The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Udeni B.r. Balasuriya - One of the best experts on this subject based on the ideXlab platform.
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Reproductive effects of arteriViruses: Equine Arteritis Virus and porcine reproductive and respiratory syndrome Virus infections.
Current opinion in virology, 2017Co-Authors: Udeni B.r. Balasuriya, Mariano CarossinoAbstract:Equine Arteritis Virus (EAV) and porcine reproductive and respiratory syndrome Virus (PRRSV) are the most economically important members of the family Arteriviridae. EAV and PRRSV cause reproductive and respiratory disease in equids and swine, respectively and constitute a significant economic burden to Equine and swine industries around the world. Furthermore, they both cause abortion in pregnant animals and establish persistent infection in their natural hosts, which fosters viral shedding in semen leading to sexual transmission. The primary focus of this article is to provide an update on the effects of these two Viruses on the reproductive tract of their natural hosts and provide a comparative analysis of clinical signs, Virus-host interactions, mechanisms of viral pathogenesis and viral persistence.
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Development and Characterization of an Infectious cDNA Clone of Equine Arteritis Virus.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Udeni B.r. Balasuriya, Jianqiang ZhangAbstract:Development and characterization of several infectious cDNA clones of Equine Arteritis Virus (EAV) have been described in the literature. Here we describe the assembly of the full-length infectious cDNA clone of the virulent Bucyrus strain (VBS; ATCC VR-796) of EAV in a plasmid vector. This system allows generation of infectious in vitro-transcribed (IVT) RNA from the linearized plasmid that can be transfected or electroporated into mammalian cells to produce infectious recombinant progeny Virus. This is an efficient reverse genetics system that allows easy manipulation of EAV genomes to study molecular biology of the Virus and pathogenesis of Equine viral Arteritis.
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Research Article Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist
2016Co-Authors: Zhenhai Chen, Mingyuan Han, Dongwan Yoo, Ying Fang, Udeni B.r. BalasuriyaAbstract:Copyright © 2014 Yun Young Go et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The objective of this study was to investigate the effect of Equine Arteritis Virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN
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Experiences with infectious cDNA clones of Equine Arteritis Virus: lessons learned and insights gained.
Virology, 2014Co-Authors: Udeni B.r. Balasuriya, Jianqiang Zhang, N. James MaclachlanAbstract:The advent of recombinant DNA technology, development of infectious cDNA clones of RNA Viruses, and reverse genetic technologies have revolutionized how Viruses are studied. Genetic manipulation of full-length cDNA clones has become an especially important and widely used tool to study the biology, pathogenesis, and virulence determinants of both positive and negative stranded RNA Viruses. The first full-length infectious cDNA clone of Equine Arteritis Virus (EAV) was developed in 1996 and was also the first full-length infectious cDNA clone constructed from a member of the order Nidovirales. This clone was extensively used to characterize the molecular biology of EAV and other NidoViruses. The objective of this review is to summarize the characterization of the virulence (or attenuation) phenotype of the recombinant Viruses derived from several infectious cDNA clones of EAV in horses, as well as their application for characterization of the molecular basis of viral neutralization, persistence, and cellular tropism.
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Equine Arteritis Virus.
Veterinary Microbiology, 2013Co-Authors: Udeni B.r. Balasuriya, N. James MaclachlanAbstract:Equine Arteritis Virus (EAV) is the causative agent of Equine viral Arteritis (EVA), a respiratory and reproductive disease of equids. There has been significant recent progress in understanding the molecular biology of EAV and the pathogenesis of its infection in horses. In particular, the use of contemporary genomic techniques, along with the development and reverse genetic manipulation of infectious cDNA clones of several strains of EAV, has generated significant novel information regarding the basic molecular biology of the Virus. Therefore, the objective of this review is to summarize current understanding of EAV virion architecture, replication, evolution, molecular epidemiology and genetic variation, pathogenesis including the influence of host genetics on disease susceptibility, host immune response, and potential vaccination and treatment strategies.
María Gabriela Echeverría - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of apoptosis markers in different cell lines infected with Equine Arteritis Virus
Biotechnic & histochemistry : official publication of the Biological Stain Commission, 2018Co-Authors: Germán Ernesto Metz, María Soledad Serena, Carlos Javier Panei, María Mercedes Abeyá, María Gabriela EcheverríaAbstract:Equine Arteritis Virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis v...
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Intrinsic, extrinsic and endoplasmic reticulum stress-induced apoptosis in RK13 cells infected with Equine Arteritis Virus.
Virus research, 2015Co-Authors: Germán Ernesto Metz, María Gabriela Echeverría, María Mercedes Abeyá, Inmaculada Galindo, Covadonga AlonsoAbstract:The modulation of the expression of caspases by Viruses influences the cell survival of different cell types. Equine Arteritis Virus (EAV) induces apoptosis of BHK21 and Vero cell lines, but it is not known whether EAV induces apoptosis in RK13 cells, a common cell line routinely used in EAV diagnosis and research. In this study, we determined that caspase-3 expression was triggered after infection of RK13 cells with EAV in a time- and dose-dependent manner. We also detected caspase-8 and caspase-9 activation, indicating the stimulation of both extrinsic and intrinsic apoptosis pathways. Finally, we found caspase-12 activation, an indicator of endoplasmic reticulum stress-induced apoptosis. The variability observed in the apoptotic response in the different cell lines demonstrates that apoptosis depends on the distinctive sensitivity of each cell line used for investigation.
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The Equine Arteritis Virus isolate from the 2010 Argentinian outbreak.
Revue scientifique et technique (International Office of Epizootics), 2014Co-Authors: Germán Ernesto Metz, María Soledad Serena, Carlos Javier Panei, Edgardo Nosetto, María Gabriela EcheverríaAbstract:Summary A semen sample from a stallion infected during the 2010 Equine Arteritis Virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The Virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE TM 1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum
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Development of a peptide ELISA for the diagnosis of Equine Arteritis Virus
Journal of virological methods, 2014Co-Authors: Germán Ernesto Metz, Silvina L. Diaz, María Soledad Serena, Carlos Javier Panei, Esteban N. Lorenzón, Santiago Gerardo Corva, Eduardo Maffud Cilli, María Gabriela EcheverríaAbstract:A peptide-based indirect ELISA was developed to detect antibodies against Equine Arteritis Virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral Arteritis outbreak, analyzed previously by Virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed Virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.
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Equine Arteritis Virus gP5 protein induces apoptosis in cultured insect cells
Virus research, 2014Co-Authors: Germán Ernesto Metz, Silvina L. Diaz, María Soledad Serena, María Mercedes Abeyá, Andrea Belén Dulbecco, Adriana Raquel Massone, María Gabriela EcheverríaAbstract:Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculoVirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.
E. D. Chirnside - One of the best experts on this subject based on the ideXlab platform.
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Equine Arteritis Virus.
Theriogenology, 1997Co-Authors: A.l. Glaser, E. D. Chirnside, Marian C. Horzinek, A. A. F. De VriesAbstract:Abstract Equine Arteritis Virus (EAV) is a small, enveloped, positive-stranded RNA Virus, in the family Arteriviridae , W.H.ich can infect both horses and donkeys. While the majority of EAV infections are asymptomatic, acutely infected animals may develop a wide range of clinical signs, including pyrexia, limb and ventral edema, depression, rhinitis, and conjunctivitis. The Virus may cause abortion and has caused mortality in neonates. After natural EAV infection, most horses develop a solid, long-term immunity to the disease. Marzz and geldings eliminate the Virus within 60 days, but 30 to 60% of acutely infected stallions will become persistently infected. These persistently infected animals maintain EAV within the reproductive tract, shed Virus continuously in the semen, and can transmit the Virus venereally. Mares infected venereally may not have clinical signs, but they shed large amounts of Virus in nasopharyngeal secretions and in urine, which may result in lateral spread of the infection by an aerosol route. The consequences of venereally acquired infection are minimal, with no known effects on conception rate, but mares infected at a late stages of gestation may abort. Identification of carrier stallions is crucial to control the dissemination of EAV. The stallions can be identified by serological screening using a Virus neutralization (VN) test. If positive at a titer of ≥ 1:4, the stallion should be tested for persistent infection by Virus isolation from the sperm-rich fraction of the ejaculate, or by test mating Shedding stallions should not be used for breeding, or should be bred only to mares seropositive from a natural infection or from vaccination, the mares should be subsequently isolated from seronegative horses for three weeks after natural or artificial insemination. A live attenuated (ARVAC) and a formalin-inactivated (ARTERVAC) vaccine are available. Both vaccines induce Virus-neutralizing antibodies, the presence of which correlates with protection from disease, abortion, and the development of a persistent infection. Serological investigations indicate that EAV has a worldwide distribution and that its prevalence is increasing. As a consequence, an increasing number of Equine viral Arteritis (EVA) outbreaks is being reported. This trend is likely to continue unless action is taken to slow or halt the transmission of this agent through semen.
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A survey for antibodies to Equine Arteritis Virus in donkeys, mules and zebra using Virus neutralisation (VN) and enzyme linked immunosorbent assay (ELISA)
Equine veterinary journal, 1997Co-Authors: J. T. Paweska, Matthew M. Binns, P S. A. Woods, E. D. ChirnsideAbstract:Summary A seroepidemiological survey of donkeys in South Africa (n = 4300) indicated a wide distribution and increasing prevalence of antibodies to Equine Arteritis Virus (EAV). Donkey sera inhibited Equine Arteritis Virus infection in Virus neutralisation (VN) tests and in ELISA specifically bound to a recombinant antigen derived from the Bucyrus isolate of EAV. These results suggest that donkeys have been exposed to the same serotype of this Virus as circulates among horses. A good correlation existed between EAV neutralising antibody titres and ELISA absorbance values (0.8631); the ELISA was sensitive and specific (99.2% and 80.3% respectively) for donkey sera when compared to the VN test and the recombinant ELISA antigen did not cross-react with sera positive for common African Equine pathogens. VN+ ELISA+ donkeys were also found in Morocco and Zimbabwe and seropositive mules in both South Africa and Morocco. No seropositive zebra (n = 266) were detected from game reserves or zoos in 9 countries. The results confirm that in addition to horses and donkeys, mules are naturally infected with EAV.
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Expression cloning and antigenic analysis of the nucleocapsid protein of Equine Arteritis Virus
Virus research, 1995Co-Authors: E. D. Chirnside, P.m. Francis, J.a. MumfordAbstract:Abstract A series of recombinant fusion proteins derived from Equine Arteritis Virus (EAV) open reading frame (ORF) 7 have been used to define the immunoreactive region of the viral nucleocapsid (N) protein. Reactivities of recombinant N fusion proteins with post-infection Equine sera in immunoblots and ELISAs indicate that the major nucleocapsid protein epitope is located within amino acid residues 1–69. In ELISAs two recombinant nucleocapsid fusion proteins containing residues 1–69 (rN1–69) and 1–28 (rN1–28) discriminated between pre- and post-infection, and pre- and post-vaccination serum samples. Additionally rN1–69 and rN1–28 detected seroconversions following vaccination with a killed Virus preparation, even in the absence of a detectable Virus neutralising response. Although a good correlation existed between Virus neutralising antibody and rN1–69 ELISA positive values in post-infection sera, all the rN proteins failed to induce any Virus neutralising response in immunised rabbits.
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Sexual and in-contact transmission of asinine strain of Equine Arteritis Virus among donkeys.
Journal of clinical microbiology, 1995Co-Authors: J. T. Paweska, D.h. Volkmann, B.j.h. Barnard, E. D. ChirnsideAbstract:Two in a group of five naturally seropositive donkey stallions were found to shed Equine Arteritis Virus (EAV) in their semen as demonstrated by Virus isolation. Direct intramuscular inoculation of sonicated semen from one Virus-shedding stallion (S3) caused clinical disease in two donkeys from which Virus was recovered and in which seroconversion was detected. Sexual transmission was confirmed in two mares mated to S3 when after a febrile response during which EAV was isolated from huffy coats and nasal and ocular exudates, both mares were found to have seroconverted. In-contact transmission in a susceptible stallion was demonstrated after its exposure to a sexually infected mare. The 3' end of the asinine Virus was amplified directly from donkey semen with EAV-specific primers, and its nucleotide sequence was found to be homologous to that of the prototype Bucyrus Virus isolated from horses. These results indicate that EAV and its disease transmission are analogous in donkeys and horses.
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Comparison of M and N gene sequences distinguishes variation amongst Equine Arteritis Virus isolates
Journal of General Virology, 1994Co-Authors: E. D. Chirnside, Catherine M. Wearing, Matthew M. Binns, Jennifer A. MumfordAbstract:cDNA copies of the M and N genes of Equine Arteritis Virus (EAV) isolates were synthesized by reverse transcription followed by polymerase chain reaction amplification. The cDNA was subjected to a cycle sequencing strategy using Taq polymerase, and the nucleotide and derived amino acid sequences of 10 Virus isolates were compared. The M and N genes of all isolates had the same initiation and termination sites as the prototype Bucyrus strain and the encoded proteins were conserved between Viruses. Comparison of nucleotide sequence homologies and phylogenetic tree analysis implied the existence of three EAV variants originating from the U.S.A. (Bucyrus), Austria (Vienna) and Switzerland (Bibuna), and suggested that RNA recombination between EAV isolates may have occurred.
Germán Ernesto Metz - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of apoptosis markers in different cell lines infected with Equine Arteritis Virus
Biotechnic & histochemistry : official publication of the Biological Stain Commission, 2018Co-Authors: Germán Ernesto Metz, María Soledad Serena, Carlos Javier Panei, María Mercedes Abeyá, María Gabriela EcheverríaAbstract:Equine Arteritis Virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis v...
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Intrinsic, extrinsic and endoplasmic reticulum stress-induced apoptosis in RK13 cells infected with Equine Arteritis Virus.
Virus research, 2015Co-Authors: Germán Ernesto Metz, María Gabriela Echeverría, María Mercedes Abeyá, Inmaculada Galindo, Covadonga AlonsoAbstract:The modulation of the expression of caspases by Viruses influences the cell survival of different cell types. Equine Arteritis Virus (EAV) induces apoptosis of BHK21 and Vero cell lines, but it is not known whether EAV induces apoptosis in RK13 cells, a common cell line routinely used in EAV diagnosis and research. In this study, we determined that caspase-3 expression was triggered after infection of RK13 cells with EAV in a time- and dose-dependent manner. We also detected caspase-8 and caspase-9 activation, indicating the stimulation of both extrinsic and intrinsic apoptosis pathways. Finally, we found caspase-12 activation, an indicator of endoplasmic reticulum stress-induced apoptosis. The variability observed in the apoptotic response in the different cell lines demonstrates that apoptosis depends on the distinctive sensitivity of each cell line used for investigation.
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The Equine Arteritis Virus isolate from the 2010 Argentinian outbreak.
Revue scientifique et technique (International Office of Epizootics), 2014Co-Authors: Germán Ernesto Metz, María Soledad Serena, Carlos Javier Panei, Edgardo Nosetto, María Gabriela EcheverríaAbstract:Summary A semen sample from a stallion infected during the 2010 Equine Arteritis Virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The Virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE TM 1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum
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Development of a peptide ELISA for the diagnosis of Equine Arteritis Virus
Journal of virological methods, 2014Co-Authors: Germán Ernesto Metz, Silvina L. Diaz, María Soledad Serena, Carlos Javier Panei, Esteban N. Lorenzón, Santiago Gerardo Corva, Eduardo Maffud Cilli, María Gabriela EcheverríaAbstract:A peptide-based indirect ELISA was developed to detect antibodies against Equine Arteritis Virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral Arteritis outbreak, analyzed previously by Virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed Virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.
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Equine Arteritis Virus gP5 protein induces apoptosis in cultured insect cells
Virus research, 2014Co-Authors: Germán Ernesto Metz, Silvina L. Diaz, María Soledad Serena, María Mercedes Abeyá, Andrea Belén Dulbecco, Adriana Raquel Massone, María Gabriela EcheverríaAbstract:Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculoVirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.
Peter J. Timoney - One of the best experts on this subject based on the ideXlab platform.
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Evidence that In Vitro Susceptibility of CD3+ T Lymphocytes to Equine Arteritis Virus Infection Reflects Genetic Predisposition of Naturally Infected Stallions To Become Carriers of the Virus
Journal of virology, 2012Co-Authors: Ernest Bailey, Peter J. Timoney, Kathleen M Shuck, Udeni B.r. BalasuriyaAbstract:We investigated the correlation between in vitro susceptibility of CD3(+) T lymphocytes to Equine Arteritis Virus (EAV) infection and establishment of persistent infection among 14 stallions following natural infections. The data showed that carrier stallions with a CD3(+) T lymphocyte susceptibility phenotype to in vitro EAV infection may be at higher risk of becoming carriers than those that lack this phenotype (P = 0.0002).
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evaluation of two magnetic bead based viral nucleic acid purification kits and three real time reverse transcription pcr reagent systems in two taqman assays for Equine Arteritis Virus detection in semen
Journal of Clinical Microbiology, 2011Co-Authors: Fabien Miszczak, Stéphane Pronost, Peter J. Timoney, Jianqiang Zhang, Kathleen M Shuck, Guillaume Fortier, Astrid Vabret, Stephen F Sells, Udeni B.r. BalasuriyaAbstract:This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of Virus isolation for the detection of Equine Arteritis Virus in semen.
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Identification of an additional neutralization determinant of Equine Arteritis Virus.
Virus research, 2008Co-Authors: Jianqiang Zhang, Peter J. Timoney, N. James Maclachlan, Udeni B.r. BalasuriyaAbstract:We recently established an in vitro model of Equine Arteritis Virus (EAV) persistence in HeLa cells. The objective of this study was to determine whether viral variants with novel neutralization phenotypes emerged during persistent EAV infection of HeLa cells, as occurs during viral persistence in carrier stallions. Viruses recovered from persistently infected HeLa cells had different neutralization phenotypes than the Virus in the original inoculum, as determined by neutralization assays using EAV-specific monoclonal antibodies and polyclonal Equine antisera raised against different strains of EAV. Comparative sequence analyses of the entire structural protein genes (ORFs 2a, 2b, and 3-7) of these Viruses, coupled with construction of chimeric Viruses utilizing an infectious cDNA clone of EAV, confirmed that the alterations in neutralization phenotype were caused by amino acid changes in the GP5 protein encoded by ORF5. Site-directed mutagenesis studies unequivocally confirmed that amino acid 98 in the GP5 protein was responsible for the altered neutralization phenotype of these Viruses. Amino acid 98 in the GP5 protein, which has not previously been identified as a neutralization determinant of EAV, should be included in an expanded neutralization site D (amino acids 98-106).
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persistent Equine Arteritis Virus infection in hela cells
Journal of Virology, 2008Co-Authors: Jianqiang Zhang, Peter J. Timoney, William H. Mccollum, James N Maclachlan, Udeni B.r. BalasuriyaAbstract:The horse-adapted virulent Bucyrus (VB) strain of Equine Arteritis Virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, Virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 Viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 Virus. It was further demonstrated that recombinant Viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant Viruses with combined substitutions in the E (Ser53→Cys and Val55→Ala), GP2 (Leu15→Ser, Trp31→Arg, Val87→Leu, and Ala112→Thr), GP3 (Ser115→Gly and Leu135→Pro), and GP4 (Tyr4→His and Ile109→Phe) proteins or with a single point mutation in the GP5 protein (Pro98→Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying Virus-host cell interactions, especially Virus-receptor interactions.
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detection of Equine Arteritis Virus by real time taqman reverse transcription pcr assay
Journal of Virological Methods, 2002Co-Authors: Udeni B.r. Balasuriya, Peter J. Timoney, William H. Mccollum, J. B. Topol, Christian M Leutenegger, James N MaclachlanAbstract:A one-tube real-time TaqMan® reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of Equine Arteritis Virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional Virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan® probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time TaqMan® PCR assay detected EAV RNA in all samples that were confirmed to contain infectious EAV by Virus isolation. The assay had an analytical sensitivity of 10 molecules of EAV RNA allowing the detection of EAV in clinical samples or tissue culture fluid (TCF) containing at least 200 viral RNA copies per ml. Thus, the one-tube real-time TaqMan® RT-PCR assay provides a rapid, accurate, quantitative, convenient and high sample throughput system for diagnosis of EAV infection, in a closed-tube format that minimizes the risk of cross-contamination.