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
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Abstract |
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Introduction |
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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 5151989) 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.
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Methods |
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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 freezethaw cycles. Cellular debris was removed by centrifugation (10 min at 1000 g) and supernatants containing virions were collected.
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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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.
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
).
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.
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Results |
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DNA sequence analysis
Genome sequences of the newly identified parvoviruses corresponding to nucleotides 564940 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 (8889% nucleotide identity) (Table 2
). MMVi, MPV and HaPV showed 8182% nucleotide identity with these new RMVs. In contrast, the new RMVs showed only 73% identity with RPV-1a.
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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 56117) 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 9394%. Among rodent parvoviruses, RMV-1 was least similar to the NS-1 protein of RPV-1a (86%).
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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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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 7679% when compared with that of KRV, H-1, HaPV, MPV and MMV, and 68% similar to that of RPV-1a.
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Discussion |
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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 (7783% 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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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.
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Acknowledgments |
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Footnotes |
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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.
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References |
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Received 6 September 2001;
accepted 9 April 2002.
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