INTRODUCTION

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Sialodacryoadenitis virus (SDAV) is distributed worldwide in laboratory rats. SDAV infects the lacrimal and salivary glands and the upper and lower respiratory tracts of rats, causing the clinical manifestations of enlarged salivary glands, sialoadenitis, dacryoadenitis, rhinitis, tracheitis, and bronchoalveolitis (3, 9, 10). SDAV can also cause reproductive disorders and behavioral changes in the infected animals. Serologic surveys indicate that coronavirus infections are common in laboratory rats housed in research facilities (11, 16), and several outbreaks of SDAV in rat colonies have been reported (2, 6, 12, 22, 32; J. Storz, personal communication). Therefore, SDAV is an important viral pathogen in comparative laboratory medicine.

SDAV is antigenically related to the mouse hepatitis virus (MHV) serogroup of the family Coronaviridae in the order of Nidovirales (20). The MHV serogroup includes Parker's rat coronavirus (PRCV), bovine coronavirus (BCV), and human coronavirus (HCV) strain OC43. Coronavirus is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 31 kb. The 5'-most 22 kb of the coronavirus genome encodes the nonstructural RNA-dependent RNA polymerase, while all the structural proteins are encoded in the 3' terminal 9 kb of the genome (15). Although a large amount of genetic information has been accumulated for MHV and other coronaviruses, such information is not available for any rat coronavirus, mainly due to the difficulty with propagation of the virus in cell cultures. Percy and coworkers (25) reported that a subclone of L2 cells produced relatively higher titers of the virus, and subsequently, Baker et al. (1) used these cells to identify at least three structural proteins associated with the virion: spike (S) protein, membrane (M) protein, and nucleocapsid (N) protein. Antibodies specific for MHV structural proteins were able to recognize both SDAV and PRCV proteins on immunoblots. However, it has not been possible to differentiate coronaviruses from each rat coronavirus in the laboratory with antibodies (26). Defined genetic information will be of help in developing a differential diagnostic method and in providing a better understanding of this group of coronaviruses. To these ends, we have performed cDNA cloning of the entire structural-protein region of the sialodacryoadenitis rat coronavirus genome, and in this communication we report the complete sequence of the 3' terminal 9.8 kb of the SDAV genomic RNA.

	MATERIALS AND METHODS

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Cells and virus. SDAV strain 681 and L2(Percy) cells were provided by D. H. Percy (Ontario Veterinary College, Guelph, Ontario, Canada) (25). SDAV strain 681 was originally obtained from P. N. Bhatt (Yale University, New Haven, Conn.) (2). L2(Percy) cells were maintained as monolayers at 37°C with 5% CO₂ in a humidified incubator. The virus was propagated in L2(Percy) cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (CanSera, Mississauga, Ontario, Canada).

Viral RNA preparation and the first-strand cDNA synthesis. Cells were infected at a multiplicity of infection of 1 to 3 and were incubated for 3 days. The supernatant was collected and clarified with a benchtop centrifuge. Virus was pelleted through a 30% sucrose cushion with an ultracentrifuge (Beckman model XL-90) at 25,000 rpm for 2 h in an SW28 rotor. The pellets were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]), and viral RNA was extracted with the QIAmp viral RNA extract kit (Qiagen, Mississauga, Ontario, Canada). Specific procedures for viral RNA extraction were followed, as described in the manufacturer's instructions. For the first-strand cDNA synthesis, total cellular RNA was extracted from the virus-infected cells, and approximately 5 µg of the total cellular RNA was incubated at 70°C for 10 min with 0.5 µg of specific oligonucleotide. After chilling of the reaction mixture on ice, 200 U of SuperScript II RNase H reverse transcriptase (Gibco BRL, Mississauga, Ontario, Canada) was added. The reverse transcription (RT) reaction was carried out for 1 h at 37°C in the presence of 1 mM each dCTP, dGTP, dTTP, and dATP, 10 mM dithiothreitol, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl₂ in a reaction volume of 20 µl. The second strand was synthesized by PCR amplification.

PCR amplification. Four microliters of the first-strand cDNA reaction was added to the PCR mixture containing final concentrations of 0.15 µg of each forward and reverse primer, 20 mM Tris-HCl (pH 8.4), 5 mM MgCl₂, 50 mM KCl, 1 mM each deoxynucleoside triphosphate, and 0.5 U of Taq DNA polymerase (Gibco BRL). The PCR was performed in a Perkin-Elmer thermocycler (model PE 2400) for 30 cycles as follows: 94°C for 30 s for denaturation, 62°C for 30 s for annealing, and 72°C for 2.5 min for extension, followed by a 10-min elongation at 72°C after the final cycle. The 3' end of the viral genome was synthesized with a 3' Marathon rapid amplification cDNA ends kit purchased from Clontech (Palo Alto, Calif.). The PCR products were analyzed by agarose gel electrophoresis, and the residual primers were removed from the amplified products by gel filtration column chromatography (QIAquick oligonucleotide removal kit; Qiagen).

Cloning and sequencing. The cDNA fragments were cloned into the SmaI site of the cloning vector pGEM3zf(+) (Promega, Madison, Wis.). Manipulation of DNA and general cloning procedures were followed, as described by Sambrook et al. (27). Nucleotide sequences were determined either by direct sequencing of the PCR fragments with the specific primers or by sequencing of the cloned plasmid with the universal primers. New sets of primers were designed on the basis of the sequence information obtained and were used to sequence the internal region of the clone. Nucleotide sequences were assembled and analyzed with the GeneRunner software program and the SeqWeb (version 1.1) interface connected to the GCG Wisconsin sequence analysis package provided through the Canadian Bioinformatics Resource, National Research Council (http://www.cbr.nrc.ca; Halifax, Nova Scotia, Canada).

Nucleotide sequence accession numbers. The sequences reported in this work have been deposited in the GenBank database under accession numbers AF188191, AF188192, AF188193, AF188194, AF188195, and AF207551.

	RESULTS AND DISCUSSION

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Genome organization. A total of seven cDNA fragments were generated by RT-PCR cDNA cloning to represent the 3' terminal 9.8 kb of the SDAV RNA genome. The oligonucleotide primer sets used for these amplifications are listed in Table 1. Selection of the sequences used to design these primers for RT-PCR of the SDAV genome was based on the known genomic information of several strains of coronaviruses. First, the 3' terminal 1.6-kb fragment of the SDAV genome was cloned by the 3' rapid amplification cDNA ends method, and the nucleotide sequence of the cloned fragment was determined. On the basis of the sequence obtained, the reverse primer n1-rev-nc was designed to clone upstream fragment e. For the forward primer to be used as part of a pair with n1-rev-nc, amino acid sequences of the spike protein gene of BCV, HCV OC43, MHV A59, and MHV DVIM (GenBank accession numbers D00662, Z21849, AF029248, and AB008940, respectively) were compared, and a highly conserved region was identified. Nucleotide sequences that corresponded to the conserved region of the amino acid sequence were then examined to design the upstream primer, S2-fwd-nc. By using the primer pair of S2-fwd-nc and n1-rev-nc, the e fragment was synthesized (Table 1). Similar approaches were used for other fragment amplifications. When one or two mismatches were identified in the sequence, degenerate primers were designed, as indicated in Table 1. The contiguous overlapping sequences were assembled as a single genome. Nine major open reading frames (ORFs) were identified in the cloned 9.8-kb region of the viral genome, and these ORFs represented the coding sequences for all the structural and nonstructural proteins of the virus except the polymerase protein (Fig. 1A). The polymerase 1b coding sequence is presented in the +2 frame, and the frames for other genes are listed in Fig. 1B.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Genome structure and the coding assignments of SDAV. (A) ORFs identified in the 3' terminal 9.8 kb of the viral genome. (B) The gene products of the coding sequences and their structural characteristics. nts, nucleotides; aa, amino acid.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Intergenic consensus transcription signals for SDAV. The putative intergenic consensus sequences are indicated as boldface characters, and the first AUG methionine codon for the ORF following the intergenic consensus promoter sequence is underlined. Numbers in parentheses indicate the number of nucleotides between the consensus sequence and the start of the gene.

Sequence comparisons with other coronaviruses. The overall sequence similarities of the SDAV genes to the sequences of the genes of other coronaviruses within the same serogroup are presented in Table 2. The SDAV genome was more similar to MHV A59 than BCV, with overall homologies of 86.1 and 66.8%, respectively. More specifically, the NS2, HE, M-protein and N-protein genes including the 3' UTR were highly conserved between the rat coronavirus and mouse coronavirus, with similarities of 91 to 99%. The sequence homologies of the sM-protein genes were also high between SDAV and MHV A59, with an amino acid identity of 85.2%, while the similarities of the S-protein genes were 76.2%. The relatively low degrees of similarity of the S-protein genes were largely due to the addition of sequences to the N-terminal half of the S-protein gene in SDAV. In MHV, the sequence of the N-terminal half of the S-protein gene is known to be heterogeneous, and this heterogeneous region has been suggested to be associated with viral pathogenesis (8, 23, 24, 31). When the SDAV S-protein sequence was compared to the corresponding sequences of MHV 2, MHV JHM, and MHV A59, a hypervariable region was identified that included a large addition of 172, 127, and 44 amino acid residues, respectively (Fig. 3). In contrast, a relatively small but distinct insertion (amino acid positions 474 to 478) and deletions (positions 497 to 498 and 543 to 545) were identified within the S-protein gene sequence in the SDAV genome when the sequence was compared to that of the S-protein gene sequence of the MHV 4 genome.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Sequence heterogeneity in the N-terminal half of the S protein of SDAV. Numbers in parentheses indicate amino acid positions of the spike protein. Asterisks indicate conservation of the cysteine residues, and hyphens indicate amino acid deletions. GenBank accession numbers are as follows: for SDAV, AF188193; for MHV A59, M18379; for MHV JHM, D00093; for MHV 2, AF107212; for MHV-4, S51114; for BCV, D00662; for HCV OC43 S62886.

Differential PCR for SDAV and MHV. Rat coronavirus and mouse coronavirus share antigenic similarities, and the antisera raised against SDAV cross-react with MHV strains. The sensitive indirect fluorescent-antibody test and the enzyme immunoassay were not able to differentiate antibodies to MHV or SDAV. Thus, at present no diagnostic method is available for differentiation of rat coronavirus and mouse coronavirus. The hypervariable region identified in the SDAV S sequence may be used as a genetic marker to develop a reliable differential diagnostic PCR. On the basis of the sequence divergence observed in the S-protein gene of the rat coronavirus SDAV and mouse coronavirus MHV strains, we attempted to demonstrate a principle of PCR differentiation for rodent coronaviruses. Using a primer pair that encompasses the hypervariable region (1stf and 1str in Table 1), we amplified part of the S-protein genes of SDAV and MHV strains (Fig. 4A). SDAV was amplified as a 1,010-bp fragment (lane 2), while MHV A59, MHV JHM, and MHV 2 were amplified as 893-, 626-, and 590-bp fragments, respectively (lanes 3, 4, and 5, respectively). BCV and HCV OC43 were amplified as fragments of 1,046 and 1,016 bp, respectively (lanes 6 and 7, respectively). The BCV and HCV OC43 fragments were similar in size to the SDAV S-protein fragment. Since rodents are not naturally susceptible to BCV and HCV OC43, the similar sizes of the BCV and HCV OC43 fragments that were amplified may not be a practical matter of concern in the development of a differential diagnostic PCR for rodent coronaviruses. However, it may still be possible that BCV and HCV OC43 may cross the species barrier and infect rodent species. To further differentiate SDAV infection from BCV or HCV OC43 infection, we used a restriction analysis of the PCR products. By digestion with KpnI, the SDAV product generated 485- and 526-bp fragments (Fig. 4B, lane 1), whereas both the BCV and HCV OC43 products remained as uncleaved forms of 1,010 and 1,045 bp, respectively (Fig. 4B, lanes 2 and 3, respectively). Furthermore, the MfeI digestion yielded four cleaved fragments of 663, 215, 127, and 6 bp for SDAV (Fig. 4B, lane 4), while only two fragments were generated by the same enzyme for both BCV (821 and 189 bp) and HCV OC43 (856 and 189 bp) (Fig. 4B, lanes 5 and 6, respectively). The 6-bp fragment of SDAV generated by MfeI digestion is not seen on the gel in Fig. 4B. Therefore, the amplification of the hypervariable region of the S-protein gene by PCR and the subsequent digestion with KpnI or/and MfeI allows us to differentiate SDAV from the other coronaviruses.

View larger version (42K): [in this window] [in a new window]   FIG. 4.   (A) Differential amplifications of the hypervariable region of rodent coronaviruses. Total RNA was extracted from virus-infected cells 24 h postinfection. RT-PCR was performed with the primer pair which was used for amplification of fragment a (1stf and 1str in Table 1), and the PCR products were electrophoresed on a 1% agarose gel. Lanes: M, molecular weight marker; 1, uninfected cells; 2, SDAV-infected cells; 3, MHV A59-infected cells; 4, MHV JHM-infected cells; 5, MHV 2-infected cells; 6, BCV-infected cells; 7, HCV OC43 infected cells. (B) Restriction patterns of PCR fragment a from SDAV (lanes 1 and 4), BCV (lanes 2 and 5), and HCV OC43 (lanes 3 and 6). Lanes 1, 2, and 3, digestion patterns obtained with restriction endonuclease KpnI; lanes 4, 5, and 6, digestion patterns obtained with restriction endonuclease MfeI; lane M, 100-bp ladder molecular weight marker. For the sizes of the digested fragments, see the text.