1 Bernard and Gloria Salick Equine Viral Disease Laboratory, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
2 Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA
3 Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands
Correspondence
Udeni B. R. Balasuriya
ubbalasuriya{at}ucdavis.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nucleotide sequences of CW96 and CW01 are deposited in GenBank under the accession numbers AY349167 and AY349168, respectively.
Present address: Molecular Biology, Montana State University, 19th and Lincoln, Bozeman, MT 59717, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EAV is the prototype virus in the family Arteriviridae, genus Arterivirus, which also includes porcine reproductive and respiratory syndrome virus (PRRSV), simian haemorrhagic fever virus, and lactate dehydrogenase elevating virus of mice (Snijder, 2001). The family Arteriviridae is included in the order Nidovirales, together with the Coronaviridae and Roniviridae (Cowley & Walker, 2002
; de Vries et al., 1997
; Cavanagh, 1997
; Jitrapakdee et al., 2003
; Snijder, 2001
; Snijder et al., 2001
). The only available full-length EAV genome sequence is derived from a highly cell culture adapted, North American isolate (den Boon et al., 1991
; GenBank accession number X53459) and was subsequently used to engineer an EAV infectious cDNA clone (pEAV030, GenBank accession number Y07862; van Dinten et al., 1997
). In this paper this virus will be referred to as EAV030. The EAV genome is a positive-stranded RNA molecule (approx. 12·7 kb) and includes nine open reading frames (ORFs) (den Boon et al., 1991
; van Dinten et al., 1997
). The two most 5'-proximal ORFs (1a and 1b) occupy approximately three-quarters of the genome and encode two replicase proteins (1a and 1ab) that are post-translationally processed into at least 12 nonstructural proteins (nsp112; van Dinten, 1999
; Snijder & Meulenberg, 1998
; Ziebuhr et al., 2000
). The remaining seven ORFs (2a, 2b and 37) are located in the 3'-proximal quarter of the genome and encode seven structural proteins (E, GP2, GP3, GP4, GP5, M and N respectively; de Vries et al., 1992
; Snijder et al., 1999
; Snijder, 2001
; Wieringa et al., 2003
). The EAV particle consists of a nucleocapsid (N) protein that forms the icosahedral core, and six envelope proteins. The envelope contains two principal viral proteins, the GP5 major envelope glycoprotein (3044 kDa; encoded by ORF5) and the unglycosylated membrane protein M (17 kDa; encoded by ORF6). The M and GP5 proteins form a disulfide-linked heterodimer, and this interaction is critical for virus infectivity and expression of the neutralization determinants in authentic form (Balasuriya et al., 1993
, 1995a
, 1997
, 2000
, 2002
; Deregt et al., 1994
; de Vries et al., 1995
; Glaser et al., 1995
; Snijder et al., 2003
). In addition, the EAV envelope contains a heterotrimer of three minor membrane glycoproteins designated as GP2, GP3 and GP4 (25 kDa, 3642 kDa and 28 kDa; encoded by ORFs 2b, 3 and 4 respectively) and an unglycosylated envelope protein E (8 kDa; encoded by ORF2a; Snijder et al., 1999
; Wieringa et al., 2003
).
EAV causes a long-term persistent infection that is specifically localized to the ampulla of the reproductive tract of carrier stallions (Timoney & McCollum, 1993; Timoney et al., 1986
, 1987
). Infectious virus is continuously shed in the semen of carrier stallions, despite high titres of neutralizing antibodies in their serum. Persistently infected stallions act as a natural reservoir of EAV and transmit the virus to susceptible mares during artificial or natural breeding (McCollum et al., 1999
; Balasuriya et al., 1998
, 1999
). Persistently infected carrier stallions can cease to shed virus in their semen weeks to years after infection with no apparent later reversion to a shedding state; however, the mechanism responsible for this spontaneous clearance of EAV from persistently infected stallions is undefined (Timoney & McCollum, 1993
). EAV evolves in the course of persistent infection at a rate of approximately 1 % nucleotide substitutions per 2822 nt (ORFs 27) per year, leading to emergence of novel genotypic and phenotypic variants (Hedges et al., 1999b
). ORF5 varies markedly between strains of EAV, and large numbers of point mutations and occasional deletions were previously identified in ORF5 amplified from the semen of persistently infected stallions (Balasuriya et al., 1999
; Glaser et al., 1995
; Hedges et al., 1999b
). The objectives of this study were to characterize the evolution of the entire virus genome during persistent EAV infection, and the possible emergence of defective interfering (DI) virus particles. Although DI particles have been implicated in the pathogenesis of persistent infections caused by a number of RNA viruses (Roux et al., 1991
), their true significance in maintaining or terminating persistent virus infections of animals is still unclear (Drolet et al., 1995
; Huang & Baltimore, 1970
, 1977
; Tautz et al., 1994
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stallions and viruses.
Archived semen samples previously collected from EAV carrier stallions were evaluated (Table 1), including a Holsteiner stallion (CW) imported to the United States from Europe in 1995, as well as two Standardbred (FLS and KS), a Westfalen (RS), a pony (P) and five Thoroughbred stallions (D, E, RC, SS and WHB) that all were resident in North America. Semen samples were diluted (10-1, 10-2 and 10-3) and inoculated onto RK-13 cell monolayers for virus isolation, as previously described (Balasuriya et al., 1998
). EAV isolated from these semen samples was identified by microneutralization assay using monoclonal antibodies (mAbs) and polyclonal equine sera to the virus. Viruses contained in individual semen samples are identified by stallion and year of isolation (e.g. CW96).
|
The prototype Bucyrus strain [ATCC (ATCC VR-796)] of EAV was used as a control virus in the neutralization assay, and for extraction of RNA for the development of Northern hybridization probes. The DI virus derived from the pED1 clone (Molenkamp et al., 2000) was also used as a control in the Northern hybridization assay.
Microneutralization assay.
The EAV microneutralization assay was performed as previously described (Balasuriya et al., 1995a, 1997
). Neutralization titres were determined for each virus isolated from the semen of individual carrier stallions using a panel of EAV-specific mAbs and polyclonal equine antisera.
Isolation of viral RNA, RT-PCR amplification and sequencing.
Viral RNA was directly isolated from infected BHK-21 cell cultures or from the semen of persistently infected stallions using the QIAmp Viral RNA isolation kit (Qiagen). ORF 5 with flanking portions of ORFs 4 and 6 from each strain of EAV was amplified by RT-PCR. The entire genome of the CW96 and CW01 strains of EAV contained in the semen of stallion CW (in 1996 and 2001 respectively) were amplified in nine overlapping segments. RT-PCR amplifications were performed with Superscript II (Invitrogen Life Science Products) and Pfu Turbo DNA polymerase (Stratagene) as previously described (Balasuriya et al., 1999). The 5' and 3' ends of CW96 and CW01 were amplified with the SMART Race cDNA amplification kit (Clontech) according to the manufacturer's protocol. The PCR products were gel-purified and both sense and anti-sense strands were sequenced with the PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems) as previously described.
Sequence and phylogenetic analysis.
Sequence data were analysed with the Sequencher 3.0 (Gene Codes), HIBIO MacDNASIS pro version 3.5 (Hitachi) and Vector NTI Suite V.6 (InforMax) software programs. Phylogenetic analyses were performed as previously described (Balasuriya et al., 1995b, 1999
), using the Phylogenetic Inference Package (PHYLIP; Felsenstein, 1993
). Estimates of the number of synonymous and nonsynonymous nucleotide changes and the number of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS) were estimated using the MEGA 2.0 program (Kumar et al., 2001
). The dN/dS ratios were calculated for each nonstructrural and structural protein gene by the LiWuLuo method (Li et al., 1985
; Yang & Nielsen, 2000
).
Analysis of viral RNA by Northern hybridization assay.
The possible emergence of DI viruses in the semen of EAV carrier stallions was evaluated by Northern hybridization assay. Two anti-sense RNA probes (nt 1300 and 212550) were developed to the leader sequence and the 5' end of the ORF1a of the ATCC strain of EAV for use in the Northern hybridization assay. The first probe reacts with the common leader sequence (nt 1224) that is present in all seven viral mRNAs and also in the published EAV DI RNAs (Molenkamp et al., 2000). The second probe is expected to react only with genomic RNA, and with DI RNAs which are likely to have retained this region (nt 212550) of the genome because it contains important replication signals (Tijms et al., 2001
). Viral RNA was directly isolated from tissue culture fluid containing the EAV ATCC virus and the leader sequence was amplified with two primer pairs (Table 2
) using the QIAgen OneStep RT-PCR kit. The gel-purified RT-PCR products were cloned into a linearized plasmid vector with overhanging 3' T residues (TOPO TA cloning kit, Invitrogen Life Science Products). The plasmids were purified using a commercial kit (QIAmp Miniprep kit) and the authenticity and orientation of the leader sequence were determined by sequencing of both strands of DNA with M13 reverse and forward primers. Plasmid DNA (1 µg) was linearized with BamHI, phenol/chloroform extracted and resuspended in nuclease-free water. Digoxigenin (DIG)-labelled RNA probes were made using the DIG RNA labelling kit according to the manufacturer's instructions (Roche). The plasmid DNA was removed by digestion with RNase-free DNase I (Pharmacia) for 15 min at 37 °C.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Comparative nucleotide and amino acid sequence analysis of viruses contained in the semen of stallion CW
To estimate the genetic variation of EAV during persistent infection, the complete nucleodide sequence of the virus contained in the semen of stallion CW in 2001 (CW01) was compared to the sequence of the 1996 virus from the same stallion (CW96). There were 178 nucleotide differences between the CW96 and CW01 consensus sequences (1·4 % difference). The replicase gene had only 1·2 % nucleotide variation between 1996 and 2001, and eight of the nonstructural proteins did not show any amino acid changes (nsp3, nsp510 and nsp12). Two of the four viral proteinases (nsp1 and nsp4) in CW01 had single amino acid substitutions as compared to CW96, whereas the other two proteinases (nsp2 and nsp11) respectively had three and two substitutions. The E, GP2b and N proteins were highly conserved during persistent infection, whereas the GP3, GP4 and GP5 proteins were more variable, although the GP4 protein was more conserved than either the GP3 or GP5 proteins.
To compare the selective pressure on individual EAV proteins the ratios of the number of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS) were estimated. The fixation of advantageous nonsynonymous substitutions will increase the dN/dS ratio (>1·0). In contrast, the presence of a large number of synonymous substitutions will decrease the dN/dS ratio (<1·0) and lead to negative (purifying) selection indicative of a reduced rate of evolution. In this study the dN/dS ratios estimated by pair-wise comparison of all nonstructural and structural coding regions of these three viruses were consistently less than 1·0 (Table 3). Therefore, these data suggest that positive selection did not significantly contribute to the nucleotide diversity of these viruses or to the establishment of persistent infection in stallion CW.
Emergence of novel phenotypic variants during persistent EAV infection of stallion CW
The GP5 protein, encoded by ORF5, contains the known neutralization determinants of EAV (Balasuriya et al., 1993, 1995a
, 1997
). In our previous studies we have shown that substitution of individual amino acids within the GP5 ectodomain occurs during persistent infection of stallions, leading to the emergence of viruses of different neutralization phenotype (Balasuriya et al., 1999
, 2001
; Hedges et al., 1999b
). Neutralization assays with a large panel of well-characterized mAbs and polyclonal equine sera confirmed that the nucleotide variation in ORF5 of the strains of EAV that were isolated from the semen of stallion CW (CW96 and CW01 respectively) correlated with phenotypic differences between these viruses (Table 4
). For example, mAb 6A2 did not neutralize the CW01 virus but neutralized the CW96 virus to a high titre (128). Similarly, some of the EAV strain-specific polyclonal equine sera neutralized the CW96 virus to a significantly higher titre (512) than the CW01 virus (Table 4
). Interestingly, the GP5 protein of the CW01 virus had three amino acid substitutions in neutralization site B (aa 61; Val
Phe) and C (aa 6790; 71 Glu
Asp and 84 Phe
Ile) as compared to the CW96 virus. These data further confirm that novel phenotypic variants of EAV emerge during persistent infection of stallions.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies have demonstrated that phylogenetic analysis based on ORF5 sequences can be used to trace the origin of EAV strains (Balasuriya et al., 1995b, 1998
, 1999
; Larsen et al., 2001
; Stadejek et al., 1999
). The phylogenetic analyses of virus strains present in the semen of stallion CW confirmed that this stallion was infected with a European strain of EAV, which is consistent with the fact that stallion CW was imported to North America from Europe as a mature stallion. Full-length nucleotide sequences of viruses contained in the semen of stallion CW were only 85·6 % (CW96) and 85·7 % (CW01) identical to the published sequence of the prototype laboratory strain of EAV (EAV030), which is derived from the original isolate of EAV that was made from an aborted fetus in Bucyrus, Ohio (Doll et al., 1957
). We previously have shown that European strains of EAV are
85 % identical to North American viruses based on their ORF5 sequences (Balasuriya et al., 1995b
). Thus, data from this present study further confirm that North American and European strains of EAV are genetically distinct, but that they also are more closely related to each other than the two porcine arterivirus genotypes isolated from these two continents. In contrast to EAV, there is only 5570 % nucleotide identity between the European and North American genotypes of PRRSV (Nelsen et al., 1999
; Shen et al., 2000
).
Genetic variation among the viruses contained in the semen of stallion CW and the prototype EAV030 virus was especially marked in the ORF1a gene, particularly the region that encodes the nsp2 replicase subunit. An amino acid insertion and several highly variable regions were identified at aa 388480 of the ORF1a protein. Interestingly, this region overlaps with the region in which a host cell-specific, internal cleavage site of nsp2 was previously observed (Snijder et al., 2001), although its biological significance remains to be determined. This region of the nsp2 protein varies markedly in both size and sequence in other arteriviruses (Nelsen et al., 1999
; Shen et al., 2000
). For example, a 36 aa insertion was previously described in the nsp2 protein of a vaccine strain of PRRSV (Shen et al., 2000
).
The sequence data obtained in this study clearly demonstrate that each of the nine ORFs of EAV may evolve at a different rate during persistent infection and that the greatest variation amongst ORFs encoding structural viral proteins occurs in ORFs 3 and 5 (which respectively encode the GP3 and GP5 envelope proteins of the virus; Table 3). The GP4 protein encoded by ORF4 was more conserved than GP3, which is consistent with our previous sequence analysis of ORFs 3 and 4 of EAV isolates from persistently infected stallions and from outbreaks of EVA (Hedges et al., 1999a
, 2001
; J. F. Hedges & U. B. R. Balasuriya, unpublished results). In a previous study we also demonstrated that loss or acquisition of glycosylation sites may occur in different EAV structural proteins during persistent infection of stallions (Balasuriya et al., 1999
), and that amino acid changes in the GP5 protein alter the neutralization phenotype of EAV (Hedges et al., 1999b
; Balasuriya et al., 1997
, 2001
). The neutralization phenotype data obtained in this study with different viruses further confirm that novel phenotypic variants emerge during persistent EAV infection of carrier stallions.
Although small (340 nt) deletions have occasionally been detected by the sequencing of cloned and uncloned PCR products containing ORF5 from different EAV stains (Balasuriya et al., 1995a, 1997
, 1999
; Glaser et al., 1995
; Hedges et al., 1999
), there are no reports of naturally occurring EAV DI particles. Molenkamp et al. (2000)
documented the first arterivirus DI RNA (5·6 kb) by serial passage of a laboratory strain of EAV in BHK-21 cells. In this present study, nucleotide deletions were not detected after direct RT-PCR amplification and sequencing of ORF5 of strains of EAV contained in the semen of 10 persistently infected carrier stallions. Furthermore, Northern hybridization assay of intracellular RNA from BHK-21 cells infected with these EAV strains did not show any evidence of DI RNA even after serial cell culture passage at high m.o.i. These data suggest that viruses with substantial genome deletions are uncommon during persistent infection of the stallion, and that DI viruses are unlikely to be involved in either the maintenance or clearance of persistent EAV infection of the reproductive tract of carrier stallions.
In summary, the data from the present study confirm that the genomic sequence of North American and European strains of EAV can differ by approximately 15 %. The data further confirm that EAV evolves in the course of persistent infection of carrier stallions and that variation is especially marked in the nsp2, GP3 and GP5 proteins. With the notable exception of GP5, however, the biological significance of this variation in individual EAV proteins is undetermined.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balasuriya, U. B. R., MacLachlan, N. J., de Vries, A. A. F., Rossitto, P. V. & Rottier, P. J. M. (1995a). Identification of a neutralization site in the major envelope glycoprotein (GL) of equine arteritis virus. Virology 207, 518527.[CrossRef][Medline]
Balasuriya, U. B. R., Timoney, P. J., McCollum, W. H. & MacLachlan, N. J. (1995b). Phylogenetic analysis of open reading frame 5 of field isolates of equine arteritis virus and identification of conserved and nonconserved regions in the GL envelope glycoprotein. Virology 214, 690697.[CrossRef][Medline]
Balasuriya, U. B. R., Patton, J. F., Rossitto, P. V., Timoney, P. J., McCollum, W. H. & MacLachlan, N. J. (1997). Neutralization determinants of laboratory strains and field isolates of equine arteritis virus: identification of four neutralization sites in the amino-terminal ectodomain of the GL envelope glycoprotein. Virology 232, 114128.[CrossRef][Medline]
Balasuriya, U. B. R., Evermann, J. F., Hedges, J. F., McKeirnan, A. J., Mitten, J. Q., Beyer, J. C., McCollum, W. H., Timoney, P. J. & MacLachlan, N. J. (1998). Serologic and molecular characterization of an abortigenic strain of equine arteritis virus derived from infective frozen semen and an aborted equine fetus. J Am Vet Med Assoc 213, 15861589.[Medline]
Balasuriya, U. B. R., Hedges, J. F., Timoney, P. J., McCollum, W. H. & MacLachlan, N. J. (1999). Genetic stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis. J Gen Virol 80, 19491958.
Balasuriya, U. B. R., Heidner, H. W., Hedges, J. F., Williams, J. C., Davis, N. L., Johnston, R. E. & MacLachlan, N. J. (2000). Expression of two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles. J Virol 74, 1062310630.
Balasuriya, U. B. R., Hedges, J. F. & MacLachlan, N. J. (2001). Molecular epidemiology and evolution of equine arteritis virus. Adv Exp Med Biol 494, 1924.[Medline]
Balasuriya, U. B. R., Heidner, H. W., Davis, N. L. & 7 other authors (2002). Alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses. Vaccine 20, 16091627.[CrossRef][Medline]
Cavanagh, D. (1997). Nidovirales: a new order comprising coronaviridae and arteriviridae. Arch Virol 142, 629633.[Medline]
Cowley, J. A. & Walker, P. J. (2002). The complete sequence of gill-associated virus of Penacus monodon prawns indicates a gene organization unique amongst nidoviruses. Arch Virol 147, 19771987.[CrossRef][Medline]
den Boon, J. A., Snijder, E. J., Chirnside, E. D., de Vries, A. A. F., Horzinek, M. C. & Spaan, W. J. M. (1991). Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily. J Virol 65, 29102920.[Medline]
Deregt, D., de Vries, A. A. F., Raamsman, M. J. B., Elmgren, L. D. & Rottier, P. J. M. (1994). Monoclonal antibodies to equine arteritis virus proteins identify the GL protein as a target for virus neutralization. J Gen Virol 75, 24392444.[Abstract]
de Vries, A. A. F., Chirnside, E. D., Horzinek, M. C. & Rottier, P. J. M. (1992). Structural proteins of equine arteritis virus. J Virol 66, 62946303.[Abstract]
de Vries, A. A. F., Post, S. M., Raamsman, M. J. B., Horzinek, M. C. & Rottier, P. J. M. (1995). The two major envelope proteins of equine arteritis virus associate into disulfide-linked heterodimers. J Virol 69, 46684674.[Abstract]
de Vries, A. A. F., Horzinek, M. C., Rottier, P. J. M. & de Groot, R. J. (1997). The genome organization of the nidovirales: similarities and differences between arteri-, toro- and coronaviruses. Semin Virol 8, 3347.[CrossRef]
Doll, E. R., Bryans, J. T., McCollum, W. H. & Crowe, M. E. W. (1957). Isolation of a filterable agent causing arteritis of horses and abortion by mares. Its differentiation from the equine abortion (influenza) virus. Cornell Vet 47, 341.
Drolet, B. S., Chiou, P. P., Heidel, J. & Leong, J. C. (1995). Detection of truncated virus particles in a persistent RNA virus infection in vivo. J Virol 69, 21402147.[Abstract]
Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) 3.5c Manual. Department of Genetics SK-50, University of Washington, Seattle, WA.
Frank, S. A. (2000). Within-host spatial dynamics of viruses and defective interfering particles. J Theor Biol 206, 279290.[CrossRef][Medline]
Glaser, A. L., de Vries, A. A. F. & Dubovi, E. J. (1995). Comparison of equine arteritis virus isolates using neutralizing monoclonal antibodies and identification of sequence changes in GL associated with neutralization resistance. J Gen Virol 76, 22232233.[Abstract]
Glaser, A. L., Rottier, P. J. M., Horzinek, M. C. & Colenbrander, B. (1996). Equine arteritis virus: a review of clinical features and management aspects. Vet Q 18, 9599.[Medline]
Glaser, A. L., Chirnside, E. D., Horzinek, M. C. & de Vries, A. A. F. (1997). Equine arteritis virus. Theriogenology 47, 12751295.[CrossRef]
Hedges, J. F., Balasuriya, U. B. R., Timoney, P. J., McCollum, W. H. & MacLachlan, N. J. (1999). Genetic divergence with emergence of phenotypic variants of equine arteritis virus during persistent infection of stallions. J Virol 73, 36723681.
Hedges, J. F., Balasuriya, U. B. R., Topol, J. & MacLachlan, N. J. (2001). Genetic variation of ORFs 3 and 4 of equine arteritis virus. Adv Exp Med Biol 494, 6972.[Medline]
Huang, A. S. & Baltimore, D. (1970). Defective viral particles and viral disease processes. Science 226, 325327.
Huang, A. S. & Baltimore, D. (1977). Defective interfering animal viruses. In Comprehensive Virology, pp. 73116. Edited by H. Fraenkel-Conrat & R. R. Wagner. New York: Plenum.
Jitrapakdee, S., Unajak, S., Sittidilokranta, N., Hodgson, R. A. J., Cowley, J. A., Walker, P. J., Panyim, S. & Boonsaeng, V. (2003). Identification and analysis of gp116 and gp64 structural glycoproteins of yellow head nidovirus of Penaeus monodon shrimp. J Gen Virol 84, 863873.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2.0: Molecular Evolutionary Genetics Analysis software. Tempe, AZ: Arizona State University.
Larsen, L. E., Storgaard, T. & Holmback, K. (2001). Phylogenetic characterization of the GL sequence of equine arteritis virus isolated from semen of asymptomatic stallions and fatal cases of equine viral arteritis. Vet Microbiol 80, 339346.[CrossRef][Medline]
Li, W.-H., Wu, C.-I. & Luo, C.-C. (1985). A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Mol Biol Evol 2, 150174.[Abstract]
McCollum, W. H. & Timoney, P. J. (1999). Experimental observation on the virulence of isolates of equine arteritis virus. In Equine Infectious Diseases VIII, pp. 558559. Edited by U. Wernery, J. F. Wade, J. A. Mumford & O.-R. Kaaden. Newmarket, UK: R & W Publications.
McCollum, W. H., Timoney, P. J., Lee, J. W., Jr, Habacker, P. L., Balasuriya, U. B. R. & MacLachlan, N. J. (1999). Features of an outbreak of equine viral arteritis on a breeding farm associated with abortion and fatal interstitial pneumonia in neonatal foals. In Equine Infectious Diseases VIII, pp. 559560. Edited by U. Wernery, J. F. Wade, J. A. Mumford & O.-R. Kaaden. Newmarket, UK: R & W Publications.
Molenkamp, R., Rozier, B. C. D., Greve, S., Spaan, W. J. M. & Snijder, E. J. (2000). Isolation and characterization of an arterivirus defective interfering RNA genome. J Virol 74, 31563165.
Nelsen, C. J., Murtaugh, M. P. & Faaberg, K. S. (1999). Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents. J Virol 73, 270280.
Patton, J. F., Balasuriya, U. B. R., Hedges, J. F., Schweidler, T. M., Hullinger, P. J. & MacLachlan, N. J. (1999). Phylogenetic characterization of a highly attenuated strain of equine arteritis virus from the semen of a persistently infected standardbred stallion. Arch Virol 144, 817827.[CrossRef][Medline]
Roux, L., Simon, A. E. & Holland, J. J. (1991). Effects of defective interfering viruses on virus replication and pathogenesis in vitro and in vivo. Adv Virus Res 40, 181211.[Medline]
Shen, S., Kwang, J., Liu, W. & Liu, D. X. (2000). Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion. Arch Virol 145, 871883.[CrossRef][Medline]
Snijder, E. J. (2001). Arteriviruses. In Fields Virology, pp. 12051220. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Snijder, E. J. & Meulenberg, J. J. M. (1998). The molecular biology of arteriviruses. J Gen Virol 79, 961979.
Snijder, E. J., van Tol, H., Pedersen, K. W., Raamsman, M. J. B. & de Vries, A. A. F. (1999). Identification of a novel structural protein of arteriviruses. J Virol 73, 63356345.
Snijder, E. J., van Tol, H., Roos, N. & Pedersen, K. W. (2001). Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex. J Gen Virol 82, 985994.
Snijder, E. J., Dobbe, J. C. & Spaan, W. J. M. (2003). Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity. J Virol 77, 97104.
Stadejek, T., Bjorklund, H., Bascunana, C. R. & 9 other authors (1999). Genetic diversity of equine arteritis virus. J Gen Virol 80, 691699.[Abstract]
Tautz, N., Theil, H.-J., Dubovi, E. J. & Meyers, G. (1994). Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion. J Virol 68, 32893297.[Abstract]
Tijms, M. A., van Dinten, L. C., Gorbalenya, A. E. & Snijder, E. J. (2001). A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in positive-stranded RNA virus. Proc Natl Acad Sci U S A 98, 18891894.
Timoney, P. J. (1999). Equids and equine semen: international trade vs. disease control. In Equine Infectious Diseases VIII, pp. 328331. Edited by U. Wernery, J. F. Wade, J. A. Mumford & O.-R. Kaaden. Newmarket: R & W Publications.
Timoney, P. J. & McCollum, W. H. (1993). Equine viral arteritis. Vet Clin North Am Equine Pract 9, 295309.[Medline]
Timoney, P. J., McCollum, W. H., Roberts, A. W. & Murphy, T. W. (1986). Demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion. Res Vet Sci 41, 279280.[Medline]
Timoney, P. J., McCollum, W. H., Murphy, T. W., Roberts, A. W., Willard, J. G. & Carswell, G. D. (1987). The carrier state in equine arteritis virus infection in the stallion with specific emphasis on the venereal mode of virus transmission. J Reprod Fertil Suppl 35, 95102.[Medline]
van Dinten, L. C. (1999). Equine arteritis virus replicative proteins. PhD dissertation, University of Leiden, The Netherlands, pp. 1113.
van Dinten, L. C., den Boon, J. A., Wassenaar, A. L. M., Spaan, W. J. M. & Snijder, E. J. (1997). An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc Natl Acad Sci U S A 94, 991996.
Wieringa, R., de Vries, A. A. F., Raamsman, M. J. & Rottier, P. J. (2003). Characterization of two new structural glycoproteins, GP(3) and GP(4), of equine arteritis virus. J Virol 76, 1082910840.
Yang, Z. & Nielsen, R. (2000). Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models. Mol Biol Evol 17, 3243.
Ziebuhr, J., Snijder, E. J. & Gorbalenya, A. E. (2000). Virus-encoded proteinases and proteolytic processing in the nidoviruses. J Gen Virol 81, 853879.
Received 4 August 2003;
accepted 21 October 2003.