was isolated from the semen of a persistently infected Standardbred stallion. The CA95G virus caused subclinical infection and seroconversion in susceptible.
Arch Virol (1999) 144: 817–827
Phylogenetic characterization of a highly attenuated strain of equine arteritis virus from the semen of a persistently infected Standardbred stallion∗ Brief Report J. F. Patton1 , U. B. R. Balasuriya1 , J. F. Hedges1 , T. M. Schweidler1 , P. J. Hullinger2 , and N. J. MacLachlan1 1
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California, U.S.A. 2 California Department of Food and Agriculture, Sacramento, California, U.S.A. Accepted October 3, 1998
Summary. An avirulent, novel variant of equine arteritis virus (EAV; CA95G) was isolated from the semen of a persistently infected Standardbred stallion. The CA95G virus caused subclinical infection and seroconversion in susceptible horses, and virus was isolated only once from blood and nasal secretions collected from 6 experimentally infected horses. Sequence analysis of genes encoding the known EAV structural proteins shows that this highly attenuated strain of EAV is genetically similar to virulent field strains of EAV and, in particular, to a strain of EAV that was isolated during an outbreak of equine viral arteritis in western Canada in 1986. Not only is the carrier stallion the critical natural reservoir of EAV, but genetic diversity of the virus is generated in the course of persistent infection of carrier stallions. The subtle genetic changes that facilitate and maintain persistent EAV infection of the stallion’s reproductive tract likely influence phenotypic properties of the virus such as virulence. ∗ ∗
The GenBank accession numbers of the sequences reported in this paper are, respectively: ORF2 and OFRs 5 through 7 for each virus: U81022, U81023 (CA95G); U81026, U81027, (CA95Gs); and U81024, U81025 (CA95I); ORFs 5 through 7 of the following viruses: U81013 (VBS53); U81014 (KY63); U81018 (PA76); U81015 (KY77); U81016 (KY84); U81017 (KY93); U81021 (CAN86); U81019 (ARVAC); and U81020 (ATCC).
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Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a disease of horses that is characterized by interstitial pneumonia in very young foals, generalized influenza-like illness of adult horses, abortion in pregnant mares, and persistent infection of the reproductive tract of carrier stallions [18]. EAV is a positive-stranded RNA virus that recently was taxonomically placed in the family Arteriviridae, genus Arterivirus, in the order Nidovirales, along with porcine reproductive and respiratory syndrome virus, lactate dehydrogenaseelevating virus, and simian hemorrhagic fever virus [6]. The EAV genome is a single, stranded infectious, polyadenylated RNA molecule of approximately 12.7 kb that includes at least eight open reading frames [ORFs 1a, 1b, 2, 3, 4, 5, 6 and 7]. The 50 two thirds of the genome includes two large open reading frames (ORFs 1a and 1b) that encode the viral replicase [8]. The ORFs 2 to 7 are overlapping, located at the 30 end of the genome, and encode four known structural proteins and two proteins of unknown function. The EAV virion consists of a 14 kDa phosphorylated nucleocapsid protein (N), a 30–44 kDa N-glycosylated major membrane protein (GL ), a 25 kDa N-glycosylated minor membrane protein (GS ) and a 17 kDa unglycosylated membrane protein (M). The GS , GL , M and N proteins are encoded by ORFs 2, 5, 6 and 7 respectively [8]. The asymptomatic carrier stallion is the principal reservoir of EAV as it can harbor the virus between breeding seasons and venereally infect susceptible mares. Stallions infected with EAV via the respiratory route frequently retain the virus in the reproductive tract and shed virus in their semen for up to several years [18, 19]. Significant genetic variation of EAV occurs in the course of persistent infection of the carrier stallion, whereas the virus is remarkably stable during horizontal aerosol transmission in outbreaks of EVA [1, 12, 16]. Genetic variation of ORFs encoding the four known structural EAV proteins during persistent infection is most pronounced in specific (variable) regions of ORF5 and, similarly, previous studies have demonstrated that genetic heterogeneity of field strains of EAV also is pronounced within these same regions and correlate with differences in neutralization phenotype [3, 5, 9]. ORF5 encodes the GL envelope glycoprotein, which is the likely cell attachment protein and includes the known neutralization determinants of EAV [2, 4]. Strains of EAV also vary markedly in their virulence, although the genetic basis of viral virulence is undetermined [10]. Previous studies have almost exclusively focused on the genetic characterization of virulent strains of EAV that have been isolated during epizootics of EVA [5, 7, 11]. The goal of this study, therefore, was to compare ORFs encoding the structural proteins of virulent strains of EAV with those of a naturally occurring avirulent strain of the virus that was isolated from a carrier stallion on a farm in California with a high seroprevalence of EAV infection but no history of EVA disease [13]. A strain of EAV was isolated from semen collected from a Standardbred stallion (G) that had a high serum neutralization (SN) antibody titer to EAV (≥256). The virus was designated CA95G and a working stock was made following the fourth passage in rabbit kidney cells (RK-13; American Type Culture Collection [ATCC] CCL 37). Viral RNA was extracted from virus-infected RK-13 cells for
An attenuated strain of EAV
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reverse transcription and polymerase chain reaction (RT-PCR) amplification prior to sequencing of ORFs 2 and 5 to 7 with the QIAamp Viral RNA isolation kit. Viral RNA also was isolated from the seminal plasma of the persistently infected stallion. The sequence derived directly from viral RNA in the stallion’s semen was designated CA95Gs. Both negative and positive strands of cDNA resulting from RT-PCR of each amplicon were sequenced with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS (Perkin-Elmer, Corp.) as previously described [1, 5, 11, 12]. The nucleotide sequences of ORFs 2, 5, 6 and 7 of the strain of EAV isolated in cell culture (CA95G) and its parent virus in semen (CA95Gs) were compared to those of the published sequence of EAV (EAVUtr), 9 other field isolates of EAV (VBS53, PA76, KY63, KY77, KY84, KY93, CAN86, SWZ64 and AUT68) isolated during outreaks of EVA, an isolate of EAV from the semen of another persistently infected Standardbred stallion from the same farm (CA95I), the highly virulent U.S. prototype strain of EAV obtained from the ATCC [EAVATCC(ATCC VR-796)] and the modified-live virus (MLV) vaccine (ARVAC, Fort Dodge Laboratories). The geographical location, year of isolation, horse breed of origin, and passage history of each strain of EAV used in this study have been previously described [5, 11]. The ORFs 2, 5, 6 and 7 of CA95G differed by 34, 45, 22 and 6 ( 95.1, 94.2, 95.5 and 98.2% identity) nucleotides from the highly virulent U.S. prototype virus obtained from the ATCC, and by 17 to 62 (97.5 to 90.9% identify), 11 to 102 (98.6 to 86.7%), 11 to 43 (97.8 to 91.2%), and 1 to 17 (99.7 to 94.9%) nucleotides from the same ORFs of the other field isolates and the MLV ARVAC vaccine virus. The nucleotide sequences of ORF’s 2, 5, 6 and 7 of CA95G differed from those of a virus (CA95I) isolated from the semen of another carrier stallion from the same property by 46 (93.3% identity), 61 (92%), 28 (94.8%) and 4 (98.8%) nucleotides respectively, indicating that the two stallions were infected with different viruses. As compared to the other field isolates of EAV, therefore, CA95G varied in all four ORFs encoding structural proteins and the greatest variability occurred in ORF5. The ORF7 sequence of the cell culture adapted virus (CA95G) was identical to that of the PCR products directly amplified from the parental virus in semen (CA95Gs), whereas the sequence of ORFs 2, 5 and 6 of CA95Gs differed by 5 (99.3%), 5(99.32%) and 3 (99.4%) nucleotides respectively. Only 3 of these nucleotide differences resulted in a coding change, indicating that very little change occurred in the structural proteins of CA95G during adaptation to cell culture. The ORFs encoding the EAV structural proteins were the same length in all viruses (ORF2-684, ORF5-768, ORF6-489 and ORF7-333 nucleotides), and encoded proteins of 227 (GS ), 255 (GL ), 162 (M) and 110 (N) amino acids respectively. The deduced amino acid sequence of the GS , GL and M structural proteins of CA95G differed by between 7 and 14 (96.9 and 93.8% identity), 3 and 20 (98.8 and 92.2%) and, 1 and 13 (99.4 and 92%) amino acids, respectively, from those of the other strains of EAV included in the study and by 9, 10 and 3 (96, 96.1 and 98.2% identity) amino acids from the virulent ATCC U.S. prototype strain. The amino acid sequence of the N protein of CA95G was highly conserved and
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identical to those of all but four of the other strains of EAV (AUT68, KY93, SWZ64 and the MLV ARVAC vacine). The four structural proteins of the avirulent CA95G virus differed at only nine amino acid residues from those of the virulent CAN86 strain that was isolated during an outbreak of EVA in Alberta in 1986. Nucleotide sequence comparisons and phylogenetic analyses of ORFs 2, 5, 6 and 7 from the various viruses were done as previously described [5]. Phylogenetic relationships between the nucleotide sequence of a 1500 base pair (bp; 11,129– 12,628) region encompassing ORFs 5 through 7 of the various viruses were also determined. With this analysis, CA95G and CA95Gs clustered with the North American viruses isolated after 1976, and with the CAN86 strain in particular (Fig. 1). Phylogenetic analysis of individual ORFs 2, 5, 6 and 7 gave similar trees to those obtained using the 1500 nucleotide region that encompassed ORFs 5, 6 and 7 (Fig. 2). Differences in branch lengths obtained with each ORF indicate that individual ORFs evolved at different rates. Seven adult horses (two mares and five geldings) that were seronegative to EAV by serum neutralization (SN) assay were housed in isolation facilities. Six horses were intranasally inoculated with 1 × 105 tissue culture infectious doses (TCID50 ) of CA95G. A control horse was inoculated only with uninfected cell culture media [15]. All horses were monitored twice daily for two weeks for clinical manifestations of EVA. Serum, whole blood (acid citrate dextrose anticoagulent) and nasopharyngeal swabs were collected from each inoculated horse at 0, 4, 7, 14, 21, 28 days post-infection (DPI), and serum also was collected at 2, 4, 6 and 12 months after infection. Complete blood counts (CBC) were performed on all horses at 0, 4, 7, 9, 12 and 14 DPI. Numbers of individual blood cell types were compared using the two sample T test [17]. Coagulation profiles [(prothrombin time (PT), partial thromboplastin time (PTT) and fibrin degradation products (FDP)] also were done at 0, 4, 7, 9, 12 and 14 DPI on bloods from three of the infected horses (#’s 9 649, 9 715 and 9 131). None of the inoculated horses developed clinical manifestations of EVA at any time after infection. Horse 9131 died at 28 DPI of causes unrelated to the experimental infection with CA95G. The inoculated horses had a modest but significant lymphopenia (p
