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.     

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.     

Morphological studies on equine arteritis virus

Summary Electron microscopy of purified preparations of equine arteritis virus (EAV) revealed enveloped, spherical particles with an average diameter of 55 m. The envelope was found to carry tiny projections on the surface, 3 to 5 m in length. No detailed structure of an internal component could be seen.In sections of EAV infected BHK cells 24 hours after inoculation, viral particles were shown to bud from the cytoplasmic matrix into cisternae of the endoplasmic reticulum, the process starting in the vicinity of the Golgi apparatus. The mature particles had an average diameter of 50 m, with an inner core measuring 25m.This study was supported by grant no. B70-16X-744-05-6068-19195 from the Swedish Medical Research Council.     

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.     

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).     

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)     

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).     

Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus.

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.     

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.     

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.     

Structural proteins of HADEN virus

Purified HADEN virus was disrupted and separated into three polypeptide types in 7.5% neutral sodium dodecyl sulfate-polyacrylamide gels. The major polypeptide (HVP1) had a molecular weight of about 67,000 and accounted for 75–83% of the virion protein. The two minor components (HVP2 and HVP3) had molecular weights of about 77,000 and 85,500, respectively. When HADEN virus polypeptides were compared to the polypeptides of adenovirus-associated virus similar profiles were noted, but the polypeptides of the two viruses were distinguishable when coelectrophoresed on a single gel. Purified low density noninfectious HADEN particles were found to contain three polypeptides with the same molecular weights and concentrations as those found in the infectious virions.     

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.     

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.     

Structural proteins of Marek's disease virus

Marek's disease virus (MDV) was propagated in roller-bottle cultures of duck embryo fibroblasts and partially purified by sucrose gradient centrifugation. Analysis of viral protein by polyacrylamide gel electrophoresis revealed that at least eight proteins (designated VPI-VPVIII) were present in MDV. The VPI is the major viral protein. At least two viral proteins, VPII and VPIV, with glucosamine label could be detected. These two peaks may represent viral glycoprotein and may be associated with the viral envelope. No host cell proteins coelectrophoresed with any viral proteins. Similar electropherograms were obtained by coelectrophoresis of MDV and herpes simplex virus proteins and of MDV and pseudorabies virus proteins.