Combination of an unbiased amplification method and a resequencing microarray for detecting and genotyping equine arteritis virus.

This study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.

[Development of PCR methods for detection of EAV infection]. / Entwicklung einer PCR zum Nachweis der EAV-Infektion.

The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.

Le but de ce travail était de développer, comme alternative à l'isolation, une méthode de RT-PCR (en temps réel) pour le diagnostic rapide de l'EAV et pour la caractérisation des souches virales. Pour cela, on a adapté deux méthodes de RT-PCR conventionnelles et une de RT-PCR en temps réel, de manière à ce qu'un spectre aussi large que possible d'isolats soit démontrable. Les lignées de dilution avec la souche Bucyrus ont montré une sensibilité cent fois plus élevée avec la RT-PCR en temps réel et avec la RT-PCR heminested qu'avec la RT-PCR simple. L'efficacité des méthodes a pu être démontrée avec 11 surnageants de cultures cellulaires de divers isolats d'EAV et 7 échantillons de sperme positifs à l'EAV. L'analyse phylogénétique des séquences des échantillons par rapport à des séquences connues laisse penser qu'il existe plus de sous-groupes d'EAV que décrit jusqu'à ce jour.

Extended phylogeny of the equine arteritis virus sequences including South American sequences.

OBJECTIVE: To perform genetic analysis of the ORF5 of equine arteritis virus (EAV) may provide new insights into the genetic evolution and origin of the Argentinean EAV sequences. METHODS: A total of 76 sequences were analyzed by neighbor joining (NJ), maximum parsimony and maximum likelihood algorithms. The analysis of the selective pressures was performed using the Tajima's test. RESULTS: The trees showed similar topologies. Two clades were identified: the first clade was formed by strains isolated mainly from a donkey, whereas the second clade presented four large groups from different geographic regions exclusively from Equus caballus. In this clade, we identified a group formed by South African and another one by South American and European sequences. In the latter, the monophyletic group was formed by seven Argentinean sequences. In the NJ tree, we identified a group formed by six Argentinean sequences. The Tajima's test showed a D value of 1.73663, indicating that the sequences analyzed follow a neutral evolution model. CONCLUSION: We concluded that the Argentinean sequences have a paraphyletic origin and that the fixation of point mutation might follow the neutral model evolution; however, we identified purifying pressures that may be involved in the differentiation of the EAV sequences.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported.

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

Persistent equine arteritis virus infection in HeLa cells.

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.

Equine arteritis virus: a new isolate from the presumable first carrier stallion in Argentina and its genetic relationships among the four reported unique Argentinean strains.

Equine arteritis virus (EAV) was isolated from a testicle of the presumable first stallion infected with EAV in Argentina. This virus isolate (named LT-LP-ARG) was confirmed by GP5-specific PCR and indirect immunofluorescence assays. The PCR product was sequenced, and the phylogenetic analysis revealed that the LT-LP-ARG strain of EAV forms a monophyletic group, together with other strains previously isolated in our laboratory (LP02 group). However, all Argentinean EAV strains belong to a polyphyletic group. We believe that the virus isolate presented in this report could be the origin of EAV infection in our country.

Molecular epizootiology of equine arteritis virus isolates from Poland.

Phylogenetic analysis was performed on the sequences of 44 Polish isolates of equine arteritis virus that were isolated from the semen of stallions from national and private studs, collected during 2001--2005. These sequences were also compared with 41 reference strains previously described and commonly used in phylogenesis. On the basis of the nucleotide sequence analysis of the ORF5 gene, encoding the glycoprotein GP5, it was demonstrated that the Polish EAV isolates belonged to two subgroups and showed the closest relationship to the European strains. Similar results were obtained using the nucleotide sequences of the ORF7 gene. The nucleotide identity between the ORF5 and ORF7 sequences of all Polish isolates was in the range of 80.1-99.0% and 93.6-100%, respectively. The analysis of genetic diversity within the ORF5 sequences enabled a retrospective epizootic investigation. This study suggested that some of the EAV shedding stallions were probably infected before they were moved to Poland.

Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants.

In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, respectively. The real-time RT-PCR assay was at least 2 log(10) more sensitive than the classical RT-PCR and at least 1 log(10) more sensitive than the primer set used in the semi-nested PCR. The specificities of the amplification reactions were confirmed with biotinylated probes in the PCR-enzyme-linked immunosorbent assay (PCR-ELISA). Under the conditions described in our study, the sensitivity of the real-time RT-PCR was found to be superior to the PCR-ELISA assay. Thus, while the PCR-ELISA method was found to be both relatively demanding and time consuming, better sensitivity coupled with high specificity and speed of the assay makes the real-time RT-PCR a valuable tool for diagnosis of EAV infection.

Identification of a divergent genotype of equine arteritis virus from South American donkeys.

A novel equine arteritis virus (EAV) was isolated and sequenced from feral donkeys in Chile. Phylogenetic analysis indicates that the new virus and South African asinine strains diverged at least 100 years from equine EAV strains. The results indicate that asinine strains belonged to a different EAV genotype.

Highly diverse type of equine arteritis virus (EAV) from the semen of a South African donkey: short communication.

Equine arteritis virus (EAV) was detected by RT-nested PCR in semen samples from a naturally infected South African donkey. Sequence analysis of the amplified ORF5 fragment revealed only 60 to 70% nucleotide identity to a panel of EAV reference sequences. The unique donkey EAV sequence was also found to be stable during passage in horses. The sequence data reported in this study indicate that the South African donkey variant might represent a new genotype of EAV. The distinct genetic properties of the South African asinine strain of EAV suggest a divergent evolution of this arterivirus in various host species or, alternatively, a possible role for African donkeys in the emergence of EAV in horses.

Re-emergence of a genetic outlier strain of equine arteritis virus: Impact on phylogeny.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids, which is notifiable in some countries including the Great Britain (GB) and to the OIE. Herein, we present the case of a persistently infected stallion and the phylogenetic tracing of the virus strain isolated. Discussing EAV occurrence and phylogenetic analysis we review features, which may aid to harmonise and enhance the classification of EAV.

Genetic variation in open reading frame 2 of field isolates and laboratory strains of equine arteritis virus.

The open reading frame 2 (ORF2) of three laboratory strains, the live attenuated vaccine virus, and 18 field isolates of equine arteritis virus (EAV) from Europe and North America was sequenced. The ORF2 of EAV encodes the Gs protein that is abundantly expressed in infected cells but constitutes less than 2% of the virion protein mass. Variation of ORF2 among the isolates facilitated phylogenetic analysis that largely confirmed results of an earlier study based on sequence divergence of ORF5 of the same isolates of EAV, despite exposure of the proteins encoded by ORF2 (Gs) and ORF5 (GL) to potentially different selective pressures in vivo. The data indicate that the Gs protein is highly conserved between isolates, considerably more so than the GL protein, consistent with an important role of the Gs protein in virus replication.

Lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus.

Lelystad virus (LV) is an enveloped positive-stranded RNA virus, which causes abortions and respiratory disease in pigs. The complete nucleotide sequence of the genome of LV has been determined. This sequence is 15.1 kb in length and contains a poly(A) tail at the 3' end. Open reading frames that might encode the viral replicases (ORFs 1a and 1b), membrane-associated proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7) have been identified. Sequence comparisons have indicated that LV is distantly related to the coronaviruses and toroviruses and closely related to lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). A 3' nested set of six subgenomic RNAs is produced in LV-infected alveolar lung macrophages. These subgenomic RNAs contain a leader sequence, which is derived from the 5' end of the viral genome. Altogether, these data show that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-stranded RNA viruses, the Arteriviridae.

Phylogenetic characterisation of the G(L) sequences of equine arteritis virus isolated from semen of asymptomatic stallions and fatal cases of equine viral arteritis in Denmark.

The study describes for the first time the phylogenetic relationship between equine arteritis virus (EAV) isolated from asymptomatic virus-shedding stallions and fatal cases of equine viral arteritis (EVA) in an European country. EAV was isolated from three dead foals and an aborted foetus during three different outbreaks of EVA. From these fatalities, the complete open reading frame 5, encoding the EAV G(L) protein, was amplified by reverse transcription-polymerase chain reaction and subjected to nucleotide sequence analysis. Furthermore, DNA sequences were obtained from virus isolated from semen samples of seven virus-shedding, but clinically healthy, Danish stallions. DNA sequence alignment revealed an overall divergence of 0-14 and 0-10% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis including 24 previously published sequences revealed that European as well as North American "types" of EAV were present in the semen of asymptomatic carrier stallions and in fatal cases of EVA. Our results reveal that the presence of EAV-shedding stallions in Denmark represents a potential source of severe EVA.

Equine viral arteritis. Current status in Finland.

A serological study for antibodies against equine arteritis virus (EAV) in Finland was performed during 1996. All equine sera delivered to the Virology Unit at the National Veterinary and Food Research Institute were tested with a micro-neutralization test, using the Arvac strain as antigen. The study also included imported horses to evaluate EAV circulation in the countries of origin. Nucleocapsid gene sequences of 2 Finnish equine semen isolates were amplified with RT-PCR and sequenced. The genetic relationships of those isolates with strains isolated elsewhere in the world were analyzed. The Finnish isolates shared 98.2% nucleotide identity, and the closest relatives to the Finnish strains were isolated from the semen of 2 Norwegian horses in 1988 and 1989.

Genetic variation and phylogenetic analysis of open reading frames 3 and 4 of various equine arteritis virus isolates.

The genetic variation in equine arteritis virus (EAV) nonstructural (NS) protein-encoding open reading frames (ORF) 3 and 4 genes was investigated. Nucleotide and deduced amino acid sequences from seven different EAV isolates (one European, one American and five Canadian isolates) and the Arvac vaccine strain were compared with those of the Bucyrus reference strain. ORF 3 nucleotide and amino acid sequence identities amongst these isolates (including the Arvac vaccine strain) and the Bucyrus reference strain ranged from 85.6 to 98.8%, and 85.3 to 98.2%, respectively, whereas ORF 4 nucleotide and amino acid sequence identities ranged from 90.4 to 98.3%, and 90.8 to 97.4%, respectively. Phylogenetic tree analysis based on the ORF 3 nucleotide sequences showed that the European Vienna isolate could be classified into a genetically divergent group from all other isolates and the Arvac vaccine strain. In contrast, a phylogenetic relationship among all EAV isolates and the Arvac vaccine strain based on the ORF 4 nucleotide sequences was observed.

Lateral transmission of equine arteritis virus among Lipizzaner stallions in South Africa.

REASONS FOR PERFORMING STUDY: A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. OBJECTIVES: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. METHODS: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF 5-specific primers, subjected to sequence and phylogenetic analysis. RESULTS: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stallions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from bedding or fomites contaminated with semen. CONCLUSIONS: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. POTENTIAL RELEVANCE: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV- infected semen.

[Equine infectious arteritis: molecular biology, epidemiology and preventative measures]. / L'artérite infectieuse des équidés: biologie moléculaire, épidémiologie et mesures de prophylaxie.

After a brief historical account of the outbreak of infectious arteritis of horses which occurred in 1984 in Kentucky (United States of America), the author reports on the present state of knowledge concerning the organisation of the genome of the virus. Clinical signs of the disease are described, as well as modes and routes of transmission. Finally, currently-available vaccination procedures are discussed and their value is assessed.

Nucleotide sequence and genetic analysis of the leader region of Canadian, American and European equine arteritis virus isolates.

The extreme 5' end, the entire leader sequence of the Arvac vaccine strain, and 10 equine arteritis virus (EAV) isolates, including the ATCC Bucyrus reference strain and 5 Canadian field isolates, were determined and compared at the primary nucleotide and secondary structure levels. The leader sequence of eight EAV isolates, including the Bucyrus reference strain, and the leader sequence of the Arvac vaccine strain was determined to be 206 nt in length (not including the putative 5' cap structure-associated nucleotide) whereas those of the 86AB-A1 and 86NY-A1 isolates were found to be 205 and 207 nt in length, respectively. The sequence identity of the leader sequences, between the different isolates and the Bucyrus reference strain, ranged from 94.2 to 98.5%. Phylogenetic analysis and estimation of genetic distances, based on the leader nucleic acid sequences, showed that all EAV isolates/strains are likely to represent a large phylogenetically-related group. An AUG start codon found at position 14 in all EAV isolates/strains could initiate an open reading frame (ORF) that could produce a polypeptide of 37 amino acids, except for the 86NY-A1 isolate where the intraleader polypeptide would contain 54 amino acids. Computer-predicted RNA secondary structures were identified in the 11 EAV leader regions analyzed. All EAV isolates/strains showed 3 conserved stem-loops (designated A, B and C). An additional conserved stem-loop (D) was observed in 7 EAV isolates, including the Bucyrus reference strain. The leader region distal to stem-loop D did not contain conserved sequences or stem-loop structures common to the EAV isolates/strains.

Differentiation of strains of equine arteritis virus of differing virulence to horses by growth in equine endothelial cells.

OBJECTIVE: To compare growth characteristics of strains of equine arteritis virus (EAV) of differing virulence to horses in rabbit kidney (RK)-13 cells and equine endothelial cells (EECs) cultured from the pulmonary artery of a foal. SAMPLE POPULATION: 13 strains of EAV, including 11 field isolates of differing virulence to horses; the highly virulent, horse-adapted Bucyrus strain; and the modified-live virus (MLV) vaccine derived from it. PROCEDURE: The growth characteristics of the 13 strains were compared in EECs and RK-13 cells. Viral nucleoprotein expression, cytopathogenicity, and plaque size were compared to determine whether growth characteristics of the 13 strains were predictive of their virulence to horses. RESULTS: Cytopathogenicity, viral nucleoprotein expression, and plaque size induced by all 13 viruses were similar in RK-13 cells, whereas virulent strains of EAV caused significantly larger plaques in EECs than did the avirulent strains of EAV. Paradoxically, the highly attenuated MLV vaccine and 1 field isolate of EAV caused plaques in EECs that were larger than those caused by any of the other viruses, and sequence analysis confirmed the field isolate of EAV to be indistinguishable from the MLV vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: With the notable exception of the MLV vaccine, growth of the various strains of EAV in EECs was predictive of their individual virulence to horses. Thus, EECs provide a relevant and useful model to further characterize determinants of virulence and attenuation amongst strains of EAV.