Buoyant density studies on equine arteritis virus

Summary The buoyant density of the Bucyrus strain of equine arteritis virus was studied by isodensity centrifugation on three types of gradients. The distribution patterns of infectivity were dependent on the type of gradient used. In sucrose gradients a single peak of infectivity at densities approximating 1.17 g/ml was obtained. From this gradient the total recovery of infectious virus varied between 80 and 90%. In cesium chloride gradients a rather broad band appeared in the form of a saddled peak with a range of densities from 1.180 to 1.215 g/ml, the total recovery being 90%. In potassium tartrate (KT) gradients two peaks were formed, one at 1.17 and another at 1.24 g/ml; the total recovery was 50%. Recentrifugation of fractions from KT gradient on sucrose gradients resulted in a changed distribution of infectivity for particles of higher density (1.24 g/ml).     

Structural proteins of equine arteritis virus

Summary Polyacrylamide gel electrophoresis of purified and solubilized equine arteritis virus (EAV) revealed nine structural proteins. Two of these proteins with molecular weights of 15,000 (VP7) and 13,000 (VP8) daltons are considered as major. The molecular weights of the seven minor proteins ranged between 72,000 and 10,500 daltons.The core fraction of the virion was dissociated from the envelope fraction by sucrose gradient centrifugation of virus treated with the nonionic detergent Nonidet P40 and phospholipase C. The core fraction contained RNA and one major protein (VP8), whereas the envelope contained one major protein (VP7) and the seven minor proteins. Six of the nine proteins (VP1 through VP6) were labeled with¹⁴C-glucosamine and are thus glycoproteins. VP8, a nonglycoprotein associated with the core fraction, is apparently the nucleoprotein of EAV.     

The genome of equine arteritis virus.

Equine arteritis virus (EAV) contains an infectious RNA. [³H]uridine-labeled RNA was released from purified virus (density, 1.155 g/ml in sucrose; s_20,w, 224 ± 8 S) with sodium dodecyl sulfate and 2-mercaptoethanol. An s_20,w value of 48 S was found in isokinetic sucrose gradients in 0.1 M saline. In 1 mM saline, sedimentation was slower (33 S), in 0.1 M saline plus 1 mM MgCl₂, a value of 56 S was measured. A molecular weight of 4.0 × 10⁶ was determined by polyacrylamide-agarose-gel electrophoresis. Heating of purified RNA in the presence of 1.1 M formaldehyde and subsequent centrifugation in gradients containing formaldehyde did not result in degradation to smaller RNA's. From this procedure a molecular weight of 4.1 × 10⁶ was calculated. Buoyant density in Cs₂SO₄ of the RNA was 1.65 g/ml. In most experiments Semliki forest virus RNA was taken as a reference. It behaved almost indistinguishably from the RNA of EAV. In conclusion EAV contains an infectious, colinear molecule of single-stranded RNA with a molecular weight of about 4 million. These data justify a definite inclusion of this virus in the family Togaviridae.     

The structural proteins of equine arteritis virus.

Equine arteritis virus (EAV) grown in Vero, BHK-21, and RK-13 cells was purified by pelleting, Sepharose 6B chromatography, and sucrose gradient centrifugation. Analysis of whole gradients by polyacrylamide slab gel electrophoresis and subsequent autoradiography revealed a large number of proteins in all fractions. Comparison of protein patterns of virus grown in the different cell systems showed that only three proteins with molecular weights of 12,000 (VP1), 14,000 (VP2), and 21,000 (VP3) were virus specific. VP1 is a phosphorylated core protein, while VP3 is a glycoprotein. These findings, together with data obtained earlier about morphology and RNA of the virion, lend further support to inclusion of EAV in the family Togaviridae with a possible relationship to lactic dehydrogenase virus.     

Genetic typing of equine arteritis virus isolates from Argentina

We report the nucleotide sequence and genetic diversity of four Equine Arteritis Virus (EAV) ORF 5 and 6 from Argentina isolates, obtained from asymptomatic virus-shedding stallions. Nucleic acid recovered from the isolates were amplified by RT-PCR and sequenced. Nucleotide and deduced amino acid sequences from the Argentine isolates were compared with 17 sequences available from the GenBank. Phylogenetic analysis revealed that the Argentine isolates grouped together in a definite cluster near European strains. Despite the greater genetic variability among ORF 5 from different isolates and strains of EAV, phylogenetic trees based on ORF 5 and 6 are similar. Both trees showed that virus sequences from America and Europe segregate into distinct clades based on sequence analysis of either ORF 5 or 6. This study constitutes the first characterization of Argentine EAV isolates. M. G. Echeverría, S. Díaz, E. Nosetto Members of CONICET (Scientific Research Council)     

Characterization of equine humoral antibody response to the nonstructural proteins of equine arteritis virus

Equine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV infection. Individual nsp coding regions were cloned and expressed in both mammalian and bacterial expression systems. Each recombinant protein was used in an immunoprecipitation assay with equine serum samples from horses (n = 3) that were experimentally infected with three different EAV strains (VB, KY77, and KY84), from stallions (n = 4) that were persistently infected with EAV, and from horses (n = 4) that were vaccinated with the modified live-virus (MLV) vaccine strain. Subsequently, protein-antibody complexes were subjected to Western immunoblotting analysis with individual nsp-specific rabbit antisera, mouse anti-His antibody, or anti-FLAG tag antibody. Nsp2, nsp4, nsp5, and nsp12 were immunoprecipitated by most of the sera from experimentally or persistently infected horses, while sera from vaccinated horses did not react with nsp5 and reacted weakly with nsp4. However, serum samples from vaccinated horses were able to immunoprecipitate nsp2 and nsp12 proteins consistently. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection in horses.     

Identification of an additional neutralization determinant of equine arteritis virus

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).     

The complement-requiring neutralization of equine arteritis virus by late antisera

Neutralization of equine arteritis virus (EAV) by late antibody raised in horses, guinea pigs, rabbits, hamsters, and mice was investigated and found to be complement-dependent. The complement-dependent EAV neutralizing antibody activity was found associated with the IgG fraction of late antisera. Early antisera or their IgM and IgG fractions were ineffective. Analysis of virus-antibody interaction at 37° showed that no appreciable loss of EAV infectivity occurred following incubation with heat-inactivated late antiserum for 20 min. However, upon prolonged incubation, partial reduction in EAV infectivity was detected. The sensitization of EAV (as assessed by anti-IgG) was shown to begin following mixing with antiserum and was essentially completed after 20 min of incubation. Neutralization of sensitized EAV by complement or anti-IgG was instantaneous and not temperature dependent. Analysis of EAV infectivity (in vitro) indicated that specific antibody apparently did not interfere with the early steps of virus-cell interaction. Further studies suggested that following penetration into susceptible cells, sensitized EAV was rendered insensitive to complement inactivation.The present investigation has confirmed the existence of a complement-dependent viral neutralizing antibody in late antisera and, further, has shown that EAV provides a new model for analysis of the complement-dependent neutralization of sensitized virus.     

Neutralization of equine arteritis virus; enhancing effect of guinea pig serum

Summary Neutralization of equine arteritis virus (EAV) by antisera is usually enhanced in the presence of unheated guinea pig serum. The enhancing effect of unheated guinea pig serum could be reduced by heating or eliminated by absorption on a heterologous antigen-antibody system. Immune horse and rabbit sera were fractionated and the fractions containing IgG or IgM tested against EAV. Only IgG had a neutralizing and sensitizing effect. EAV belongs thus to viruses which are in part neutralized, in part sensitized by antiviral IgG.     

Intracellular equine arteritis virus (EAV)-specific RNAs contain common sequences

Equine arteritis virus (EAV) is a nonarthropod-borne togavirus. Six virus-specific RNA species have been found in EAV-infected cells having the following molecular weights: 4.3 X 10(6) (RNA1), 1.3 X 10(6) (RNA2), 0.9 X 10(6) (RNA3), 0.7 X 10(6) (RNA4), 0.3 X 10(6) (RNA5), and 0.2 X 10(6) (RNA6). RNA1 comigrates with the viral genome (M. F. Van Berlo, M. C. Horzinek, and B. A. M. Van der Zeijst, 1982, Virology 118, 345-352). All RNAs hybridized with a radio-labeled cDNA probe representing RNA6, indicating that they contain common sequences. To study this homology in more detail, RNase T1 oligonucleotide fingerprinting of the RNAs was undertaken. This confirmed the presence of common sequences and showed more specifically that the intracellular viral RNAs form a nested set. The number of oligonucleotides in RNA1, however, is only one-third of the expected value. In all aspects studied the replication mechanism of EAV differs from that of other known positive-stranded RNA viruses.     

Characterization of equine arteritis virus particles and demonstration of their hemolytic activity

Equine arteritis virus (EAV), a member of the newly established family Arteriviridae, is a small, positive-stranded RNA virus. It carries two protein complexes in its envelope, gp5/M and the recently described gp2b/gp3/gp4 complex. We report here on several basic features of EAV replication in cell culture and on the protein composition of virus particles. We have also characterized gp2b, gp3, and gp4 expressed using a baculovirus system in insect cells. Finally, we provide evidence that EAV possess hemagglutinating and hemolytic activity. The hemolysis assay might be useful for determining which of the surface proteins carries the receptor-binding and membrane fusion activity of EAV.     

Physicochemical studies of equine infectious anemia virus

Summary A purification procedure for equine infectious anemia (EIA) virus has been developed by combining ultracentrifugation, DEAE cellulose column chromatography and cesium chloride equilibrium density gradient centrifugation. Recovery and purity of the virus were determined at each step of the purification procedure. Using this combined method, an amount of purified and concentrated virus was prepared from a large volume of virus material. Such specimens showed an infectivity of 10^8.25 TCID₅₀/0.5 ml, a complement fixing antigenicity of 80 density of 1.146 g/ml, and proved to be suitable for electron microscopic observation of negatively stained preparations.Most of the virus particles had a spherical shape and sized between 90 and 140 m, in diameter. A well-defined outer envelope was observed in spontaneously disrupted particles, but no organized internal component could be resolved.     

Genetic variation and phylogenetic analysis of 22 French isolates of equine arteritis virus

Summary Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001–2004) were determined by sequencing open reading frames (ORFs) 2a–7. The ORFs 2a–7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (nucleotides 11296–11813) of 22 French isolates and 216 additional EAV strains available in GenBank clustered the global isolates of EAV into two distinct groups: North American and European. The latter could be further divided into two large subgroups: European subgroup 1 (EU-1) and European subgroup 2 (EU-2). Phylogenetic analysis based on 100 EAV ORF3 sequences yielded similar results. Of the 22 French EAV isolates, the 11 isolates obtained before January 28, 2003 clustered with either the EU-1 (9 isolates) or EU-2 (2 isolates) subgroup. In contrast, by the criteria used in this study, the 11 isolates obtained after January 30, 2003 belong to the North American group, strongly suggesting that these strains were recently introduced into France. The first two authors contributed equally to this work.     

Immunodiffusion studies of purified equine infectious anemia virus

Antigenicity of purified equine infectious anemia (EIA) virus was examined by immunodiffusion against sera obtained from horses experimentally infected with EIA virus. The purified virus reacted with the infected horse serum, and virus-specific precipitating antibody was demonstrated. Furthermore, it was found that purified EIA virus reacted against the serum of horses infected with all strains of EIA virus which were antigenically different from one another. From the result, group-specific components of the virus rather than strain-specific ones were considered to be involved in the reaction. Serological reactivity was lost by adding antiserum from the infected horse to the antigen. The precipitating antibody usually appeared in the serum 1 to 2 weeks after the first febrile attack of EIA and remained for a longer period. Some characteristics of the purified antigen and specificity of the reaction for EIA are described.     

Sexual and in-contact transmission of asinine strain of equine arteritis virus among donkeys.

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.