Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine1, and Department of Nematology, College of Agriculture and Environmental Sciences2, University of California, Davis, CA 95616, USA
Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, USA3
Author for correspondence: James MacLachlan.Fax +1 530 754 8124. e-mail njmaclachlan{at}ucdavis.edu
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Abstract |
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Introduction |
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The EAV genome is a linear 12·7 kb, positive-sense, polyadenylated, single-stranded RNA molecule that contains eight ORFs (den Boon et al., 1991 ; de Vries et al., 1997
). ORFs 1a and 1b encode the viral replicase and are located at the 5' end of the genome. Four ORFs (2, 5, 6 and 7) that encode the known structural proteins of EAV, and two ORFs (3 and 4) that encode two glycoproteins of unknown function, are located downstream of ORF1b at the 3' end of the genome (de Vries et al., 1992
). ORFs 5 and 6, respectively, encode the major envelope glycoprotein GL (3042 kDa) and the non-glycosylated envelope protein M (17 kDa). ORF2 encodes the minor envelope glycoprotein GS (25 kDa) and ORF7 encodes the phosphorylated N protein (14 kDa) that forms the icosahedral nucleocapsid core that encapsidates genomic RNA.
The circumstances and clinical features of an extensive outbreak of EVA that occurred on a Warmblood breeding farm in Pennsylvania, USA, during the spring of 1996 have been recently reported (McCollum et al., 1999 ). This outbreak provided an unparalleled opportunity to investigate the evolution of EAV during an outbreak of EVA and during persistent infection of stallions that became carriers of EAV following the outbreak. The objectives of the present study were to confirm the source of the outbreak and to determine the degree of virus genetic heterogeneity that was generated during the outbreak.
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Methods |
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Results |
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The virus in the lung of the first foal that died during the outbreak (BT lung) differed by 14 nt (99·5% identity) from the virus in the semen sample (A2) that was collected from stallion A 4 days prior to the outbreak (Fig. 2). With the exception of one virus (RQ), the other viruses in foal tissues collected during the outbreak, in serum from an acutely infected mare (BN) and from semen of an acutely infected stallion (R1) were identical. Virus present in the placenta of foal RQ differed from the other outbreak viruses by only one nucleotide (99·9%). Thus, there were no obvious sequence differences between viruses present in the tissues of the aborted foal, the foals that developed pneumonia or systemic illness, and in the placentas of those foals that exhibited no clinical evidence of EAV infection. The viral sequences from the semen of the two stallions (P2 and R2) that became persistently infected after the outbreak differed by 44 nt (98·4% identity) after 12 months of persistent infection, indicating that each virus evolved independently during the course of persistent infection (Fig. 1b
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Analysis of clone sequences from the outbreak and three carrier stallions
ORF5 sequences of clones derived from the RTPCR products directly amplified from the semen of carrier stallions A, P and R (samples A2, P2 and R2), and tissues from three foals (BT, HV and RC) were compared to further characterize the genetic variation of EAV during horizontal and vertical transmission (Fig. 3). A total of 78 clones that included the entire ORF5 was analysed, including 1420 clones from each semen sample and 28 clones from the various foal tissues. With the exception of one clone from stallion A (semen sample A2), sequences of the clones from the semen of the three stallions and the foal tissues segregated into two main clades (Fig. 3
). All clones from the semen of stallion R and the foal tissues grouped with a single clone from the semen of stallion A (A07) to form one clade, indicating that transfer of a viral variant in the semen of stallion A probably initiated the outbreak. Within this clade, clones from the semen of stallion R formed a monophyletic group that was reliably supported by bootstrapping. The majority of the clones from stallion A (14/16) and all of the clones from stallion P (P2) formed the second clade. All clones (14/14) from semen sample P2 and two clones from A2 (A02 and A23) formed a reliably supported monophyletic group within this clade, suggesting that stallion P might have been originally infected with a variant present in the A2 semen that was different from the variant that infected all other animals in the outbreak.
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Comparative amino acid sequence analysis of GS, GP3, GP4, GL, M and N proteins
The nucleotide sequences of each ORF (27) of the various viruses present during and following the outbreak were translated into amino acid sequences (Fig. 4). The individual viral proteins of the outbreak viruses were compared to those of the virus present in the semen of stallion A at the time of the outbreak (A2). Most of the viruses had one or two amino acid changes in four proteins (GL, GS, M and GP4) compared to A2, whereas the N and GP3 proteins were conserved during the outbreak. The GP4 and M proteins of the virus present in the placenta of foal RQ had unique changes at amino acid positions 37 and 114, respectively. There was a single amino acid change (K to N) at position 61 of the GL protein of all the outbreak viruses and two of the semen viruses (R1 and R2). In contrast, the amino acid at position 61 of the P1 and P2 viruses was maintained in its original status (K). This site has previously been identified as being important for virus neutralization (Balasuriya et al., 1997
).
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Discussion |
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Previous sequence analyses indicated that EAV behaves as a quasispecies and that genetic and phenotypic divergence can occur during persistent EAV infection of carrier stallions (Hedges et al., 1999 ). Major EVA outbreaks are the result of periodic emergence of novel genetic and phenotypic variants of the virus. However, the mechanisms involved in selection and emergence of virulent viral variants remain unclear. Selection of such variants might occur either during initial virus transmission or at some time thereafter. Data from this study indicate that selective amplification and transmission of a viral variant present in the semen of carrier stallion A was the source of this outbreak of EVA. This viral variant apparently was spread directly by contact to infect another stallion on the property (McCollum et al., 1999
), after which it was efficiently transmitted horizontally by aerosol to other horses, perhaps because of an enhanced ability to replicate in susceptible cells within the respiratory tract. In contrast, many viral variants present in semen may be better adapted to persist in the reproductive tract of the stallion and replicate only poorly in the respiratory tract (Patton et al., 1999
). Variants with the capacity to replicate efficiently in the respiratory tract would be expected to dominate during horizontal aerosol transmission, and so emerge as the dominant strain during an outbreak of EVA. Another plausible explanation for the genetic homogeneity of EAV during the outbreak would be that the contact stallion was infected with a single variant by chance, and that this variant was amplified and subsequently circulated as the dominant strain during the outbreak.
It has been previously reported that selection of variants also occurs following experimental lactate dehydrogenase-elevating virus (LDV) infection of mice and during natural infection of monkeys with simian haemorrhagic fever virus (SHFV; Plagemann, 1996 ). LDV and SHFV are also arteriviruses (Plagemann, 1996
). During epizootics of SHF in macaque monkeys, viral variants are selected that are virulent for both macaques as well as patas monkeys. In contrast, variants with lower virulence and immunogenicity are selected during persistent SHFV infection of patas monkeys. Our findings indicate that EVA outbreaks can be associated with selection of virulent variants from the EAV quasispecies generated during persistent infection of the carrier stallion.
Examination of multiple clones of viruses present in foal tissues and the semen of carrier stallions clearly indicates that the genetic heterogeneity of EAV during persistent infection in the stallion is considerably greater than that generated during this outbreak of EVA (Hedges et al., 1999 ). The presence of deletion mutant viruses in the semen of stallion P, and the rapid increase in micro-heterogeneity of viruses within the quasispecies, may have led to a highly unstable, unfit virus population (loss of population equilibrium) in the reproductive tract of this stallion. Generation of defective interfering particles could reflect either a mechanism responsible for virus persistence in the carrier stallion or more likely, a loss of virus fitness that contributed to the clearance of the virus from the reproductive tract of stallion P after one year of persistent infection (Domingo et al., 1998
).
The variable glycosylation of the GS, GP3, GP4 and GL proteins observed in this study suggests that loss or acquisition of glycosylation sites in different EAV proteins might facilitate transmission and persistence of a virus that has a relatively limited capacity to generate genetic diversity. The loss of two N-glycosylation sites in the VP-3 envelope glycoprotein of LDV-C and LDV-v is associated with increased neurovirulence, whereas LDV-P has all three N-glycosylation sites in the VP-3 protein and replicates in macrophages during persistent infection without producing neurological signs (Anderson et al., 1995 ; Faaberg et al., 1995
; Plagemann, 1996
). Variable glycosylation of individual structural proteins, therefore, can influence the pathogenesis and virulence of arterivirus infections.
In summary, the nucleotide and phylogenetic analyses included in this study confirm the epidemiological data that virus present in the semen of imported carrier stallion A initiated the outbreak of EVA on this farm in Pennsylvania. The data indicate that a variant(s) of EAV that was present in the semen of stallion A was selected and efficiently transmitted horizontally by aerosol. Sequence data obtained from multiple clones derived from foal tissues and the semen of persistently infected stallions are consistent with the hypothesis that the carrier stallion can be a source of genetic and phenotypic diversity of EAV (Hedges et al., 1999 ), and that specific viral variants present in semen can be efficiently transmitted by aerosol to initiate outbreaks of EVA.
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Acknowledgments |
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Footnotes |
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References |
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Received 22 January 1999;
accepted 9 April 1999.