Isolates of cytomegalovirus (CMV) from the black rat Rattus rattus form a distinct group of rat CMV

L. M. Smith, J. N. Tonkin, M. A. Lawson and G. R. Shellam

Discipline of Microbiology, School of Biomedical & Chemical Sciences, M502, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia

Correspondence
L. M. Smith
LMSmith{at}cyllene.uwa.edu.au


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Two different betaherpesviruses, the English and Maastricht species of rat cytomegalovirus (CMV), have previously been isolated from Rattus norvegicus. CMVs were isolated from both the brown rat, R. norvegicus, and the black rat, R. rattus, within Australia. The viruses isolated from R. norvegicus appeared to be genetically related to the English species of rat CMV by PCR, RFLP, and sequencing, but the viruses isolated from R. rattus were distinct from both prototype virus species, although more closely genetically related to the Maastricht virus. This is the first genetic characterization of cytomegaloviruses from R. rattus, and the first isolation of CMVs from Australian rats.


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Cytomegaloviruses (CMVs) are species-specific betaherpesviruses capable of causing both acute and latent infections. They infect a broad range of species with individual virus strains exhibiting strict species specificity. Consequently, a species-specific CMV can be found in a diverse range of animals from mice to pigs, apes and humans (Staczek, 1990).

CMVs have previously been isolated from the brown rat Rattus norvegicus [the Maastricht and English species of rat CMV (RCMV)] (Bruggeman et al., 1982; Priscott & Tyrrell, 1982). Whilst RFLP and sequence analysis has shown that the genomes of these viruses are collinear with the genomes of other CMVs, analysis of their genome size, growth characteristics, and sequencing of the major immediate-early locus suggests that they are not simply different strains of RCMV, but are in fact different betaherpesvirus species (Beisser et al., 1998). Sequencing of random clones derived from the English RCMV genome in our laboratory has confirmed the variation in sequence homology and possibly gene content between this virus and the Maastricht species (L. M. Smith & J. Redwood, unpublished data).

The genome of the Maastricht species of RCMV has been fully sequenced (Vink et al., 2000). It is 230 138 bp in length, and contains a predicted 166 genes, 53 of which contain no significant homology to open reading frames in either murine (MCMV) or human (HCMV) cytomegaloviruses. This species of RCMV has been extensively studied as a model for cardiovascular disease, atherogenesis and transplant rejection caused by HCMV in humans (Streblow et al., 2003; Bruggeman et al., 1999; Orloff et al., 2002).

We have isolated and characterized CMVs from several wild brown and black rats, as the isolation of betaherpesviruses from two closely related species would provide us with an opportunity to examine the evolutionary relationship between host and virus. The brown rat is also the only species apart from humans from which multiple betaherpesviruses have been isolated, allowing us to examine virus–virus interactions within the same host, and to more closely examine the immune-evasion genes found in each virus. The RCMVs isolated in this laboratory are the only CMVs isolated from R. norvegicus in Australia, and we report here the first genetic analysis of CMV isolates from the black rat, R. rattus, anywhere. Whilst CMV has previously been identified in R. rattus, characterization was limited to visualization by electron microscopy of cytomegalic virions and in vitro growth in fibroblasts (Rabson et al., 1969; Berezesky et al., 1971).

Wild brown and black rats were trapped in Monash, Victoria, Australia by Grant Singleton (Division of Sustainable Ecosystems, CSIRO, Canberra) and identified using morphological characteristics (G. Singleton, personal communication). Homogenates of salivary glands were inoculated onto rat embryo fibroblasts (REF) prepared from Wistar rats (R. norvegicus) and overlaid with 1x MEM (2 % NCS) 3·5 % methylcellulose, and incubated at 37 °C, 5 % CO2 until CPE was visible (or for 7 days). CPE was seen in samples from both R. norvegicus (RN1 and 2), and from four of the six R. rattus samples (RR3, 5, 7 and 8). The CPE seen in samples from RN2 and from RR3, 5, 7 and 8 was similar to that produced by the reference English species of RCMV, however the plaques formed by isolate RN1 were smaller and included fewer cells (Fig. 1A). One plaque from each of samples RN1, RR3, RR5 and RR8 was selected to produce virus stocks. To analyse the genetic heterogeneity of virus populations within single animals, multiple plaques were picked from samples RN2 and RR7 (identified a–e) and stocks produced from each. Viruses were plaque-purified in this way three times, prior to infection of REF in 75 cm2 flasks to produce a master stock. None of the isolates produced CPE on mouse embryo fibroblasts (data not shown), as would be expected with Maastricht RCMV (Bruggeman et al., 1982).



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Fig. 1. Characterization of RCMV isolates. (A) Plaques formed by isolates on REF, stained with neutral red (RN1, RN2a and RR7a) or methylene blue (English). RN1 exhibited unusual plaque morphology, but other isolates induced the formation of typical cytomegalic plaques. (B) Electron micrographs (16 000x–30 000x) of purified virions. All isolates had typical CMV morphology, including multi-capsid virions. (C) Single-step growth curve of RCMV isolates on REFs, at an m.o.i. of 5. Isolates RN1 ({blacklozenge}) and RN2a ({blacksquare}) exhibited comparable growth characteristics to the English strain (x), whilst isolate RR7a ({blacktriangleup}) had a slightly longer lag phase, but still reached similar titres.

 
To analyse virion structure, purified virus was pelleted by centrifugation through a 30 % sucrose cushion at 40 000 r.p.m. in an SW41 rotor (200 000 g) for 3 h at 4 °C. Virions were resuspended in 100 µl mouse osmolality-buffered saline (MOBS), stained using sodium silicotungstate, and examined by transmission electron microscopy. Electron micrographs of virus stocks from isolates RN1, RN2a and RR7a showed typical cytomegalovirus morphology, with virions consisting of icosahedral capsids surrounded by a pleiomorphic envelope (Fig. 1B). Multi-capsid virions, consisting of 2–5 capsids within a single envelope, were frequently observed in all samples. Mean capsid sizes were determined for isolates RN1 (104·1±2 nm), RN2a (97·9±2 nm), RR7a (99·3±3 nm) and the English strain (112·2±2 nm) compared to the published English (111 nm) and Maastricht (100 nm) strain capsid sizes (Bruggeman et al., 1982; Priscott & Tyrrell, 1982).

Growth characteristics for isolates RR7a, RN1, RN2a and the English strain of virus on REFs were similar, although isolate RR7a exhibited a longer lag phase (22 h as compared to 18 h) and took longer to reach peak titres (35 h as compared to approximately 25 h) than the other isolates (Fig. 1C). This may be due to the in vitro analyses being performed on fibroblasts from R. norvegicus rather than R. rattus, as the latter were not available.

The genomes of the new RCMV isolates were analysed by three different methods (Table 1). As the methods used vary, it is not unexpected that some of the novel viruses classify differently according to the method used. However, none of these classifications is inherently contradictory.


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Table 1. Classification of novel RCMV isolates depending on the methods

ND, Not determined (no available virus); +, classified with this virus; –, does not classify with this virus.

 
A standard method of typing CMVs is by RFLP analysis of viral DNA purified from pelleted virions. When analysed by digestion with EcoRI, isolates RN2a, RN2d and RR3 gave identical RFLP patterns, as did isolates RR5, RR7a and RR8. Isolate RN1 had a unique RFLP pattern, as did the reference English strain of RCMV (Fig. 2A). These virus groupings were confirmed by SacI RFLP analysis (data not shown).



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Fig. 2. Genetic analysis of RCMV isolates. (A) EcoRI RFLP patterns from RCMV isolates, showing most RR isolates are similar, and different from RN isolates. DNA purified from pelleted virions by guanidine thiocyanate lysis followed by phenol/chloroform extraction. (B) PCR amplification of RCMV isolates with primers designed against conserved Maastricht RCMV and MCMV sequences, suggesting gross genomic differences between RR and RN isolates. (C) Phylogenetic analysis of derived amino acid sequences of R87 gene DNA sequences. Maximum parsimony method used, Bootstrap replicates=500, analysis performed using MEGA version 2.1 (Kumar et al., 2001). Bootstrap values shown.

 
PCR was performed on DNA from all isolates using degenerate primers which were designed against conserved sequences within all betaherpesviruses (designed to amplify the R87 gene: R87for, 5'-CCAACTTCCCCTGCATGATC3'; R87rev, 5'-TGTGYTTGATCTTGGTGTGCGC-3') or against conserved sequences from MCMV (Smith) and RCMV (Maastricht) (R44 gene and R50 gene primers: R44for, 5'-GTGCACGCAGGCSGAGCTGAA-3'; R44rev, 5'-TACAAGRCCGCCATCCAGCAG-3'; R50for, 5'-AAGACGAACTGGTGCAGGTC-3'; R50rev, 5'-CATCCGTGAGCTCTTCGGG-3'). The R87 primers amplified all the RCMV isolates and MCMV, whilst the R44 and R50 primers designed to amplify the published RCMV and MCMV sequences amplified most of the R. rattus isolates (except for RR3) and the RN1 isolate, but failed to amplify isolate RN2a and the English strain suggesting that these isolates are genomically distinct from the Maastricht virus at these loci (Fig. 2B).

A third method of typing CMV isolates is by direct sequencing of specific loci. The 294 bp PCR product amplified from all viruses by the R87 primers was sequenced and the resultant sequences aligned using CLUSTALW (www.ebi.ac.uk). The reference English strain has between 99 % and 100 % sequence identity (i.e. between 0 and 3 nt changes) within this region to all isolates from RN2 and to isolates RN1 and RR3, and 69·4 % nt sequence identity (83·9 % amino acid sequence identity) to the published Maastricht sequence. The sequence of four of the isolates from R. rattus (RR5, RR7a, RR7d and RR8) was identical in this region, sharing 87 % nt sequence identity with the published sequence from the Maastricht virus and 73·9 % identity with the English virus. In comparison, the level of amino acid sequence identity between the RR isolates and the Maastricht sequence was 98·1 %, and between the RR isolates and the English strain was 83·9 %. This was as a result of a high rate of synonymous nucleotide changes, suggesting functional conservation between the R87 gene product in these viruses (data not shown). Phylogenetic analysis of the derived amino acid sequences from the R87 gene of these RCMVs was performed using maximum parsimony (sequences aligned using CLUSTALW) with tree stability determined using bootstrap analysis (500 replicates). Also included were derived amino acid sequences of gene homologues from published CMV sequences: the Smith strain of MCMV (accession no. U68299), the AD169 strain of HCMV (accession no. X17403), and the Maastricht strain of RCMV (accession no. AF232689). Rooting the phylogenetic tree on HCMV, all of the isolates from R. norvegicus, in addition to isolate RR3, grouped with the English strain. The remaining R. rattus isolates grouped together, related to but separate from the Maastricht species of virus (Fig. 2C). Sequences were deposited in the EMBL database (accession nos AJ536159–AJ536170). As the region to be amplified and sequenced was selected because of its low sequence variation in published sequences, the genetic distances identified between these viruses may be artificially low compared with other genomic loci. The grouping of most of the RR isolates as being distinct from but related to the Maastricht species of virus, was confirmed by sequence analysis of the PCR product amplified from the region 3' to the R50 gene (data not shown). The relationships of these viruses to the English and RN isolates at this genomic locus could not be analysed, as this region was not amplifiable from these viruses. Random cloning of the RR7a, RN2a and English virus genomes also supports the grouping shown here, as almost all (20/23) of the clones from RR7a have nt sequence identity (58–84 %) to the Maastricht virus, whilst many of the clones from the English virus (10/21) contain novel, unrelated sequences (data not shown).

Ideally, this analysis should be repeated with regions of the genome which are both conserved and divergent between the English and Maastricht species, however the lack of sequence data from the English virus makes this difficult. This approach would, however, allow us to examine isolates for evidence of recombination between the extant strains.

Also of interest is an isolate from R. norvegicus, RN2a, which appears to be genetically identical (by PCR, sequencing and RFLP) to an isolate from R. rattus, RR3, raising the possibility that these isolates can infect both species.

BAC clones of several of the isolates are currently being constructed to facilitate genomic analysis; however, future work is dependent on obtaining a laboratory colony of R. rattus for in vivo work.


   Acknowledgements
 
Electron microscopy performed by Marina Silich-Carrara (Discipline of Microbiology, UWA). This work was supported financially by the Pest Animal Control CRC, and by the Grains Research and Development Corporation.


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Since acceptance of this paper, an isolation of cytomegalovirus from Rattus rattus has been described by Loh et al. (2003). Characterization of a novel rat cytomegalovirus (RCMV) infecting placenta-uterus of Rattus rattus diardii. Arch Virol 148, 2353–2367.


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Received 24 November 2003; accepted 14 January 2004.