Equine Arteritis Virus Derived from an Infectious cDNA Clone Is Attenuated and Genetically Stable in Infected Stallions

Virology 260, 201–208 (1999) Article ID viro.1999.9817, available online at http://www.idealibrary.com on

Equine Arteritis Virus Derived from an Infectious cDNA Clone Is Attenuated and Genetically Stable in Infected Stallions Udeni B. R. Balasuriya,* Eric J. Snijder,† Leonie C. van Dinten,† Hans W. Heidner,‡ W. David Wilson,§ Jodi F. Hedges,* Pamela J. Hullinger, ¶ and N. James MacLachlan* ,1 *Department of Pathology, Microbiology and Immunology and §Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, California 95616; †Department of Virology, Institute of Medical Microbiology, Leiden University, AZL L4-Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands; ‡Division of Life Sciences, University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, Texas 78249; and ¶California Department of Food and Agriculture, 1220 N Street, Sacramento, California 95814 Received April 21, 1999; returned to author for final revision May 6, 1999; accepted May 20, 1999 Virus derived from an infectious cDNA clone of equine arteritis virus (EAV030H) was intranasally inoculated into two stallions, neither of which subsequently developed clinical manifestations of equine viral arteritis (EVA). Virus was isolated from nasal swabs and mononuclear cells collected from both stallions #14 days p.i. and from the semen of one stallion only at 7 days p.i. Similarly, viral RNA was detected by RT nested-PCR in nasal swabs and mononuclear cells for #14 days p.i. and at 7 days p.i. in the semen of the one stallion. Both stallions seroconverted to EAV by 10 days p.i. and maintained high neutralizing antibody titers thereafter. Sequence and restriction digestion analysis demonstrated that the recombinant virus present in nasal swabs, mononuclear cells, and semen from the two stallions was identical to the infectious clone-derived virus that was used to inoculate them. Furthermore analysis of multiple clones derived by RT nested-PCR amplification from several samples indicated that the recombinant EAV030H virus was stable during replication in horses. These studies document for the first time that a recombinant virus derived from an infectious cDNA clone of a member of the order Nidovirales is replication competent in animals, and the genetic stability of the recombinant virus during in vivo replication indicates that it will be useful for the characterization of genetic determinants of virulence and persistence of EAV. The genetic conservation of the cloned recombinant virus during in vivo infection is similar to that which occurs during natural horizontal and vertical transmission of EAV in horses and contrasts with the heterogeneous virus population (quasispecies) that occurs in the semen of carrier stallions. © 1999 Academic Press

the carrier stallion as a quasispecies of closely related genomes (Hedges et al., 1999). We have recently demonstrated that specific viral variants present in the semen of carrier stallions can initiate outbreaks of EVA during which the virus spreads by aerosol (Balasuriya et al., 1999). In contrast to the heterogeneic viral population present in the semen of persistently infected stallions, viruses that circulated during an outbreak of EVA were relatively homogeneous (Balasuriya et al., 1999). EAV is the prototype virus in the family Arteriviridae (genus Arterivirus, order Nidovirales), which also includes lactate dehydrogenase elevating virus of mice, simian hemorrhagic fever virus, and porcine reproductive and respiratory syndrome virus (Cavanagh, 1997; Snijder and Meulenberg, 1998). EAV is an enveloped positive-stranded RNA virus. The EAV genome is ;12,700 nucleotides in length with a 39 polyadenylated tail and includes nine open reading frames [ORFs; 1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7 (den Boon et al., 1991; Snijder et al., 1999)]. The ORFs 1a and 1b encode the viral replicase (den Boon et al., 1991; Snijder and Meulenberg, 1998). ORFs 2b, 5, and 6, respectively, encode the 25-kDa minor envelope glycoprotein (G S), the 30- to 42-kDa major envelope glycoprotein (G L), and the 17-kDa unglycosylated

INTRODUCTION Equine arteritis virus (EAV), the causative agent of equine viral arteritis (EVA) in horses, is distributed throughout the world, although the prevalence of infection varies between countries and horse breeds (McCollum and Bryans, 1973; Timoney and McCollum, 1993; Glaser et al., 1997). EAV was first isolated from the lung of an aborted fetus following an extensive outbreak of respiratory disease and abortion on a Standardbred breeding farm near Bucyrus, OH in 1953 (Doll et al., 1957). The consequences of EAV infection of horses range from subclinical infection to clinical disease characterized by systemic influenza-like illness in adult horses, abortion of pregnant mares, and interstitial pneumonia in neonatal foals (Timoney and McCollum, 1993; Glaser et al., 1996, 1997). Some 60% of stallions infected with EAV subsequently become persistently infected carriers and play a critical role in maintenance and dissemination of the virus (Timoney et al., 1987; Timoney and McCollum, 1993). EAV persists in the reproductive tract of

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0042-6822/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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envelope protein (M). The G L envelope glycoprotein expresses the known neutralization determinants of the virus. It recently has been demonstrated that ORF2a encodes a 67-amino-acid unglycosylated structural protein (E) of EAV (Snijder et al., 1999). ORFs 3 and 4 are predicted to encode membrane glycoproteins (GP3 and GP4) of unknown function. The ORF 7 encodes the 12kDa nucleocapsid protein (N), which forms an icosahedral core that encapsidates the genomic RNA. There is only one serotype of EAV; however, geographically and temporally distinct EAV isolates differ substantially in their phenotypic properties such as virulence and neutralization determinants (Timoney and McCollum, 1993; Balasuriya et al., 1997). Experimental infection studies with different strains have identified lentogenic (avirulent), mesogenic (moderately virulent), and velogenic (highly virulent) strains of EAV (McCollum and Swerczek, 1978; McCollum and Timoney, 1984, 1998; MacLachlan et al., 1996; Patton et al., 1999). Previous studies have demonstrated that geographically and temporally distinct strains of EAV also differ significantly in their structural protein genes (Balasuriya et al., 1995b, 1999; Hedges et al., 1996, 1999; Patton et al., 1999). The significance of such changes demonstrated in retrospective studies, however, can only be unequivocally established with prospective genome manipulation using infectious cDNA clones. The genome of positive-stranded RNA viruses functions as a mRNA from which viral proteins necessary for virus replication are translated, thus in vitro RNA transcripts from full-length cDNA clones of positive-stranded RNA viruses are infectious (Racaniello and Baltimore, 1981; Boyer and Haenni, 1994). Infectious cDNA clones of a number of animal and human viruses have been developed, including an infectious cDNA clone of EAV (Rice et al., 1987; Sumiyoshi et al., 1992; Meyers et al., 1996; van Dinten et al., 1997; Meulenberg et al., 1998). The full-length infectious clone of EAV provides a novel tool to better characterize the replication, pathogenesis, and evolution of EAV. The objective of this study was to determine the pathogenicity and genetic stability in horses of the virus derived from an infectious cDNA clone developed from a highly cell culture-adapted laboratory variant of the original Bucyrus strain of EAV. RESULTS AND DISCUSSION An immunofluorescence staining assay was performed to demonstrate the expression of EAV proteins in cells transfected with the full-length in vitro RNA transcripts derived from the infectious cDNA clone (pEAV030H; van Dinten et al., 1997). Immunofluorescence staining of BHK-21 cells 20–22 h after transfection showed expression of nonstructural proteins nsp2 and nsp7–8, and of the G L and N structural proteins, suggesting that in vitro RNA transcripts were infectious to the

FIG. 1. Restriction digestion analysis of the RT–PCR amplified 901-bp segment containing the marker HindIII site in ORF1b of the recombinant EAV030H virus. Lanes 1 and 3, respectively, contain the undigested 901-bp segment from EAV030H virus and (as a representative example) the first tissue culture passage of EAV isolated from the nasal swab collected from stallion E at 6 days p.i. Lanes 2, 4, and 5, respectively, contain the HindIII digested 901-bp segment from the original recombinant EAV030H, the first tissue culture passage of EAV isolated from the nasal swab collected from stallion E at 6 days p.i., and from the mononuclear cells collected from stallion O at 6 days p.i. Sizes of the fragments are indicated on the left margin.

transfected cells (data not shown). A virus stock made from the tissue culture supernatant of the transfected cells was confirmed to be EAV by microneutralization assay using both EAV-specific neutralizing monoclonal antibodies and polyclonal equine antiserum (data not shown). The identity of the virus derived from transfected RNA was unequivocally confirmed by amplification of a 901-bp segment (nucleotide 6590–7451) containing an engineered marker HindIII restriction site (nucleotide number 6973) in ORF 1b of the recombinant EAV030H virus (Fig. 1; van Dinten et al., 1997). The recombinant EAV030H virus derived from the infectious cDNA clone was intranasally inoculated into two stallions. Neither of the two stallions developed severe clinical manifestations of EVA, but both became mildly febrile and developed mild to moderate lymphopenia and thrombocytopenia although there were no significant changes in their clotting parameters (Fig. 2). Virus was isolated from nasal swabs and mononuclear cells collected from both stallions and from the semen of one stallion (Table 1). Viral RNA was detected by direct reverse transcription and nested PCR (RT nested-PCR) amplification of ORF5 from nasal swab, mononuclear cell, serum, and semen samples collected from the stallions (Table 1 and Fig. 2E). The 901-bp segment containing the marker HindIII restriction site in ORF 1b was also amplified by RT–PCR from the first tissue culture passage of virus isolated from both stallions [6 days p.i. nasal swab (stallion E) and 6 days p.i. buffy coat (stallion O); Fig. 1]. Digestion with HindIII showed that the marker restriction site in ORF1b of the recombinant EAV030H virus was present in the amplicon generated from each sample, thus confirming that the virus in the sample indeed was derived from the recombinant EAV030H virus that was used to inoculate the two stallions. EAV is not usually isolated beyond 28 days p.i. from

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FIG. 2. Body temperature (A), hematological parameters [blood lymphocyte counts per microliter (B) and platelet counts per microliter (C)], neutralizing antibody titers [(D); expressed as the reciprocal of the highest final dilution that provided $50% protection of the RK-13 cell monolayer], and presence (indicated by shaded boxes) or absence (indicated by open boxes) of virus (viremia) or viral RNA in the blood of stallions O and E after experimental inoculation with the recombinant EAV030H virus.

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BALASURIYA ET AL. TABLE 1 Virus Isolation and Detection of Viral RNA in Samples Collected after Inoculation of Stallions with Recombinant EAV 030H Virus Stallion O (Days postinoculation) Method

Virus isolation

Sample name

Nasal swabs Mononuclear cells Serum Plasma Semen RT-Nested PCR Nasal swabs Mononuclear cells Serum Plasma Whole blood Semen

Stallion E (Days postinoculation)

0

2

4

6

7

8

10

12

14

21

0

2

4

6

7

8

10

12

14

21

2* 2* 2* 2* 2* 2* 2* 2* 2* 2* 2*

1 1 2 2 NC 1** 2 2 2 2 NC

1 1 2 2 NC 1 2 1 2 2 NC

1 1 2 2 NC 1 1** 1 2 2 NC

NC NC NC NC 1 NC NC NC NC NC 1**

1 1 2 2 NC 1 1 2 2 2 NC

2 1 2 2 NC 1 1 2 2 2 NC

2 2 2 2 NC 1 1 2 2 2 NC

1 1 2 2 2 1** 1** 2 2 2 2

2 2 2 2 2 2 2 2 2 2 2

2 1 2 2 2* 2 2 2 2 2 2*

1 1 2 2 NC 1** 2 2 2 2 NC

1 1 2 2 NC 1 2 2 2 2 NC

1 NC 2 2 NC 2 1** 2 2 2 NC

NC 2 NC NC 2 NC NC NC NC NC 2

2 2 2 2 NC 2 1 1 2 2 NC

2 2 2 2 NC 2 1** 2 2 2 NC

2 2 2 2 NC 2 2 2 2 2 NC

2 2 2 2 2 1** 2 2 2 2 2

2 2 2 2 2 2 2 2 2 2

* Preinoculation sample, ** Samples also used for direct sequencing. NC, Not collected. 1, virus isolation or RT nested-PCR positive; 2, virus isolation or RT nested-PCR negative.

infected horses other than from the semen of persistently infected stallions. In this study, we did not demonstrate either infectious virus or viral RNA in nasal secretions or blood after 14 days p.i. of the stallions (Table 1, Fig. 2E). The minor discrepancies that occurred between virus isolation and RT nested-PCR results may be due to differences in the original volume used for each assay (1 ml vs. 140–500 ml of sample, respectively). The data demonstrate that false negative results can occur with RT nested-PCR as compared with virus isolation, thus results should be carefully interpreted in any diagnostic RT nested-PCR assay. The patterns of viremia and association of recombinant virus with blood mononuclear cells were similar to those observed in horses that were naturally or experimentally infected with other strains of EAV (McCollum et al., 1971; McCollum, 1981; Timoney and McCollum, 1993; McCollum and Timoney, 1984; MacLachlan et al., 1996). The recombinant virus failed to establish persistent infection in either stallion; however, stallion O shed virus in semen at 7 days p.i. when it also was viremic. Both stallions seroconverted to EAV by 10 days p.i., and titers of neutralizing antibody in their serum increased to 256 by 42 days p.i. in stallion O and to 64 by 14 days p.i. in stallion E and remained constant thereafter (Fig. 2D). The clearance of virus from blood mononuclear cells coincided with the appearance of neutralizing antibodies (Figs. 2D and 2E). EAV strains vary significantly in their pathogenicity. The majority of field strains cause subclinical or inapparent infection, whereas some strains cause obvious clinical signs of EVA (Timoney and McCollum, 1993; Glaser et al., 1996; McCollum and Timoney, 1998). The pathogenesis of EVA has been characterized by inoculation of horses with virulent and avirulent strains of EAV by the intranasal, intramuscular, or intravenous routes, as well

as by careful evaluation of natural outbreaks of EVA (McCollum, 1970; McCollum et al., 1971, 1995, 1998; 1981; Timoney, 1984; Glaser et al., 1996). These studies have identified lentogenic, mesogenic, and velogenic strains of EAV (McCollum and Timoney, 1998). The highly virulent VBS53 strain of EAV was derived by serial passage of the original Bucyrus strain of EAV in horses (McCollum and Timoney, 1998). The VBS53 virus is considered the prototype strain of EAV and causes severe clinical disease or death in inoculated horses (MacLachlan et al., 1996; McCollum and Timoney, 1998). Most of the available EAV laboratory strains, including EAV030H, are derived from this highly virulent virus. The parent virus of recombinant EAV030H was passaged extensively in cell culture and cloned by end point dilution and plaque purification in African green monkey cells (Vero cells), prior to propagation of the working virus stocks. Viral RNA from this highly cell culture adapted laboratory strain of EAV was used to generate the genomic cDNA library that was used construct the infectious cDNA clone of EAV. Experimental infection of two stallions with the recombinant EAV030H virus derived from this infectious cDNA clone led to subclinical infection, which indicates that an attenuated virus was selected during cell culture passage or during construction of the recombinant virus based on the consensus sequence derived from the cDNA library. Alternatively, the plaque purification and cell culture propagation likely subjected the original virus to a bottleneck leading to the selection of an attenuated variant. We previously have shown that ORF 5 is the most variable structural protein gene of EAV (Balasuriya et al., 1995; Hedges et al., 1996). To determine the genetic variability of the recombinant virus following infection, the master sequence of the virus directly amplified from the EAV030H tissue culture fluid (that was used to inoculate the two stallions) as well as those from the nine

CLONE-DERIVED EAV IS REPLICATION COMPETENT

samples collected during the course of infection of the two stallions was determined by cycle sequencing (Table 1). Sequence analysis of ORF5 demonstrated that eight of nine master sequences of viruses present in the different samples were identical to that of the original 030H virus, whereas that of one virus (6 days p.i. buffy coat from stallion E) differed by just one nucleotide at position 11859 (C3T). The nucleotide substitution was noncoding, and the amino acid sequences of all G L proteins were identical. Thus sequence analysis of ORF5 and restriction analysis of ORF 1b demonstrated that the recombinant virus recovered from nasal swabs, mononuclear cells, and semen of the two stallions was identical to the original cloned virus that was used to inoculate them, which clearly indicates that the recombinant virus derived from the infectious cDNA clone of EAV was stable during replication in the two stallions. RNA virus genomes are prone to error due to the lack of proofreading-repair activities and low fidelity of their RNA replicases and transcriptases (Domingo et al., 1985). Therefore RNA virus populations do not consist of a single genome species with a single sequence but rather of heterogeneous mixtures of related genomes known as a viral quasispecies. We previously have demonstrated that EAV behaves as a quasispecies in the reproductive tract of the carrier stallion (Balasuriya et al., 1999; Hedges et al., 1999). To investigate the generation of viral variants following in vivo replication of the EAV30H virus, the ORF 5 sequence of 80 clones derived by direct RT nested-PCR from various samples were compared. These included 20 clones from the original 030H virus derived from the RNA-transfected cell culture fluid and 40 clones from two mononuclear cell samples (stallion O: 14 days p.i., stallion E: 8 days p.i.; 20 each) and 20 clones from the single semen sample (stallion O: 7 days p.i.). To reduce artifactual substitutions, the PCR amplification reactions were carried out with the highfidelity Pfu DNA polymerase enzyme (Stratagene; Smith et al., 1997). The sequences of 60 of the 80 clones (75%) were identical to that of the original EAV030H virus. Sixteen of the clones had a single nucleotide change, and 4 clones had two changes. The majority of the changes in these clones were transitional (22/24) and randomly scattered throughout ORF 5. Sequence differences in these clones included a unique transitional change (A3G) at nucleotide 11,365 in 11 clones from the 14 days p.i. mononuclear cell sample from stallion O. Most changes were also nonsynonymous (22/24) but conservative; however, three changes resulted in nonconservative amino acid changes in the G L protein (nonpolar to uncharged polar [amino acid 124 F to Y and 213 P to S] and basic to nonpolar [amino acid 39 R to L]). Overall, the data indicate that the recombinant EAV030H virus derived from the infectious cDNA clone is stable during replication in vivo. The conserved nature of the recombinant virus is similar to that which occurs during

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natural horizontal and vertical transmission of EAV in horses and contrasts markedly with the heterogeneous virus population (quasispecies) that occurs in the semen of carrier stallions (Balasuriya et al., 1999; Hedges et al., 1999). In conclusion, recombinant EAV derived from the recently developed infectious cDNA clone is attenuated and genetically stable during replication in vivo. These studies document for the first time that a recombinant virus derived from an infectious cDNA clone of a member of the order Nidovirales is replication competent in animals. The genetic stability of this recombinant virus during in vivo replication indicates that it will be useful to the characterization of genetic determinants of virulence and persistence of EAV. The approach used to engineer this clone now should be used to construct full-length infectious clones of mesogenic and velogenic strains as well as recombinant chimeric viruses of these strains of EAV. Development of such recombinant chimeric clones from different EAV strains will facilitate characterization of genetic determinants of critical phenotypic properties of EAV. METHODS Cells and virus Baby hamster kidney cells (BHK-21; ATCC CCL10) were maintained in Eagle’s medium (EMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories Inc.), 10% tryptose phosphate broth, and 1% penicillin and streptomycin. Rabbit kidney 13 (RK-13; ATCC CCL 37) cells were maintained in EMEM supplemented with 10% calf serum (CS; Hyclone Laboratories Inc.) and antibiotics. Full-length run off RNA transcripts were generated in vitro with modification to a previously described protocol from XhoI-linearized pEAV030H plasmid, which has an engineered marker HindIII site at nucleotide 6973 in the ORF 1b region (van Dinten et al., 1997). Briefly, a 50-ml reaction containing 2 mg of linearized plasmid DNA, RNA guard (37 U/ml; Pharmacia), m 7G(59)PPP(59)G RNA cap structure analogue (New England BioLabs), 5 ml of rATP, rCTP, rGTP, and rUTP (10 mM mix), 2.5 ml of 100 mM DTT, 2.5 ml of T7 RNA polymerase, and 13 transcription buffer (Promega) was incubated at 37°C for 1 h. The in vitro transcribed RNA was stored at 280°C until use. In vitro generated RNA transcripts were transfected into BHK-21 cells by electroporation. Briefly, BHK-21 cells were grown to subconfluence in regular growth medium, and the cells were trypsinized, washed, and resuspended in phosphate-buffered saline (PBS; pH 7.4) at a concentration of 1 310 7 cells/ml. RNA transcript (;10–20 mg) was added to 500 ml of BHK-21 cell suspension (5 3 10 6 cells) in an electroporation cuvette (0.2-cm electrode gap; BioRad). Two pulses of 1500 V, infinity V (pulse controller), and 25-mF capacitance were given with a

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Gene Pulser II electroporator unit (BioRad). Cells were incubated at room temperature for 10 min after electroporation and mixed with 10–15 ml of complete BHK-21 medium warmed to room temperature. The cells were seeded onto 10-cm-diameter tissue culture plates (Falcon) and incubated 37°C for 60 h until 100% cytopathic effect (CPE) was evident. Tissue culture fluid was harvested, centrifuged at 1600 g for 10 min at 4°C. The supernatant was aliquoted into 1.5 ml tubes and stored at 280°C until use. The virus was designated as EAV030H, and titers were determined by both Reed and Munch method [(Reed and Muench, 1938); 2.0 3 10 6.25 tissue culture infectious doses (TCID 50)/50 ml] and plaque assay (3.25 3 10 6 PFU/ml). For immunofluorescence assays, electroporated BHK-21 cells were directly plated onto chamber slides and incubated at 37°C for 20–22 h. Immunofluorescence assays Monospecific rabbit anti-peptide serum to the nonstructural proteins nsp2 and nsp7–8, and monoclonal antibodies to G L and N proteins were used to detect the expression of specific proteins in the electroporated BHK-21 cells as previously described (van Dinten et al., 1996, 1997). Intranasal inoculation of horses and sample collection Two 2- to 3-year-old stallions [O (Thoroughbred) and E (Tennessee Walker) seronegative to EAV by serum neutralization and Western immunoblotting assays were housed in an isolation facility. Each stallion was intranasally inoculated with 3.25 3 10 6 PFU/ml (2 3 10 6.25 TCID 50) of recombinant EAV030H virus that was delivered in 4.0 ml of EMEM using a fenestrated catheter. The stallions were monitored twice daily for 4 weeks for clinical manifestations of EVA. Whole blood for hematology [in buffered sodium citrate (Monojet) and thrombin soya bean trypsin (Becton Dickinson) anticoagulants] from both stallions were collected at 0, 2, 4, 6, 8, 10, 12, and 14 days p.i. These samples were used for complete blood counts (CBC), differential counts, and coagulation profiles [prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen degradation products (FDP)]. Blood for virus isolation from plasma (heparin anticoagulant) and serum were collected at 0, 2, 4, 6, 8, 10, 12,14, 21, 28, 35, and 42 days p.i.. Whole blood samples were also collected into VACCUTAINER CPT cell preparation tubes (Becton Dickinson) for the separation of mononuclear cells for virus isolation and viral RNA extraction. The nasopharyngeal regions of both stallions were thoroughly swabbed at 0, 2, 4, 6, 8, 10, 12, 14, 21, and 28 days p.i. using sterile gauze sponges at the end of a 64-cmlong stainless steel wire. The gauze swabs were removed after collection and placed in 5 ml of transport medium [EMEM, 2% FBS, 1% penicillin/streptomycin, 1%

gentamicin, and 2% fungizone (Gibco BRL)]. Semen samples were also collected from both stallions before and after infection (7, 14, 28, and 60 days p.i.). Virus isolation Virus isolation was attempted from mononuclear cells, plasma, serum, nasal swabs, and semen samples. Mononuclear cells were separated by centrifugation. Briefly, blood samples in VACCUTAINER CPT cell preparation tubes were centrifuged at room temperature (18– 25°C) at 1600 g (2800 rpm) for 7 min, and the cell monolayer layer was collected and washed twice in sterile PBS. The final cell pellet was resuspended in 6.5 ml of sterile PBS at ;1.5 3 10 7–3.0 3 10 7 cells/ml. Individual nasal swabs (gauze sponges) and transport medium were transferred to the barrel of a 12-ml disposable syringe and expressed through 0.45-mm syringe filter (Millipore). Semen samples were pretreated by sonication and centrifugation at 1000 g for 10 min at 4°C to sediment the spermatozoa. Virus isolation was attempted on RK-13 cells. Briefly, confluent monolayers (24-h old) of RK-13 cells in 12-well plates or in 25-cm 2 flasks (for semen) were inoculated with serial 10-fold dilutions (10 0–10 5 in duplicate) and overlaid with RK-13 gowth medium containing 0.75% caboxymethyl cellulose. The cells were incubated at 37°C for 8–9 days, and plaques were visualized by staining of the monolayer with crystal violet. A second passage was performed 4–5 days after the initial passage. Virus isolates were confirmed as EAV by microneutralization assay using EAVspecific antiserum and neutralizing monoclonal antibodies. Microneutralization assay Serum neutralizing antibodies to EAV were detected by microneutralization assay using EAV030H as the challenge virus in the presence of 5% guinea pig complement, as previously described (Balasuriya et al., 1995). Antibody titers were recorded as the reciprocal of the highest final dilution that provided $50% protection of the RK-13 cell monolayer. RNA isolation Viral RNA was directly isolated using the QIAmp Viral RNA isolation kit (QIAgen) from 140 ml of nasopharyngeal swab filtrate, plasma, serum, and semen samples, as previously described (Balasuriya et al., 1999). Total RNA was also isolated from 500 ml of whole blood and mononuclear cells (;1.5 3 10 7 cells) using the RNA STAT-50 LS(Test Tel Inc.) according to the manufactures instructions. The purified RNA samples were stored at 280°C until used.

CLONE-DERIVED EAV IS REPLICATION COMPETENT

RT nested-PCR and sequencing ORF 5 and flanking portions of ORF 4 and ORF 6 of the EAV in the various samples (882 bp) were amplified by reverse transcriptase polymerase chain reaction (RT– PCR) using MuLV reverse transcriptase (Perkin–Elmer) and AmpliTaq DNA polymerase (Perkin–Elmer) enzymes, as previously described (Balasuriya et al., 1995). The external primers 11,978–11,998 (59CTA ACC CAG ATG CTA CAT ACC 39; antisense primer) and 11,023–11,043 (59 AGG ACA AGA GGC ATC CTT AC 39; sense primer) were used for the first 35 cycles of PCR. A second round of amplification (nested PCR) was performed with 10 ml of the first round product with two internal primers at positions 11,940–11,961 (59 GAG TGG GAC GGA CAG AAT AAA G 39; antisense primer) and 11,080–111,001 (59 TTG TGG CTA TAG TTT ATG TTC 39; sense primer). The reaction mixtures were cycled 40 times in the second round with the same thermal profiles as those described previously. Twenty microliters of the final product was electrphoresed on a 1% agarose gel and visualized by ethidium bromide staining. Twelve nested PCR reactions (100 ml per reaction) derived from each sample were pooled, gel purified, and both sense and non-sense strands were sequenced with the PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystem) primed by internal sequence-specific primers as previously described (Hedges et al., 1996, 1999). The 901-bp segment containing an engineered marker HindIII restriction site in ORF 1b was RT-PCR amplified (7468–7487 antisense primer 59 AAG TGG AGC GGT ACA TGA TG 39; 6587–6607 sense primer 59 GAC CTG GAG AGT TGT GAT CG 39) from RNA purified from the first tissue culture passage of the 6 days p.i. nasal swab (stallion E) and 6 days p.i. mononuclear cells (stallion O). The fragment was digested with HindIII (Boehringer Mannheim GmbH) and subjected to electrophoresis on a 1% agarose gel, and visualized by ethidium bromide staining. Molecular cloning ORF 5 and flanking portions of ORF 4 and ORF 6 of EAV in the original 030H virus-infected cell culture fluid and selected mononuclear cell samples (stallion O: 14 days p.i., stallion E: 8 days p.i.), and a single semen sample (stallion O: 7 days p.i.) were reverse transcribed and nested-PCR amplified with Superscript II (Gibco BRL) and Pfu DNA polymerase (Stratagene) enzymes respectively for cloning. First-strand DNA was synthesized with oligo (dT) primers (Gibco BRL) and purified with GlassMAX DNA isolation system after digestion with RNase H and T1 (Gibco BRL). The high-fidelity Pfu DNA polymerase was used for PCR amplification to reduce artifactual substitutions (Smith et al., 1997). The first 35 cycles of PCR were performed using the same external primers as

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described above. The reactions were initially heated at 96°C for 45 s and each cycle of amplification consisted of 45 s of denaturation at 96°C, 45 s of annealing at 60°C, and 2-min extension at 72°C. After 35 cycles, the reactions were subjected to a 10-min final extension at 72°C. A second round of amplification was performed with 10 ml of the first-round product with the same two internal primers described above. The reaction mixtures were cycled 40 times in the second round with the same thermal profiles as those described above for Pfu DNA polymerase (Stratagene). Twelve different PCR reactions (100 ml/reaction) were carried out with each RNA sample, and the reaction products were pooled and concentrated (Centricon 30; Amicon). The 882-bp fragment containing ORF5 was agarose gel purified with a commercial kit (Qiaquick; Qiagen) and was cloned into the pCR2.1-TOPO cloning vector according to the manufacturer’s instructions (TOPO TA Cloning; Invitrogen Corp.). Twenty clones from each sample were sequenced as previously described (Balasuriya et al., 1999; Hedges et al., 1999). The estimated rate of misincorporations in RT nested-PCR and cloning is 0.5 artifactual substitutions per 768 bp [based on the error rate of retrovirus RT enzymes (5.5 3 10 24 substitutions/base pair/cycle) and Pfu DNA polymerase (1.3 3 10 26)]. Sequence analysis Sequence data were analyzed with the Sequencher 3.0 (Gene Codes Corp.) and HIBIO MacDNASIS pro version 3.5 (Hitachi) software programs using a Macintosh Power PC. ACKNOWLEDGMENTS The authors gratefully acknowledge Connie Littrell, Steve Lampman, and Dr. Jeff Lakritz, School of Veterinary Medicine, at the University of California, Davis for excellent animal care. The authors would also like to thank Travis Thayer and Dave Pettigrew for PCR and sequencing assistance. These studies were supported by USDA National Research Initiative Competitive Grant 97-35204-4736 and the Center for Equine Health, University of California, Davis with funds provided by the Oak Tree Racing Association, the State of California Satellite Wagering Fund, and contributions by private donors.

REFERENCES Balasuriya, U. B. R., Hedges, J. F., Timoney, P. J., McCollum, W. H., and 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., in press. Balasuriya, U. B. R., MacLachlan, N. J., de Vries, A. A. F., Rossitto, P. V., and Rottier, P. J. M. (1995a). Identification of a neutralization site in the major envelope glycoprotein (G L) of equine arteritis virus. Virology 207, 518–527. Balasuriya, U. B. R., Patton, J. F., Rossitto, P. V., Timoney, P. J., McCollum, W. H., and 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 G L envelope glycoprotein. Virology 232, 114–128. Balasuriya, U. B. R., Timoney, P. J., McCollum, W. H., and MacLachlan,

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