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PubMed1994

Comparison of M and N gene sequences distinguishes variation amongst equine arteritis virus isolates.

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.

Base Sequence↗
PubMed1994

Monoclonal antibodies to equine arteritis virus proteins identify the GL protein as a target for virus neutralization.

Monoclonal antibodies (MAbs) to equine arteritis virus (EAV) proteins were produced and characterized. The protein specificities of eight MAbs were determined definitively by immunoprecipitation of EAV proteins expressed from vaccinia virus recombinants (VVRs). Included were two new VVRs produced for this study, expressing the M and the GL proteins, respectively. Three MAbs were determined to be N-specific and five MAbs recognized the GL protein. One GL-specific MAb, 17F5, of the IgA class, efficiently neutralized EAV infectivity. In competitive binding assays (CBAs), the N-specific MAbs defined a single antigenic domain on this protein. Four GL-specific MAbs, including MAb 17F5, demonstrated strong reciprocal competition in binding to the GL protein but differed in their virus-neutralizing ability. Thus the antigenic domain defined by these MAbs is probably composed of overlapping or closely adjacent epitopes. The fifth GL-specific MAb, a non-neutralizing antibody, may define an epitope adjacent to this antigenic domain as reciprocal CBAs demonstrated lower competition.

Animals↗
PubMed1995

Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL.

Complementary DNAs encoding ORFs 2 to 7 equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immuno-dominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-specific synthetic peptide (residues 75 through 97) induced EAV-neutralizing antibody in vaccinated horses. The defined antigenic region of GL is likely to be exposed on the surface of the native EAV virion and consequently may be useful in the development of diagnostic tests and vaccines.

Animals↗
PubMed1995

Comparison of equine arteritis virus isolates using neutralizing monoclonal antibodies and identification of sequence changes in GL associated with neutralization resistance.

Three murine monoclonal antibodies (MAbs) that neutralize equine arteritis virus (EAV) infectivity were identified and characterized. The antibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprotein (GL) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants with the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize virus isolates indicated that they recognize non-identical epitopes. Susceptibility to virus neutralization could not be used to distinguish viruses from acutely and persistently infected horses. Comparison of the ORF 5 nucleotide and deduced amino acid sequence from NR and neutralization-sensitive virus isolates revealed amino acid sequence changes at positions 99 and 100 which correlate with the NR phenotype. Additional unique changes in the amino acid sequence of MAb NR viruses at positions 96 and 113 may also contribute to neutralization resistance. The sequence data further showed that the Bucyrus-derived viruses contain one N-glycosylation site, whereas the field isolates DL8 and DL11 possess two sites, both of which are used. Most of the non-conservative amino acid sequence changes were located within the second half of the N-terminal hydrophilic domain. Sequence changes within the first half of the N-terminal ectodomain, the predicted transmembrane domain and the C-terminal hydrophilic domain were mainly silent base substitutions or resulted in conservative amino acid substitutions, suggesting that these regions of the protein are functionally conserved.

Amino Acid Sequence↗
PubMed1999

Genetic diversity of equine arteritis virus.

Equine arteritis viruses (EAV) from Europe and America were compared by phylogenetic analysis of 43 isolates obtained over four decades. An additional 22 virus sequences were retrieved from GenBank. Fragments of the glycoprotein G(L) and the replicase genes were amplified by RT-PCR, prior to sequencing and construction of phylogenetic trees. The trees revealed many distinctive lineages, consistent with prolonged diversification within geographically separated host populations. Two large groups and five subgroups were distinguished. Group I consisted mainly of viruses from North America, whilst group II consisted mainly of European isolates. In most instances, where the geographic origin of the viruses appeared to be at variance with the phylogenetically predicted relationships, the horses from which the viruses were recovered had been transported between Europe and America or vice versa. Analysis of the replicase gene revealed similar phylogenetic relationships although not all of the groups were as clearly defined. Virus strains CH1 (Switzerland, 1964) and S1 (Sweden, 1989) represented separate 'outgroups' based on analysis of both genomic regions. The results of this study confirm the value of the G(L) gene of EAV for estimating virus genetic diversity and as a useful tool for tracing routes by which EAV is spread. In addition, computer-assisted predictions of antigenic sites on the G(L) protein revealed considerable variability among the isolates, especially with respect to regions associated with neutralization domains.

Amino Acid Sequence↗
PubMed1999

Genetic stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis.

An imported carrier stallion (A) from Europe was implicated in causing an extensive outbreak of equine viral arteritis (EVA) on a Warmblood breeding farm in Pennsylvania, USA. Strains of equine arteritis virus (EAV) present in the semen of two carrier stallions (A and G) on the farm were compared to those in tissues of foals born during the outbreak, as well as viruses present in the semen of two other stallions that became persistently infected carriers of EAV following infection during the outbreak. The 2822 bp segment encompassing ORFs 2-7 (nt 9807-12628; which encode the G(S), GP3, GP4, G(L), M and N proteins, respectively) was directly amplified by RT-PCR from semen samples and foal tissues. Nucleotide and phylogenetic analyses confirmed that virus present in the semen of stallion A initiated the outbreak. The genomes of viruses present in most foal tissues (10/11) and serum from an acutely infected mare collected during the outbreak were identical to that of virus present in the lung of the first foal that died of EVA. Virus in the placenta of one foal differed by one nucleotide (99.9% identity) from the predominant outbreak virus. The relative genetic stability of viruses that circulated during the outbreak contrasts markedly with the heterogeneous virus populations variously present in the semen of persistently infected stallions on the farm. These findings are consistent with the hypothesis that the carrier stallion can be a source of genetic diversity of EAV, and that outbreaks of EVA can be initiated by the horizontal aerosol transmission of specific viral variants that occur in the semen of particular carrier stallions.

Amino Acid Sequence↗
PubMed2001

Evaluation of a prototype sub-unit vaccine against equine arteritis virus comprising the entire ectodomain of the virus large envelope glycoprotein (G(L)): induction of virus-neutralizing antibody and assessment of protection in ponies.

An Escherichia coli-expressed recombinant protein (6hisG(L)ecto) comprising the entire ectodomain (aa 18-122) of equine arteritis virus (EAV) glycoprotein G(L), the immunodominant viral antigen, induced higher neutralizing antibody titres than other G(L)-derived polypeptides when compared in an immunization study in ponies. The potential of the recombinant G(L) ectodomain to act as a sub-unit vaccine against EAV was evaluated further in three groups of four ponies vaccinated with doses of 35, 70 or 140 microg of protein. All vaccinated animals developed a virus-neutralizing antibody (VNAb) response with peak titres 1-2 weeks after the administration of a booster on week 5 (VNAb titres of 1.8-3.1), 13 (VNAb titres of 1.4-2.9) or 53 (VNAb titres of 1.2-2.3). Vaccinated and unvaccinated control ponies were infected with EAV at different times post-vaccination to obtain information about the degree of protection relative to the levels of pre-challenge VNAb. Vaccination conferred varying levels of protection, as indicated by reduced or absent pyrexia, viraemia and virus excretion from the nasopharynx. The degree of protection correlated well with the levels of pre-challenge VNAb and, in particular, with levels of virus excretion. These results provide the first evidence that a sub-unit vaccine protects horses against EAV. The use of the sub-unit vaccine in combination with a differential diagnostic test based on other EAV antigens would enable serological discrimination between naturally infected and vaccinated equines.

Amino Acid Sequence↗
PubMed2001

Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex.

The replicase polyproteins of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are processed by three viral proteases to yield 12 non-structural proteins (nsps). The nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. Remarkably, upon infection of Vero cells, but not of BHK-21 or RK-13 cells, EAV nsp2 is now shown to be subject to an additional, internal, cleavage. In Vero cells, approximately 50% of nsp2 (61 kDa) was cleaved into an 18 kDa N-terminal part and a 44 kDa C-terminal part, most likely by a host cell protease that is absent in BHK-21 and RK-13 cells. Although the functional consequences of this additional processing step are unknown, the experiments in Vero cells revealed that the C-terminal part of nsp2 interacts with nsp3. Most EAV nsps localize to virus-induced double-membrane structures in the perinuclear region of the infected cell, where virus RNA synthesis takes place. It is now shown that, in an expression system, the co-expression of nsp2 and nsp3 is both necessary and sufficient to induce the formation of double-membrane structures that strikingly resemble those found in infected cells. Thus, the nsp2 and nsp3 cleavage products play a crucial role in two processes that are common to positive-strand RNA viruses that replicate in mammalian cells: controlled proteolysis of replicase precursors and membrane association of the virus replication complex.

Animals↗
PubMed2002

Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus.

RNA synthesis (genome replication and subgenomic mRNA transcription) directed by equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) occurs on modified cytoplasmic membranes to which most viral replicase subunits localize. Remarkably, a fraction of non-structural protein 1 (nsp1), a protein essential for transcription but dispensable for genome replication, is present in the host cell nucleus, in particular during the earlier stages of infection. Expression of GFP-tagged fusion proteins revealed that nsp1 is actively imported into the nucleus. Although the signals responsible for nsp1 transport could not be identified, our studies revealed that another EAV protein with a partially nuclear localization, the nucleocapsid (N) protein, utilizes the CRM1-mediated nuclear export pathway. Inactivation of this pathway with the drug leptomycin B resulted in the unexpected and immediate nuclear retention of all N protein molecules, thus revealing that the protein shuttles between cytoplasm and nucleus before playing its role in cytoplasmic virus assembly.

Active Transport, Cell Nucleus↗
PubMed2003

Detection of equine arteritis virus (EAV)-specific cytotoxic CD8+ T lymphocyte precursors from EAV-infected ponies.

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a (51)Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain (51)Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8(+)/CD4(+) ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8(+) T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.

Animals↗
PubMed2003

Equine arteritis virus non-structural protein 1, an essential factor for viral subgenomic mRNA synthesis, interacts with the cellular transcription co-factor p100.

Non-structural protein 1 (nsp1), the N-terminal subunit of the replicase polyprotein of the arterivirus Equine arteritis virus (EAV), is essential for viral subgenomic mRNA synthesis, but fully dispensable for genome replication. However, at the molecular level, the role of nsp1 in EAV subgenomic mRNA synthesis is poorly understood. A yeast two-hybrid screen did not reveal interactions between EAV nsp1 and other viral non-structural proteins or the nucleocapsid protein, although both nsp1 and the nucleocapsid protein were found to form homomers. Subsequently, a yeast two-hybrid screen of a HeLa cell cDNA library was performed using nsp1 as bait. Remarkably, this analysis revealed (potential) interactions between EAV nsp1 and factors that are involved in host cell transcriptional regulation. The interaction of nsp1 with one of these proteins, p100, a transcription co-activator that also interacts with regulatory proteins of other viruses, was confirmed by mutual co-immunoprecipitation from lysates of EAV-susceptible mammalian cells.

Amino Acid Sequence↗
PubMed2004

Genetic characterization of equine arteritis virus during persistent infection of stallions.

Equine arteritis virus (EAV) causes a persistent infection of the reproductive tract of carrier stallions. The authors determined the complete genome sequences of viruses (CW96 and CW01) that were present 5 years apart in the semen of a carrier stallion (CW). The CW96 and CW01 viruses respectively had only 85.6 % and 85.7 % nucleotide identity to the published sequence of EAV (EAV030). The CW96 and CW01 viruses had two 1 nt insertions and a single 1 nt deletion in the leader sequence, and a 3 nt coding insertion in ORF1a; thus their genomes included 12 708 nt as compared to the 12 704 nt in EAV030. Variation between viruses present in the semen of stallion CW and EAV030 was especially marked in the replicase gene (ORF1a and 1b), and the greatest variation occurred in the portion of ORF1a encoding the nsp2 protein. The ORFs 3 and 5, which respectively encode the GP3 and GP5 envelope proteins, showed greatest variation amongst ORFs encoding structural EAV proteins. Comparative sequence analyses of CW96 and CW01 indicated that ORFs 1a, 1b and 7 were highly conserved during persistent infection, whereas there was substantial variation in ORFs 3 and 5. Although the variation that occurs in ORF5 results in the emergence of novel phenotypic viral variants as determined by neutralization assay, all variants were neutralized by high-titre polyclonal equine antisera, suggesting that immune evasion is unlikely to be responsible for the establishment of persistent EAV infection of carrier stallions. Northern blot analyses of RNA extracted from cell culture propagated viruses isolated from 10 different persistently infected stallions failed to demonstrate any large genomic deletions, suggesting that defective interfering particles are also unlikely to be important in either the maintenance or clearance of persistent EAV infection of the reproductive tract of carrier stallions.

Amino Acid Sequence↗
PubMed2004

Rescue of disabled infectious single-cycle (DISC) equine arteritis virus by using complementing cell lines that express minor structural glycoproteins.

Equine arteritis virus (EAV) contains seven structural proteins that are all required to produce infectious progeny. Alphavirus-based expression vectors have been generated for each of these proteins to explore the possibilities for their constitutive expression in cell lines. This approach was successful for minor glycoproteins GP(2b), GP(3) and GP(4) and for the E protein. Subsequently, it was demonstrated that cell lines expressing these proteins could rescue EAV mutants that were disabled in the expression of the corresponding gene, resulting in the production of virus particles carrying the mutant genome. This system was particularly efficient for GP(2b)- and GP(4)-knockout mutants. Upon infection of non-complementing cells with these mutants, a self-limiting single cycle of replication was initiated, resulting in the expression of all but one of the viral proteins. These disabled infectious single-cycle (DISC) arteriviruses can also be used to express foreign sequences and are potentially useful in both fundamental research and vaccine development.

Animals↗
PubMed2005

Antiviral activity of morpholino oligomers designed to block various aspects of Equine arteritis virus amplification in cell culture.

The antiviral efficacy of ten antisense phosphorodiamidate morpholino oligomers (PMOs) directed against Equine arteritis virus (EAV), a nidovirus belonging to the family Arteriviridae, was evaluated in mammalian (Vero-E6) cells. Peptide-conjugated PMOs (P-PMOs) supplied in cell culture medium at micromolar concentrations were efficiently taken up by Vero-E6 cells and were minimally cytotoxic. The P-PMOs were designed to base pair to RNA sequences involved in different aspects of EAV amplification: genome replication, subgenomic mRNA synthesis, and translation of genome and subgenomic mRNAs. A novel recombinant EAV, expressing green fluorescent protein as part of its replicase polyproteins, was used to facilitate drug screening. A moderate reduction of EAV amplification was observed with relatively high concentrations of P-PMOs designed to anneal to the 3'-terminal regions of the viral genome or antigenome. To determine if the synthesis of subgenomic mRNAs could be specifically reduced, transcription-regulating sequences essential for their production, but not for the production of genomic RNA, were targeted, but these P-PMOs were found to be ineffective at transcription interference. In contrast, all four P-PMOs designed to base pair with targets in the genomic 5' untranslated region markedly reduced virus amplification in a sequence-specific and dose-responsive manner. At concentrations in the low micromolar range, some of the P-PMOs tested completely inhibited virus amplification. In vitro translation assays showed that these P-PMOs were potent inhibitors of translation. The data suggest that these compounds could be useful as reagents for exploring the molecular mechanics of nidovirus translation and have anti-EAV potential at relatively low concentrations.

Animals↗
PubMed2006

RNA signals in the 3' terminus of the genome of Equine arteritis virus are required for viral RNA synthesis.

RNA virus genomes contain cis-acting sequences and structural elements involved in virus replication. Both full-length and subgenomic negative-strand RNA synthesis are initiated at the 3' terminus of the positive-strand genomic RNA of Equine arteritis virus (EAV). To investigate the molecular mechanism of EAV RNA synthesis, the RNA secondary structure of the 3'-proximal region of the genome was analysed by chemical and enzymic probing. Based on the RNA secondary structure model derived from this analysis, several deletions were engineered in a full-length cDNA copy of the viral genome. Two RNA domains were identified that are essential for virus replication and most likely play a key role in viral RNA synthesis. The first domain, located directly upstream of the 3' untranslated region (UTR) (nt 12610-12654 of the genome), is mainly single-stranded but contains one small stem-loop structure. The second domain is located within the 3' UTR (nt 12661-12690) and folds into a prominent stem-loop structure with a large loop region. The location of this stem-loop structure near the 3' terminus of the genome suggests that it may act as a recognition signal during the initiation of minus-strand RNA synthesis.

Animals↗
PubMed2006

Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7.

The positive-stranded RNA genome of the arterivirus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and pp1ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of pp1a (the nsp3-8 region) was postulated to occur following either of two alternative proteolytic pathways (the 'major' and 'minor' pathways). Here, the importance of these two pathways was investigated by using a reverse-genetics system and inactivating each of the cleavage sites by site-directed mutagenesis. For all of these pp1a cleavage sites, mutations that prevented cleavage by the nsp4 proteinase were found to block or severely inhibit EAV RNA synthesis. Furthermore, our studies identified a novel nsp4 cleavage site (Glu-1575/Ala-1576) that is located within nsp7 and is conserved in arteriviruses. The N-terminal nsp7 fragment (nsp7alpha) derived from this cleavage was detected in lysates of both EAV-infected cells and cells transiently expressing pp1a. Mutagenesis of the novel cleavage site in the context of an EAV full-length cDNA clone proved to be lethal, underlining the fact that the highly regulated, nsp4-mediated processing of the C-terminal half of pp1a is a crucial event in the arterivirus life cycle.

Animals↗