M P; Hoefert, L L

1977-07-01

The occurrence of annulate lamellae (AL) in differentiating phloem of Sonchus oleraceus (Compositae) singly infected with sowthistle yellow vein virus (SYVV) and doubly infected with a combination of SYVV and beet yellow stunt virus is documented by electron microscopy. Cell types in which AL were found were immature sieve elements and phloem parenchyma cells. AL were found only in cells that also contained SYVV particles although a direct association between the virus and AL was not apparent. The substructure of the AL and the relationships between the AL and the nuclear envelope and endoplasmic reticulum are similar to those reported in other descriptions of this organelle in the literature. This report appears to be the first one concerning the association of AL with a plant virus disease.

XMRV: A New Virus in Prostate Cancer?

Science.gov (United States)

Aloia, Amanda L.; Sfanos, Karen S.; Isaacs, William B.; Zheng, Qizhi; Maldarelli, Frank; De Marzo, Angelo M.; Rein, Alan

2010-01-01

Several recent papers have reported the presence of a gammaretrovirus, termed “XMRV” (xenotropic murine leukemia virus-related virus) in prostate cancers (PCa). If confirmed, this could have enormous implications for the detection, prevention, and treatment of PCa. However, other papers report failure to detect XMRV in PCa. We tested nearly 800 PCa samples, using a combination of real-time PCR and immunohistochemistry (IHC). The PCR reactions were simultaneously monitored for amplification of a single-copy human gene, in order to confirm the quality of the sample DNA and its suitability for PCR. Controls demonstrated that the PCR assay could detect the XMRV in a single infected cell, even in the presence of a 10,000-fold excess of uninfected human cells. The IHC used two rabbit polyclonal antisera, each prepared against a purified MLV protein. Both antisera always stained XMRV-infected or – transfected cells, but never stained control cells. No evidence for XMRV in PCa was obtained in these experiments. We discuss possible explanations for the discrepancies in the results from different laboratories. It is possible that XMRV is not actually circulating in the human population; even if it is, the data do not seem to support a causal role for this virus in PCa. PMID:20966126

Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection.

Science.gov (United States)

Fernandez, Estefania; Dejnirattisai, Wanwisa; Cao, Bin; Scheaffer, Suzanne M; Supasa, Piyada; Wongwiwat, Wiyada; Esakky, Prabagaran; Drury, Andrea; Mongkolsapaya, Juthathip; Moley, Kelle H; Mysorekar, Indira U; Screaton, Gavin R; Diamond, Michael S

2017-11-01

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.

Virus-Like-Vaccines against HIV.

Science.gov (United States)

Andersson, Anne-Marie C; Schwerdtfeger, Melanie; Holst, Peter J

2018-02-11

Protection against chronic infections has necessitated the development of ever-more potent vaccination tools. HIV seems to be the most challenging foe, with a remarkable, poorly immunogenic and fragile surface glycoprotein and the ability to overpower the cell immune system. Virus-like-particle (VLP) vaccines have emerged as potent inducers of antibody and helper T cell responses, while replication-deficient viral vectors have yielded potent cytotoxic T cell responses. Here, we review the emerging concept of merging these two technologies into virus-like-vaccines (VLVs) for the targeting of HIV. Such vaccines are immunologically perceived as viruses, as they infect cells and produce VLPs in situ, but they only resemble viruses, as the replication defective vectors and VLPs cannot propagate an infection. The inherent safety of such a platform, despite robust particle production, is a distinct advantage over live-attenuated vaccines that must balance safety and immunogenicity. Previous studies have delivered VLVs encoded in modified Vaccinia Ankara vectors and we have developed the concept into a single-reading adenovirus-based technology capable of eliciting robust CD8⁺ and CD4⁺ T cells responses and trimer binding antibody responses. Such vaccines offer the potential to display the naturally produced immunogen directly and induce an integrated humoral and cellular immune response.

Quantitative Image Restoration in Bright Field Optical Microscopy.

Science.gov (United States)

Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

2017-11-07

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

Science.gov (United States)

Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

2016-01-01

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs

NARCIS (Netherlands)

Sullivan, Nancy J.; Geisbert, Thomas W.; Geisbert, Joan B.; Shedlock, Devon J.; Xu, Ling; Lamoreaux, Laurie; Custers, Jerome H. H. V.; Popernack, Paul M.; Yang, Zhi-Yong; Pau, Maria G.; Roederer, Mario; Koup, Richard A.; Goudsmit, Jaap; Jahrling, Peter B.; Nabel, Gary J.

2006-01-01

BACKGROUND: Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or

Antivirion Effects of Streptovaricin Complex Against Friend Virus

Science.gov (United States)

Horoszewicz, Julius S.; Leong, Susan S.; Byrd, Daniel M.; Carter, William A.

1974-01-01

The in vitro antivirion activities of five different streptovaricin complex lots against the polycythemic strain of the Friend virus were evaluated. The assay system was based on the inhibition of the Friend virus-induced spleen foci. The virus inactivation process was shown to be susceptible to variation in temperature, pH, and time. The antivirion activity and the acute toxicity for mice, as well as the optical properties of these streptovaricin complexes, do not co-vary; this suggests that their biological activities are not associated with a single molecular structure. In addition, the antivirion activity of the five preparations of streptovaricin complex differs about 30-fold, indicating that this activity does not reside in a major component of the complex. PMID:15825311

Genomic screening for blood-borne viruses in transfusion settings.

Science.gov (United States)

Allain, J P

2000-02-01

The residual risk of post-transfusion human immunodeficiency virus (HIV) infection is low but slightly higher for hepatitis B virus (HBV) and hepatitis C virus (HCV), the main reason being viraemia during the window period preceding antibody or antigen detection by enzyme immunoassays. Immunosilent-infected individuals and carriers of distant viral variants also play an unquantifiable role. Multiple techniques, e.g. reverse transcription-polymerase chain reaction (RT-PCR), PCR, ligase-chain reaction, nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA) have been developed to amplify and detect viral genomes as single or multiplex assays. Equipment providing various degrees of automation has been adapted to these techniques. Applying nucleic acid amplification techniques (NAT) to blood screening, two main approaches have been advocated: plasma pool and single-donation testing. Pool testing presents the advantage of lower cost and readily available equipment although it is prone to false negative and positive reactions. The time required to identify infected donations is incompatible with blood component release, and may lead to product waste. Single-unit testing, although appealing, is not yet fully automated and potentially very costly unless a systematic multiplex approach is taken. Although technically feasible, NAT applied to the blood supply needs to be clinically evaluated and its cost efficiency assessed in the general public health context. However, pool NAT is currently implemented in continental Europe and the USA.

Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines

Directory of Open Access Journals (Sweden)

Rahmberg Andrew R

2011-05-01

Full Text Available Abstract Background Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. Results Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. Conclusions Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent TH1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

KAUST Repository

Mahfouz, Magdy M.

2017-12-22

CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

A single mutation in the E2 glycoprotein important for neurovirulence influences binding of Sindbis virus to neuroblastoma cells

NARCIS (Netherlands)

Lee, PY; Knight, R; Smit, JM; Wilschut, J; Griffin, DE

The amino acid at position 55 of the E2 glycoprotein (E2(55)) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E2(55) = histidine) differs only at this position from virus strain 633 (E2(55) = glutamine), yet TE is considerably more

Quantification and imaging of HER2 protein using nanocrystals conjugated with single-domain antibodies

International Nuclear Information System (INIS)

Glukhov, S; Berestovoy, M; Nabiev, I; Sukhanova, A; Chames, P; Baty, D

2017-01-01

This study dealt with quantification and imaging of human epidermal growth factor receptor 2 (HER2), an important prognostic marker for cancer diagnosis and treatment, using specific quantum-dot-based conjugates. Fluorescent inorganic nanocrystals or quantum dots (QDs) are extremely highly resistant to photobleaching and have a high emission quantum yield and a continuous range of emission spectra, from the ultraviolet to the infrared regions. Ultrasmall nanoprobes consisting of highly affine anti-HER2 single-domain antibodies (sdAbs or 'nanobodies') conjugated with QDs in a strictly oriented manner have been designed. QDs with a fluorescence peak maxima at wavelengths of 562 nm, 569 nm, 570 nm or in the near-infrared region were used. Here, we present our results of ISA quantification of HER2 protein, in situ imaging of HER2 protein on the surface of HER2-positive SK-BR-3 cells in immunohistochemical experiments, and counting of stained with anti-HER2 conjugates HER2-positive SK-BR-3 cells in their mixture with unstained cells of the same culture in flow cytometry experiments. The data demonstrate that the anti-HER2 QD–sdAb conjugates obtained are highly specific and sensitive and could be used in numerous applications for advanced integrated diagnosis. (paper)

Quantification and imaging of HER2 protein using nanocrystals conjugated with single-domain antibodies

Science.gov (United States)

Glukhov, S.; Berestovoy, M.; Chames, P.; Baty, D.; Nabiev, I.; Sukhanova, A.

2017-01-01

This study dealt with quantification and imaging of human epidermal growth factor receptor 2 (HER2), an important prognostic marker for cancer diagnosis and treatment, using specific quantum-dot-based conjugates. Fluorescent inorganic nanocrystals or quantum dots (QDs) are extremely highly resistant to photobleaching and have a high emission quantum yield and a continuous range of emission spectra, from the ultraviolet to the infrared regions. Ultrasmall nanoprobes consisting of highly affine anti-HER2 single-domain antibodies (sdAbs or "nanobodies") conjugated with QDs in a strictly oriented manner have been designed. QDs with a fluorescence peak maxima at wavelengths of 562 nm, 569 nm, 570 nm or in the near-infrared region were used. Here, we present our results of ISA quantification of HER2 protein, in situ imaging of HER2 protein on the surface of HER2-positive SK-BR-3 cells in immunohistochemical experiments, and counting of stained with anti-HER2 conjugates HER2-positive SK-BR-3 cells in their mixture with unstained cells of the same culture in flow cytometry experiments. The data demonstrate that the anti-HER2 QD-sdAb conjugates obtained are highly specific and sensitive and could be used in numerous applications for advanced integrated diagnosis.

Diversity in the enteric viruses detected in outbreaks of gastroenteritis from Mumbai, Western India.

Science.gov (United States)

Chitambar, Shobha; Gopalkrishna, Varanasi; Chhabra, Preeti; Patil, Pooja; Verma, Harsha; Lahon, Anismrita; Arora, Ritu; Tatte, Vaishali; Ranshing, Sujata; Dhale, Ganesh; Kolhapure, Rajendra; Tikute, Sanjay; Kulkarni, Jagannath; Bhardwaj, Renu; Akarte, Sulbha; Pawar, Sashikant

2012-03-01

Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6%) were positive, 73 (76.8%) for a single virus and 22 (23.2%) for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40%) and rotavirus A (RVA, 33.3% and 25%). The other viruses detected in these months were norovirus (NoV, 12.1% and 10%), rotavirus B (RVB, 12.1% and 10%), enteric adenovirus (AdV, 6.1% and 7.5%), Aichivirus (AiV, 3% and 7.5%) and human astrovirus (HAstV, 3% and 0%). Mixed viral infections were largely represented by two viruses (84.6% and 88.9%), a small proportion showed presence of three (7.7% and 11%) and four (7.7% and 0%) viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage), NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India.

Native nucleic acid electrophoresis as an efficient alternative for genotyping method of influenza virus.

Science.gov (United States)

Pajak, Beata; Lepek, Krzysztof

2014-01-01

Influenza viruses are the worldwide major causative agents of human and animal acute respiratory infections. Some of the influenza subtypes have caused epidemics and pandemics among humans. The varieties of methods are available for the rapid isolation and identification of influenza viruses in clinical and environmental samples. Since nucleic acids amplification techniques such as RT-PCR have been adapted, fast and sensitive influenza type and subtype determination is possible. However, in some ambiguous cases other, more detailed assay might be desired. The genetic material of influenza virus is highly unstable and constantly mutates. It is known that single nucleotide polymorphisms (SNPs) results in resistance to commercially available anti-viral drugs. The genetic drift of the virus could also result in weakening of immune response to infection. Finally, in a substantial number of patients co-infection with various virus strains or types has been confirmed. Although the detection of co-infection or presence of minor genetic variants within flu-infected patients is not a routine procedure, a rapid and wide spectrum diagnostics of influenza virus infections could reveal an accurate picture of the disease and more importantly, is crucial for choosing the appropriate therapeutics and virus monitoring. Herein we present the evidences that native gel electrophoresis and MSSCP--a method based on multitemperature single strand conformation polymorphism could furnish a useful technique for minor variants, which escape discovery by conventional diagnostic assays.

Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

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Steven M Valles

Full Text Available Solenopsis invicta virus 3 (SINV-3 is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV, an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

Titration of a cytoplasmic polyhedrosis virus by a tissue microculture assay: some applications.

Science.gov (United States)

Belloncik, S; Chagnon, A

1980-01-01

A simple tissue microculture technique was developed for the titration of a cytoplasmic polyhedrosis virus (CPV) from Euxoa scandens. The procedure was similar to the 50% tissue culture infectious dose assay, but a single infected cell, detected by the presence of cytoplasmic polyhedra, was scored rather than the degeneration of cell monolayers. The filtration of CPV suspensions resulted in decreased virus titers under certain conditions. This microculture assay was used to determine the effect of cell disruption methods on virus yields. Sonication of infected cells was more efficient than freeze-thawing for the recovery of nonoccluded virus.

Specificity in the association of tomato black ring virus satellite RNA with helper virus.

Science.gov (United States)

Oncino, C; Hemmer, O; Fritsch, C

1995-10-20

The satellite RNAs (sat-RNAs) associated with some isolates of tomato black ring virus (TBRV) consist of single-stranded molecules of about 1375 nucleotides, encoding a nonstructural protein of 48K which has been shown to be involved in the replication of the sat-RNA. The TBRV sat-RNAs are also dependent for their replication and for their encapsidation on the helper virus. To characterize the nature of the association between sat-RNA and helper virus, transcripts of sat-RNA from TBRV isolates C and L (respectively, of serotypes G and S) have been prepared and inoculated onto Chenopodium quinoa leaves or protoplasts. Transcript of the TBRV sat-RNA C is efficiently multiplied when coinoculated with the genomic RNAs of TBRV isolate G (used instead of TBRV isolate C, because isolate G was depleted of sat-RNA), but does not multiply with TBRV isolate L. On the other hand, transcript of the sat-RNA L is able to multiply with the cognate helper virus and, less efficiently, with grapevine chrome mosaic virus (another nepovirus, 80% similar to TBRV), but does not multiply with TBRV G. The specificity of the association resides at the level of sat-RNA replication. Analysis of the multiplication of chimeric sat-RNAs, obtained by exchanging different regions between the two sat-RNAs C and L, showed that the 5' and the 3' noncoding regions of the sat-RNA, although important for replication, are not implicated in specificity. The results suggest that the determinants of the specificity are contained in the 48K sat-RNA-encoded protein.

Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

Science.gov (United States)

Maassen, Stan J; de Ruiter, Mark V; Lindhoud, Saskia; Cornelissen, Jeroen J L M

2018-05-23

Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reverse genetics with animal viruses. NSV reverse genetics

International Nuclear Information System (INIS)

Mebatsion, T.

2005-01-01

New strategies to genetically manipulate the genomes of several important animal pathogens have been established in recent years. This article focuses on the reverse genetics techniques, which enables genetic manipulation of the genomes of non-segmented negative-sense RNA viruses. Recovery of a negative-sense RNA virus entirely from cDNA was first achieved for rabies virus in 1994. Since then, reverse genetic systems have been established for several pathogens of medical and veterinary importance. Based on the reverse genetics technique, it is now possible to design safe and more effective live attenuated vaccines against important viral agents. In addition, genetically tagged recombinant viruses can be designed to facilitate serological differentiation of vaccinated animals from infected animals. The approach of delivering protective immunogens of different pathogens using a single vector was made possible with the introduction of the reverse genetics system, and these novel broad-spectrum vaccine vectors have potential applications in improving animal health in developing countries. (author)

Discovery and Early Development of AVI-7537 and AVI-7288 for the Treatment of Ebola Virus and Marburg Virus Infections

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Sina Bavari

2012-11-01

Full Text Available There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs AVI-6002 (composed of AVI-7357 and AVI-7539 and AVI-6003 (composed of AVI-7287 and AVI-7288 targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO transitioning to cell penetrating peptide conjugated PMOs (PPMO and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables.

Science.gov (United States)

Sánchez, G; Elizaquível, P; Aznar, R

2012-01-03

Fresh-cut vegetables are prone to be contaminated with foodborne pathogens during growth, harvest, transport and further processing and handling. As most of these products are generally eaten raw or mildly treated, there is an increase in the number of outbreaks caused by viruses and bacteria associated with fresh vegetables. Foodborne pathogens are usually present at very low levels and have to be concentrated (i.e. viruses) or enriched (i.e. bacteria) to enhance their detection. With this aim, a rapid concentration method has been developed for the simultaneous recovery of hepatitis A virus (HAV), norovirus (NV), murine norovirus (MNV) as a surrogate for NV, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica. Initial experiments focused on evaluating the elution conditions suitable for virus release from vegetables. Finally, elution with buffered peptone water (BPW), using a Pulsifier, and concentration by polyethylene glycol (PEG) precipitation were the methods selected for the elution and concentration of both, enteric viruses and bacteria, from three different types of fresh-cut vegetables by quantitative PCR (qPCR) using specific primers. The average recoveries from inoculated parsley, spinach and salad, were ca. 9.2%, 43.5%, and 20.7% for NV, MNV, and HAV, respectively. Detection limits were 132 RT-PCR units (PCRU), 1.5 50% tissue culture infectious dose (TCID₅₀), and 6.6 TCID₅₀ for NV, MNV, and HAV, respectively. This protocol resulted in average recoveries of 57.4%, 64.5% and 64.6% in three vegetables for E. coli O157:H7, L. monocytogenes and Salmonella with corresponding detection limits of 10³, 10² and 10³ CFU/g, respectively. Based on these results, it can be concluded that the procedure herein is suitable to recover, detect and quantify enteric viruses and foodborne pathogenic bacteria within 5 h and can be applied for the simultaneous detection of both types of foodborne pathogens in fresh-cut vegetables. Copyright �

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.

The administration of a single dose of a multivalent (DHPPiL4R vaccine prevents clinical signs and mortality following virulent challenge with canine distemper virus, canine adenovirus or canine parvovirus

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Stephen Wilson

2014-01-01

In conclusion, we demonstrated that a single administration of a minimum titre, multivalent vaccine to dogs of six weeks of age is efficacious and prevents clinical signs and mortality caused by CAV-1 and CDV; prevents clinical signs and significantly reduces virus shedding caused by CAV-2; and prevents clinical signs, leucopoenia and viral excretion caused by CPV.

Detection and sequencing of Potato virus Y (PVY and Potato leafroll virus (PLRV in a volunteer plant of Solanum tuberosum L. cv. Diacol-Capiro

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Héctor Camilo Medina Cárdenas

2017-10-01

Full Text Available Viral diseases are among the most limiting factors in the production of potato in Colombia and the rest of the world. The best strategy to control plant viruses consists on the use of certified seed tubers, control of arthropod vectors and the use of adequate crop management practices that reduce mechanical transmission and the presence of viral reservoirs like weeds and volun-teer plants. However, the successful implementation of these practices relies on the availability of highly sensitive techniques that allow for the asymptomatic detection of viruses. In this work, we tested the performance of Next-generation sequencing (NGS and real time RT-PCR (RT-qPCR on a single volunteer potato plant (cv. Diacol-Capiro growing naturally in a seed-tuber storage facility in Yarumal (Antioquia. Our NGS results demonstrate a mixed infection with Potato virus Y (PVY and Potato leafroll virus (PLRV. RT-qPCR was performed in roots, main stolons, crown (root collar and upper, middle and lower leaves using specific primers for PVY, PLRV, Potato virus S (PVS, Potato virus V (PVV, Potato virus X (PVX and Potato yellow vein virus (PYVV. Only PVY and PLRV were detected in good agreement with the NGS data. This work demonstrates the use-fulness of both techniques for supporting integrated management of plant viruses in potato, in-cluding virus detection in natural reservoirs such as volunteer plants and weeds.

A single tube PCR assay for simultaneous amplification of HSV-1/-2, VZV, CMV, HHV-6A/-6B, and EBV DNAs in cerebrospinal fluid from patients with virus-related neurological diseases.

Science.gov (United States)

Yamamoto, T; Nakamura, Y

2000-10-01

Cerebrospinal fluid (CSF) specimens from 27 patients with encephalitis, meningitis, and other neurological diseases were studied for the presence of herpes simplex virus types 1 and 2 (HSV-1/-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesviruses 6A and 6B (HHV-6A/-6B) and Epstein-Barr virus (EBV) DNA using the polymerase chain reaction (PCR) method. The DNAs were amplified using two sets of consensus primer pairs in a single tube, bringing simultaneous amplification of the herpesviruses. The PCR products were analyzed by agarose gel electrophoresis, and Southern blot hybridization with virus-type specific probes, thus allowing discrimination between the different types of herpesviruses to be made. Each virus-specific probe was highly specific for identifying the PCR product. Thirty CSF specimens from 13 patients with encephalitis and 10 specimens from 10 patients with meningitis, respectively, were examined using this method. Eight patients with encephalitis and six with meningitis were positive for different herpesviruses, including patients with coinfections (HSV-1/-2 and VZV, VZV and CMV). Among four CSF specimens from four patients with other neurological disorders, dual amplification of CMV and EBV was present. Since identification of the types of herpesviruses in this system requires a very small amount of CSF, and is completed with one PCR, it is useful for routine diagnosis of herpesvirus infections in diagnostic laboratories. The viruses responsible for central nervous system infection are easily detected with various coinfection and serial patterns of herpesviruses, by this consensus primer-based PCR method. This may give an insight into the relationship between virus-related neurological diseases (VRNDS) and herpesvirus infections.

Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

DEFF Research Database (Denmark)

Russell, Rodney S; Meunier, Jean-Christophe; Takikawa, Shingo

2008-01-01

mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7......The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five...

Cloned defective interfering influenza virus protects ferrets from pandemic 2009 influenza A virus and allows protective immunity to be established.

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Nigel J Dimmock

Full Text Available Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1. Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.

Understanding Zika virus.

Science.gov (United States)

Murray, John S

2017-01-01

This article describes what pediatric healthcare professionals should know about Zika virus (ZIKV). ZIKV is classified as an arthropod-borne, single-stranded RNA virus of the Flaviviridae family and genus Flavivirus. ZIKV is not new. The virus was first discovered almost 70 years ago in Uganda. The first isolate of the virus was found in rhesus monkeys in the Zika Forrest, hence the nomenclature. The primary route of ZIKV transmission to humans is through the bite of an infected Aedes species mosquito-primarily Aedes aegypti. When the mosquito bites individuals infected with the virus, mosquitos then become the vector of transmitting the infection to others. Women can also pass ZIKV to their fetus during pregnancy and at the time of delivery. ZIKV can also be transmitted through sexual activity from an individual who is infected with the virus to his or her partners. It is estimated that approximately 18% of individuals infected with ZIKV will go on to develop symptoms. When symptoms develop, it is usually within 3-12 days, although this may vary. Most often, symptoms are mild and self-limited. The most common symptoms are fever, arthralgia, maculopapular rash, and conjunctivitis lasting up to seven days. Less frequent symptoms include headache, vertigo, myalgia, vomiting, and diarrhea. At present, there is no vaccine available to prevent ZIKV and no specific antiviral treatment. Supportive care consisting of rest, hydration, analgesics, antihistamines, and antipyretics is recommended as needed. Given that there is no vaccine or treatment for ZIKV, considerable efforts must be focused on prevention. One of the most effective ways of preventing ZIKV infection is through avoiding mosquito bites, especially when traveling to or residing in areas where transmission is present. Precautions should include wearing appropriate attire with the objective of having as little skin exposed as possible, use of screens for windows and doors, and use of insect repellent. What is

Molecular characterization of viruses associated with gastrointestinal infection in HIV-positive patients

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Raquel C Silva

Full Text Available BACKGROUND: Diarrhea is a major cause of morbidity and mortality among HIV-infected patients worldwide. OBJECTIVE: We sought to determine the frequency of viral gastrointestinal infections among Brazilian HIV-infected patients with diarrhea. METHODS: A collection of 90 fecal specimens from HIV-infected individuals with diarrhea, previously tested for the presence of bacteria and parasite was analyzed by polymerase chain reaction and sequence analysis for the presence of enteric viruses such as astrovirus, norovirus, rotavirus groups A, B and C, adenovirus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus, and human bocavirus. RESULTS: Twenty patients (22.2%; n = 90 were infected with parasites (11 single infections and nine coinfected with virus. Enteropathogenic bacteria were not found. Virus infections were detected in 28.9% (26/90 of the specimens. Cytomegalovirus was the most common virus detected (24.4%; 22/90. Coinfections with viruses and/or parasite were observed in 10 (11.1% samples. CONCLUSION: Gastrointestinal virus infections were more frequent than parasitic or bacterial infections in this patient population.

Successful topical respiratory tract immunization of primates against Ebola virus.

Science.gov (United States)

Bukreyev, Alexander; Rollin, Pierre E; Tate, Mallory K; Yang, Lijuan; Zaki, Sherif R; Shieh, Wun-Ju; Murphy, Brian R; Collins, Peter L; Sanchez, Anthony

2007-06-01

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.

Epidemiological, immunological and virological aspects of acute and chronic hepatitis C virus infections

NARCIS (Netherlands)

Thomas, X.V.

2015-01-01

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus classified as a member of the Hepacivirus genus in the family Flavirviridae, and was first described by Choo et al. in 1989 as the causative agent of non-A-non-B post-transfusion hepatitis. HCV is a major cause of blood-borne

Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range.

Science.gov (United States)

Vasilakis, Nikos; Guzman, Hilda; Firth, Cadhla; Forrester, Naomi L; Widen, Steven G; Wood, Thomas G; Rossi, Shannan L; Ghedin, Elodie; Popov, Vsevolov; Blasdell, Kim R; Walker, Peter J; Tesh, Robert B

2014-05-20

The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes. Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d'Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 - 588 nt) of unknown function in the 5' region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication. In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.

Concern over Zika virus outbreak: another alarming global threat

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Hafiz MY

2016-06-01

Full Text Available Muhammad Yusuf Hafiz,1 Syed Uzair Mahmood,2 Maria Shoaib,1 Farah Hafiz Yusuf1 1Dow Medical College, Dow University of Health Sciences, 2Sindh Medical College, Jinnah Sindh Medical University, Karachi, Pakistan Abstract: Zika virus, as highlighted by the World Health Organization in February 2016, has emerged as a public health emergency of international concern. Zika virus is enveloped and icosahedral, and has a nonsegmented, single-stranded, positive-sense RNA genome. It belongs to Flaviviridae family. Aedes aegypticus mosquito is the known vector. Transmission is anthroponotic (human to vector to human during outbreaks, or occurs perinatally in utero, sexually, and via transfusion of infected blood. Zika virus is turning out to be a major public health concern. Not only has it shown dramatic teratogenic association and caused serious neurological concerns but it has also spread around the globe. Countries that have not yet been affected by Zika virus should adopt proper preventive methods to limit its spread in the population. Keywords: Zika virus, global spread, teratogenic, neurological anomalies, public health emergency

Host-Specific and Segment-Specific Evolutionary Dynamics of Avian and Human Influenza A Viruses: A Systematic Review

KAUST Repository

Kim, Kiyeon

2016-01-13

Understanding the evolutionary dynamics of influenza viruses is essential to control both avian and human influenza. Here, we analyze host-specific and segment-specific Tajima’s D trends of influenza A virus through a systematic review using viral sequences registered in the National Center for Biotechnology Information. To avoid bias from viral population subdivision, viral sequences were stratified according to their sampling locations and sampling years. As a result, we obtained a total of 580 datasets each of which consists of nucleotide sequences of influenza A viruses isolated from a single population of hosts at a single sampling site within a single year. By analyzing nucleotide sequences in the datasets, we found that Tajima’s D values of viral sequences were different depending on hosts and gene segments. Tajima’s D values of viruses isolated from chicken and human samples showed negative, suggesting purifying selection or a rapid population growth of the viruses. The negative Tajima’s D values in rapidly growing viral population were also observed in computer simulations. Tajima’s D values of PB2, PB1, PA, NP, and M genes of the viruses circulating in wild mallards were close to zero, suggesting that these genes have undergone neutral selection in constant-sized population. On the other hand, Tajima’s D values of HA and NA genes of these viruses were positive, indicating HA and NA have undergone balancing selection in wild mallards. Taken together, these results indicated the existence of unknown factors that maintain viral subtypes in wild mallards.

Host-Specific and Segment-Specific Evolutionary Dynamics of Avian and Human Influenza A Viruses: A Systematic Review

KAUST Repository

Kim, Kiyeon; Omori, Ryosuke; Ueno, Keisuke; Iida, Sayaka; Ito, Kimihito

2016-01-01

Understanding the evolutionary dynamics of influenza viruses is essential to control both avian and human influenza. Here, we analyze host-specific and segment-specific Tajima’s D trends of influenza A virus through a systematic review using viral sequences registered in the National Center for Biotechnology Information. To avoid bias from viral population subdivision, viral sequences were stratified according to their sampling locations and sampling years. As a result, we obtained a total of 580 datasets each of which consists of nucleotide sequences of influenza A viruses isolated from a single population of hosts at a single sampling site within a single year. By analyzing nucleotide sequences in the datasets, we found that Tajima’s D values of viral sequences were different depending on hosts and gene segments. Tajima’s D values of viruses isolated from chicken and human samples showed negative, suggesting purifying selection or a rapid population growth of the viruses. The negative Tajima’s D values in rapidly growing viral population were also observed in computer simulations. Tajima’s D values of PB2, PB1, PA, NP, and M genes of the viruses circulating in wild mallards were close to zero, suggesting that these genes have undergone neutral selection in constant-sized population. On the other hand, Tajima’s D values of HA and NA genes of these viruses were positive, indicating HA and NA have undergone balancing selection in wild mallards. Taken together, these results indicated the existence of unknown factors that maintain viral subtypes in wild mallards.

Mechanism of attenuation of a chimeric influenza A/B transfectant virus.

Science.gov (United States)

Luo, G; Bergmann, M; Garcia-Sastre, A; Palese, P

1992-08-01

The ribonucleoprotein transfection system for influenza virus allowed us to construct an influenza A virus containing a chimeric neuraminidase (NA) gene in which the noncoding sequence is derived from the NS gene of influenza B virus (T. Muster, E. K. Subbarao, M. Enami, B. P. Murphy, and P. Palese, Proc. Natl. Acad. Sci. USA 88:5177-5181, 1991). This transfectant virus is attenuated in mice and grows to lower titers in tissue culture than wild-type virus. Since such a virus has characteristics desirable for a live attenuated vaccine strain, attempts were made to characterize this virus at the molecular level. Our analysis suggests that the attenuation of the virus is due to changes in the cis signal sequences, which resulted in a reduction of transcription and replication of the chimeric NA gene. The major finding concerns a sixfold reduction in NA-specific viral RNA in the virion, causing a reduction in the ratio of infectious particles to physical particles compared with the ratio in wild-type virus. Although the NA-specific mRNA level is also reduced in transfectant virus-infected cells, it does not appear to contribute to the attenuation characteristics of the virus. The levels of the other RNAs and their expression appear to be unchanged for the transfectant virus. It is suggested that downregulation of the synthesis of one viral RNA segment leads to the generation of defective viruses during each replication cycle. We believe that this represents a general principle for attenuation which may be applied to other segmented viruses containing either single-stranded or double-stranded RNA.

Diversity in the Enteric Viruses Detected in Outbreaks of Gastroenteritis from Mumbai, Western India

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Renu Bhardwaj

2012-03-01

Full Text Available Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6% were positive, 73 (76.8% for a single virus and 22 (23.2% for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40% and rotavirus A (RVA, 33.3% and 25%. The other viruses detected in these months were norovirus (NoV, 12.1% and 10%, rotavirus B (RVB, 12.1% and 10%, enteric adenovirus (AdV, 6.1% and 7.5%, Aichivirus (AiV, 3% and 7.5% and human astrovirus (HAstV, 3% and 0%. Mixed viral infections were largely represented by two viruses (84.6% and 88.9%, a small proportion showed presence of three (7.7% and 11% and four (7.7% and 0% viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage, NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India.

Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses.

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Pascua, Philippe Noriel Q; Song, Min-Suk; Lee, Jun Han; Baek, Yun Hee; Kwon, Hyeok-il; Park, Su-Jin; Choi, Eun Hye; Lim, Gyo-Jin; Lee, Ok-Jun; Kim, Si-Wook; Kim, Chul-Joong; Sung, Moon Hee; Kim, Myung Hee; Yoon, Sun-Woo; Govorkova, Elena A; Webby, Richard J; Webster, Robert G; Choi, Young-Ki

2012-09-25

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA(225G) and NA(315N) substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA(225G) and NA(315N) as potential virulence markers in mammals.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

International Nuclear Information System (INIS)

Koonin, Eugene V.; Dolja, Valerian V.; Krupovic, Mart

2015-01-01

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources

Origins and evolution of viruses of eukaryotes: The ultimate modularity

Energy Technology Data Exchange (ETDEWEB)

Koonin, Eugene V., E-mail: koonin@ncbi.nlm.nih.gov [National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 (United States); Dolja, Valerian V., E-mail: doljav@science.oregonstate.edu [Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 (United States); Krupovic, Mart, E-mail: krupovic@pasteur.fr [Institut Pasteur, Unité Biologie Moléculaire du Gène chez les Extrêmophiles, Department of Microbiology, Paris 75015 (France)

2015-05-15

Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources

Molecular Characterization of Viruses from Clinical Respiratory Samples Producing Unidentified Cytopathic Effects in Cell Culture

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Guy Boivin

2009-07-01

Full Text Available The sequence-independent single primer amplification (SISPA method was performed to identify a virus in 17 clinical respiratory samples producing uncharacterized cytopathic effects in LLC-MK2 cells. Sequence analysis of 600-1600 bp amplicons allowed the identification of six viruses (one influenza C, two parechovirus-3 and three cardioviruses. Genomic sequences of the cardioviruses showed similarities with those of the recently-described Saffold virus strain although significant variation was present in the viral surface EF and CD loops. These results demonstrate the usefulness of SISPA for identifying emerging viruses and also known viruses not easily identified by standard virological methods.

Strawberry crinkle virus, a Cytorhabdovirus needing more attention from virologists.

Science.gov (United States)

Posthuma, K I; Adams, A N; Hong, Y

2000-11-01

Summary Taxonomic relationship: A member of nonsegmented, negative-sense, single-stranded RNA viruses of the genus Cytorhabdovirus (type member: Lettuce necrotic yellows virus), family Rhabdoviridae, order Mononegavirales. Members of the family Rhabdoviridae can infect vertebrates, invertebrates and plants. Physical properties: Virions are bacilliform, 74-88 nm in diameter and 163-383 nm in length with surface projections probably composed of trimers of the glycoprotein G, occurring in the cytoplasm in either the coated or the uncoated form (Fig. 1). The nucleocapsid is enclosed in a host-derived envelope. Within the virion, the SCV genome consists of a single negative-sense single-stranded RNA molecule of approximately 13 kb. Viral proteins: The SCV genome encodes at least five proteins: the nucleocapsid (N) protein (45 kDa), the matrix (M) protein (77 kDa), the nonstructural protein [Ns, 55 kDa, also known as phosphoprotein (P)], the glycoprotein (G, 23 kDa) and the large (L) protein. Hosts: The natural host range of SCV is limited to species of the genus Fragaria L. Experimental hosts include Physalis pubescens L., P. floridana Rydb., Nicotiana occidentalis, N. glutinosa L. and N. clevelandi Gray. SCV also replicates in its insect vectors Chaetosiphon fragaefolii Cockerell and C. jacobi Hille Ris Lamberts. When injected as purified virus, SCV replicates in aphids Hyperomyzus lactucae (L.), Macrosiphon euphorbiae Thomas, Myzus ornatus Laing, Megoura viciae Buckton, and Acyrthosiphoa pisum (Harris).

Coinfections of Sudanese dairy cattle with bovine herpes virus 1, bovine viral diarrhea virus, bluetongue virus and bovine herpes virus 4 and their relation to reproductive disorders

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Amira M. Elhassan

2016-12-01

Reults: The meta-analysis of the data indicated high seroprevalence of coinfections with various combinations of these agents; only few animals were singly infected. An infection with BHV-1 was observed to be higher than the prevalence of associations between BHV-1 and the other three viral agents. Prevalence of seropositivities to coinfection with BHV-1/BTV; BHV-1/BVD; BHV-1/BTV/BVD were the highest while seropositivities prevalences that involved BHV-4 were much lower. The highest abortion rates were encountered in coinfections with BHV-1/BVD/BTV (31% and BHV-1/BVD/BTV/BHV-4 (30% while most infertility cases were noticed in coinfection with BHV-1/BVD/BTV (44% and BHV-1/BVD/BTV/BHV-4 (21%, and coinfections with the four viruses were encountered in most of the death after birth cases (25%. Overall mixed infections with BHV-1/BVD/BTV (34% and BHV-1/BVD/BTV/BHV-4 (22.5% were involved in the majority of reproductive problems studied. Conclusion: Mixed infections constitutes the vast majority of cases and are involved in the majority of reproductive disorders investigated. The high prevalence of seropositivity to all of the four viruses should call for an intervention strategy to reduce the impact of these viruses. [J Adv Vet Anim Res 2016; 3(4.000: 332-337

Ability of Aphis gossypii and Myzus persicae to Transmit Cucumber mosaic virus in Single and Mixed Infection with Two Potyviruses to Zucchini Squash Eficiência dos afídeos Aphis gossypii e Myzus persicae na transmissão do Cucumber mosaic virus em infecção simples e mista com dois Potyvirus para abobrinha de moita

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Zayame Vegette Pinto

2008-06-01

Full Text Available The main objective of this work was to investigate the ability of Aphis gossypii and Myzus persicae to transmit Cucumber mosaic virus (CMV singly and mixed with two potyviruses (Papaya ringspot virus - type W, PRSV-W and Zucchini yellow mosaic virus, ZYMV, to zucchini squash plants (Cucurbita pepo. The results showed that the potyviruses in general were more efficiently transmitted by both species of aphids as compared to CMV. The transmission of PRSV-W, ZYMV and CMV separately was more efficient than in mixture.O objetivo desse trabalho foi estudar a eficiência de Aphis gossypii e Myzus persicae na transmissão do vírus do mosaico do pepino (Cucumber mosaic virus, CMV, isoladamente e em mistura com duas espécies de potyvirus (Vírus do mosaico do mamoeiro = Papaya ringspot virus - type W, PRSV-W e Vírus do mosaico amarelo da abobrinha = Zucchini yellow mosaic virus, ZYMV, para planta-testes de abobrinha de moita (Cucurbita pepo. Os dois potyvirus em geral foram transmitidos com mais eficiência pelas duas espécies de afídeos do que o CMV. A transmissão do PRSV-W, ZYMV e CMV, separadamente, foi mais eficiente do que em mistura.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

Energy Technology Data Exchange (ETDEWEB)

Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

2016-03-15

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

International Nuclear Information System (INIS)

Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

2016-01-01

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

A review on architecture of the gag-pol ribosomal frameshifting RNA in human immunodeficiency virus: a variability survey of virus genotypes.

Science.gov (United States)

Qiao, Qi; Yan, Yanhua; Guo, Jinmei; Du, Shuqiang; Zhang, Jiangtao; Jia, Ruyue; Ren, Haimin; Qiao, Yuanbiao; Li, Qingshan

2017-06-01

Programmed '-1' ribosomal frameshifting is necessary for expressing the pol gene overlapped from a gag of human immunodeficiency virus. A viral RNA structure that requires base pairing across the overlapping sequence region suggests a mechanism of regulating ribosome and helicase traffic during expression. To get precise roles of an element around the frameshift site, a review on architecture of the frameshifting RNA is performed in combination of reported information with augments of a representative set of 19 viral samples. In spite of a different length for the viral RNAs, a canonical comparison on the element sequence allocation is performed for viewing variability associations between virus genotypes. Additionally, recent and historical insights recognized in frameshifting regulation are looked back as for indel and single nucleotide polymorphism of RNA. As specially noted, structural changes at a frameshift site, the spacer sequence, and a three-helix junction element, as well as two Watson-Crick base pairs near a bulge and a C-G pair close a loop, are the most vital strategies for the virus frameshifting regulations. All of structural changes, which are dependent upon specific sequence variations, facilitate an elucidation about the RNA element conformation-dependent mechanism for frameshifting. These facts on disrupting base pair interactions also allow solving the problem of competition between ribosome and helicase on a same RNA template, common to single-stranded RNA viruses. In a broad perspective, each new insight of frameshifting regulation in the competition systems introduced by the RNA element construct changes will offer a compelling target for antiviral therapy.

Deep sequencing as a method of typing bluetongue virus isolates.

Science.gov (United States)

Rao, Pavuluri Panduranga; Reddy, Yella Narasimha; Ganesh, Kapila; Nair, Shreeja G; Niranjan, Vidya; Hegde, Nagendra R

2013-11-01

Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E. Copyright © 2013 Elsevier B.V. All rights reserved.

Viruses in Marine Animals: Discovery, Detection, and Characterization

Science.gov (United States)

Fahsbender, Elizabeth

Diseases in marine animals are emerging at an increasing rate. Disease forecasting enabled by virus surveillance presents a proactive solution for managing emerging diseases. Broad viral surveys aid in disease forecasting by providing baseline data on viral diversity associated with various hosts, including many that are not associated with disease. However, these viruses can become pathogens due to expansion in host or geographic range, as well as when changing conditions shift the balance between commensal viruses and the host immune system. Therefore, it is extremely valuable to identify and characterize viruses present in many different hosts in a variety of environments, regardless of whether the hosts are symptomatic or not. The lack of a universal gene shared by all viruses makes virus surveillance difficult, because no single assay exists that can detect the enormous diversity of viruses. Viral metagenomics circumvents this issue by purifying viral particles directly from host tissues and sequencing the nucleic acids, allowing for virus identification. However, virus identification is only the first step, which should ideally be followed by complete sequencing of the viral genome to identify genes of interest and develop assays to reveal viral prevalence, tropism, ecology, and pathogenicity. This dissertation focuses on the discovery of novel viruses in marine animals, characterization of complete viral genomes, and the development of subsequent diagnostic assays for further analysis of virus ecology. First, viral metagenomics was used to explore the viruses present in the healthy Weddell seal (Leptonychotes weddellii) population in Antarctica, which led to the discovery of highly prevalent small, circular single-stranded DNA (ssDNA) viruses. The lack of knowledge regarding the viruses of Antarctic wildlife warrants this study to determine baseline viral communities in healthy animals that can be used to survey changes over time. From the healthy Weddell

Zika virus infection: The resurgence of a neglected disease

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Tushar Kambale

2016-01-01

Full Text Available "Zika virus" (ZIKV is an enveloped, icosahedral virus and has a positive-sense, single-stranded RNA genome approximately 11 kb in length. Genetic studies have revealed three ZIKV lineages: East African, West African, and Asian. Serologic studies and virus isolations have demonstrated that the virus has a wide geographic distribution, spanning East and West Africa, the Americas, Indian subcontinent, and Southeast Asia. ZIKV can cause complications such as Guillain-Barré syndrome, meningitis, meningoencephalitis, and myelitis. During pregnancy ZIKV infection can lead to miscarriages and microcephaly, cerebral calcifications, macular neuroretinal atrophy, and loss of foveal reflex in the fetus. A clinically suspected case of infection with dengue negative result should be further tested for Flavivirus, including Zika. Immunofluorescence or enzyme-linked immunosorbent assay is used to detect specific IgM or IgG antibodies against ZIKV. In cases of positive ZIKV infection, symptomatic treatment should be given after excluding other condition such as dengue, malaria, and bacterial infections.

Nairobi sheep disease virus/Ganjam virus.

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M D, Baron; B, Holzer

2015-08-01

Nairobi sheep disease virus (NSDV) is a tick-borne virus which causes a severe disease in sheep and goats, and has been responsible for several outbreaks of disease in East Africa. The virus is also found in the Indian subcontinent, where it is known as Ganjam virus. The virus only spreads through the feeding of competent infected ticks, and is therefore limited in its geographic distribution by the distribution of those ticks, Rhipicephalus appendiculata in Africa and Haemaphysalis intermedia in India. Animals bred in endemic areas do not normally develop disease, and the impact is therefore primarily on animals being moved for trade or breeding purposes. The disease caused by NSDV has similarities to several other ruminant diseases, and laboratory diagnosis is necessary for confirmation. There are published methods for diagnosis based on polymerase chain reaction, for virus growth in cell culture and for other simple diagnostic tests, though none has been commercialised. There is no established vaccine against NSDV, although cell-culture attenuated strains have been developed which show promise and could be put into field trials if it were deemed necessary. The virus is closely related to Crimean-Congo haemorrhagic fever virus, and studies on NSDV may therefore be useful in understanding this important human pathogen.

A new global threat for the public safety: Zika virus

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Simona Bicheru

2016-04-01

Full Text Available Zika virus, the etiological agent of Zika fever, is transmitted by mosquitoes and has been affecting the South American continent starting with 2015. It was reported in several European countries, carried by the people who returned from Latin America, as reported by the health authorities in those countries. Today, according to the World Health Organization (WHO, the virus suspected to cause serious birth defects in the fetus has also been confirmed in 21 of the 55 countries of South America, but also in other states from Europe and North America. Zika virus is a single stranded positive sense RNA virus belonging to Flavivirus genus (family Flaviviridae and was first identified in 1947 in Uganda rainforest Zika. The increased number of cases of microcephaly, in children from northern Brazil, suggested a connection with Zika virus, but it has not yet been proven. Also, the virus can be transmitted sexually and through blood or blood products. Diagnosis of the infection is made using Polymerase Chain Reaction (PCR. So far, there is no specific antiviral treatment or vaccine against the infection with Zika virus. The best form of prevention is to avoid mosquito bites. WHO has estimated that the spread of Zika virus, transmitted through mosquito bite, is “a global public health emergency”. The priority is to protect pregnant women and to control the mosquitoes.

Reverse Transcription Polymerase Chain Reaction-based System for Simultaneous Detection of Multiple Lily-infecting Viruses

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Ji Yeon Kwon

2013-09-01

Full Text Available A detection system based on a multiplex reverse transcription (RT polymerase chain reaction (PCR was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV, lily mottle virus (LMoV, lily symptomless virus (LSV. Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single-or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles.

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Moeschler, Sarah; Locher, Samira; Conzelmann, Karl-Klaus; Krämer, Beate; Zimmer, Gert

2016-09-16

Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses.

Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles

Directory of Open Access Journals (Sweden)

Sarah Moeschler

2016-09-01

Full Text Available Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT. Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses.

How to perform a nanoindentation experiment on a virus

NARCIS (Netherlands)

Roos, Wouter H.

2011-01-01

To broaden our knowledge on virus structure and function, a profound insight into their mechanical properties is required. Nanoindentation measurements with an atomic force microscope (AFM) are increasingly being performed to probe such material properties. This single-particle approach allows for

Brain Endothelial- and Epithelial-Specific Interferon Receptor Chain 1 Drives Virus-Induced Sickness Behavior and Cognitive Impairment

NARCIS (Netherlands)

Blank, Thomas; Detje, Claudia N.; Spiess, Alena; Hagemeyer, Nora; Brendecke, Stefanie M.; Wolfart, Jakob; Staszewski, Ori; Zoeller, Tanja; Papageorgiou, Ismini; Schneider, Justus; Paricio-Montesinos, Ricardo; Eisel, Ulrich L. M.; Manahan-Vaughan, Denise; Jansen, Stephan; Lienenklaus, Stefan; Lu, Bao; Imai, Yumiko; Mueller, Marcus; Goelz, Susan E.; Baker, Darren P.; Schwaninger, Markus; Kann, Oliver; Heikenwalder, Mathias; Kalinke, Ulrich; Prinz, Marco

2016-01-01

Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded

A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

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Alvaro Díaz-Badillo

2014-04-01

Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production.

Science.gov (United States)

Gouklani, H; Beyer, C; Drummer, H; Gowans, E J; Netter, H J; Haqshenas, G

2013-04-01

The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV. © 2012 Blackwell Publishing Ltd.

The archetype enhancer of simian virus 40 DNA is duplicated during virus growth in human cells and rhesus monkey kidney cells but not in green monkey kidney cells

International Nuclear Information System (INIS)

O'Neill, Frank J.; Greenlee, John E.; Carney, Helen

2003-01-01

Archetype SV40, obtained directly from its natural host, is characterized by a single 72-bp enhancer element. In contrast, SV40 grown in cell culture almost invariably exhibits partial or complete duplication of the enhancer region. This distinction has been considered important in studies of human tumor material, since SV40-associated tumor isolates have been described having a single enhancer region, suggesting natural infection as opposed to possible contamination by laboratory strains of virus. However, the behavior of archetypal SV40 in cultured cells has never been methodically studied. In this study we reengineered nonarchetypal 776-SV40 to contain a single 72-bp enhancer region and used this reengineered archetypal DNA to transfect a number of simian and human cell lines. SV40 DNA recovered from these cells was analyzed by restriction endonuclease analysis, PCR, and DNA sequencing. Reengineered archetype SV40 propagated in green monkey TC-7 or BSC-1 kidney cells remained without enhancer region duplication even after extensive serial virus passage. Archetype SV40 grown in all but one of the rhesus or human cell lines initially appeared exclusively archetypal. However, when virus from these cell types was transferred to green monkey cells, variants with partial enhancer duplication appeared after as little as a single passage. These findings suggest (1) that virus with a single 72-bp enhancer may persist in cultured cells of simian and human origin; (2) that variants with partially duplicated enhancer regions may arise within cell lines in quantities below limits of detection; (3) that these variants may enjoy a selective advantage in cell types other than those from which they arose (e.g., green monkey kidney cells); and (4) that certain cell lines may support a selective growth advantage for the variants without supporting their formation. Our data indicate that enhancer duplication may also occur in human as well as rhesus kidney cells. Thus, detection of

Specific Mutations in the PB2 Protein of Influenza A Virus Compensate for the Lack of Efficient Interferon Antagonism of the NS1 Protein of Bat Influenza A-Like Viruses.

Science.gov (United States)

Aydillo, Teresa; Ayllon, Juan; Pavlisin, Amzie; Martinez-Romero, Carles; Tripathi, Shashank; Mena, Ignacio; Moreira-Soto, Andrés; Vicente-Santos, Amanda; Corrales-Aguilar, Eugenia; Schwemmle, Martin; García-Sastre, Adolfo

2018-04-01

Recently, two new influenza A-like viruses have been discovered in bats, A/little yellow-shouldered bat/Guatemala/060/2010 (HL17NL10) and A/flat-faced bat/Peru/033/2010 (HL18NL11). The hemagglutinin (HA)-like (HL) and neuraminidase (NA)-like (NL) proteins of these viruses lack hemagglutination and neuraminidase activities, despite their sequence and structural homologies with the HA and NA proteins of conventional influenza A viruses. We have now investigated whether the NS1 proteins of the HL17NL10 and HL18NL11 viruses can functionally replace the NS1 protein of a conventional influenza A virus. For this purpose, we generated recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing the NS1 protein of the PR8 wild-type, HL17NL10, and HL18NL11 viruses. These viruses (r/NS1PR8, r/NS1HL17, and r/NS1HL18, respectively) were tested for replication in bat and nonbat mammalian cells and in mice. Our results demonstrate that the r/NS1HL17 and r/NS1HL18 viruses are attenuated in vitro and in vivo However, the bat NS1 recombinant viruses showed a phenotype similar to that of the r/NS1PR8 virus in STAT1 -/- human A549 cells and mice, both in vitro and in vivo systems being unable to respond to interferon (IFN). Interestingly, multiple mouse passages of the r/NS1HL17 and r/NS1HL18 viruses resulted in selection of mutant viruses containing single amino acid mutations in the viral PB2 protein. In contrast to the parental viruses, virulence and IFN antagonism were restored in the selected PB2 mutants. Our results indicate that the NS1 protein of bat influenza A-like viruses is less efficient than the NS1 protein of its conventional influenza A virus NS1 counterpart in antagonizing the IFN response and that this deficiency can be overcome by the influenza virus PB2 protein. IMPORTANCE Significant gaps in our understanding of the basic features of the recently discovered bat influenza A-like viruses HL17NL10 and HL18NL11 remain. The basic biology of these unique

First molecular detection of Chronic Bee Paralysis Virus (CBPV in Iran

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Modirrousta, H.

2015-12-01

Full Text Available Among the viruses infecting honey bees, chronic bee paralysis virus (CBPV is known to induce significant losses in honey bee colonies. CBPV is an unclassified polymorphic single stranded RNA virus. Using RT-PCR, the virus infections in honey bees can be detected in a rapid and accurate manner. Bee samples were collected from 23 provinces of Iran, between July-September 2011 and 2012. A total of 160 apiaries were sampled and submitted for virus screening. RNA extraction and RT-PCR were performed with QIAGEN kits. The primers lead to a fragment of 315 bp. The PCR products were electrophoresed in a 1.2 % agarose gel. Following the RT-PCR reaction with the specific primers, out of the 160 apiaries examined, 12 (7.5 % were infected with CBPV. This is the first study of CBPV detection in Iranian apiaries. We identified CBPV in the collected samples from different geographic regions of Iran.

AFM review study on pox viruses and living cells.

Science.gov (United States)

Ohnesorge, F M; Hörber, J K; Häberle, W; Czerny, C P; Smith, D P; Binnig, G

1997-10-01

Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm. The cell was held by a micropipette mounted onto the scanner-piezo as shown in Häberle, W., J. K. H. Hörber, and G. Binnig. 1991. Force microscopy on living cells. J. Vac. Sci. Technol. B9:1210-0000. To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Häberle, W., J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, and G. Binnig. 1992. In situ investigations of single living cells infected by viruses. Ultramicroscopy. 42-44:1161-0000; Hörber, J. K. H., W. Häberle, F. Ohnesorge, G. Binnig, H. G. Liebich, C. P. Czerny, H. Mahnel, and A. Mayr. 1992. Investigation of living cells in the nanometer regime with the atomic force microscope. Scanning Microscopy. 6:919-930). Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level. Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (small dark spots in the light microscope, that we believed to be the regions in the cell plasma where viruses are assembled; this is known from the literature on electron microscopy on pox-infected cells and referred to there as "virus factories" (e.g., Moss, B. 1986. Replication of pox viruses. In Fundamental Virology, B. N. Fields and D. M. Knape, editors. Raven Press, New York. 637-655). Therefore, we assume that the cells stay alive during imaging, in our experience for approximately 30-45 h p.i.).

Replication kinetics and shedding of very virulent Marek's disease virus and vaccinal Rispens/CVI988 virus during single and mixed infections varying in order and interval between infections.

Science.gov (United States)

Islam, Tanzila; Walkden-Brown, Stephen W; Renz, Katrin G; Islam, A F M Fakhrul; Ralapanawe, Sithara

2014-10-10

Vaccination is thought to contribute to an evolution in virulence of the Marek's disease virus (MDV) as vaccines prevent disease but not infection. We investigated the effects of co-infections at various intervals between Rispens/CVI988 vaccine virus (Rispens) and very virulent MDV (vvMDV) on the replication and shedding of each virus. The experiment used 600 ISA Brown layer chickens in 24 isolators with all treatments replicated in two isolators. Chickens were vaccinated with Rispens and/or challenged with the vvMDV isolate 02LAR on days 0, 5, or 10 post hatching providing vaccination to challenge intervals (VCI) of -10, -5, 0, 5 or 10 days with the negative values indicating challenge prior to vaccination. Peripheral blood lymphocytes (PBL), feathers and isolator exhaust dust were sampled between 7 and 56 days post infection (dpi) and subjected to quantitative real-time polymerase chain reaction (qPCR) to differentiate the two viruses. Overall Rispens significantly reduced the viral load of vvMDV in PBL and feather cells and shedding in dust. Similarly vvMDV significantly reduced the viral load of Rispens in PBL and feather cells but not in dust. VCI significantly influenced these relationships having strong positive and negative associations with load of vvMDV and Rispens respectively. Differences between the two viruses and their effects on each other were greatest in PBL and feathers, and least in dust. This study expands our understanding of the interaction between pathogenic and vaccinal viruses following vaccination with imperfect vaccines and has implications for selection of appropriate samples to test for vaccination success. Copyright © 2014 Elsevier B.V. All rights reserved.

Spliced RNA of woodchuck hepatitis virus.

Science.gov (United States)

Ogston, C W; Razman, D G

1992-07-01

Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

DIAGNOSTICS OF VIRUS PHYTOPATHOGENS FRUIT TREE PLUM POX VIRUS, PRUNUS NECROTIC RINGSPOT VIRUS AND PRUNUS DWARF VIRUS BY BIOLOGICAL AND MOLECULAR DIAGNOSTICS

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Július Rozák

2013-02-01

Full Text Available The aim of this study was to determine the incidence of viral phytopathogen Plum pox virus, Prunus necrotic ringspot virus and Prunus dwarf virus in selected localities of Slovakia and diagnose them using a molecular and biological methods. Forty samples of fruit trees of the genus Prunus, twenty samples from intensive plantings and twenty samples from wild subject were analysed. Biological diagnostic by using biological indicators Prunus persica cv. GF 305, Prunus serrulata cv. Schirofugen and molecular diagnostic by mRT-PCR were applied. Five samples with Plum pox virus were infected. The two samples positive for Prunus necrotic ringspot virus and one sample for Prunus dwarf virus were confirmed. The two samples were found to be infected with two viruses Prunus necrotic ringspot virus and Prunus dwarf virus. This work focuses on two techniques, their application to the diagnosis of stone fruit viruses and their routinely used for sanitary and certification programmes.

Can VHS virus bypass the protective immunity induced by DNA vaccination in rainbow trout?

DEFF Research Database (Denmark)

Sepúlveda, Dagoberto; Lorenzen, Niels

2016-01-01

DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability...... and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly...... pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus...

Real-time single airborne nanoparticle detection with nanomechanical resonant filter-fiber

DEFF Research Database (Denmark)

Schmid, Silvan; Kurek, Maksymilian; Adolphsen, Jens Q

2013-01-01

Nanomechanical resonators have an unprecedented mass sensitivity sufficient to detect single molecules, viruses or nanoparticles. The challenge with nanomechanical mass sensors is the direction of nano-sized samples onto the resonator. In this work we present an efficient inertial sampling...... study of single filter-fiber behavior. We present the direct measurement of diffusive nanoparticle collection on a single filter-fiber qualitatively confirming Langmuir's model from 1942....

Evidence for repair of ultraviolet light-damaged herpes virus in human fibroblasts by a recombination mechanism

International Nuclear Information System (INIS)

Hall, J.D.; Featherston, J.D.; Almy, R.E.

1980-01-01

Human cells were either singly or multiply infected with herpes simplex virus (HSV-1) damaged by ultraviolet (uv) light, and the fraction of cells able to produce infectious virus was measured. The fraction of virus-producing cells was considerably greater for multiply infected cells than for singly infected cells at each uv dose examined. These high survival levels of uv-irradiated virus in multiply infected cells demonstrated that multiplicity-dependent repair, possibly due to genetic exchanges between damaged HSV-1 genomes, was occurring in these cells. To test whether uv light is recombinogenic for HSV-1, the effect of uv irradiation on the yield of temperature-resistant viral recombinants in cells infected with pairs of temperature-sensitive mutants was also investigated. The results of these experiments showed that the defective functions in these mutant host cells are not required for multiplicity-dependent repair or uv-stimulated viral recombination in herpes-infected cells

Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus

International Nuclear Information System (INIS)

Stapleton, J.T.; Lemon, S.M.

1987-01-01

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development

Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

Science.gov (United States)

Lung, O; Furukawa-Stoffer, T; Burton Hughes, K; Pasick, J; King, D P; Hodko, D

2017-12-01

Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID 50 /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site. © 2016 Her Majesty the Queen in Right of Canada.

Characterization of Posa and Posa-like virus genomes in fecal samples from humans, pigs, rats, and bats collected from a single location in Vietnam

NARCIS (Netherlands)

Oude Munnink, Bas B.; Phan, My V. T.; Simmonds, Peter; Koopmans, Marion P. G.; Kellam, Paul; van der Hoek, Lia; Cotten, Matthew

2017-01-01

Porcine stool-associated RNA virus (posavirus), and Human stool-associated RNA virus (husavirus) are viruses in the order Picornavirales recently described in porcine and human fecal samples. The tentative group (Posa and Posa-like viruses: PPLVs) also includes fish stool-associated RNA virus

Oncogenic Viruses and Breast Cancer: Mouse Mammary Tumor Virus (MMTV, Bovine Leukemia Virus (BLV, Human Papilloma Virus (HPV, and Epstein–Barr Virus (EBV

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James S. Lawson

2018-01-01

Full Text Available BackgroundAlthough the risk factors for breast cancer are well established, namely female gender, early menarche and late menopause plus the protective influence of early pregnancy, the underlying causes of breast cancer remain unknown. The development of substantial recent evidence indicates that a handful of viruses may have a role in breast cancer. These viruses are mouse mammary tumor virus (MMTV, bovine leukemia virus (BLV, human papilloma viruses (HPVs, and Epstein–Barr virus (EBV-also known as human herpes virus type 4. Each of these viruses has documented oncogenic potential. The aim of this review is to inform the scientific and general community about this recent evidence.The evidenceMMTV and human breast cancer—the evidence is detailed and comprehensive but cannot be regarded as conclusive. BLV and human breast cancer—the evidence is limited. However, in view of the emerging information about BLV in human breast cancer, it is prudent to encourage the elimination of BLV in cattle, particularly in the dairy industry. HPVs and breast cancer—the evidence is substantial but not conclusive. The availability of effective preventive vaccines is a major advantage and their use should be encouraged. EBV and breast cancer—the evidence is also substantial but not conclusive. Currently, there are no practical means of either prevention or treatment. Although there is evidence of genetic predisposition, and cancer in general is a culmination of events, there is no evidence that inherited genetic traits are causal.ConclusionThe influence of oncogenic viruses is currently the major plausible hypothesis for a direct cause of human breast cancer.

Oncogenic Viruses and Breast Cancer: Mouse Mammary Tumor Virus (MMTV), Bovine Leukemia Virus (BLV), Human Papilloma Virus (HPV), and Epstein-Barr Virus (EBV).

Science.gov (United States)

Lawson, James S; Salmons, Brian; Glenn, Wendy K

2018-01-01

Although the risk factors for breast cancer are well established, namely female gender, early menarche and late menopause plus the protective influence of early pregnancy, the underlying causes of breast cancer remain unknown. The development of substantial recent evidence indicates that a handful of viruses may have a role in breast cancer. These viruses are mouse mammary tumor virus (MMTV), bovine leukemia virus (BLV), human papilloma viruses (HPVs), and Epstein-Barr virus (EBV-also known as human herpes virus type 4). Each of these viruses has documented oncogenic potential. The aim of this review is to inform the scientific and general community about this recent evidence. MMTV and human breast cancer-the evidence is detailed and comprehensive but cannot be regarded as conclusive. BLV and human breast cancer-the evidence is limited. However, in view of the emerging information about BLV in human breast cancer, it is prudent to encourage the elimination of BLV in cattle, particularly in the dairy industry. HPVs and breast cancer-the evidence is substantial but not conclusive. The availability of effective preventive vaccines is a major advantage and their use should be encouraged. EBV and breast cancer-the evidence is also substantial but not conclusive. Currently, there are no practical means of either prevention or treatment. Although there is evidence of genetic predisposition, and cancer in general is a culmination of events, there is no evidence that inherited genetic traits are causal. The influence of oncogenic viruses is currently the major plausible hypothesis for a direct cause of human breast cancer.

The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox.

Science.gov (United States)

Gubser, Caroline; Smith, Geoffrey L

2002-04-01

Camelpox virus (CMPV) and variola virus (VAR) are orthopoxviruses (OPVs) that share several biological features and cause high mortality and morbidity in their single host species. The sequence of a virulent CMPV strain was determined; it is 202182 bp long, with inverted terminal repeats (ITRs) of 6045 bp and has 206 predicted open reading frames (ORFs). As for other poxviruses, the genes are tightly packed with little non-coding sequence. Most genes within 25 kb of each terminus are transcribed outwards towards the terminus, whereas genes within the centre of the genome are transcribed from either DNA strand. The central region of the genome contains genes that are highly conserved in other OPVs and 87 of these are conserved in all sequenced chordopoxviruses. In contrast, genes towards either terminus are more variable and encode proteins involved in host range, virulence or immunomodulation. In some cases, these are broken versions of genes found in other OPVs. The relationship of CMPV to other OPVs was analysed by comparisons of DNA and predicted protein sequences, repeats within the ITRs and arrangement of ORFs within the terminal regions. Each comparison gave the same conclusion: CMPV is the closest known virus to variola virus, the cause of smallpox.

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

International Nuclear Information System (INIS)

Lamb, Kristen; Lokesh, G.L.; Sherman, Michael; Watowich, Stanley

2010-01-01

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.

Mixed-genotype white spot syndrome virus infections of shrimp are inversely correlated with disease outbreaks in ponds

NARCIS (Netherlands)

Tuyet Hoa, T.T.; Zwart, M.P.; Phuong, N.T.; Oanh, D.T.H.; Jong, de M.C.M.; Vlak, J.M.

2011-01-01

Outbreaks of white spot syndrome virus (WSSV) in shrimp culture and its relation to virus virulence are not well understood. Here we provide evidence that the presence of WSSV mixed-genotype infections correlate with lower outbreak incidence and that disease outbreaks correlate with single-genotype

Hepatitis A Virus Genome Organization and Replication Strategy.

Science.gov (United States)

McKnight, Kevin L; Lemon, Stanley M

2018-04-02

Hepatitis A virus (HAV) is a positive-strand RNA virus classified in the genus Hepatovirus of the family Picornaviridae It is an ancient virus with a long evolutionary history and multiple features of its capsid structure, genome organization, and replication cycle that distinguish it from other mammalian picornaviruses. HAV proteins are produced by cap-independent translation of a single, long open reading frame under direction of an inefficient, upstream internal ribosome entry site (IRES). Genome replication occurs slowly and is noncytopathic, with transcription likely primed by a uridylated protein primer as in other picornaviruses. Newly produced quasi-enveloped virions (eHAV) are released from cells in a nonlytic fashion in a unique process mediated by interactions of capsid proteins with components of the host cell endosomal sorting complexes required for transport (ESCRT) system. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Role of Electrostatics in the assembly pathway of a single-stranded RNA virus

NARCIS (Netherlands)

Garmann, R.F.; Comas-Garcia, M.; Koay, M.S.T.; Cornelissen, Jeroen Johannes Lambertus Maria; Knobler, C.M.; Gelbart, W.M.

2014-01-01

We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318–3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of

Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

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Duborjal Hervé

2008-02-01

Full Text Available Abstract Background Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus. Results Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population. Using engineered allelic markers in the genome of Cauliflower mosaic virus, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1. Conclusion This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized

Food and environmental routes of Hepatitis E virus transmission

NARCIS (Netherlands)

Poel, van der W.H.M.

2014-01-01

Abstract Hepatitis E virus (HEV), genus Hepevirus, family hepeviridae is a main cause of epidemic hepatitis in developing countries and single cases of hepatitis in higher income countries. There are at least four HEV genotypes which have different epidemiologic and clinical features. Hepatitis E

Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments

NARCIS (Netherlands)

Wichgers Schreur, Paul J.; Kortekaas, Jeroen

2016-01-01

The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging.

A quantitative quasispecies theory-based model of virus escape mutation under immune selection.

Science.gov (United States)

Woo, Hyung-June; Reifman, Jaques

2012-08-07

Viral infections involve a complex interplay of the immune response and escape mutation of the virus quasispecies inside a single host. Although fundamental aspects of such a balance of mutation and selection pressure have been established by the quasispecies theory decades ago, its implications have largely remained qualitative. Here, we present a quantitative approach to model the virus evolution under cytotoxic T-lymphocyte immune response. The virus quasispecies dynamics are explicitly represented by mutations in the combined sequence space of a set of epitopes within the viral genome. We stochastically simulated the growth of a viral population originating from a single wild-type founder virus and its recognition and clearance by the immune response, as well as the expansion of its genetic diversity. Applied to the immune escape of a simian immunodeficiency virus epitope, model predictions were quantitatively comparable to the experimental data. Within the model parameter space, we found two qualitatively different regimes of infectious disease pathogenesis, each representing alternative fates of the immune response: It can clear the infection in finite time or eventually be overwhelmed by viral growth and escape mutation. The latter regime exhibits the characteristic disease progression pattern of human immunodeficiency virus, while the former is bounded by maximum mutation rates that can be suppressed by the immune response. Our results demonstrate that, by explicitly representing epitope mutations and thus providing a genotype-phenotype map, the quasispecies theory can form the basis of a detailed sequence-specific model of real-world viral pathogens evolving under immune selection.

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

OpenAIRE

Savithri, HS; Murthy, MRN

1983-01-01

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses - cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2...

Differences in the Selection Bottleneck between Modes of Sexual Transmission Influence the Genetic Composition of the HIV-1 Founder Virus.

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Damien C Tully

2016-05-01

Full Text Available Due to the stringent population bottleneck that occurs during sexual HIV-1 transmission, systemic infection is typically established by a limited number of founder viruses. Elucidation of the precise forces influencing the selection of founder viruses may reveal key vulnerabilities that could aid in the development of a vaccine or other clinical interventions. Here, we utilize deep sequencing data and apply a genetic distance-based method to investigate whether the mode of sexual transmission shapes the nascent founder viral genome. Analysis of 74 acute and early HIV-1 infected subjects revealed that 83% of men who have sex with men (MSM exhibit a single founder virus, levels similar to those previously observed in heterosexual (HSX transmission. In a metadata analysis of a total of 354 subjects, including HSX, MSM and injecting drug users (IDU, we also observed no significant differences in the frequency of single founder virus infections between HSX and MSM transmissions. However, comparison of HIV-1 envelope sequences revealed that HSX founder viruses exhibited a greater number of codon sites under positive selection, as well as stronger transmission indices possibly reflective of higher fitness variants. Moreover, specific genetic "signatures" within MSM and HSX founder viruses were identified, with single polymorphisms within gp41 enriched among HSX viruses while more complex patterns, including clustered polymorphisms surrounding the CD4 binding site, were enriched in MSM viruses. While our findings do not support an influence of the mode of sexual transmission on the number of founder viruses, they do demonstrate that there are marked differences in the selection bottleneck that can significantly shape their genetic composition. This study illustrates the complex dynamics of the transmission bottleneck and reveals that distinct genetic bottleneck processes exist dependent upon the mode of HIV-1 transmission.

Structure of the Triatoma virus capsid.

Science.gov (United States)

Squires, Gaëlle; Pous, Joan; Agirre, Jon; Rozas-Dennis, Gabriela S; Costabel, Marcelo D; Marti, Gerardo A; Navaza, Jorge; Bressanelli, Stéphane; Guérin, Diego M A; Rey, Felix A

2013-06-01

The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

Safety and immunogenicity of a live attenuated Japanese encephalitis chimeric virus vaccine (IMOJEV®) in children.

Science.gov (United States)

Chokephaibulkit, K; Houillon, G; Feroldi, E; Bouckenooghe, A

2016-01-01

JE-CV (IMOJEV®, Sanofi Pasteur, France) is a live attenuated virus vaccine constructed by inserting coding sequences of the prM and E structural proteins of the Japanese encephalitis SA14-14-2 virus into the genome of yellow fever 17D virus. Primary immunization with JE-CV requires a single dose of the vaccine. This article reviews clinical trials of JE-CV in children aged up to 6 years conducted in countries across South-East Asia. Strong and persistent antibody responses were observed after single primary and booster doses, with 97% of children seroprotected up to five years after booster vaccination. Models of long-term antibody persistence predict a median duration of protection of approximately 30 years after a booster dose. The safety and reactogenicity profiles of JE-CV primary and booster doses are comparable to other widely used childhood vaccines.

Previously unknown and highly divergent ssDNA viruses populate the oceans.

Science.gov (United States)

Labonté, Jessica M; Suttle, Curtis A

2013-11-01

Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.

Survey of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus incidence in Korea by Duplex RT-PCR

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Seung-Yeol Lee

2014-12-01

Full Text Available The incidence of Cherry necrotic rusty mottle virus (CNRMV and Cherry green ring mottle virus (CGRMV have recently been occurred in Korea, posing a problem for sweet cherry cultivation. Since infected trees have symptomless leaves or ring-like spots on the pericarp, it is difficult to identify a viral infection. In this study, the incidence of CNRMV and CGRMV in sweet cherry in Gyeongbuk province was surveyed using a newly developed duplex reverse transcriptase polymerase chain reaction (RT-PCR method that can detect both viruses in a single reaction. CNRMV and CGRMV co-infection rates were 29.6%, 53.6%, and 17.6%, respectively, in samples collected from three different sites (Daegu, Gyeongju and Gyeongsan in Gyeongbuk province during 2012 and 2013. This duplex RT-PCR method offers a simple, rapid, and effective way of identifying CNRMV and CGRMV simultaneously in sweet cherry trees, which can aid in the management of viral infections that could undermine yield.

Genetic mapping of xenotropic murine leukemia virus-inducing loci in five mouse strains.

Science.gov (United States)

Kozak, C A; Rowe, W P

1980-07-01

A single mendelian gene was identified for induction of the endogenous xenotropic murine leukemia virus in five mouse strains (C57BL/10, C57L, C57BR, AKR, and BALB/c). This locus, designated Bxv-1, mapped to the same site on chromosome 1 in all strains: Id-1-Pep-3-[Bxv-1-Lp]. Thus, inducibility loci for xenotropic virus are more limited in number and chromosomal distribution than ecotropic inducibility loci. Virus expression in mice with Bxv-1 was induced by treatment of fibroblasts with 5-iododeoxyuridine or by exposure of spleen cells to a B cell mitogen, bacterial lipopolysaccharide. An analysis of the hamster X mouse somatic cell hybrids indicated that chromosome 1, alone, was sufficient for virus induction.

A novel small molecule inhibitor of influenza A viruses that targets polymerase function and indirectly induces interferon.

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Mila Brum Ortigoza

Full Text Available Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.

Increased hepatitis E virus seroprevalence correlates with lower CD4+ cell counts in HIV-infected persons in Argentina

NARCIS (Netherlands)

J.D. Debes; Martínez Wassaf, M. (Maribel); Pisano, M.B. (María Belén); Isa, M.B. (María Beatriz); Lotto, M. (Martin); Marianelli, L.G. (Leonardo G.); Frassone, N. (Natalia); Ballari, E. (Estefania); Bohjanen, P.R. (Paul R.); B.E. Hansen (Bettina); Ré, V. (Viviana)

2016-01-01

textabstractHepatitis E virus (HEV) is a single-stranded RNA virus that can cause hepatitis in an epidemic fashion. HEV usually causes asymptomatic or limited acute infections in immunocompetent individuals, whereas in immunosuppressed individuals such as transplant recipients, HEV can cause chronic

Correlation between particle multiplicity and location on virion RNA of the assembly initiation site for viruses of the tobacco mosaic virus group.

Science.gov (United States)

Fukuda, M; Meshi, T; Okada, Y; Otsuki, Y; Takebe, I

1981-07-01

The initiation site for reconstitution on genome RNA was determined by electron microscopic serology for a watermelon strain of cucumber green mottle mosaic virus (CGMMV-W), which is chemically and serologically related to tobacco mosaic virus (TMV). The initiation site was located at the same position as that of the cowpea strain, a virus that produces short rods of encapsidated subgenomic messenger RNA for the coat protein (a two-component TMV), being about 320 nucleotides away from the 3' terminus, and hence within the coat protein cistron. Although CGMMV-W was until now believed to be a single-component TMV, the location of the initiation site indicated the presence of short rods containing coat protein messenger RNA in CGMMV-W-infected tissue, as in the case for the cowpea strain. We found such short rods in CGMMV-W-infected tissue. The results confirmed our previous hypothesis that the site of the initiation region for reconstitution determines the rod multiplicity of TMV. The finding of the second two-component TMV, CGMMV, indicates that the cowpea strain of TMV is not unique in being a two-component virus and that the location of the assembly initiation site on the genome RNA can be a criterion for grouping of viruses.

Respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza A virus

DEFF Research Database (Denmark)

White, Mitchell R; Crouch, Erika; Vesona, Jenny

2005-01-01

of IAV with SP-D in vitro strongly increases neutrophil respiratory burst responses to the virus. Several factors are shown to modify this apparent proinflammatory effect of SP-D. Although multimeric forms of SP-D show dose-dependent augmentation of respiratory burst responses, trimeric, single-arm forms...... of IAV while reducing the respiratory burst response to virus....

Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses

DEFF Research Database (Denmark)

Gottwein, Judith M; Jensen, Sanne B; Li, Yi-Ping

2013-01-01

With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi...... to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations...

pH-Controlled Two-Step Uncoating of Influenza Virus

Science.gov (United States)

Li, Sai; Sieben, Christian; Ludwig, Kai; Höfer, Chris T.; Chiantia, Salvatore; Herrmann, Andreas; Eghiaian, Frederic; Schaap, Iwan A.T.

2014-01-01

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5–6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection. PMID:24703306

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

Energy Technology Data Exchange (ETDEWEB)

Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

2007-07-26

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

Science.gov (United States)

Murphy, John E.; Zhou, Shangzhen; Giese, Klaus; Williams, Lewis T.; Escobedo, Jaime A.; Dwarki, Varavani J.

1997-01-01

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2–5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity. PMID:9391128

Frequency and Molecular Characterization of Watermelon Mosaic Virus from Serbia

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Ana Vučurović

2010-01-01

Full Text Available Watermelon mosaic virus (WMV is widespread in cucurbit crops, most commonly occuring in temperate and Mediterranean regions. In Serbia WMV has been detected in single and mixed infections with Zucchini yellow mosaic virus and Cucumber mosaic virus in field-grown pumpkin and squash crops. Among pumpkin-affecting viruses WMV is the most frequent one, both by the number of localities and its incidence at each location. During the growing season of 2009, samples from 583 plants of Cucurbita pepo cvs. Olinka, Belgrade zucchini and Tosca (Zucchini group, as well as from C. maxima and C. moschata showing symptoms of virus infection were collected from 12 commercial fields at eight localities and analyzed by DAS-ELISA using polyclonal antisera specific to six most important cucurbit viruses. Interestingly, WMV was detected at fewer sites and had lower ncidence rate than in two previous years. In single infections, WMV was found in 11% of tested plants in three fields; in mixed infections with ZYMV, it was recorded in 9.9% of plants in five fields and with CMV in only 0.2% in one field. The partial coat protein gene and 3’ non-translated region from two representativeisolates of WMV originating from different localities and host plant species were amplified by RT-PCR, sequenced, and compared with the sequences available in GenBank database. The PCR-amplified fragment of predicted size of approximately 1017 bp was obtained. The sequences of isolates 137-08 (Acc. No. GQ259958 and 159-08 (GU144020 proved to be 94-99% identical at the nucleotide level with those from other parts of the world. The sequences of these two isolates differed from each other only at two nucleotide positions, without any amino acid substitution. Phylogenetic analysis of 57 isolates based on 750 bp sequences of the coat protein gene showed no correlation between isolates and their geographic origin, and italso indicated that these isolates fell into three molecular groups of

A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus.

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Yoshimi Tsuda

2011-08-01

Full Text Available Human outbreaks of Ebola virus (EBOV are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV vector expressing a CD8+ T cell epitope from the nucleoprotein (NP of Zaire ebolavirus (ZEBOV (MCMV/ZEBOV-NP(CTL. MCMV/ZEBOV-NP(CTL induced high levels of long-lasting (>8 months CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.

Double-label immunofluorescence method for simultaneous detection of adenovirus and herpes simplex virus from the eye.

Science.gov (United States)

Walpita, P; Darougar, S

1989-07-01

The development and application of a double-label immunofluorescence method which has the potential to screen for single or dual infections from any site, in single shell vial cultures, is described. In this study, a total of 1,141 ocular specimens were inoculated in shell vials, centrifuged at 15,000 X g for 1 h, incubated at 37 degrees C for 48 h, and fixed in methanol at room temperature for 15 min. The virus inclusions were detected by staining with a double-label indirect immunofluorescence procedure using mixtures of appropriate first antibodies, followed by fluorescein- and rhodamine-conjugated second antibodies. Each specimen was also inoculated in parallel by the conventional virus isolation method. The sensitivity and specificity of the double-label shell vial procedure were comparable to those with the conventional method, and the former test took only 48 h to complete. The test offers a rapid and simple single-vial procedure which allows for individual or simultaneous detection of multiple pathogens. It results in savings in time and cost over the conventional virus isolation method and other shell vial procedures.

Phylogenetic analysis of Dengue virus 1 isolated from South Minas Gerais, Brazil

OpenAIRE

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; Silveira, Nelson José Freitas da; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

2016-01-01

Abstract 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...

Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X.

Science.gov (United States)

Choi, Hoseong; Jo, Yeonhwa; Lian, Sen; Jo, Kyoung-Min; Chu, Hyosub; Yoon, Ju-Yeon; Choi, Seung-Kook; Kim, Kook-Hyung; Cho, Won Kyong

2015-06-01

The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

Characterisation and Identification of Avian Influenza Virus (AI

Directory of Open Access Journals (Sweden)

Dyah Ayu Hewajuli

2008-06-01

Full Text Available Avian Influenza is caused by Influenza A virus which is a member of Orthomyxoviridae family. Influenza A virus is enveloped single stranded RNA with eight-segmented, negative polarity and filament or oval form, 50 – 120 by 200 – 300 nm diameters. Influenza A viruses have been found to infect birds, human, pig, horse and sometimes in the other mammalian such as seal and whale. The viruses are divided into different subtypes based on the antigenic protein which covers the virus surface i.e. Haemaglutinin (HA and Neuraminidase (NA. In addition, the nomenclature of subtype virus is based on HA and NA i.e HxNx, for example H5N1, H9N2 and the others. According to pathogenic, it could be divided into two distinct groups, they are Highly Pathogenic Avian Influenza (HPAI and Low Pathogenic Avian Influenza (LPAI. The Avian Influenza viruses have been continuously occurred and spread out in some continents such us America, Europe, Africa and Asian countries. The outbreak of Avian Influenza caused high mortality on birds and it has been reported that in human case Avian Influenza subtype H5N1 virus has caused several deaths. To anticipate this condition, an effort to prevent the transmission of Avian Influenza is needed. These strategic attempts include biosecurity, depopulation, vaccination, control of virus movement, monitoring and evaluation. Laboratory diagnostic plays an important role for successful prevention, control and eradication programs of Avian Influenza. Recently, there are two diagnostic methods for Avian Influenza. They are conventional (virological diagnosis and molecular methods. The conventional method is usually used for initial diagnostic of Avian Influenza. The conventional method takes more time and more costly, whereas the molecular method is more effective than conventional method. Based on the available diagnostic technique, basically diagnostic of Avian Influenza is done by serology test, isolation and identification as well

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

International Nuclear Information System (INIS)

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.; Selvapandiyan, Angamuthu; Hewlett, Indira; Duncan, Robert; Puri, Raj K.; Nakhasi, Hira L.; Kaplan, Gerardo G.

2007-01-01

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients

Reemerging Sudan Ebola Virus Disease in Uganda, 2011

Science.gov (United States)

Shoemaker, Trevor; Balinandi, Stephen; Campbell, Shelley; Wamala, Joseph Francis; McMullan, Laura K.; Downing, Robert; Lutwama, Julius; Mbidde, Edward; Ströher, Ute; Rollin, Pierre E.; Nichol, Stuart T.

2012-01-01

Two large outbreaks of Ebola hemorrhagic fever occurred in Uganda in 2000 and 2007. In May 2011, we identified a single case of Sudan Ebola virus disease in Luwero District. The establishment of a permanent in-country laboratory and cooperation between international public health entities facilitated rapid outbreak response and control activities. PMID:22931687

Enteric viruses in a mangrove lagoon, survival and shellfish incidence

Energy Technology Data Exchange (ETDEWEB)

Lopez de Cardona, I.; Bermudez, M.; Billmire, E.; Hazen, T.C. [Univ. of Puerto Rico, Rio Piedras (Puerto Rico)

1988-12-31

Mangrove oysters (Crassostrea rhizophorae) were screened for enteric viruses. For 18 months oysters were collected from Cano Boqueron, a tropical mangrove lagoon on the southwest coast of Puerto Rico. This popular tourist resort has two primary sewage treatment plants which service 158 single family cabanas. In spite of the heavy seasonal input of sewage to Cano Boqueron and high densities of fecal coliform bacteria, enteric viruses were not detected in shellfish meat. Because no viruses were detected in the oysters, a virus survival study was performed. Poliovirus type 1 was placed in diffusion chambers in situ at two sites in Cano Boqueron. More than 95% of the poliovirus inactivation occurred within 24 h. Virus inactivation was significantly different by site, indicating different inactivation rates within the lagoon. Chamber studies done simultaneously with Escherichia coli did not reveal differences between sites. It is suggested that the sewage effluent had an antiviral effect in the absence of an antibacterial effect. This study demonstrates the importance for establishing microbial contamination standards for shellfish growing waters in the tropics based upon in situ studies with tropical species, e.g. mangrove oyster.

pLoc-mVirus: Predict subcellular localization of multi-location virus proteins via incorporating the optimal GO information into general PseAAC.

Science.gov (United States)

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-09-10

Knowledge of subcellular locations of proteins is crucially important for in-depth understanding their functions in a cell. With the explosive growth of protein sequences generated in the postgenomic age, it is highly demanded to develop computational tools for timely annotating their subcellular locations based on the sequence information alone. The current study is focused on virus proteins. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex proteins is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mVirus" by extracting the optimal GO (Gene Ontology) information into the general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on a same stringent benchmark dataset indicated that the proposed pLoc-mVirus predictor is remarkably superior to iLoc-Virus, the state-of-the-art method in predicting virus protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc-mVirus/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier B.V. All rights reserved.

Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes.

Science.gov (United States)

Le Coupanec, Alain; Tchankouo-Nguetcheu, Stéphane; Roux, Pascal; Khun, Huot; Huerre, Michel; Morales-Vargas, Ronald; Enguehard, Margot; Lavillette, Dimitri; Missé, Dorothée; Choumet, Valérie

2017-08-04

Arthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti . The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells.

Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

Science.gov (United States)

Milrot, Elad; Mutsafi, Yael; Fridmann-Sirkis, Yael; Shimoni, Eyal; Rechav, Katya; Gurnon, James R; Van Etten, James L; Minsky, Abraham

2016-01-01

The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway. © 2015 John Wiley & Sons Ltd.

Heparin binding sites on Ross River virus revealed by electron cryo-microscopy

International Nuclear Information System (INIS)

Zhang Wei; Heil, Marintha; Kuhn, Richard J.; Baker, Timothy S.

2005-01-01

Cell surface glycosaminoglycans play important roles in cell adhesion and viral entry. Laboratory strains of two alphaviruses, Sindbis and Semliki Forest virus, have been shown to utilize heparan sulfate as an attachment receptor, whereas Ross River virus (RRV) does not significantly interact with it. However, a single amino acid substitution at residue 218 in the RRV E2 glycoprotein adapts the virus to heparan sulfate binding and expands the host range of the virus into chicken embryo fibroblasts. Structures of the RRV mutant, E2 N218R, and its complex with heparin were determined through the use of electron cryo-microscopy and image reconstruction methods. Heparin was found to bind at the distal end of the RRV spikes, in a region of the E2 glycoprotein that has been previously implicated in cell-receptor recognition and antibody binding

Structural analysis of radiation-induced chromosome aberrations by atomic force microscope (AFM) before and after Giemsa staining

International Nuclear Information System (INIS)

Murakami, M.; Kanda, R.; Minamihisamatsu, M.; Hayata, I.

2003-01-01

Full text: We have studied structures of chromosome aberration induced by ionizing radiation by an atomic force microscope (AFM). The AFM could visualize the fine structure of chromosomes on Giemsa stained or unstained samples, although it was difficult to visualize unstained chromosomes by light microscope. The height data of chromosomes obtained by AFM provided useful information to describe detailed structure of chromatid gaps induced by heavy ion irradiation. A fibrous structure was observed on the unstained chromosome and these structures were considered to be the 30nm fibers on the chromosome. These types of structures were observed in the gaps as well as on surface of the chromosome. Further more, other types of chromosome aberration induced by ionizing radiation visualized by AFM will be presented

Evaluation of the genetic diversity of Plum pox virus in a single plum tree.

Science.gov (United States)

Predajňa, Lukáš; Šubr, Zdeno; Candresse, Thierry; Glasa, Miroslav

2012-07-01

Genetic diversity of Plum pox virus (PPV) and its distribution within a single perennial woody host (plum, Prunus domestica) has been evaluated. A plum tree was triply infected by chip-budding with PPV-M, PPV-D and PPV-Rec isolates in 2003 and left to develop untreated under open field conditions. In September 2010 leaf and fruit samples were collected from different parts of the tree canopy. A 745-bp NIb-CP fragment of PPV genome, containing the hypervariable region encoding the CP N-terminal end was amplified by RT-PCR from each sample and directly sequenced to determine the dominant sequence. In parallel, the PCR products were cloned and a total of 105 individual clones were sequenced. Sequence analysis revealed that after 7 years of infection, only PPV-M was still detectable in the tree and that the two other isolates (PPV-Rec and PPV-D) had been displaced. Despite the fact that the analysis targeted a relatively short portion of the genome, a substantial amount of intra-isolate variability was observed for PPV-M. A total of 51 different haplotypes could be identified from the 105 individual sequences, two of which were largely dominant. However, no clear-cut structuration of the viral population by the tree architecture could be highlighted although the results obtained suggest the possibility of intra-leaf/fruit differentiation of the viral population. Comparison of the consensus sequence with the original source isolate showed no difference, suggesting within-plant stability of this original isolate under open field conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

Delineating morbillivirus entry, dissemination and airborne transmission by studying in vivo competition of multicolor canine distemper viruses in ferrets.

Directory of Open Access Journals (Sweden)

Rory D de Vries

2017-05-01

Full Text Available Identification of cellular receptors and characterization of viral tropism in animal models have vastly improved our understanding of morbillivirus pathogenesis. However, specific aspects of viral entry, dissemination and transmission remain difficult to recapitulate in animal models. Here, we used three virologically identical but phenotypically distinct recombinant (r canine distemper viruses (CDV expressing different fluorescent reporter proteins for in vivo competition and airborne transmission studies in ferrets (Mustela putorius furo. Six donor ferrets simultaneously received three rCDVs expressing green, red or blue fluorescent proteins via conjunctival (ocular, Oc, intra-nasal (IN or intra-tracheal (IT inoculation. Two days post-inoculation sentinel ferrets were placed in physically separated adjacent cages to assess airborne transmission. All donor ferrets developed lymphopenia, fever and lethargy, showed progressively increasing systemic viral loads and were euthanized 14 to 16 days post-inoculation. Systemic replication of virus inoculated via the Oc, IN and IT routes was detected in 2/6, 5/6 and 6/6 ferrets, respectively. In five donor ferrets the IT delivered virus dominated, although replication of two or three different viruses was detected in 5/6 animals. Single lymphocytes expressing multiple fluorescent proteins were abundant in peripheral blood and lymphoid tissues, demonstrating the occurrence of double and triple virus infections. Transmission occurred efficiently and all recipient ferrets showed evidence of infection between 18 and 22 days post-inoculation of the donor ferrets. In all cases, airborne transmission resulted in replication of a single-colored virus, which was the dominant virus in the donor ferret. This study demonstrates that morbilliviruses can use multiple entry routes in parallel, and co-infection of cells during viral dissemination in the host is common. Airborne transmission was efficient, although

Delineating morbillivirus entry, dissemination and airborne transmission by studying in vivo competition of multicolor canine distemper viruses in ferrets.

Science.gov (United States)

de Vries, Rory D; Ludlow, Martin; de Jong, Alwin; Rennick, Linda J; Verburgh, R Joyce; van Amerongen, Geert; van Riel, Debby; van Run, Peter R W A; Herfst, Sander; Kuiken, Thijs; Fouchier, Ron A M; Osterhaus, Albert D M E; de Swart, Rik L; Duprex, W Paul

2017-05-01

Identification of cellular receptors and characterization of viral tropism in animal models have vastly improved our understanding of morbillivirus pathogenesis. However, specific aspects of viral entry, dissemination and transmission remain difficult to recapitulate in animal models. Here, we used three virologically identical but phenotypically distinct recombinant (r) canine distemper viruses (CDV) expressing different fluorescent reporter proteins for in vivo competition and airborne transmission studies in ferrets (Mustela putorius furo). Six donor ferrets simultaneously received three rCDVs expressing green, red or blue fluorescent proteins via conjunctival (ocular, Oc), intra-nasal (IN) or intra-tracheal (IT) inoculation. Two days post-inoculation sentinel ferrets were placed in physically separated adjacent cages to assess airborne transmission. All donor ferrets developed lymphopenia, fever and lethargy, showed progressively increasing systemic viral loads and were euthanized 14 to 16 days post-inoculation. Systemic replication of virus inoculated via the Oc, IN and IT routes was detected in 2/6, 5/6 and 6/6 ferrets, respectively. In five donor ferrets the IT delivered virus dominated, although replication of two or three different viruses was detected in 5/6 animals. Single lymphocytes expressing multiple fluorescent proteins were abundant in peripheral blood and lymphoid tissues, demonstrating the occurrence of double and triple virus infections. Transmission occurred efficiently and all recipient ferrets showed evidence of infection between 18 and 22 days post-inoculation of the donor ferrets. In all cases, airborne transmission resulted in replication of a single-colored virus, which was the dominant virus in the donor ferret. This study demonstrates that morbilliviruses can use multiple entry routes in parallel, and co-infection of cells during viral dissemination in the host is common. Airborne transmission was efficient, although transmission of

Peste des petits ruminants in Benin: Persistence of a single virus genotype in the country for over 42 years

International Nuclear Information System (INIS)

Adombi, C.M.; Waqas, A.; Dundon, W.G.; Li, S.; Daojin, Y.; Kakpo, L.; Aplogan, G.L.; Diop, M.; Lo, M.M.; Silber, R.; Loitsch, A.; Diallo, A.

2016-01-01

Full text: Peste des petits ruminants (PPR) is a contagious and often fatal disease affecting sheep and goats. Currently, it is endemic in Africa, the Middle and Near East, the Indian subcontinent and China. Understanding the molecular epidemiology and evolution of PPR virus (PPRV) can assist in the control of the transboundary spread of this economically important disease. We isolated PPRV from pathological and swab samples collected 42 years apart (1969 and 2011) in Benin, West Africa, and sequenced the full genome of two isolates (Benin/B1/1969 and Benin/ 10/2011). Phylogenetic analysis showed that all of the characterized isolates clustered within viral lineage II and that the 2011 isolates fell into two distinct subgroups. Comparison of the full genome sequences revealed a 95.3% identity at the nucleotide level, while at the protein level, the matrix protein was the most conserved between the two viruses with an identity of 99.7% and only one amino acid substitution over the 42-year sampling period. An analysis of specific amino acid residues of known or putative function did not identify any significant changes between the two viruses. A molecular clock analysis of complete PPRV genomes revealed that the lineage II viruses sampled here arose in the early 1960s and that these viruses have likely persisted in Benin since this time. (author)

Canine distemper virus (CDV) in another big cat: should CDV be renamed carnivore distemper virus?

Science.gov (United States)

Terio, Karen A; Craft, Meggan E

2013-09-17

One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or "spillover" hosts but, rather, a normal part of the complex ecology of this infectious disease.

Canine Distemper Virus (CDV) in Another Big Cat: Should CDV Be Renamed Carnivore Distemper Virus?

Science.gov (United States)

Terio, Karen A.; Craft, Meggan E.

2013-01-01

ABSTRACT One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or “spillover” hosts but, rather, a normal part of the complex ecology of this infectious disease. PMID:24045642

Resistance of Aerosolized Bacterial Viruses to Four Germicidal Products.

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Nathalie Turgeon

Full Text Available Viral diseases can spread through a variety of routes including aerosols. Yet, limited data are available on the efficacy of aerosolized chemicals to reduce viral loads in the air. Bacteriophages (phages are often used as surrogates for hazardous viruses in aerosol studies because they are inexpensive, easy to handle, and safe for laboratory workers. Moreover, several of these bacterial viruses display physical characteristics similar to pathogenic human and animal viruses, like morphological size, type of nucleic acids, capsid morphology, and the presence of an envelope. In this study, the efficacy of four chemicals was evaluated on four airborne phages at two different relative humidity levels. Non-tailed bacteriophages MS2 (single-stranded RNA, ϕ6 (double-stranded RNA, enveloped, PR772 (double-stranded DNA, and ϕX174 (single-stranded DNA were first aerosolized in a 55L rotative environmental chamber at 19°C with 25% and 50% relative humidity. Then, hydrogen peroxide, Eugenol (phenylpropene used in commercial perfumes and flavorings, Mist® (automobile disinfectant containing Triethylene glycol, and Pledge® (multisurface disinfectant containing Isopropanol, n-Alkyl Dimethyl Benzyl Amonium Chlorides, and n-Alkyl Dimethyl Ethylbenzyl Ammonium Chloride were nebulized with the phages using a separate nebulizer. Aerosols were maintained in suspension during 10 minutes, 1 hour, and 2 hours. Viral aerosols were sampled using an SKC BioSampler and samples were analyzed using qPCR and plaque assays. The resistance levels of the four phages varied depending on the relative humidity (RH and germicidal products tested. Phage MS2 was the most stable airborne virus under the environmental conditions tested while phage PR772 was the least stable. Pledge® and Eugenol reduced the infectivity of all airborne phages tested. At 25% RH, Pledge® and Eugenol were more effective at reducing infectivity of RNA phages ϕ6 and MS2. At 50% RH, Pledge® was the most

Extensive sequence divergence among bovine respiratory syncytial viruses isolated during recurrent outbreaks in closed herds

DEFF Research Database (Denmark)

Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

2000-01-01

and veal calf production units) in different years and from all confirmed outbreaks in Denmark within a short period. The results showed that identical viruses were isolated within a herd during outbreaks and that viruses from recurrent infections varied by up to 11% in sequence even in closed herds......The nucleotides coding for the extracellular part of the G glycoprotein and the full SH protein of bovine respiratory syncytial virus (BRSV) were sequenced from viruses isolated from numerous outbreaks of BRSV infection. The isolates included viruses isolated from the same herd (closed dairy farms....... It is possible that a quasispecies variant swarm of BRSV persisted in some of the calves in each herd and that a new and different highly fit virus type (master and consensus sequence) became dominant and spread from a single animal in connection with each new outbreak. Based on the high level of diversity...

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

Energy Technology Data Exchange (ETDEWEB)

Stenger, Drake C., E-mail: drake.stenger@ars.usda.gov [USDA, Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Ave., Parlier, CA 93648-9757 (United States); Krugner, Rodrigo [USDA, Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Ave., Parlier, CA 93648-9757 (United States); Nouri, Shahideh; Ferriol, Inmaculada; Falk, Bryce W. [Department of Plant Pathology, University of California, Davis, CA 95616 (United States); Sisterson, Mark S. [USDA, Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Ave., Parlier, CA 93648-9757 (United States)

2016-11-15

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of the corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection. -- Highlights: •Sampling protocol minimized differential selection/history among isolates. •Polymorphism among consensus sequences dominated by negative/purifying selection. •Within-isolate N:S ratio corrected for RT-PCR error by bootstrapping. •Within-isolate mutant spectrum dominated by new mutations yet to undergo selection.

VP3 is crucial for the stability of Nora virus virions.

Science.gov (United States)

Sadanandan, Sajna Anand; Ekström, Jens-Ola; Jonna, Venkateswara Rao; Hofer, Anders; Hultmark, Dan

2016-09-02

Nora virus is an enteric virus that causes persistent, non-pathological infection in Drosophila melanogaster. It replicates in the fly gut and is transmitted via the fecal-oral route. Nora virus has a single-stranded positive-sense RNA genome, which is translated in four open reading frames. Reading frame three encodes the VP3 protein, the structure and function of which we have investigated in this work. We have shown that VP3 is a trimer that has an α-helical secondary structure, with a functionally important coiled-coil domain. In order to identify the role of VP3 in the Nora virus life cycle, we constructed VP3-mutants using the cDNA clone of the virus. Our results show that VP3 does not have a role in the actual assembly of the virus particles, but virions that lack VP3 or harbor VP3 with a disrupted coiled coil domain are incapable of transmission via the fecal-oral route. Removing the region downstream of the putative coiled coil appears to have an effect on the fitness of the virus but does not hamper its replication or transmission. We also found that the VP3 protein and particularly the coiled coil domain are crucial for the stability of Nora virus virions when exposed to heat or proteases. Hence, we propose that VP3 is imperative to Nora virus virions as it confers stability to the viral capsid. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Experimental Inoculation of Egyptian Rousette Bats (Rousettus aegyptiacus with Viruses of the Ebolavirus and Marburgvirus Genera

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Megan E.B. Jones

2015-06-01

Full Text Available The Egyptian rousette bat (Rousettus aegyptiacus is a natural reservoir for marburgviruses and a consistent source of virus spillover to humans. Cumulative evidence suggests various bat species may also transmit ebolaviruses. We investigated the susceptibility of Egyptian rousettes to each of the five known ebolaviruses (Sudan, Ebola, Bundibugyo, Taï Forest, and Reston, and compared findings with Marburg virus. In a pilot study, groups of four juvenile bats were inoculated with one of the ebolaviruses or Marburg virus. In ebolavirus groups, viral RNA tissue distribution was limited, and no bat became viremic. Sudan viral RNA was slightly more widespread, spurring a second, 15-day Sudan virus serial euthanasia study. Low levels of Sudan viral RNA disseminated to multiple tissues at early time points, but there was no viremia or shedding. In contrast, Marburg virus RNA was widely disseminated, with viremia, oral and rectal shedding, and antigen in spleen and liver. This is the first experimental infection study comparing tissue tropism, viral shedding, and clinical and pathologic effects of six different filoviruses in the Egyptian rousette, a known marburgvirus reservoir. Our results suggest Egyptian rousettes are unlikely sources for ebolaviruses in nature, and support a possible single filovirus—single reservoir host relationship.

Effects of RNA branching on the electrostatic stabilization of viruses

NARCIS (Netherlands)

Erdemci-Tandogan, Gonca; Wagner, Jef; Schoot, Paul van der|info:eu-repo/dai/nl/102140618; Podgornik, Rudolf; Zandi, Roya

2016-01-01

Many single-stranded (ss) RNA viruses self assemble from capsid protein subunits and the nucleic acid to form an infectious virion. It is believed that the electrostatic interactions between the negatively charged RNA and the positively charged viral capsid proteins drive the encapsidation, although

Measles Outbreak with Unique Virus Genotyping, Ontario, Canada, 2015.

Science.gov (United States)

Thomas, Shari; Hiebert, Joanne; Gubbay, Jonathan B; Gournis, Effie; Sharron, Jennifer; Severini, Alberto; Jiaravuthisan, Manisa; Shane, Amanda; Jaeger, Valerie; Crowcroft, Natasha S; Fediurek, Jill; Sander, Beate; Mazzulli, Tony; Schulz, Helene; Deeks, Shelley L

2017-07-01

The province of Ontario continues to experience measles virus transmissions despite the elimination of measles in Canada. We describe an unusual outbreak of measles in Ontario, Canada, in early 2015 that involved cases with a unique strain of virus and no known association among primary case-patients. A total of 18 cases of measles were reported from 4 public health units during the outbreak period (January 25-March 23, 2015); none of these cases occurred in persons who had recently traveled. Despite enhancements to case-patient interview methods and epidemiologic analyses, a source patient was not identified. However, the molecular epidemiologic analysis, which included extended sequencing, strongly suggested that all cases derived from a single importation of measles virus genotype D4. The use of timely genotype sequencing, rigorous epidemiologic investigation, and a better understanding of the gaps in surveillance are needed to maintain Ontario's measles elimination status.

Evaluation of ViroCyt® Virus Counter for Rapid Filovirus Quantitation

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Cynthia A. Rossi

2015-03-01

Full Text Available Development and evaluation of medical countermeasures for diagnostics, vaccines, and therapeutics requires production of standardized, reproducible, and well characterized virus preparations. For filoviruses this includes plaque assay for quantitation of infectious virus, transmission electron microscopy (TEM for morphology and quantitation of virus particles, and real-time reverse transcription PCR for quantitation of viral RNA (qRT-PCR. The ViroCyt® Virus Counter (VC 2100 (ViroCyt, Boulder, CO, USA is a flow-based instrument capable of quantifying virus particles in solution. Using a proprietary combination of fluorescent dyes that stain both nucleic acid and protein in a single 30 min step, rapid, reproducible, and cost-effective quantification of filovirus particles was demonstrated. Using a seed stock of Ebola virus variant Kikwit, the linear range of the instrument was determined to be 2.8E+06 to 1.0E+09 virus particles per mL with coefficient of variation ranging from 9.4% to 31.5% for samples tested in triplicate. VC particle counts for various filovirus stocks were within one log of TEM particle counts. A linear relationship was established between the plaque assay, qRT-PCR, and the VC. VC results significantly correlated with both plaque assay and qRT-PCR. These results demonstrated that the VC is an easy, fast, and consistent method to quantify filoviruses in stock preparations.

Evaluation of ViroCyt® Virus Counter for Rapid Filovirus Quantitation

Directory of Open Access Journals (Sweden)

Cynthia A. Rossi

2015-02-01

Full Text Available Development and evaluation of medical countermeasures for diagnostics, vaccines, and therapeutics requires production of standardized, reproducible, and well characterized virus preparations. For filoviruses this includes plaque assay for quantitation of infectious virus, transmission electron microscopy (TEM for morphology and quantitation of virus particles, and real-time reverse transcription PCR for quantitation of viral RNA (qRT-PCR. The ViroCyt® Virus Counter (VC 2100 (ViroCyt, Boulder, CO, USA is a flow-based instrument capable of quantifying virus particles in solution. Using a proprietary combination of fluorescent dyes that stain both nucleic acid and protein in a single 30 min step, rapid, reproducible, and cost-effective quantification of filovirus particles was demonstrated. Using a seed stock of Ebola virus variant Kikwit, the linear range of the instrument was determined to be 2.8E+06 to 1.0E+09 virus particles per mL with coefficient of variation ranging from 9.4% to 31.5% for samples tested in triplicate. VC particle counts for various filovirus stocks were within one log of TEM particle counts. A linear relationship was established between the plaque assay, qRT-PCR, and the VC. VC results significantly correlated with both plaque assay and qRT-PCR. These results demonstrated that the VC is an easy, fast, and consistent method to quantify filoviruses in stock preparations.

Genome-wide evolutionary dynamics of influenza B viruses on a global scale.

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Pinky Langat

2017-12-01

Full Text Available The global-scale epidemiology and genome-wide evolutionary dynamics of influenza B remain poorly understood compared with influenza A viruses. We compiled a spatio-temporally comprehensive dataset of influenza B viruses, comprising over 2,500 genomes sampled worldwide between 1987 and 2015, including 382 newly-sequenced genomes that fill substantial gaps in previous molecular surveillance studies. Our contributed data increase the number of available influenza B virus genomes in Europe, Africa and Central Asia, improving the global context to study influenza B viruses. We reveal Yamagata-lineage diversity results from co-circulation of two antigenically-distinct groups that also segregate genetically across the entire genome, without evidence of intra-lineage reassortment. In contrast, Victoria-lineage diversity stems from geographic segregation of different genetic clades, with variability in the degree of geographic spread among clades. Differences between the lineages are reflected in their antigenic dynamics, as Yamagata-lineage viruses show alternating dominance between antigenic groups, while Victoria-lineage viruses show antigenic drift of a single lineage. Structural mapping of amino acid substitutions on trunk branches of influenza B gene phylogenies further supports these antigenic differences and highlights two potential mechanisms of adaptation for polymerase activity. Our study provides new insights into the epidemiological and molecular processes shaping influenza B virus evolution globally.

Genome-wide evolutionary dynamics of influenza B viruses on a global scale

Science.gov (United States)

Langat, Pinky; Bowden, Thomas A.; Edwards, Stephanie; Gall, Astrid; Rambaut, Andrew; Daniels, Rodney S.; Russell, Colin A.; Pybus, Oliver G.; McCauley, John

2017-01-01

The global-scale epidemiology and genome-wide evolutionary dynamics of influenza B remain poorly understood compared with influenza A viruses. We compiled a spatio-temporally comprehensive dataset of influenza B viruses, comprising over 2,500 genomes sampled worldwide between 1987 and 2015, including 382 newly-sequenced genomes that fill substantial gaps in previous molecular surveillance studies. Our contributed data increase the number of available influenza B virus genomes in Europe, Africa and Central Asia, improving the global context to study influenza B viruses. We reveal Yamagata-lineage diversity results from co-circulation of two antigenically-distinct groups that also segregate genetically across the entire genome, without evidence of intra-lineage reassortment. In contrast, Victoria-lineage diversity stems from geographic segregation of different genetic clades, with variability in the degree of geographic spread among clades. Differences between the lineages are reflected in their antigenic dynamics, as Yamagata-lineage viruses show alternating dominance between antigenic groups, while Victoria-lineage viruses show antigenic drift of a single lineage. Structural mapping of amino acid substitutions on trunk branches of influenza B gene phylogenies further supports these antigenic differences and highlights two potential mechanisms of adaptation for polymerase activity. Our study provides new insights into the epidemiological and molecular processes shaping influenza B virus evolution globally. PMID:29284042

In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity.

Science.gov (United States)

Chutinimitkul, Salin; van Riel, Debby; Munster, Vincent J; van den Brand, Judith M A; Rimmelzwaan, Guus F; Kuiken, Thijs; Osterhaus, Albert D M E; Fouchier, Ron A M; de Wit, Emmie

2010-07-01

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

In Vitro Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity▿

Science.gov (United States)

Chutinimitkul, Salin; van Riel, Debby; Munster, Vincent J.; van den Brand, Judith M. A.; Rimmelzwaan, Guus F.; Kuiken, Thijs; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.; de Wit, Emmie

2010-01-01

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished. PMID:20392847

Potential role of viruses in white plague coral disease.

Science.gov (United States)

Soffer, Nitzan; Brandt, Marilyn E; Correa, Adrienne M S; Smith, Tyler B; Thurber, Rebecca Vega

2014-02-01

White plague (WP)-like diseases of tropical corals are implicated in reef decline worldwide, although their etiological cause is generally unknown. Studies thus far have focused on bacterial or eukaryotic pathogens as the source of these diseases; no studies have examined the role of viruses. Using a combination of transmission electron microscopy (TEM) and 454 pyrosequencing, we compared 24 viral metagenomes generated from Montastraea annularis corals showing signs of WP-like disease and/or bleaching, control conspecific corals, and adjacent seawater. TEM was used for visual inspection of diseased coral tissue. No bacteria were visually identified within diseased coral tissues, but viral particles and sequence similarities to eukaryotic circular Rep-encoding single-stranded DNA viruses and their associated satellites (SCSDVs) were abundant in WP diseased tissues. In contrast, sequence similarities to SCSDVs were not found in any healthy coral tissues, suggesting SCSDVs might have a role in WP disease. Furthermore, Herpesviridae gene signatures dominated healthy tissues, corroborating reports that herpes-like viruses infect all corals. Nucleocytoplasmic large DNA virus (NCLDV) sequences, similar to those recently identified in cultures of Symbiodinium (the algal symbionts of corals), were most common in bleached corals. This finding further implicates that these NCLDV viruses may have a role in bleaching, as suggested in previous studies. This study determined that a specific group of viruses is associated with diseased Caribbean corals and highlights the potential for viral disease in regional coral reef decline.

Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

OpenAIRE

Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

2010-01-01

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-tra...

Sensitive detection of multiple hepatitis A virus genotypes with a single polony-based assay

Science.gov (United States)

Hepatitis A virus (HAV) is one of the major causes of non-bacterial gastroenteritis in humans worldwide. HAV is mostly transmitted via direct person-to-person contact, or by consumption of contaminated foods and water. Since only a few viral particles may cause disease, detection of low levels of HA...

EPSTEIN-BARR VIRUS INFECTIONS – AVIDITY TEST FOR IgG ANTIBODIES

Directory of Open Access Journals (Sweden)

Katja Strašek

2001-06-01

Full Text Available Background. We wish to introduce specific IgG avidity test as a supplementary assay in serological screening for Epstein-Barr virus infection if the status of patient cannot be resolved from a single serum sample with routine testing.Methods. Avidity of IgG antibodies was determined in sera of 57 patients with different stage of Epstein-Barr virus infection. Enzyme-immuno assay was used with a short incubation of 6-molar urea included in the procedure. Urea should remove low avidity antibodies. Avidity was expressed as the avidity index. Avidity testing with commercial kit was done as well.Results. Low avidity index was found for IgG antibodies of acute phase sera and high for those of past infection, recent infection and reactivation of endogenic virus.Conclusions. Avidity test for IgG antibodies might be supplementary assay to prove acute infection but also to resolve some other clinical states related to Epstein-Barr virus.

RNA virus interference via CRISPR/Cas13a system in plants

KAUST Repository

Aman, Rashid

2017-11-04

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

DEFF Research Database (Denmark)

Sweeney, Trevor; Cisnetto, Valentina; Bose, Daniel

2010-01-01

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-termin...

Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle.

Science.gov (United States)

Biedenkopf, Nadine; Lier, Clemens; Becker, Stephan

2016-05-15

Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle. The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that

Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity.

Science.gov (United States)

Rai, Devendra K; Diaz-San Segundo, Fayna; Campagnola, Grace; Keith, Anna; Schafer, Elizabeth A; Kloc, Anna; de Los Santos, Teresa; Peersen, Olve; Rieder, Elizabeth

2017-08-01

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3D pol ) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3D pol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A 24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237F HF ) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237I LF ) and W237L LF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates. IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3D pol mutations that affect polymerase

Competitive virus assay method for titration of noncytopathogenic bovine viral diarrhea viruses (END⁺ and END⁻ viruses).

Science.gov (United States)

Muhsen, Mahmod; Ohi, Kota; Aoki, Hiroshi; Ikeda, Hidetoshi; Fukusho, Akio

2013-03-01

A new, reliable and secure virus assay method, named the competitive virus assay (CVA) method, has been established for the titration of bovine viral diarrhea viruses (BVDVs) that either show the exaltation of Newcastle disease virus (END) phenomenon or heterologous interference phenomenon (but not the END phenomenon). This method is based on the principle of (1) homologous interference between BVDVs, by using BVDV RK13/E(-) or BVDV RK13/E(+) strains as competitor virus, and (2) END phenomenon and heterologous interference, by using attenuated Newcastle disease virus (NDV) TCND strain as challenge virus. In titration of BVDV END(+) and BVDV END(-) viruses, no significant difference in estimated virus titer was observed between CVA and conventional methods. CVA method demonstrated comparable levels of sensitivity and accuracy as conventional END and interference methods, which require the use of a velogenic Miyadera strain of NDV and vesicular stomatitis virus (VSV), both of which are agents of high-risk diseases. As such, the CVA method is a safer alternative, with increased bio-safety and bio-containment, through avoidance of virulent strains that are commonly employed with conventional methods. Copyright © 2012 Elsevier B.V. All rights reserved.

Radioactive probes as diagnostic tools for rice tungro viruses

International Nuclear Information System (INIS)

Azzam, O.; Arboleda, M.; Reyes. J. de los

1996-01-01

Rice tungro bacilliform (RTBV) and rice tungro spherical viruses (RTSV) are the two viral components responsible for rice tungro disease which has seriously affected the irrigated rice ecosystem in Southeast Asia for the last 30 years. RTBV has an 8 Kb double-stranded DNA circular genome, and it is primarily responsible for induction of symptoms in infected plants. RTSV has a 12 kb single-stranded RNA genome. It does not induce any apparent symptoms in the infected plant, and it is transmitted by greenleafhopper. RTBV depends upon RTSV for its own transmission. The two viruses are limited to the vascular tissue of the rice plant and are present at a low titer. Most of the detection methods used for the identification of these viruses have relied on the virus protein properties and therefore, early detection of the virus activity was not possible. We were interested in evaluating tissue printing, dot blot, and southern techniques for early detection of virus nucleic acids in rice plant using radioactive and non radioactive probes. 32 P-labeled T7 or SP6 RNA polymerase transcripts complementary to the RTBV genome and RTSV coat protein genes were used as probes of the positive stand of both viruses. For nonradioactive probes, RTBV DNA genome was labeled using the ECL detection kit (Amersham). Preliminary results show that viral nucleic acids of RTBV and RTSV could be detected using both labelling systems. Non radioactive probes were comparable in their sensitivity to the radioactive probes. Less than 100 pg of viral DNA was detected in the dot-blot assays. More data will be presented to compare the efficiency and reliability of these two techniques in detecting early virus activity in the rice plant. (author)

Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses.

Science.gov (United States)

Holtsberg, Frederick W; Shulenin, Sergey; Vu, Hong; Howell, Katie A; Patel, Sonal J; Gunn, Bronwyn; Karim, Marcus; Lai, Jonathan R; Frei, Julia C; Nyakatura, Elisabeth K; Zeitlin, Larry; Douglas, Robin; Fusco, Marnie L; Froude, Jeffrey W; Saphire, Erica Ollmann; Herbert, Andrew S; Wirchnianski, Ariel S; Lear-Rooney, Calli M; Alter, Galit; Dye, John M; Glass, Pamela J; Warfield, Kelly L; Aman, M Javad

2016-01-01

The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross

Preliminary X-ray Data Analysis of Crystalline Hibiscus Chlorotic Ringspot Virus

Energy Technology Data Exchange (ETDEWEB)

Cheng, A.; Speir, J; Yuan, Y; Johnson, J; Wong, S

2009-01-01

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3Synchrotron .2 A resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 . Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV.

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

Science.gov (United States)

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

Science.gov (United States)

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

Vaccines in Development against West Nile Virus

Directory of Open Access Journals (Sweden)

Frederic Tangy

2013-09-01

Full Text Available West Nile encephalitis emerged in 1999 in the United States, then rapidly spread through the North American continent causing severe disease in human and horses. Since then, outbreaks appeared in Europe, and in 2012, the United States experienced a new severe outbreak reporting a total of 5,387 cases of West Nile virus (WNV disease in humans, including 243 deaths. So far, no human vaccine is available to control new WNV outbreaks and to avoid worldwide spreading. In this review, we discuss the state-of-the-art of West Nile vaccine development and the potential of a novel safe and effective approach based on recombinant live attenuated measles virus (MV vaccine. MV vaccine is a live attenuated negative-stranded RNA virus proven as one of the safest, most stable and effective human vaccines. We previously described a vector derived from the Schwarz MV vaccine strain that stably expresses antigens from emerging arboviruses, such as dengue, West Nile or chikungunya viruses, and is strongly immunogenic in animal models, even in the presence of MV pre-existing immunity. A single administration of a recombinant MV vaccine expressing the secreted form of WNV envelope glycoprotein elicited protective immunity in mice and non-human primates as early as two weeks after immunization, indicating its potential as a human vaccine.

Virus Database and Online Inquiry System Based on Natural Vectors.

Science.gov (United States)

Dong, Rui; Zheng, Hui; Tian, Kun; Yau, Shek-Chung; Mao, Weiguang; Yu, Wenping; Yin, Changchuan; Yu, Chenglong; He, Rong Lucy; Yang, Jie; Yau, Stephen St

2017-01-01

We construct a virus database called VirusDB (http://yaulab.math.tsinghua.edu.cn/VirusDB/) and an online inquiry system to serve people who are interested in viral classification and prediction. The database stores all viral genomes, their corresponding natural vectors, and the classification information of the single/multiple-segmented viral reference sequences downloaded from National Center for Biotechnology Information. The online inquiry system serves the purpose of computing natural vectors and their distances based on submitted genomes, providing an online interface for accessing and using the database for viral classification and prediction, and back-end processes for automatic and manual updating of database content to synchronize with GenBank. Submitted genomes data in FASTA format will be carried out and the prediction results with 5 closest neighbors and their classifications will be returned by email. Considering the one-to-one correspondence between sequence and natural vector, time efficiency, and high accuracy, natural vector is a significant advance compared with alignment methods, which makes VirusDB a useful database in further research.

Off-the-Shelf Virus-Specific T Cells to Treat BK Virus, Human Herpesvirus 6, Cytomegalovirus, Epstein-Barr Virus, and Adenovirus Infections After Allogeneic Hematopoietic Stem-Cell Transplantation.

Science.gov (United States)

Tzannou, Ifigeneia; Papadopoulou, Anastasia; Naik, Swati; Leung, Kathryn; Martinez, Caridad A; Ramos, Carlos A; Carrum, George; Sasa, Ghadir; Lulla, Premal; Watanabe, Ayumi; Kuvalekar, Manik; Gee, Adrian P; Wu, Meng-Fen; Liu, Hao; Grilley, Bambi J; Krance, Robert A; Gottschalk, Stephen; Brenner, Malcolm K; Rooney, Cliona M; Heslop, Helen E; Leen, Ann M; Omer, Bilal

2017-11-01

Purpose Improvement of cure rates for patients treated with allogeneic hematopoietic stem-cell transplantation (HSCT) will require efforts to decrease treatment-related mortality from severe viral infections. Adoptively transferred virus-specific T cells (VSTs) generated from eligible, third-party donors could provide broad antiviral protection to recipients of HSCT as an immediately available off-the-shelf product. Patient and Methods We generated a bank of VSTs that recognized five common viral pathogens: Epstein-Barr virus (EBV), adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), and human herpesvirus 6 (HHV-6). The VSTs were administered to 38 patients with 45 infections in a phase II clinical trial. Results A single infusion produced a cumulative complete or partial response rate of 92% (95% CI, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKV (n = 16), 94% for CMV (n = 17), 71% for AdV (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). Clinical benefit was achieved in 31 patients treated for one infection and in seven patients treated for multiple coincident infections. Thirteen of 14 patients treated for BKV-associated hemorrhagic cystitis experienced complete resolution of gross hematuria by week 6. Infusions were safe, and only two occurrences of de novo graft-versus host disease (grade 1) were observed. VST tracking by epitope profiling revealed persistence of functional VSTs of third-party origin for up to 12 weeks. Conclusion The use of banked VSTs is a feasible, safe, and effective approach to treat severe and drug-refractory infections after HSCT, including infections from two viruses (BKV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the VSTs ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.

Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals

Science.gov (United States)

Baker, Steven F.; Nogales, Aitor; Finch, Courtney; Tuffy, Kevin M.; Domm, William; Perez, Daniel R.; Topham, David J.

2014-01-01

ABSTRACT Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that the full-length hemagglutinin (HA) of prototype influenza B viruses can complement the function of multiple influenza A viruses. We show that viral noncoding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B virus HA segment could not be incorporated into influenza A virus virions. However, by adding the influenza A virus packaging signals to full-length influenza B virus glycoproteins, we rescued influenza A viruses that possessed HA, NA, or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be attributed at least in part to incompatibilities in the virus-specific packaging signals required for effective segment incorporation into nascent virions. IMPORTANCE Reassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment or

Molecular characterization of a novel cryptic virus infecting pigeonpea plants.

Directory of Open Access Journals (Sweden)

Surender Kumar

Full Text Available A new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L. Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1. The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5' non-coding region (5'-GATAATGATCCAAGGA-3'. The largest dsRNA (dsRNA-1 was identified as the viral RNA dependent RNA polymerase (replicase, predicted to encode a putative 55.34 kDa protein (P1. The two other smaller dsRNAs (dsRNA-2 and dsRNA-3 predicted to encode for putative capsid proteins of 38.50kDa (P2 and 38.51kDa (P3, respectively. Phylogenetic analysis indicated that ArCV-1 formed a clade together with Fragaria chiloensis cryptic virus, Rosa multiflora cryptic virus and Rose cryptic virus-1, indicating that ArCV-1 could be a new member of the genus Deltapartitivirus. ArCV-1 3Dpol structure revealed several interesting features. The 3Dpol in its full-length shares structural similarities with members of the family Caliciviridaeand family Picornaviridae. In addition, fourth dsRNA molecule (dsRNA-2A, not related to ArCV-1 genome, was found in the same plant tissue. The dsRNA-2A (1.6 kb encodes a protein (P4, with a predicted size of 44.5 kDa. P4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including Raphanus sativus cryptic virus-3. This is the first report of occurrence of a cryptic virus in pigeonpea plants.

Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

Science.gov (United States)

Herbert, Andrew S; Kuehne, Ana I; Barth, James F; Ortiz, Ramon A; Nichols, Donald K; Zak, Samantha E; Stonier, Spencer W; Muhammad, Majidat A; Bakken, Russell R; Prugar, Laura I; Olinger, Gene G; Groebner, Jennifer L; Lee, John S; Pratt, William D; Custer, Max; Kamrud, Kurt I; Smith, Jonathan F; Hart, Mary Kate; Dye, John M

2013-05-01

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

Virus wars: using one virus to block the spread of another

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Matthew L. Paff

2016-06-01

Full Text Available The failure of traditional interventions to block and cure HIV infections has led to novel proposals that involve treating infections with therapeutic viruses–infectious viruses that specifically inhibit HIV propagation in the host. Early efforts in evaluating these proposals have been limited chiefly to mathematical models of dynamics, for lack of suitable empirical systems. Here we propose, develop and analyze an empirical system of a therapeutic virus that protects a host cell population against a lethal virus. The empirical system uses E. coli bacteria as the host cell population, an RNA phage as the lethal virus and a filamentous phage as the therapeutic virus. Basic dynamic properties are established for each virus alone and then together. Observed dynamics broadly agree with those predicted by a computer simulation model, although some differences are noted. Two cases of dynamics are contrasted, differing in whether the therapeutic virus is introduced before the lethal virus or after the lethal virus. The therapeutic virus increases in both cases but by different mechanisms. With the therapeutic virus introduced first, it spreads infectiously without any appreciable change in host dynamics. With the therapeutic virus introduced second, host abundance is depressed at the time therapy is applied; following an initial period of therapeutic virus spread by infection, the subsequent rise of protection is through reproduction by hosts already protected. This latter outcome is due to inheritance of the therapeutic virus state when the protected cell divides. Overall, the work establishes the feasibility and robustness to details of a viral interference using a therapeutic virus.

Next Generation Sequencing of Elite Berry Germplasm and Data Analysis Using a Bioinformatics Pipeline for Virus Detection and Discovery

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Berry crops (members of the genera Fragaria, Ribes, Rubus, Sambucus and Vaccinium) are known hosts for more than 70 viruses and new ones are identified continually. In modern berry cultivars, viruses tend to be be asymptomatic in single infections and symptoms only develop after plants accumulate m...

Next-Generation Sequencing of Elite Berry Germplasm and Data Analysis Using a Bioinformatics Pipeline for Virus Detection and Discovery

Science.gov (United States)

Berry crops (members of the genera Fragaria, Ribes, Rubus, Sambucus and Vaccinium) are known hosts for more than 70 viruses and new ones are identified frequently. In modern berry cultivars, viruses tend to be asymptomatic in single infections and symptoms only develop after plants accumulate multip...

A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone

Science.gov (United States)

Zhuang, Jun; Cai, Guiqin; Lin, Qiying; Wu, Zujian; Xie, Lianhui

2010-01-01

Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria. PMID:21079776

A bacteriophage-related chimeric marine virus infecting abalone.

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Jun Zhuang

Full Text Available Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin. The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs, eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria.

Zika virus disease

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Adel I Al-Afaleq

2017-01-01

Full Text Available The Zika virus is an arbovirus belonging to the virus family Flaviviridae. The virus was isolated in 1947 from a rhesus monkey in the Zika Forest of Uganda. The virus causes sporadic mild human infections in Africa and later in Asia. However, by 2007 a major shift in its infection pattern was noticed and thousands of human infections were reported in the State of Yap and Federated States of Micronesia. In the last 3 years, major outbreaks have continued to occur and the virus has spread to several Pacific and American countries. These outbreaks were mostly asymptomatic; however, there were more severe clinical signs associated with the infections. Those signs included microcephaly and Guillain–Barre syndrome. It is believed that various species of mosquitoes can biologically transmit the virus. However, Aedes aegypti is most widely associated with the Zika virus. Recently, new modes of virus transmission have been reported, including mother-to-fetus, sexual, blood transfusion, animal bites, laboratory exposure and breast milk. Differential diagnosis is very important as some other arboviruses such as yellow fever virus, West Nile virus, dengue virus, and chikungunya virus have similar clinical manifestations to the Zika virus infection as well as relating serologically to some of these viruses. Established laboratory diagnostic tests to detect the Zika virus are limited, with reverse transcription polymerase chain reaction being the most widely used test. Taking into consideration the quickness of the spread of infection, size of the infected population and change of the infection severity pattern, the Zika virus infection merits collective efforts on all levels to prevent and control the disease. Limited research work and data, concurrent infection with other arboviruses, involvement of biological vectors, mass crowd events, human and trade movements and lack of vaccines are some of the challenges that we face in our efforts to prevent and

Dengue virus receptor

OpenAIRE

Hidari, Kazuya I.P.J.; Suzuki, Takashi

2011-01-01

Dengue virus is an arthropod-borne virus transmitted by Aedes mosquitoes. Dengue virus causes fever and hemorrhagic disorders in humans and non-human primates. Direct interaction of the virus introduced by a mosquito bite with host receptor molecule(s) is crucial for virus propagation and the pathological progression of dengue diseases. Therefore, elucidation of the molecular mechanisms underlying the interaction between dengue virus and its receptor(s) in both humans and mosquitoes is essent...

Complete nucleotide sequence of the RNA-2 of grapevine deformation and Grapevine Anatolian ringspot viruses.

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Ghanem-Sabanadzovic, Nina Abou; Sabanadzovic, Sead; Digiaro, Michele; Martelli, Giovanni P

2005-05-01

The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71-73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62-64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5' non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively.

Multisegment one-step RT-PCR fluorescent labeling of influenza A virus genome for use in diagnostic microarray applications

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Vasin, A V; Plotnikova, M A; Klotchenko, S A; Elpaeva, E A; Komissarov, A B; Egorov, V V; Kiselev, O I [Research Institute of Influenza of the Ministry of Health and Social Development of the Russian Federation, 15/17 Prof. Popova St., St. Petersburg (Russian Federation); Sandybaev, N T; Chervyakova, O V; Strochkov, V M; Taylakova, E T; Koshemetov, J K; Mamadaliev, S M, E-mail: vasin@influenza.spb.ru [Research Institute for Biological Safety Problems of the RK NBC/SC ME and S RK, Gvardeiskiy (Kazakhstan)

2011-04-01

Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins - hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization

International Nuclear Information System (INIS)

Lawrence, J.B.; Marselle, L.M.; Byron, K.S.; Johnson, C.V.; Sullivan, J.L.; Singer, R.H.

1990-01-01

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell

Differentiation among isolates of prunus necrotic ringspot virus by transcript conformation polymorphism.

Science.gov (United States)

Rosner, A; Maslenin, L; Spiegel, S

1998-09-01

A method based on differences in electrophoretic mobility of RNA transcripts made from polymerase chain reaction (PCR) products was used for differentiation among virus isolates. A T7 RNA polymerase promoter was attached to amplified prunus necrotic ringspot virus (PNRSV) sequences by PCR. The PCR products then served as a template for transcription. Single-stranded transcripts originated from different PNRSV isolates varied in electrophoretic mobility in polyacrylamide gels, presumably because of transcript conformation polymorphism (TCP). This procedure was applied for the differentiation of PNRSV isolates.

Respiratory Viruses in Febrile Neutropenic Patients with Respiratory Symptoms

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Mohsen Meidani

2018-01-01

Full Text Available Background: Respiratory infections are a frequent cause of fever in neutropenic patients, whereas respiratory viral infections are not frequently considered as a diagnosis, which causes high morbidity and mortality in these patients. Materials and Methods: This prospective study was performed on 36 patients with neutropenia who admitted to hospital were eligible for inclusion with fever (single temperature of >38.3°C or a sustained temperature of >38°C for more than 1 h, upper and lower respiratory symptoms. Sampling was performed from the throat of the patient by the sterile swab. All materials were analyzed by quantitative real-time multiplex polymerase chain reaction covering the following viruses; influenza, parainfluenza virus (PIV, rhinovirus (RV, human metapneumovirus, and respiratory syncytial virus (RSV. Results: RV was the most frequently detected virus and then RSV was the most. PIV was not present in any of the tested samples. Furthermore, no substantial differences in the distribution of specific viral species were observed based on age, sex, neutropenia duration, hematological disorder, and respiratory tract symptoms and signs (P > 0.05. Conclusion: Our prospective study supports the hypothesis that respiratory viruses play an important role in the development of neutropenic fever, and thus has the potential to individualize infection treatment and to reduce the extensive use of antibiotics in immunocompromised patients with neutropenia.

ECHO virus

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... page: //medlineplus.gov/ency/article/001340.htm ECHO virus To use the sharing features on this page, please enable JavaScript. Enteric cytopathic human orphan (ECHO) viruses are a group of viruses that can lead ...

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

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Luiz Tadeu M Figueiredo

1997-05-01

Full Text Available We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Multiple viral infections in Agaricus bisporus - Characterisation of 18 unique RNA viruses and 8 ORFans identified by deep sequencing

OpenAIRE

Deakin, Gregory; Dobbs, Edward; Bennett, Julie M.; Jones, Ian M.; Grogan, Helen M.; Burton, Kerry S.

2017-01-01

Thirty unique non-host RNAs were sequenced in the cultivated fungus, Agaricus bisporus, comprising 18 viruses each encoding an RdRp domain with an additional 8 ORFans (non-host RNAs with no similarity to known sequences). Two viruses were multipartite with component RNAs showing correlative abundances and common 3′ motifs. The viruses, all positive sense single-stranded, were classified into diverse orders/families. Multiple infections of Agaricus may represent a diverse, dynamic and interact...

Biology and genomics of viruses within the genus Gammabaculovirus.

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Arif, Basil; Escasa, Shannon; Pavlik, Lillian

2011-11-01

Hymenoptera is a very large and ancient insect order encompassing bees, wasps, ants and sawflies. Fossil records indicate that they existed over 200 million years ago and about 100 million years before the appearance of Lepidoptera. Sawflies have been major pests in many parts of the world and some have caused serious forest defoliation in North America. All baculoviruses isolated from sawflies are of the single nucleocapsids phenotype and appear to replicate in midgut cells only. This group of viruses has been shown to be excellent pest control agents and three have been registered in Canada and Britain for this purpose. Sawfly baculoviruses contain the smallest genome of all baculoviruses sequenced so far. Gene orders among sequenced sawfly baculoviruses are co-linear but this is not shared with the genomes of lepidopteran baculoviruses. One distinguishing feature among all sequenced sawfly viruses is the lack of a gene encoding a membrane fusion protein, which brought into question the role of the budded virus phenotype in Gammabaculovirus biology.

Simultaneous Detection of Both RNA and DNA Viruses Infecting Dry Bean and Occurrence of Mixed Infections by BGYMV, BCMV and BCMNV in the Central-West Region of Mexico

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Elizabeth Chiquito-Almanza

2017-03-01

Full Text Available A multiplex reverse transcription polymerase chain reaction (RT-PCR assay was developed to simultaneously detect bean common mosaic virus (BCMV, bean common mosaic necrotic virus (BCMNV, and bean golden yellow mosaic virus (BGYMV from common bean leaves dried with silica gel using a single total nucleic acid extraction cetyl trimethyl ammonium bromide (CTAB method. A mixture of five specific primers was used to amplify three distinct fragments corresponding to 272 bp from the AC1 gene of BGYMV as well as 469 bp and 746 bp from the CP gene of BCMV and BCMNV, respectively. The three viruses were detected in a single plant or in a bulk of five plants. The multiplex RT-PCR was successfully applied to detect these three viruses from 187 field samples collected from 23 municipalities from the states of Guanajuato, Nayarit and Jalisco, Mexico. Rates of single infections were 14/187 (7.5%, 41/187 (21.9%, and 35/187 (18.7%, for BGYMV, BCMV, and BCMNV, respectively; 29/187 (15.5% samples were co-infected with two of these viruses and 10/187 (5.3% with the three viruses. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting these viruses in the common bean and can be used for routine molecular diagnosis and epidemiological studies.

A liquid phase blocking ELISA for the detection of antibodies against infectious bronchitis virus

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Cardoso T.C.

1999-01-01

Full Text Available A liquid phase blocking ELISA (LPB-ELISA was developed for the detection and measurement of antibodies against infectious bronchitis virus (IBV. The purified and nonpurified virus used as antigen, the capture and detector antibodies, and the chicken hyperimmune sera were prepared and standardized for this purpose. A total of 156 sera from vaccinated and 100 from specific pathogen-free chickens with no recorded contact with the virus were tested. The respective serum titers obtained in the serum neutralization test (SNT were compared with those obtained in the LPB-ELISA. There was a high correlation (r2 = 0.8926 between the two tests. The LPB-ELISA represents a single test suitable for the rapid detection of antibodies against bronchitis virus in chicken sera, with good sensitivity (88%, specificity (100% and agreement (95.31%.

Simultaneous detection of and differentiation between herpes simplex and varicella-zoster viruses with two fluorescent probes in the same test system.

Science.gov (United States)

Brumback, B G; Farthing, P G; Castellino, S N

1993-12-01

Specimens from skin lesions were examined simultaneously for herpes simplex virus (HSV) and varicella-zoster virus (VZV) by direct specimen testing and shell vial culture in single-test systems. For direct testing, cells in a single specimen well were stained with a combination direct-indirect immunofluorescence stain by using two fluorescent tags. A total of 203 fresh specimens were tested in parallel. Of these, 100 specimens contained too few cells for the direct VZV comparison and 91 contained too few cells for the HSV comparison. After these specimens were eliminated, the sensitivities and specificities, respectively, of the dual direct test were 86.1 and 97.3% for HSV compared with single culture and 92.2 and 100% for VZV compared with single direct testing. Shell vial monolayers in the combined cultures were stained for both viruses by the same method. A total of 305 fresh specimens were cultured in parallel by dual- and single-culture methods. The sensitivities and specificities, respectively, of the combined culture compared with separate cultures were 100 and 98.4% for HSV and 87.9 and 99.2% for VZV. The combined methods gave a performance comparable to those of single tests, required less specimen volume, and were less costly to perform.

Hepatitis E virus coinfection with hepatotropic viruses in Egyptian children.

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Zaki, Maysaa El Sayed; Salama, Osama Saad; Mansour, Fathy Awaad; Hossein, Shaimaa

2008-06-01

Major hepatotropic viruses continue to be important causes of acute viral hepatitis in developing countries. This work was carried out to detect the seroprevalence of hepatitis E virus (HEV) markers in children with acute viral hepatitis due to hepatotropic viruses (A, B and C) and non-A, non-B, non-C acute hepatitis, and to ascertain the influence of HEV superinfection in individuals infected with hepatitis viruses (A, B and C). We studied prospectively 162 children with sporadic acute hepatitis who reported to our hospital. Thirteen healthy controls were also included in the study. Laboratory investigations were performed, including complete liver function tests. Complete serological profiles for hepatitis viruses A, B, C and E were evaluated. HEV immunoglobulin G was detected with highest percentage among patients with hepatitis B (56.7%), followed by patients with hepatitis C virus (52.0%), hepatitis A virus (34.1%) and combined hepatitis B and C viruses (30.0%). The detection rate among patients with non-A, non-B, non-C hepatitis was 7.1%. HEV immunoglobulin M was found in 4.5% of hepatitis A virus patients and in 3.3% of hepatitis B patients. The prevalence of HEV immunoglobulin G and immunoglobulin M correlated with the levels of hepatic aspartate aminotransferase and alanine aminotransferase in patients with dual markers of infection with hepatitis E and other viruses compared to patients with acute hepatitis due to A and C viruses. HEV serological markers are common among children with acute viral hepatitis, especially from hepatitis C and B viruses. There may be increased sensitivity to HEV coinfection in association with hepatitis B and C infections. Dual infection with HEV and other hepatotropic viruses was associated with greater elevation of aspartate and alanine aminotransferases.

A universal next generation sequencing protocol to generate non-infectious barcoded cDNA libraries from high containment RNA viruses

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Several biosafety level (BSL)-3/4 pathogens are high consequence, single-stranded RNA viruses and their genomes, when introduced into permissive cells, are infectious. Moreover many of these viruses are Select Agents (SAs), and their genomes are also considered SAs. For this reason cDNAs and/or th...

Human papilloma virus, herpes simplex virus and epstein barr virus in oral squamous cell carcinoma from eight different countries.

Science.gov (United States)

Jalouli, Jamshid; Jalouli, Miranda M; Sapkota, Dipak; Ibrahim, Salah O; Larsson, Per-Anders; Sand, Lars

2012-02-01

Oral squamous cell carcinoma (OSCC) is a major health problem in many parts of the world, and the major causative agents are thought to be the use of alcohol and tobacco. Oncogenic viruses have also been suggested to be involved in OSCC development. This study investigated the prevalence of human papillomaviruses (HPV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) in 155 OSCC from eight different countries from different ethnic groups, continents and with different socioeconomic backgrounds. 41 A total of OSCCs were diagnosed in the tongue (26%) and 23 in the floor of the mouth (15%); the other 91 OSCCs were diagnosed in other locations (59%). The patients were also investigated regarding the use of alcohol and smoking and smokeless tobacco habits. Tissue samples were obtained from formalin-fixed, paraffin-embedded samples of the OSCC. DNA was extracted and the viral genome was examined by single, nested and semi-nested PCR assays. Sequencing of double-stranded DNA from the PCR product was carried out. Following sequencing of the HPV-, HSV- and EBV-positive PCR products, 100% homology between the sampels was found. Of all the 155 OSCCs examined, 85 (55%) were positive for EBV, 54 (35%) for HPV and 24 (15%) for HSV. The highest prevalence of HPV was seen in Sudan (65%), while HSV (55%) and EBV (80%) were most prevalent in the UK. In 34% (52/155) of all the samples examined, co-infection by two (46/155=30%) or three (6/155=4%) virus specimens was detected. The most frequent double infection was HPV with EBV in 21% (32/155) of all OSCCs. There was a statistically significant higher proportion of samples with HSV (p=0.026) and EBV (p=0.015) in industrialized countries (Sweden, Norway, UK and USA) as compared to developing countries (Sudan, India, Sri Lanka and Yemen). Furthermore, there was a statistically significant higher co-infection of HSV and EBV in samples from industrialized countries (p=0.00031). No firm conclusions could be drawn regarding the

Individual mediodorsal thalamic neurons project to multiple areas of the rat prefrontal cortex: A single neuron-tracing study using virus vectors.

Science.gov (United States)

Kuramoto, Eriko; Pan, Shixiu; Furuta, Takahiro; Tanaka, Yasuhiro R; Iwai, Haruki; Yamanaka, Atsushi; Ohno, Sachi; Kaneko, Takeshi; Goto, Tetsuya; Hioki, Hiroyuki

2017-01-01

The prefrontal cortex has an important role in a variety of cognitive and executive processes, and is generally defined by its reciprocal connections with the mediodorsal thalamic nucleus (MD). The rat MD is mainly subdivided into three segments, the medial (MDm), central (MDc), and lateral (MDl) divisions, on the basis of the cytoarchitecture and chemoarchitecture. The MD segments are known to topographically project to multiple prefrontal areas at the population level: the MDm mainly to the prelimbic, infralimbic, and agranular insular areas; the MDc to the orbital and agranular insular areas; and the MDl to the prelimbic and anterior cingulate areas. However, it is unknown whether individual MD neurons project to single or multiple prefrontal cortical areas. In the present study, we visualized individual MD neurons with Sindbis virus vectors, and reconstructed whole structures of MD neurons. While the main cortical projection targets of MDm, MDc, and MDl neurons were generally consistent with those of previous results, it was found that individual MD neurons sent their axon fibers to multiple prefrontal areas, and displayed various projection patterns in the target areas. Furthermore, the axons of single MD neurons were not homogeneously spread, but were rather distributed to form patchy axon arbors approximately 1 mm in diameter. The multiple-area projections and patchy axon arbors of single MD neurons might be able to coactivate cortical neuron groups in distant prefrontal areas simultaneously. Furthermore, considerable heterogeneity of the projection patterns is likely, to recruit the different sets of cortical neurons, and thus contributes to a variety of prefrontal functions. J. Comp. Neurol. 525:166-185, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Phytophthora viruses.

Science.gov (United States)

Cai, Guohong; Hillman, Bradley I

2013-01-01

Phytophthora sp. is a genus in the oomycetes, which are similar to filamentous fungi in morphology and habitat, but phylogenetically more closely related to brown algae and diatoms and fall in the kingdom Stramenopila. In the past few years, several viruses have been characterized in Phytophthora species, including four viruses from Phytophthora infestans, the late blight pathogen, and an endornavirus from an unnamed Phytophthora species from Douglas fir. Studies on Phytophthora viruses have revealed several interesting systems. Phytophthora infestans RNA virus 1 (PiRV-1) and PiRV-2 are likely the first members of two new virus families; studies on PiRV-3 support the establishment of a new virus genus that is not affiliated with established virus families; PiRV-4 is a member of Narnaviridae, most likely in the genus Narnavirus; and Phytophthora endornavirus 1 (PEV1) was the first nonplant endornavirus at the time of reporting. Viral capsids have not been found in any of the above-mentioned viruses. PiRV-1 demonstrated a unique genome organization that requires further examination, and PiRV-2 may have played a role in late blight resurgence in 1980s-1990s. Copyright © 2013 Elsevier Inc. All rights reserved.

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

International Nuclear Information System (INIS)

Valles, Steven M.; Hashimoto, Yoshifumi

2009-01-01

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number (FJ528584)), comprised of 10,386 nucleotides, and polyadenylated at the 3' terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5' proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3' proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.

Viruses infecting reptiles.