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The soluble form of EIAV receptor encoded by an alternative splicing variant inhibits EIAV infection on target cells
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
clinical signs. Five were from France and five came from Romania. Positive horses identified in 2010 were from 7 different premises divided in two different episodes. The first episode detected in South West of France consisted of 4 related premises where 5 French Trotters were tested positives and euthanized. The index case has been tested as part of a control before exportation. Primary premise located in the Dordogne County was a breeding farm where 2 mares were tested positive for EIA. Others premises, where two positive horses were detected, were located in the Lot-et-Garonne and Gironde County. All Infected animals (5) came from the breeding farm in Dordogne and have been kept over there several years where they have been probably infected since it is the only source of infection identified. Epidemiological investigations lead to test more than 400 horses, in 38 different Counties, which have been trained in the Dordogne Farm between 1990 and 2010. No other positive animals have been identified during this seroepidemiological study. The second EIA episode identified in 2010 was from horses which have been imported from Romania. Indeed, following EIA cases identified in Belgium and in UK at the end of 2009, French Ministry of Agriculture decided to test horses that came from Romania since January 2007. Investigation detected 80 equids, among those horses 38 were slaughtered or re-exported. Only 35 horses were still on the French territory at the date of testing and five of them exhibited a positive result for EIA using AGID test. Horses kept at the vicinity of those positive horses were tested but no transmission was seen. In this context the European commission (EU) has modified in June 2010 the regulation regarding importation of horses from Romania in other European member states. EU enforced a quarantine and a testing at destination for horses coming from Romania.
S89
2007, an outbreak of EVA occurred in Normandy, France. Only draught and saddle horses were affected. This 2007 outbreak occurred following the used of semen contaminated by EAV for insemination. The objectives of this study were to undertake a thorough epidemiological investigation to identify shedders stallions following the 2007 EVA outbreak as well as the molecular characterization of EAV isolates encountered in France. Positive semen samples were coming from stallions collected in 2007 and 2008. Those samples were tested by Quantitative RT-PCR and by Virus isolation on cell culture as describe in the OIE manual Chapter 2.5.10. Phylogenetic analysis were performed by amplification and sequencing open reading frames 2a-7 (ORFs 2a-7) encoding the viral structural proteins. The new governmental directive leads to the testing of nearly 4500 stallions in 2008 compared to the 450 tested in 2007. Among them 39 stallions were found positive and shed EAV in their semen. Phylogenetic analysis using the ORFs2a-7 sequences and others from GenBank grouped the viruses in two groups: the North American and the European which is divided in subgroups 1 and 2. The majority of viruses (20) isolated and characterized in France were members of the European subgroup 2, six of them belong to the European subgroup 1 and one isolate belongs to the American lineage. Moreover, isolates from the 2007 outbreaks grouped in a distinct cluster, named Normandy cluster, inside the European subgroup 2. The EVA strain responsible of the 2007 outbreak grouped, inside the European subgroup 2, in a new distinct cluster and seems to be different to those used to be isolated in France previously. Appearance of this new virus might explain the speed of the viral spread in horse population observed as well as the severity of clinical signs associated with the infection as shown by the death of 5 foals recorded during this outbreak.
Molecular epidemiology of Equine Arteritis Virus in France following the 2007 outbreak A. Hans 1, D. Gaudaire 1, S. Pronost 2, B. Ferry-Abitbol 3, C. Laugier 1, and S. Zientara 4 1 Anses, Laboratory for equine diseases, 14430, Goustranville, France, 2 Frank Duncombe laboratory, EA 4655 U2RM Caen University Lower Normandy, Caen, France, 3 Institut Français du Cheval et de l’Equitation (IFCE), 49411 Saumur, France, 4 Anses, Laboratory for Animal Health, UMR 1161 ANSES/INRA/ENVA, 94703 Maisons-Alfort, France
Equine Viral Arteritis (EVA) is characterized by a broad range of clinical signs such as hyperthermia, anorexia, oedema and depression. It may cause abortion in mares and a temporary infertility in stallions. The virus, Equine arteritis virus (EAV) that belongs to the Arteriviridae family, can be transmitted either by respiratory or veneral route. Following the initial infection, stallions may become asymptomatic carriers and can shed the virus in their semen. Those stallions are reservoir for EVA and have to be properly handled to prevent any viral spread in horse population. During the summer of
The soluble form of EIAV receptor encoded by an alternative splicing variant inhibits EIAV infection on target cells Yue-Zhi Lin 1, Fei Yang 1, Shu-Qin Zhang 2, Cheng Du 1, and Jian-Hua Zhou 1 1 Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Science, Harbin 150001, China, 2 Institute of Special Wild Economic Animal and Plant Science, Chinese Academy of Agricultural Science, Changchun 130112, China
Equine lentivirus receptor 1 (ELR1) was found as the solo receptor for equine infectious anemia virus (EIAV) and identified as a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously published membrane-binding form of ELR1, two other major alternative splicing variants of ELR1 were identified from mRNAs of equine macrophages. One spliced species (ELR1-IN) contained an insertion of 153 nt between the sites of 786-787 nt of the published
S90
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
ELR1 cDNA sequence, which resulted in a premature stop signal 813 codons downstream. This translational termination was predicted truncating the receptor at the C-terminus of the transmembrane domain. The other species (ELR1-DE) had a deletion of 109 nt and caused a shift of the open reading frame and an appearance of a stop codon 312 nt downstream. Because ELR1-DE presumably encoded a peptide of mere 23 residues, only ELR1-IN was further analyzed for potential functions on EIAV infection of target cells. Firstly, the expression of the soluble form ELR1 (sELR1) by ELR1-IN was confirmed by Western-blot and immunofluorescence analysis. Like ELR1, the transcription level of ELR1-IN varied in different individuals of horse and at different time points in same individuals. The radio of ELR1-IN mRNA species to that of ELR1 was approximate 1:2. However, the expressions of both forms of the receptor were significantly regulated by the infection of EIAV. Additionally, pre-incubation of the recombinant sELR1 with EIAV significantly inhibited the infection of EIAV on equine macrophages, which is the primary in vivo target cell of the virus. Fetal horse dermal (FHD) cells are susceptible to EIAV in vitro. The replication of EIAV in FHD cells transiently transfected with ELR1-IN was markedly reduced when compared with the replication in cells transfected with the empty vector. Taken together, our data implicate that sELR1 is an important cellular factor that inhibits the infection of EIAV on host cells.
Equine Infectious Anemia Virus (EIAV) gag gene evolution in vivo Z.A. Willand 1, X. Yu 2, S.J. Cook 1, J.K. Craigo 3, 4, R.C. Montelaro 3, 4, C.J. Issel 1, and R.F. Cook 1 1 Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA, 2 Department of Statistics, University of Kentucky, Lexington, KY 40506, USA, 3 Center for Vaccine
Research, 4 Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15261, USA
Introduction: There is less than 26% predicted amino acid identity in Gag p9 sequences between different EIAV isolates. This heterogeneity is probably associated with the unstructured configuration adopted by regions of this protein that may broaden the range of permissible amino acid substitutions at specific sites. Therefore, p9 coding sequences are hypothesized to evolve more rapidly in EIAV infected equids than those specifying Gag antigens where there is significantly more conservation between isolates. Material and methods: The complete viral gag gene from two ponies (564, 567) infected with a laboratory adapted (EIAVPV) strain and two horses (9807, E72) infected with different field isolates (EIAVPA, EIAVFL) of EIAV were amplified using a PCR-based technique and sequenced with overlapping primers. All sequences were assembled using ContigExpress (Vector NTI Advance 11, Invitrogen Corporation, Carlsbad, CA) and aligned using AlignX (Vector NTI Advance 11). Results and Discussion: Nucleotide (nt) substitution rates in gag as a whole varied between subjects from 0.143 in 567 to 2.062 substitutions per year per 100 nt in 98-07 with the ratio of synonymous to non-synonymous changes for most animals being >2:1. The nucleotide substitution rate in p9 was not significantly different from the other gag coding sequences in EIAVPV or EIAVFL infected animals but did show significant differences from p26 (p ¼ 0.0018) and p11 (p ¼ 0.003) but not p15 (p ¼ 0.5153) in the EIAVPA infected horse 98-07. Conclusions: Host and or viral strain factors are important determinants of gag gene mutation rates in EIAV infected equids with the rate of non-synonymous changes being 10-fold higher in 98-07 than in all other subjects. With the exception of 98-07, gag gene sequences including those encoding p9 were relatively conserved over time.
Biosecurity Biosecurity at Equine Events K.A. Flynn 1, E.M. Wilson 1, and J.L. Traub-Dargatz 2 1 California Department of Food and Agriculture, Animal Health Branch, Sacramento, CA 95816, 2 Animal Population Health Institute, College of Veterinary Medicine, Colorado State University, Fort Collins, CO 80523 and USDA:APHIS:VS Centers for Epidemiology and Animal Health, Fort Collins, CO, 80525
The May 2011 Equine Herpesvirus-1 outbreak associated with the Western National Cutting Horse Event in Ogden, UT increased awareness and need for biosecurity measures at equine events. During the outbreak, the California
Department of Food and Agriculture, Animal Health Branch (CDFA AHB), received numerous requests from equine industry stakeholders in the state for guidance on keeping horses healthy at equine events. The California Equine Medication Monitoring Program (EMMP) Advisory Committee, representing a broad range of equine disciplines regulated by the program, is responsible for addressing concerns of the California horse show industry. With more than 1600 shows that register with the EMMP each year, the EMMP Advisory Committee made a formal request for CDFA AHB development of a toolkit to help event organizers identify infectious disease risks for their event venue and determine the best mitigation measures. This industry request was key in the initiation of toolkit development to enhance biosecurity at equine events in
clinical signs. Five were from France and five came from Romania. Positive horses identified in 2010 were from 7 different premises divided in two different episodes. The first episode detected in South West of France consisted of 4 related premises where 5 French Trotters were tested positives and euthanized. The index case has been tested as part of a control before exportation. Primary premise located in the Dordogne County was a breeding farm where 2 mares were tested positive for EIA. Others premises, where two positive horses were detected, were located in the Lot-et-Garonne and Gironde County. All Infected animals (5) came from the breeding farm in Dordogne and have been kept over there several years where they have been probably infected since it is the only source of infection identified. Epidemiological investigations lead to test more than 400 horses, in 38 different Counties, which have been trained in the Dordogne Farm between 1990 and 2010. No other positive animals have been identified during this seroepidemiological study. The second EIA episode identified in 2010 was from horses which have been imported from Romania. Indeed, following EIA cases identified in Belgium and in UK at the end of 2009, French Ministry of Agriculture decided to test horses that came from Romania since January 2007. Investigation detected 80 equids, among those horses 38 were slaughtered or re-exported. Only 35 horses were still on the French territory at the date of testing and five of them exhibited a positive result for EIA using AGID test. Horses kept at the vicinity of those positive horses were tested but no transmission was seen. In this context the European commission (EU) has modified in June 2010 the regulation regarding importation of horses from Romania in other European member states. EU enforced a quarantine and a testing at destination for horses coming from Romania.
S89
2007, an outbreak of EVA occurred in Normandy, France. Only draught and saddle horses were affected. This 2007 outbreak occurred following the used of semen contaminated by EAV for insemination. The objectives of this study were to undertake a thorough epidemiological investigation to identify shedders stallions following the 2007 EVA outbreak as well as the molecular characterization of EAV isolates encountered in France. Positive semen samples were coming from stallions collected in 2007 and 2008. Those samples were tested by Quantitative RT-PCR and by Virus isolation on cell culture as describe in the OIE manual Chapter 2.5.10. Phylogenetic analysis were performed by amplification and sequencing open reading frames 2a-7 (ORFs 2a-7) encoding the viral structural proteins. The new governmental directive leads to the testing of nearly 4500 stallions in 2008 compared to the 450 tested in 2007. Among them 39 stallions were found positive and shed EAV in their semen. Phylogenetic analysis using the ORFs2a-7 sequences and others from GenBank grouped the viruses in two groups: the North American and the European which is divided in subgroups 1 and 2. The majority of viruses (20) isolated and characterized in France were members of the European subgroup 2, six of them belong to the European subgroup 1 and one isolate belongs to the American lineage. Moreover, isolates from the 2007 outbreaks grouped in a distinct cluster, named Normandy cluster, inside the European subgroup 2. The EVA strain responsible of the 2007 outbreak grouped, inside the European subgroup 2, in a new distinct cluster and seems to be different to those used to be isolated in France previously. Appearance of this new virus might explain the speed of the viral spread in horse population observed as well as the severity of clinical signs associated with the infection as shown by the death of 5 foals recorded during this outbreak.
Molecular epidemiology of Equine Arteritis Virus in France following the 2007 outbreak A. Hans 1, D. Gaudaire 1, S. Pronost 2, B. Ferry-Abitbol 3, C. Laugier 1, and S. Zientara 4 1 Anses, Laboratory for equine diseases, 14430, Goustranville, France, 2 Frank Duncombe laboratory, EA 4655 U2RM Caen University Lower Normandy, Caen, France, 3 Institut Français du Cheval et de l’Equitation (IFCE), 49411 Saumur, France, 4 Anses, Laboratory for Animal Health, UMR 1161 ANSES/INRA/ENVA, 94703 Maisons-Alfort, France
Equine Viral Arteritis (EVA) is characterized by a broad range of clinical signs such as hyperthermia, anorexia, oedema and depression. It may cause abortion in mares and a temporary infertility in stallions. The virus, Equine arteritis virus (EAV) that belongs to the Arteriviridae family, can be transmitted either by respiratory or veneral route. Following the initial infection, stallions may become asymptomatic carriers and can shed the virus in their semen. Those stallions are reservoir for EVA and have to be properly handled to prevent any viral spread in horse population. During the summer of
The soluble form of EIAV receptor encoded by an alternative splicing variant inhibits EIAV infection on target cells Yue-Zhi Lin 1, Fei Yang 1, Shu-Qin Zhang 2, Cheng Du 1, and Jian-Hua Zhou 1 1 Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Science, Harbin 150001, China, 2 Institute of Special Wild Economic Animal and Plant Science, Chinese Academy of Agricultural Science, Changchun 130112, China
Equine lentivirus receptor 1 (ELR1) was found as the solo receptor for equine infectious anemia virus (EIAV) and identified as a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously published membrane-binding form of ELR1, two other major alternative splicing variants of ELR1 were identified from mRNAs of equine macrophages. One spliced species (ELR1-IN) contained an insertion of 153 nt between the sites of 786-787 nt of the published
S90
9th ICEID Abstracts / Journal of Equine Veterinary Science 32 (2012) S3-S95
ELR1 cDNA sequence, which resulted in a premature stop signal 813 codons downstream. This translational termination was predicted truncating the receptor at the C-terminus of the transmembrane domain. The other species (ELR1-DE) had a deletion of 109 nt and caused a shift of the open reading frame and an appearance of a stop codon 312 nt downstream. Because ELR1-DE presumably encoded a peptide of mere 23 residues, only ELR1-IN was further analyzed for potential functions on EIAV infection of target cells. Firstly, the expression of the soluble form ELR1 (sELR1) by ELR1-IN was confirmed by Western-blot and immunofluorescence analysis. Like ELR1, the transcription level of ELR1-IN varied in different individuals of horse and at different time points in same individuals. The radio of ELR1-IN mRNA species to that of ELR1 was approximate 1:2. However, the expressions of both forms of the receptor were significantly regulated by the infection of EIAV. Additionally, pre-incubation of the recombinant sELR1 with EIAV significantly inhibited the infection of EIAV on equine macrophages, which is the primary in vivo target cell of the virus. Fetal horse dermal (FHD) cells are susceptible to EIAV in vitro. The replication of EIAV in FHD cells transiently transfected with ELR1-IN was markedly reduced when compared with the replication in cells transfected with the empty vector. Taken together, our data implicate that sELR1 is an important cellular factor that inhibits the infection of EIAV on host cells.
Equine Infectious Anemia Virus (EIAV) gag gene evolution in vivo Z.A. Willand 1, X. Yu 2, S.J. Cook 1, J.K. Craigo 3, 4, R.C. Montelaro 3, 4, C.J. Issel 1, and R.F. Cook 1 1 Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA, 2 Department of Statistics, University of Kentucky, Lexington, KY 40506, USA, 3 Center for Vaccine
Research, 4 Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15261, USA
Introduction: There is less than 26% predicted amino acid identity in Gag p9 sequences between different EIAV isolates. This heterogeneity is probably associated with the unstructured configuration adopted by regions of this protein that may broaden the range of permissible amino acid substitutions at specific sites. Therefore, p9 coding sequences are hypothesized to evolve more rapidly in EIAV infected equids than those specifying Gag antigens where there is significantly more conservation between isolates. Material and methods: The complete viral gag gene from two ponies (564, 567) infected with a laboratory adapted (EIAVPV) strain and two horses (9807, E72) infected with different field isolates (EIAVPA, EIAVFL) of EIAV were amplified using a PCR-based technique and sequenced with overlapping primers. All sequences were assembled using ContigExpress (Vector NTI Advance 11, Invitrogen Corporation, Carlsbad, CA) and aligned using AlignX (Vector NTI Advance 11). Results and Discussion: Nucleotide (nt) substitution rates in gag as a whole varied between subjects from 0.143 in 567 to 2.062 substitutions per year per 100 nt in 98-07 with the ratio of synonymous to non-synonymous changes for most animals being >2:1. The nucleotide substitution rate in p9 was not significantly different from the other gag coding sequences in EIAVPV or EIAVFL infected animals but did show significant differences from p26 (p ¼ 0.0018) and p11 (p ¼ 0.003) but not p15 (p ¼ 0.5153) in the EIAVPA infected horse 98-07. Conclusions: Host and or viral strain factors are important determinants of gag gene mutation rates in EIAV infected equids with the rate of non-synonymous changes being 10-fold higher in 98-07 than in all other subjects. With the exception of 98-07, gag gene sequences including those encoding p9 were relatively conserved over time.
Biosecurity Biosecurity at Equine Events K.A. Flynn 1, E.M. Wilson 1, and J.L. Traub-Dargatz 2 1 California Department of Food and Agriculture, Animal Health Branch, Sacramento, CA 95816, 2 Animal Population Health Institute, College of Veterinary Medicine, Colorado State University, Fort Collins, CO 80523 and USDA:APHIS:VS Centers for Epidemiology and Animal Health, Fort Collins, CO, 80525
The May 2011 Equine Herpesvirus-1 outbreak associated with the Western National Cutting Horse Event in Ogden, UT increased awareness and need for biosecurity measures at equine events. During the outbreak, the California
Department of Food and Agriculture, Animal Health Branch (CDFA AHB), received numerous requests from equine industry stakeholders in the state for guidance on keeping horses healthy at equine events. The California Equine Medication Monitoring Program (EMMP) Advisory Committee, representing a broad range of equine disciplines regulated by the program, is responsible for addressing concerns of the California horse show industry. With more than 1600 shows that register with the EMMP each year, the EMMP Advisory Committee made a formal request for CDFA AHB development of a toolkit to help event organizers identify infectious disease risks for their event venue and determine the best mitigation measures. This industry request was key in the initiation of toolkit development to enhance biosecurity at equine events in