Molecular characterization of three newly recognized rat parvoviruses

Cho-Hua Wanb,1, Maria Söderlund-Venermoc,1, David J. Pintel2 and Lela K. Riley1

Department of Veterinary Pathobiology1 and Department of Molecular Microbiology & Immunology2, University of Missouri, Columbia, MO 65211, USA

Author for correspondence: Lela Riley. Present address: E111 Veterinary Medicine Building, 1600 E. Rollins Road, Columbia, MO 65211, USA. Fax +1 573 884 7521. e-mail RileyL{at}missouri.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Rodent parvoviruses have been documented to interfere with both in vivo and in vitro research. In this study, three rat parvoviruses distinct from previously characterized rodent parvoviruses were identified from naturally infected rats obtained from four discrete sources. These three newly recognized parvoviruses were designated rat minute virus (RMV)-1a, -1b and -1c. In this study, the genomic nucleotide sequence and the predicted amino acid sequences of proteins for each of the three RMV-1 variants and Kilham rat virus (KRV) were determined and compared with previously characterized rodent parvoviruses. The three RMV-1 variants were shown to be closely related to each other, to be distinct from but closely related to KRV and H-1 virus, and to be significantly different from the previously identified rat parvovirus isolate, RPV-1a.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Autonomous parvoviruses are small (15–28 nm) non-enveloped DNA viruses of the family Parvoviridae, which contain a single-stranded genome of about 5 kb in length. Replication of autonomous parvoviruses requires the presence of host factors expressed during cellular S phase. Several rodent parvoviruses were discovered in the late 1950s and early 1960s, including Kilham rat virus (KRV) and H-1 virus (H-1) in rats (Kilham & Olivier, 1959 ; Toolan et al., 1960 ) and the minute virus of mice (MVM), recently renamed mice minute virus (MMV) (Crawford, 1966 ; International Union of Microbiological Societies, 2000 ). More recently, several additional rodent parvovirus species have been isolated from infected cell lines and rodents and subsequently characterized. These include mouse parvovirus (MPV), hamster parvovirus (HaPV) and rat parvovirus 1a (RPV-1a) (Ball-Goodrich & Johnson, 1994 ; Ball-Goodrich et al., 1998 ; Besselsen et al., 1996 ; McKisic et al., 1993 ).

The molecular biology of MMV has been well characterized and has been used as the model for other autonomous parvoviruses (Cotmore & Tattersall, 1987 ). Parvoviruses have palindrome sequences at both the 5' and 3' termini of the genome and these palindromes are involved in virus replication (Cotmore & Tattersall, 1987 ). All rodent parvoviruses replicate with monomer and dimer DNA intermediates and encapsidate monomer single-stranded DNA, which is predominantly minus sense. Rodent parvoviruses encode two non-structural proteins, NS-1 and NS-2, and three capsid proteins, VP-1, VP-2 and VP-3. The NS proteins, involved in transcription and virus replication, are quite conserved among different rodent parvoviruses, whereas the VP proteins exhibit heterogeneity among different species of parvoviruses. Replication of autonomous parvoviruses is generally host-specific and parvoviruses of laboratory rodents are typically grouped according to the results of haemagglutination-inhibition (HAI) or serum-neutralization assays.

Regulation of transcription and translation in MMV has also been well studied. MMV produces three mRNA transcripts, R1, R2 and R3, which all terminate at a single polyadenylation site at genetic map unit (m.u.) 95 (Clemens & Pintel, 1987 ). R1 is generated by the P4 promoter (m.u. 4) and encodes the NS-1 protein, which is required for viral DNA replication and transactivation of the capsid gene promoter P38. R2 also is generated by the P4 promoter, but undergoes an additional splicing event, which removes a large intron (nt 515–1989) within the NS coding region (Cotmore & Tattersall, 1986 ; Naeger et al., 1990 ). Alternative splicing of the small intron from R2 generates three isoforms of the NS-2 protein (major, minor and rare NS-2 protein). The three isoforms of NS-2 protein are identical in the first 182 amino acids, but differ at their C termini. R3 arises from the P38 promoter and encodes two structural viral proteins, the VP-1 and VP-2 proteins. VP-2 is the major capsid protein and its amino acid sequences are contained within VP-1. A third structural viral protein, VP-3, is produced by proteolytic processing of VP-2 near the trypsin-sensitive REVR motif (Tattersall et al., 1976 ).

Pathogenesis of rat parvoviruses, including KRV, H-1 and RPV-1a, has been well studied. In natural and experimental infections of fetal and infant rats, KRV causes highly pathogenic infections, especially in liver and cerebellum (Kilham & Margolis, 1966 ). In experimental infections in rats, H-1 can cause lesions similar to those caused by KRV (Moore & Nicastri, 1965 ). RPV-1a, the most recently characterized rodent parvovirus, appears non-pathogenic for experimentally infected infant rats (Ball-Goodrich et al., 1998 ).

This report describes the identification and characterization of three novel parvoviruses of rat origin that are antigenically and molecularly distinct from KRV, H-1, RPV-1a and other previously identified rodent parvoviruses. These newly recognized viruses have been named rat minute virus (RMV)-1a, -1b and -1c.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Tissues and viral DNA.
Kidney or liver were collected from two wild rats obtained from St Louis County Vector Control (St Louis, Missouri, USA) and from 90 laboratory rats representing 19 different strains and 17 geographically different research colonies in the USA and Europe. Tissue samples were stored at -80 °C until processed. Of the 92 rats, sera were collected from 80 immunocompetent individuals and examined by the Research Animal Diagnostic Laboratory (RADIL), University of Missouri.

DNA was extracted from tissue samples with the QIAamp tissue kit (Qiagen) according to the protocol provided by the manufacturer. The DNA concentration and purity of the tissue DNA extracts were determined by A260 and A280 values.

KRV (ATCC VR-235) was grown in rat glial tumour cells (C6 Glial, ATCC CCL 107) in Dulbecco's modified Eagle's medium (Hazleton) containing 10% Serum-plus (JRH Biosciences) at 37 °C as described previously (Besselsen et al., 1995a ). Cell pellets were collected by centrifugation (10 min at 500 g) when approximately 90% of the cells exhibited cytopathic effect. Crude cell lysates were prepared by resuspending cell pellets in culture medium and subjecting infected cells and medium to four freeze–thaw cycles. Cellular debris was removed by centrifugation (10 min at 1000 g) and supernatants containing virions were collected.

{blacksquare} Oligonucleotide primers.
Oligonucleotide primers were synthesized at Gibco BRL, Life Technologies or the DNA Core Facility, University of Missouri. Primer sequences (Table 1) were selected on the basis of alignments performed with Genetics Computer Group (GCG) analysis programs (Genetics Computer Group, Madison, WI, USA). Primers were initially designed from sequences that are highly conserved among MMV, MPV, HaPV, KRV, H-1 and RPV-1a. As sequence data for new parvovirus isolates from rat tissues became available, these data were applied in the design of additional primers.


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Table 1. Oligonucleotide primers used for parvovirus PCR assays

 
{blacksquare} DNA amplification by PCR and nested PCR.
All reactions were performed in a 50 µl volume in an automated thermocycler (GeneAmp PCR System 2400, Perkin-Elmer). Nested PCR was applied to amplify new parvoviruses from rat tissues to increase the sensitivity of amplification. For reactions in which the size of expected products was less than 1·2 kb, each PCR mixture contained 1·25 µg of tissue DNA extract as template DNA, 1 µM of each oligonucleotide primer, 10 mM Tris–HCl (pH 8·3), 1·5 mM MgCl2, 50 mM KCl, 0·2 mM each dATP, dCTP, dGTP and dTTP and 1 U of Taq DNA polymerase (Boehringer Mannheim). PCR consisted of 30 s denaturation at 94 °C, followed by 35 cycles of 2 s denaturation at 94 °C, 2 s annealing at 57, 55 or 50 °C and 30 s to 1·5 min elongation at 72 °C. Annealing temperature and length of elongation period varied depending on the primers used and the sizes of expected amplified products. In reactions with expected amplified products longer than 1·2 kb, the Expand High Fidelity PCR System (Boehringer Mannheim) was applied and reaction mixtures contained 1·25 µg of tissue DNA extract as template DNA, 0·5 µM each oligonucleotide primer, 1x Expand High Fidelity Buffer with 1·5 mM MgCl2, 0·2 mM each dATP, dCTP, dGTP and dTTP, and 1·8 U of Expand High Fidelity PCR System enzyme mix. PCR consisted of 2 min denaturation at 95 °C, followed by 35 cycles of 3 s denaturation at 94 °C, 3 s annealing at 57 or 55 °C, and 1·5–3 min elongation at 72 °C, followed by 7 min final elongation at 72 °C. DMSO was added in some expanded-PCR reaction mixtures to a final concentration of 1% (v/v) to enhance DNA amplification. Primary PCR products (16 µl) were electrophoretically separated in 2% NuSieve agarose gel (FMC BioProducts), stained with ethidium bromide and visualized by UV light.

Nested PCR was performed as described for primary PCR, except annealing temperature and elongation period were modified depending on the primers used and the sizes of expected amplified products. Primary PCR product (1 µl) was used as the template DNA for nested PCR. Nested PCR products (7 µl) were analysed and detected as described for primary PCR products. Conditions for DNA amplification of KRV viral DNA from cell culture were the same as those used for amplification of viral DNA extracted from rat tissues, except that only primary PCR was performed.

{blacksquare} DNA sequencing and analysis.
The PCR-amplified DNA fragments were purified by the QIAquick PCR Purification Kit (Qiagen) or the QIAquick Gel Extraction Kit (Qiagen) using protocols recommended by the manufacturer. Nucleotide sequences were determined by dideoxy-chain termination with a commercially available kit (ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit; Perkin-Elmer) in the DNA Core Facility, University of Missouri. Sequence data were analysed with the GCG analysis programs PILEUP, Pretty, Gap and Translate, using default settings. The sequences used for comparison were those of H-1 virus (accession nos X01457 and J02198; Rhode & Paradiso, 1983 ), MMVi (an immunosuppressive variant of MMV) (accession no. X02481; Sahli et al., 1985 ), MPV-1a (accession no. U12469: Ball-Goodrich & Johnson, 1994 ), HaPV (accession nos U34255, AF288060 and AF288061; Besselsen et al., 1996 ; Söderlund-Venermo et al., 2001 ), RPV-1a (accession no. AF036710; Ball-Goodrich et al., 1998 ) and porcine parvovirus (PPV) (accession no. M38367; Vasudevacharya et al., 1990 ).

{blacksquare} Recombinant NS-1 (rNS1) ELISA and HAI assays.
Serum samples collected from rats were diluted 1:5 in saline, heat inactivated at 55 °C for 30 min and tested by rNS-1 ELISA, KRV HAI and H-1 HAI assays. The rNS-1 ELISA, using recombinant MMV NS-1 as antigen, was performed as described previously (Riley et al., 1996 ). The KRV and H-1 HAI assays were performed using a modification of the MPV HAI assay described by Besselsen et al. (1995b) in which 8 haemagglutination units of KRV or H-1 virus were used as the antigen and guinea pig erythrocytes were used instead of mouse erythrocytes.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Identification of non-KRV/non-H-1 parvovirus in rats
Two wild rats from St Louis County Vector Control and 90 rats representing 19 rat strains and 17 geographically different research colonies were examined. No clinical signs or external abnormalities were observed. No gross lesions were observed during necropsy and no significant lesions were identified by histopathologic evaluation of tissue sections. Sera from 80 immunocompetent rats were examined by the pan-specific parvovirus serologic assay, the rNS-1 ELISA (Riley et al., 1996 ). Antibodies against NS-1 were found in sera from the two wild rats and from 14 laboratory rats representing three rat strains and two geographically different research colonies. Specific KRV and H-1 HAI assays were negative, indicating that the rats were not infected with either KRV or H-1. No antibodies against NS-1 were found by ELISA in sera from the other 64 rats surveyed. DNA was extracted from kidney or liver from all 92 rats and was evaluated by generic parvovirus PCR (Table 1), which amplifies the conserved NS-1 gene region (Besselsen, 1998 ). An amplified DNA product was detected in kidney or liver from each of the 16 rats that were positive for NS-1 antibodies, although the intensity of the ethidium-bromide stained band was weak under UV light. Southern blot analysis using a digoxigenin-labelled HaPV DNA (nt 1458–1791) probe confirmed the homology between HaPV and PCR products amplified from all 16 rats. To compensate for a low yield of parvoviral DNA fragments by primary PCR amplification from rat tissue DNA extracts, nested PCR amplification was applied to the tissue DNA extracts. The result of the nested PCR amplification was consistent with the result of the Southern blot analysis, and the nested PCR amplification produced sufficient amplified parvovirus DNA for sequencing. In the other 76 rats examined by PCR, no parvovirus DNA was amplified from tissue DNA extracts by either generic parvovirus PCR assay or nested parvovirus PCR assay.

DNA sequence analysis
Genome sequences of the newly identified parvoviruses corresponding to nucleotides 56–4940 of H-1 (Rhode & Paradiso, 1983 ) were determined from DNA prepared from four antibody- and PCR-positive rats obtained from different sources, i.e. from the two wild rats and from two laboratory rats obtained from different geographic regions. Additionally, the genome sequence for KRV (ATCC VR-235) was also determined. The two parvoviruses from wild rats were found to be 99·98% identical in genome sequences to each other. Therefore, these two viruses were considered to be a single parvovirus strain and were designated rat minute virus 1a (RMV-1a). The parvoviruses from the two laboratory animal rats were genetically distinct from each other, showing 97% nucleotide identity. These parvoviruses were designated RMV-1b and -1c. RMV-1a was slightly more closely related to RMV-1b (98·5% nucleotide identity) than to RMV-1c (97·1% nucleotide identity) (Table 2). Comparison of the nucleotide sequence alignment of the three newly identified RMVs with other rodent parvoviruses revealed that the three new RMVs were most closely related to KRV (91% nucleotide identity) and H-1 virus (88–89% nucleotide identity) (Table 2). MMVi, MPV and HaPV showed 81–82% nucleotide identity with these new RMVs. In contrast, the new RMVs showed only 73% identity with RPV-1a.


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Table 2. Percentage nucleotide identity among rodent parvovirus isolates

 
Sequence alignment of the three new RMV-1 variants, KRV and other rodent parvoviruses further revealed that portions of the genome previously identified to be important in transcription, mRNA processing and translation of rodent parvovirus gene products were conserved. Locations of major open reading frames in these three new parvoviruses and KRV closely resembled those previously established for H-1, RPV-1a and MMV (Ball-Goodrich et al., 1998 ; Rhode & Paradiso, 1983 ; Sahli et al., 1985 ). Sequences of all splice-junction regions were also compared. Sequences flanking the large intron donor site (AA/GCAAGT), the first small intron donor site (SID-1) (AA/GTACGA) and the second small intron donor site (SID-2) (AG/GTAAGG) in the three newly identified RMV-1 genomes were identical to those found in the KRV, HaPV, MPV, MMV and RPV-1a genomes, but differed from H-1 SID-1 (AA/GTACAA) with a purine replacement (G->A) (Fig. 1a). The large intron acceptor sequences in the three newly identified RMV-1 genomes were identical to that in the H-1 genome; however, the 3' splice site polypyrimidine tracks differed from those in KRV, MPV, MMVi and RPV-1a (Fig. 1a). Slight divergence was noticed among the sequences of the first small intron acceptor (SIA-1) among the three RMV-1 isolates. Sequences of the SIA-1 site in RMV-1a and RMV-1c were identical, but differed from RMV-1b and KRV. The sequence of the SIA-1 site in RMV-1a and RMV-1c also differed from H-1, HaPV, MPV and MMV in the polypyrimidine track (Fig. 1a). The RMV-1b sequence around the SIA-1 was identical to that found in KRV, but differed from that found in RMV-1a, RMV-1c, H-1, HaPV, MPV and MMV. The sequence of the second small splice acceptor site (SIA-2) in the three newly identified RMV-1 isolates was the same as those in KRV, H-1, HaPV and MPV, but differed from MMV in one nucleotide in the polypyrimidine track of the SIA-2 (Fig. 1a). The sequences of both SIA sites in the three new RMV-1 isolates differed from the analogous sites in RPV-1a. The SIA-1 of the RMV-1 isolates differed from that of RPV-1a in one nucleotide of the splicing site (AG/GC->AG/GT) and five to seven nucleotides in the polypyrimidine tract (Fig. 1a). The RMV-1 isolates also differed from RPV-1a in three nucleotides in the polypyrimidine track of the SIA-2 (Fig. 1a).



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Fig. 1. Comparison of the nucleotide sequence of the large intron acceptor and the two small intron acceptors, SIA-1 and SIA-2 (a), and TAR elements (b) of RMV-1a, -1b, -1c and KRV with other rodent parvoviruses. Dashes indicate nucleotide identity; / indicates the splice junction; bold letters indicate nucleotide changes from the RMV-1a sequence.

 
Comparison of the sequences of the regions involved in the transcription and transcription regulation in rodent parvoviruses was also performed. The P4 promoter regions of the new RMV-1 isolates were conserved when compared with KRV, H-1, RPV-1a, HaPV, MPV and MMV. The TATAA box was identical among all these viruses. The SP1 binding site (TGGGCGTGGCT) of the three new RMV-1 isolates was identical to those of KRV and H-1, but different from the SP1 binding site of HaPV, MPV and MMV (TGGGCGTGGTT) and that of RPV-1a (AGGGCGTGGCT). At the P38 promoter region, the TATAA boxes and the SP1 binding sites were identical among the three new RMV-1 isolates and the other rodent parvoviruses (data not shown), but the transactivation responsive (TAR) elements exhibited slightly divergent sequences among different rodent parvoviruses (Fig. 1b). The TAR elements in RMV-1a, RMV-1c, KRV and H-1 were identical, but differed from those in RMV-1b, HaPV, MPV and MMV at one position and differed from that of RPV-1a at two positions. The NS1 binding site in this region was conserved among all isolates except RPV-1a.

Genomic sequences of the three new RMV-1 isolates and KRV were incomplete due to the technical difficulties in amplifying terminal hairpin structures of the parvovirus genomic DNA. The first 55 nucleotides (the outer part of the left hairpin) and the entire right hairpin of the genomic DNAs of the three RMV-1 variants and KRV were not successfully amplified by PCR so these sequences were not available for comparison analyses. The sequences of the first 62 existing nucleotides (inner part of the left hairpin) of the three new RMV-1 isolates and KRV were identical, but differed from the analogous region (nucleotides 56–117) of H-1 at one position and differed from the corresponding portion of the RPV-1a genome at four positions (data not shown). The sequences obtained from the newly identified RMV-1 isolates and KRV did not extend past the right hairpin structure when compared with the sequence of the H-1 genome. The direct repeat sequence at the 3' untranslated end of the KRV (85 nucleotides per copy) and H-1 (55 nucleotides per copy) genome was present as one copy in each of the three new RMV-1 genomes. This region in RMV-1a and -1b was 85 nucleotides, the same length as that in the KRV genome; however, in RMV-1c this region was 15 nucleotides shorter than in the KRV genome (data not shown).

Protein sequence analysis
Amino acid sequences for the NS and VP proteins were deduced from the determined nucleotide sequences and compared among the new RMV-1 variants and other autonomous rodent parvoviruses (Table 3). The RMV-1a viruses identified from the two different wild rats were identical to each other in the amino acid sequences of the NS and VP proteins. The NS-1 proteins of the three new RMV-1 variants were most similar to KRV and H-1, with greater than 97% similarity (Table 3a). The similarity of the NS-1 proteins of the newly identified RMVs to those of non-rat rodent parvoviruses (HaPV, MPV and MMV) was 93–94%. Among rodent parvoviruses, RMV-1 was least similar to the NS-1 protein of RPV-1a (86%).


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Table 3. Amino acid similarity for NS-1, the N termini of NS-2 and VP-1, and VP-2 among rodent parvovirus isolates

 
There are three isoforms of the NS-2 protein, the result of alternative splicing, produced by each parvovirus. The three NS-2 isoforms produced by one parvovirus share the same coding sequence, 546 nucleotides in length, before the second small intron, so the three isoforms of one parvovirus are identical in the first 182 amino acids at the N terminus. Comparison of the sequence of the first 182 amino acids at the N terminus of the NS-2 protein of different rodent parvoviruses showed that RMV-1a, -1c and KRV were almost identical (similarity of 99–100%) and were very similar to RMV-1b (95%) and H-1 (93–95%) (Table 3b). The protein similarity in the first 182 amino acids at the N terminus of the NS-2 protein between RMV-1b and H-1 was slightly lower (90%) (Table 3b). Comparison of these 182 amino acids of the three new RMV-1 variants with those of non-rat rodent parvoviruses (HaPV, MPV and MMV) revealed slightly less similarity (84–87%) than the similarity between RMV-1 and the other rat parvoviruses, except for RPV-1a (Table 3b). The NS-2 N-terminal protein sequences of the new RMVs exhibited the least degree of similarity (66–67%) to that of RPV-1a (Table 3b).

The three NS-2 isoforms produced by one parvovirus differ from each other in the amino acid sequences at their C termini. Comparison of the C termini of the three NS-2 isoforms from different rodent parvoviruses was performed (Fig. 2). The C terminus of the major NS-2 form (PEITWF) was identical for the three newly identified RMV-1 isolates, KRV, H-1 and the non-rat rodent parvoviruses, but differed from that of RPV-1a by two amino acids (P->Y, E->S). The C terminus of the minor NS-2 form (YDGASS) of the three new RMVs was identical to that of KRV, but differed from that of H-1 at two positions (D->N, A->T) and differed from that of HaPV, MPV and MMV with an additional serine (SS->S@). RPV-1a is distinct from the three RMV-1 variants in the C terminus of the minor NS-2 form, having amino acid replacements at two positions (A->T, S->G) and an extension of 18 amino acids. RMV-1a, -1b and KRV were identical in the C terminus of the rare NS-2 form (LGASGLQVPGTREQP), but differed from RMV-1c at one position (E->K) and differed from H-1 and HaPV by one amino acid (G->W). RPV-1a exhibited three coding changes (A->T, L->I, R->W) and three amino acids truncated (EQP) at the C terminus of the rare NS-2 form when compared with RMV-1a and -1b.



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Fig. 2. Comparison of the C termini of NS-2 in RMV-1a, -1b, -1c and KRV with other rodent parvoviruses. Dashes indicate amino acids identical to those of RMV-1a; @ indicates the termination codon.

 
The structural VP proteins exhibited more heterogeneity among different parvoviruses than NS proteins. The VP-1 protein consists of a unique N terminus (approximately 142 amino acids) with the C terminus identical to the VP-2 protein. Comparison of the unique N termini of VP-1 proteins of different rodent parvoviruses showed that the three new RMV-1 variants and KRV were very similar to each other (similarity of 97·6–99%) and were also similar to H-1 (93–94%) (Table 3c). The protein similarities in the unique N terminus of the VP-1 protein among the newly identified RMVs and the non-rat rodent parvoviruses (HaPV, MPV and MMV) were slightly lower (89–91%). Among rodent parvoviruses, the RMV-1 variants were least similar to RPV-1a in the VP-1 unique region (similarity of 78–79%).

The VP-2 protein is the major component of the parvovirus capsid. Comparison of the VP-2 protein sequence among rodent parvoviruses was performed and the results are shown in Table 3d. Conservation of the unique N terminus of the VP-1 protein resulted in higher percentage similarities for VP-1 than VP-2 (Table 3c, d). The similarity of the VP-2 protein remained 99% for RMV-1a and -1b. The VP-2 protein of RMV-1c was 95% similar to that of RMV-1a and -1b. The similarities of the VP-2 protein of the new RMV-1 variants were 76–79% when compared with that of KRV, H-1, HaPV, MPV and MMV, and 68% similar to that of RPV-1a.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
KRV and H-1 virus in rats and MMV in mice are recognized as three well-characterized parvovirus serogroups that infect rodents. Recently, additional rodent parvovirus species were discovered and characterized: RPV-1a, a third serogroup of parvovirus in rats; MPV, a second serogroup in mice; and HaPV, the first serogroup identified in hamsters. Early indication of the existence of additional parvoviruses species, such as MPV and RPV-1a, in rodent colonies was based on positive results for the presence of parvovirus antibodies by ELISA and IFA and negative HAI results for MMV, KRV and H-1 (McKisic et al., 1993 ; Ball-Goodrich et al., 1998 ; Besselsen et al., 1996 ; Rhode & Paradiso, 1983 , Sahli et al., 1985 ). In this study, a similar approach was used to identify new parvoviruses from infected rats. Three novel rat parvoviruses (RMV-1a, -1b and -1c) were identified and characterized.

Comparison of nucleotide and protein sequence alignments revealed that the three newly identified RMV-1 isolates share a common genetic organization, such as promoter and splicing regions and translation start and stop codons, with other rodent parvoviruses. RMV-1 viruses have similar but distinctly different nucleotide and protein sequences from previously characterized rodent parvoviruses. Based on nucleotide sequence identity (>=97% identity) and amino acid sequence similarity (>=95%), the three newly identified RMV-1 viruses are suggested to be variants of the same virus species. Low levels of amino acid similarity (77–83% for RMV-1 compared with KRV and H-1) in the capsid proteins (Table 3c, d), responsible for parvovirus haemagglutination activity, were consistent with the negative results of KRV and H-1 HAI assays with sera from RMV-1-infected rats. On the basis of the results of HAI assays and sequence similarity, the three newly identified RMV-1 isolates are suggested to be in a different serogroup from KRV, H-1, RPV-1a and other identified rodent parvoviruses.

Phylogenetic differences among the three newly identified RMV-1 variants and other parvoviruses also support the notion that RMV-1 is a distinct parvovirus species. Parsimony analysis of aligned amino acid sequences of NS-1 showed that the three new RMV-1 variants were most closely related to KRV and H-1 (Fig. 3a). Phylogenetic analysis of VP-2 amino acid sequences (Fig. 3b) further suggested that RMV-1 is a previously undescribed parvovirus, different from KRV and H-1. The phylogenetic differences in the VP-2 protein were also consistent with the negative results of KRV and H-1 HAI assays obtained with serum samples from RMV-1-infected rats. Parsimony analyses of NS-1 and VP-2 proteins indicated that RPV-1a, a recently identified parvovirus in rats, was the rodent parvovirus least related to RMV-1. Taken together, phylogenetic analyses of NS-1 and VP-2 sequence data supported the conclusion that all three RMV-1 viruses are parvoviruses that are distinct from other rodent parvoviruses. Thus, these viruses should be considered to be a new rodent parvovirus species.



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Fig. 3. Phylogeny using parsimony analysis with NS-1 (a) and VP-2 (b) amino acid sequences from ten different parvoviruses. The phylogeny was rooted with porcine parvovirus (PPV) as an outgroup. The scale bar represents 2% difference in amino acid sequences, determined by measuring the lengths of the horizontal lines connecting two species.

 
Potential effects of RMV-1 infection on research experiments using RMV-1-infected rats are unknown. In this study, the naturally RMV-infected rats were clinically normal and no gross or significant histopathologic lesions were observed. The diagnosis of RMV infection relied on PCR amplification. Although these data are not quantitative, the weak intensity of the primary PCR product on ethidium bromide-stained gels suggested that only limited numbers of RMV-1 viral particles were present in these naturally infected rats. It is possible that these rats are from endemically infected colonies in which active virus replication is minimal. Attempts to propagate RMV-1 in vitro were unsuccessful. This may be due to the inability to identify permissive cell lines or the paucity of RMV-1 virus particles in naturally infected rats. Attempts to develop in vitro culture systems for growth of RMV-1 are ongoing and will be important for future RMV-1 pathogenesis studies. Experimental infection of RMV-1 in animals has not been performed and will be the subject of future investigation.

The prevalence of these various rat parvovirus species in laboratory rat colonies has not been established. According to a recent serologic survey conducted by the University of Missouri Research Animal Diagnostic Laboratory (L. K. Riley, unpublished data), 2·1% of laboratory rats evaluated were positive for KRV, <0·1% were positive for H-1 and 4·4% of rats were positive for non-KRV/non-H-1 parvoviruses, a category which includes RMVs and the RPV-1a isolate. These results suggest that new rat parvovirus species (RMV-1 and RPV-1) may be more prevalent than well-described rat parvovirus species (KRV and H-1) in contemporary laboratory rat colonies. To definitively determine the prevalence of RMV and RPV infections in research rat colonies, RMV- and RPV-specific diagnostic assays are needed.

In summary, the molecular characteristics of three newly identified rat parvoviruses were determined. The three viral variants, named RMV-1a, -1b and -1c, are closely related to each other, are distinct from but closely related to KRV and H-1, and are significantly different from the previously identified rat parvovirus isolate, RPV-1a. Potential effects of RMV-1 infection on research experiments using RMV-1-infected rats are at present unknown.


   Acknowledgments
 
We thank Mr Greg Purdy for technical expertise and Mr Howard Wilson for photographic assistance. This research was supported by grant DHHS 5 R24 1177203 from the National Institutes of Health.


   Footnotes
 
The RMV-1a, RMV-1b, RMV-1c and KRV sequences have been submitted to GenBank and assigned accession nos AF332882, AF332883, AF332884 and AF321230, respectively.

b Present address: BioReliance Corporation, 14920 Medical Center Drive, Rockville, MD 20850, USA.

c Present address: Haartman Institute, Department of Virology, University of Helsinki, Finland.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Ball-Goodrich, L. J. & Johnson, E. (1994). Molecular characterization of a newly recognized mouse parvovirus. Journal of Virology 68, 6476-6486.[Abstract]

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Received 6 September 2001; accepted 9 April 2002.



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