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J Virol. 2007 August; 81(15): 8091–8100.
Published online 2007 May 16. doi:  10.1128/JVI.00255-07
PMCID: PMC1951306

Identification of Novel Rodent Herpesviruses, Including the First Gammaherpesvirus of Mus musculus[down-pointing small open triangle]

Abstract

Rodent herpesviruses such as murine cytomegalovirus (host, Mus musculus), rat cytomegalovirus (host, Rattus norvegicus), and murine gammaherpesvirus 68 (hosts, Apodemus species) are important tools for the experimental study of human herpesvirus diseases. However, alphaherpesviruses, roseoloviruses, and lymphocryptoviruses, as well as rhadinoviruses, that naturally infect Mus musculus (house mouse) and other Old World mice are unknown. To identify hitherto-unknown rodent-associated herpesviruses, we captured M. musculus, R. norvegicus, and 14 other rodent species in several locations in Germany, the United Kingdom, and Thailand. Samples of trigeminal ganglia, dorsal root ganglia, brains, spleens, and other organs, as well as blood, were analyzed with a degenerate panherpesvirus PCR targeting the DNA polymerase (DPOL) gene. Herpesvirus-positive samples were subjected to a second degenerate PCR targeting the glycoprotein B (gB) gene. The sequences located between the partial DPOL and gB sequences were amplified by long-distance PCR and sequenced, resulting in a contiguous sequence of approximately 3.5 kbp. By DPOL PCR, we detected 17 novel betaherpesviruses and 21 novel gammaherpesviruses but no alphaherpesvirus. Of these 38 novel herpesviruses, 14 were successfully analyzed by the complete bigenic approach. Most importantly, the first gammaherpesvirus of Mus musculus was discovered (Mus musculus rhadinovirus 1 [MmusRHV1]). This virus is a member of a novel group of rodent gammaherpesviruses, which is clearly distinct from murine herpesvirus 68-like rodent gammaherpesviruses. Multigenic phylogenetic analysis, using an 8-kbp locus, revealed that MmusRHV1 diverged from the other gammaherpesviruses soon after the evolutionary separation of Epstein-Barr virus-like lymphocryptoviruses from human herpesvirus 8-like rhadinoviruses and alcelaphine herpesvirus 1-like macaviruses.

Herpesviruses of small laboratory rodents, such as mice and rats, are used as surrogate models of human herpesvirus infections. They are invaluable tools for exploring various aspects of virus-host interactions, which otherwise would be difficult or even impossible to study. Human herpesviruses either do not replicate in laboratory animals or, if they do, frequently fail to cause symptoms that reflect those observed in infected humans. For example, the human varicella-zoster virus (VZV) (species, Human herpesvirus 3 [HHV-3]; subfamily, Alphaherpesvirinae) does not replicate in rodents. Experimental infection of guinea pigs is possible, but its significance is limited (14). Mice, guinea pigs, and rabbits can be infected with herpes simplex virus type 1 (HSV-1) (species, HHV-1; subfamily, Alphaherpesvirinae) but do not develop all of the facets of human pathology (13, 28). Experimental infection of New World monkeys with Epstein-Barr virus (EBV) (species, HHV-4; subfamily, Gammaherpesvirinae) (10, 20) has been reported, but these animals are endangered, rare, and expensive and as such are of limited experimental value. Infection of laboratory rodents with EBV has not been reported. Therefore, animal homologues of human herpesviruses are required for in vivo studies, and some rodent herpesviruses are currently being studied in detail.

Murine cytomegalovirus (MCMV) (species, Murid herpesvirus 1 [MuHV-1]; subfamily, Betaherpesvirinae) (Table (Table1)1) naturally infects M. musculus (5). It serves as a tool for experimental studies of human CMV (HCMV) (species, HHV-5; subfamily, Betaherpesvirinae) disease (25). For the same purpose, two strains of rat CMV (RCMV) (species, MuHV-2; subfamily, Betaherpesvirinae), RCMV strain England (RCMV-E) and RCMV strain Maastricht (RCMV-M), have been investigated in laboratory strains of Rattus norvegicus (6, 16). The viruses differ considerably in gene content and biological properties (27, 2).

TABLE 1.
Known herpesviruses of humans, rodents, and other mammals

Murine gammaherpesvirus 68 (MHV-68) (species, MuHV-4; subfamily, Gammaherpesvirinae) is a representative of the rhadinoviruses, such as the human Kaposi's sarcoma-associated herpesvirus (KSHV) (species, HHV-8; subfamily, Gammaherpesvirinae). MHV-68 is well suited for the study of gammaherpesvirus pathogenesis and was used to develop therapeutic strategies against gammaherpesviruses (reviewed in references 15, 21, and 23). However, MHV-68 pathology in mice does not entirely resemble the pathology of HHV-8 or EBV in humans. For example, MHV-68 infection does not consistently result in tumor development (reviewed in reference 17). In addition, Mus musculus (from which laboratory mice are derived) is apparently not the natural host of the virus. MHV-68 and several closely related viruses, such as MHV-60, MHV-72, and MHV-76, were isolated in Slovakia from two rodent species, Myodes glareolus (formerly Clethrionomys glareolus) and Apodemus flavicollis (4). In the United Kingdom, Apodemus sylvaticus was found to be the major natural host of MHV-68 (3).

Alphaherpesviruses, roseoloviruses, and lymphocryptoviruses that naturally infect any species of the Rodentia are currently not known. In addition, no rhadinoviruses that naturally infect M. musculus or other Old World mice have been identified (Table (Table11).

To identify hitherto-unknown rodent-associated herpesviruses, we captured M. musculus, R. norvegicus, and 14 other rodent species at several locations in Germany, the United Kingdom, and Thailand and searched by degenerate PCR methods for herpesviruses that naturally infect them. Thirty-eight novel rodent herpesviruses were detected, among them the first gammaherpesvirus of M. musculus.

MATERIALS AND METHODS

Sample collection and DNA preparation.

Free-living rodents were trapped in several rural and urban locations in Germany, the United Kingdom, and Thailand. A total of 1,132 samples from blood, brain, trigeminal ganglion, spinal ganglion, spleen, lung, intestine, liver, and inguinal lymph nodes were collected and stored at −20°C. DNA was prepared as described previously (26).

Panherpesvirus PCR with specificity for the DPOL gene of herpesviruses.

Panherpesvirus consensus PCR for amplification of 160 to 181 bp (without primer-binding sites) of the DNA polymerase (DPOL) gene (Fig. (Fig.1A)1A) (8) was carried out as described previously (7). Briefly, PCR was performed with degenerate and deoxyinosine (deg/dI)-containing primers in a nested format. Three primers were used in first-round PCR (primer 285s DFA, 5′-gayttygc[n/I]agyyt[n/I]taycc-3′; primer 285s ILK, 5′-tcctggacaagcagcar[n/I]ysgc[n/I]mt[n/I]aa-3′; primer 285as KG1, 5′-gtcttgctcaccag[n/I]tc[n/I]ac[n/I]ccytt-3′), and two primers were used in second-round PCR (primer 286s TGV, 5′-tgtaactcggtgtaygg[n/I]ttyac[n/I]gg[n/I]gt-3′; primer 286as IYG, 5′-cacagagtccgtrtc[n/I]ccrta[n/I]at-3′). Buffy coat DNA or tissue DNA (250 ng) or products of the first-round PCRs (1 μl) were used as templates in PCRs with a 25-μl reaction mixture containing a 1 μM concentration of each PCR primer (Metabion, Martinsried, Germany), a 200 μM concentration of each deoxynucleoside triphosphate, 1 unit of DNA polymerase AmpliTaq Gold, and 2.5 μl of GeneAmp 10× PCR buffer with 2 mM MgCl2 (Applied Biosystems GmbH, Darmstadt, Germany) and 5% dimethyl sulfoxide (Sigma-Aldrich Chemie GmbH). In first- and second-round PCRs, the reaction mixtures were kept for 12 min at 95°C for activation of the polymerase and then cycled 45 times with 20 s of denaturation at 95°C, 30 s of annealing at 46°C, and 30 s of strand extension at 72°C, followed by a final extension step at 72°C for 10 min.

FIG. 1.
Map of amplified genes and diagrams of PCR strategies. Degenerate nested primers (black triangles) were used to amplify part of the MDBP gene, the gB gene, or the DPOL gene. (A) The amplified fragments are represented by thin solid lines between the primer-binding ...

Samples with little or no amplification product were reanalyzed under more-relaxed conditions; i.e., the ramp time between the annealing step and the extension step was prolonged 50-fold, and the final concentration of AmpliTaq Gold was doubled (2 units/reaction mix). In each assay, a DNA of a gammaherpesvirus-positive porcine spleen sample was included as a positive control. Water samples were extracted and PCR tested as negative controls. In addition, alpha-, beta-, and gammaherpesvirus DNAs were tested at intervals to control assay performance.

Amplification of gB- and MDBP-encoding sequences with degenerate primers.

All primers are listed in Table Table2.2. For amplification of novel glycoprotein B (gB) gene sequences of members of the Betaherpesvirinae subfamily, three different deg/dI nested-primer sets were used: CM-gB1, CM-gB2, or CM-gB3. Second-round amplification products had sizes of approximately 225 bp, 265 bp, and 280 bp, respectively (without primer-binding sites [Fig. [Fig.1A]).1A]). For amplification of gB sequences of members of the Gammaherpesvirinae subfamily, the deg/dI nested-primer set RH-gB was used. Second-round amplification products had a size of approximately 450 bp (without primer-binding sites [Fig. [Fig.1A]).1A]). For amplification of sequences encoding the major DNA-binding protein (MDBP) of Mus musculus rhadinovirus 1 (MmusRHV1), the deg/dI nested-primer set RH-MDBP was used. Second-round amplification products had a size of approximately 260 bp (without primer-binding sites [Fig. [Fig.1A]).1A]). PCR was carried out at a 45°C annealing temperature. All other parameters were as described for DPOL gene amplification.

TABLE 2.
Primers used for amplification of novel gB and MDBP sequences

LD-PCR with specific primers.

Long-distance PCR (LD-PCR) (Fig. (Fig.1B)1B) was performed with the TaKaRa-Ex PCR system (Takara Bio Inc., Japan), according to the manufacturer's instructions. Amplimers were obtained by nested PCR using specific primers (not listed). For the second round, a 1-μl aliquot of the first-round reaction mix was used as a template. In case of accumulation of nonspecific products in a high-molecular-weight range (>10 kb), the first-round postcycling reaction mix was diluted 1:100 before serving as a template in the second round. Amplification products had a size of approximately 3.5 kbp.

Specific PCR assays.

The gB gene of MmusRHV1 was specifically amplified in a nested PCR with primers MmusRHVgB1 (5′-TCGGGAGTATAACTATTACAC-3′) and MmusRHVgB2 (5′-ACCTCCCGAGACTTACTC-3′) in the first round and MmusRHVgB3 (5′-GCCATCATGGAAGACCTG-3′) and MmusRHVgB4 (5′-GAAGAGGATGACGATCAC-3′) in the second round, at annealing temperatures of 53°C and 50°C, respectively. Amplification products had sizes of 317 bp and 131 bp, respectively (without primer-binding sites).

The DPOL gene of Apodemus flavicollis cytomegalovirus 3 (AflaCMV3) was specifically amplified with primers 3838s (5′-CAAAGGAAGGCGATTAGACA-3′) and 3838as (5′-ACCGTAACACGCAGTGGAT-3′) at a 57°C annealing temperature. Amplification products had a size of 254 bp (without primer-binding sites).

Cytochrome b sequences were amplified with the primer set 258s (5′-CCATCCAACATCTCAGCATGATGAAA-3′) and 258as (5′-GCCCCTCAGAATGATATTTGTCCTCA-3′) at a 58°C annealing temperature. Amplification products had a size of 307 bp (without primer-binding sites).

Sequence analysis and phylogenetic tree construction.

PCR product purification, direct sequencing with dye terminator chemistry, nucleotide sequence analysis, and amino acid sequence predictions were performed as described previously (11). Multiple-sequence alignments and phylogenetic tree construction with neighbor-joining and maximum-likelihood analysis were performed as described by Ehlers and Lowden (9).

Designation as novel rodent herpesviruses and preliminary names.

All viruses were named trinomially. The first two words are the name of the host species, and the third word indicates the grouping of the novel virus within the Herpesviridae. All MCMV- and RCMV-like betaherpesviruses were preliminarily designated cytomegaloviruses. Although they will be most likely classified as members of the genus Muromegalovirus, we named them cytomegaloviruses in analogy to the name of the type species of the genus Muromegalovirus, Murine cytomegalovirus (MuHV-1). The numbering was according to the chronological order of discovery (e.g., Apodemus flavicollis cytomegalovirus 1).

Abbreviations use the first letter of the generic host name and the first three letters of the specific host name, followed by the abbreviation of the viral genus (e.g., AflaCMV1 [Apodemus flavicollis cytomegalovirus 1]).

All rodent gammaherpesviruses were preliminarily designated rhadinoviruses (e.g., Mus musculus rhadinovirus 1 [MmusRHV1]). The novel gammaherpesviruses described here may require the definition of additional genera within the Gammaherpesvirinae. This would result in changes of the provisional virus names.

Within a rodent species, DPOL sequences of less than 95% nucleic acid sequence identity were considered to be derived from different herpesvirus species, as indicated by consecutive numbering of the same virus name (e.g., BindRHV1, BindRHV2, BindRHV3, BindRHV4). Sequences of more than 95% identity were assigned to the same virus species and named identically.

Abbreviations and nucleotide sequence accession numbers.

Abbreviations and accession numbers for the sequences of published viruses are as follows. (i) Betaherpesvirinae, genus Cytomegalovirus: CeHV-8 (Cercopithecine herpesvirus 8) = rhesus monkey cytomegalovirus (complete genome [cg], accession number [acc.] NC 006150); HHV-5 (Human herpesvirus 5) = HCMV (human cytomegalovirus) (cg, acc. NC 001347). (ii) Betaherpesvirinae, genus Muromegalovirus: MuHV-1 (Murid herpesvirus 1) = MCMV (murine cytomegalovirus) (cg, acc. NC004065); MuHV-2 (Murid herpesvirus 2) = RCMV-M (rat cytomegalovirus strain Maastricht) (cg, acc. NC 002512). (iii) Betaherpesvirinae, genus Roseolovirus: HHV-6 (Human herpesvirus 6) = HHV-6A (cg, acc. NC 001664); HHV-7 (Human herpesvirus 7) (cg, acc. NC 001716). (iv) Betaherpesvirinae, proposed genus Proboscivirus: ElHV-1 (Elephantid herpesvirus 1) = EEHV (endotheliotropic elephant herpesvirus) (partial genome, acc. AF322977). (v) Betaherpesvirinae, unassigned: CaHV-2 (Caviid herpesvirus 2) = guinea pig cytomegalovirus (complete gB and DPOL genes, acc. L25706); TuHV-1 (Tupaiid herpesvirus 1) = tree shrew herpesvirus (cg, acc. NC 002794); SuHV-2 (Suid herpesvirus 2) = PCMV (porcine cytomegalovirus) (complete gB and DPOL genes, acc. AF268039). (vi) Gammaherpesvirinae, genus Lymphocryptovirus: CalHV-3 (Callitrichine herpesvirus 3) (cg, acc. NC 004367); HHV-4 (Human herpesvirus 4) = EBV (Epstein-Barr virus) (cg, acc. NC 007605). (vii) Gammaherpesvirinae, genus Rhadinovirus: AtHV-3 (Ateline herpesvirus 3) = HVA (herpesvirus ateles) (cg, acc. AF083424); BoHV-4 (Bovine herpesvirus 4) (cg, acc. NC 002665); HHV-8 (Human herpesvirus 8) = KSHV (Kaposi's sarcoma-associated herpesvirus) (cg, acc. NC 003409); MuHV-4 (Murid herpesvirus 4) = MHV-68 (murine gammaherpesvirus 68) (cg, acc. U97553); CeHV-17 (Cercopithecine herpesvirus 17) = RRV (rhesus monkey rhadinovirus) (cg, acc. NC 003401); SaHV-2 (Saimiriine Herpesvirus 2) = HVS (herpesvirus saimiri) (cg, acc. NC 001350). (viii) Gammaherpesvirinae, proposed genus Macavirus: AlHV-1 (Alcelaphine herpesvirus 1) (cg, acc. NC 002531); SuHV-3 (Suid herpesvirus 3) = PLHV-1 (porcine lymphotropic herpesvirus 1) (partial genome, acc. AF478169). (ix) Gammaherpesvirinae, proposed genus Percavirus: EHV-2 (Equine herpesvirus 2) (cg, acc. NC 001650). (x) Unassigned herpesviruses: MuHV-3 (Murid herpesvirus 3) = MTV (mouse thymic virus); MuHV-5 (Murid herpesvirus 5) = Microtus pennsylvanicus herpesvirus; MuHV-6 (Murid herpesvirus 6) = sand rat inclusion agent (for MuHV-3, MuHV-5, and MuHV-6, no sequences are available in public databases).

Abbreviations and accession numbers for sequences of the novel viruses described here are listed in Tables Tables33 and and44.

TABLE 3.
Novel rodent betaherpesviruses
TABLE 4.
Novel rodent gammaherpesviruses

RESULTS

General findings.

Individuals of M. musculus, R. norvegicus, and 14 other rodent species were captured in several locations in Germany, the United Kingdom, and Thailand. Correct species identification was confirmed for several individuals of each rodent species by cytochrome b PCR (data not shown). Blood and tissue samples (n = 1,132 [including 289 brain and ganglion samples]) were first analyzed with panherpesvirus PCR targeting the DPOL gene. When no amplimer or an insufficient amount of amplimer accumulated, the PCR was repeated under more-relaxed conditions, as described in Materials and Methods. Of the 1,132 samples, 301 (27%) gave rise to an amplimer of herpesvirus origin, as revealed by sequence analysis. In 219/1,132 samples (19%) and 82/1,132 samples (7%), gammaherpesvirus and betaherpesvirus sequences, respectively, were found. No alphaherpesvirus was detected. For the purposes of this report, all novel herpesviruses were tentatively named, as described in Materials and Methods, and listed with GenBank accession numbers in Tables Tables33 and and44.

Genetic characterizations of novel betaherpesviruses.

The betaherpesvirus DPOL sequences, found in 82 samples, originated from 19 different DPOL genes (Fig. (Fig.1C).1C). Two of them were identical to known rodent betaherpesvirus DPOL sequences from MCMV and RCMV-E (RCMV-M was not detected). They were found in M. musculus and R. norvegicus, respectively, in accordance with published data (5, 6, 16).

The remaining 17 partial DPOL genes revealed 46% to 76% identity to MCMV DPOL (44% to 87% identity to RCMV-E and RCMV-M DPOL, respectively; values not listed) in pairwise sequence comparisons and had G+C contents of 43% to 75% (MCMV, 65%; RCMV-E, 48%; RCMV-M, 71%) (Table (Table3).3). They apparently originated from 17 novel rodent betaherpesviruses.

Next, we extended the short partial DPOL sequences toward the 5′ end of the DPOL gene and beyond, into the 3′ end of the gB gene, to allow for a more robust phylogenetic analysis. In a first step, we targeted the gB gene with degenerate primers (Fig. (Fig.1A).1A). Using different nested-primer sets (Table (Table2),2), we amplified seven novel gB sequences (Fig. (Fig.1C).1C). Second, we aimed to connect the gB/DPOL sequence pairs by LD-PCR (Fig. (Fig.1B),1B), in order to confirm that the gB and DPOL sequences found in any sample originated de facto from the same virus genome. For six of the novel viruses, this approach was successful (Fig. (Fig.1C).1C). However, it failed for AterCMV1, either because the genome copy number was insufficient to allow for successful LD-PCR or because the gB and DPOL sequences from that single sample were derived indeed from different species of betaherpesviruses. We sequenced all LD-PCR products by primer walking, resulting in contiguous sequences of approximately 3.5 kbp for each novel virus. These encoded roughly 350 amino acids (aa) of gB and 750 aa of DPOL.

Phylogenetic analysis was performed with multiple alignments of (i) the short partial DPOL sequences (<200 bp), to include all novel viruses (n = 17), or (ii) concatenated gB-DPOL amino acid sequences (~1,100 aa [with gaps removed]) from all viruses for which LD-PCR was successful (n = 6). The first tree comprised all novel rodent viruses, the known rodent viruses MCMV, RCMV-M, and RCMV-E, and other known betaherpesviruses. Nearly all rodent viruses were found in one separate clade, consisting of four subclades: subclade I, MCMV, McerCMV1, and AflaCMV2; subclade II, RCMV-M, AflaCMV1, AsylCMV1, and BindCMV-4; subclade III, RCMV-E, RratCMV1, RratCMV2, RtioCMV1, RexuCMV1, and BindCMV1 to BindCMV3; subclade IV, AterCMV1, MglaCMV1, MagrCMV1, MarvCMV1, and OzibCMV1 (not shown). In the second tree, the same topology and higher statistical significance of branching were found (Fig. (Fig.22).

FIG. 2.
Phylogenetic analysis of novel rodent betaherpesviruses. A phylogenetic tree was constructed by using the amino acid sequences encoded by the gB-DPOL segments of the novel rodent betaherpesviruses (Table (Table3)3) and of known betaherpesviruses, ...

Remarkably, one virus (AflaCMV3) branched completely separately from all other rodent betaherpesviruses in both trees. In pairwise nucleic acid and amino acid sequence comparisons, it was most closely related to another rodent herpesvirus, the guinea pig betaherpesvirus (CaHV-2). In the phylogenetic tree, a clade with a multifurcation was obtained, comprising AflaCMV3, CaHV-2, TuHV-1, and three subclades (HCMV, MCMV, and HHV-6A, with their relatives, respectively). Therefore, its evolutionary relatedness remained somewhat uncertain (Fig. (Fig.2).2). To confirm that A. flavicollis is the natural host for AflaCMV3, 31 individuals were analyzed with primers (3838-s/3838-as) specific for the AflaCMV3 DPOL gene. Six spleen, lung, and kidney samples (about 20%) were positive (Table (Table33).

Genetic characterizations of novel gammaherpesviruses.

The gammaherpesvirus DPOL sequences, detected in 219 samples with degenerate primers, originated from 22 different DPOL genes. One of them was identical to the known MHV-68 DPOL gene and was found in three Apodemus species, namely, A. sylvaticus, A. flavicollis, and A. agrarius (but not in M. glareolus, from which MHV-68 was originally isolated [4]). The other partial DPOL genes appeared to originate from 21 as-yet-unknown rodent gammaherpesviruses. Upon analysis of their G+C contents and CpG dinucleotide suppression (12), they fell into two groups. Group I had a G+C content of 40% to 49% (MHV-68, 50%) and a clearly visible CpG suppression (like MHV-68). Group II had a G+C content of 58% to 70% and no CpG suppression (Table (Table4).4). Group I comprised MHV-68 and novel viruses of Apodemus flavicollis (AflaRHV1), Apodemus sylvaticus (AsylRHV1), Myodes glareolus (MglaRHV1), Microtus agrestis (MagrRHV1), Bandicota indica (BindRHV4), and Rattus tiomanicus (RtioRHV1). Group II comprised a novel rhadinovirus of Mus musculus (MmusRHV1) and those of Mus cervicolor (McerRHV1), Apodemus agrarius (AagrRHV1), Rattus norvegicus (RnorRHV1-2), Rattus rattus (RratRHV1-3), Rattus exulans (RexuRHV1-2), Rattus tiomanicus (RtioRHV2), Bandicota indica (BindRHV1-3), and Bandicota savilei (BsavRHV1). Interestingly, representatives of both groups were detected in the same rodent species, i.e., B. indica, R. tiomanicus, and A. agrarius.

Pairwise comparisons of partial DPOL sequences revealed 58% to 85% identity (group I) and 40% to 50% identity (group II) to the corresponding MHV-68 sequence. Both the group I and group II viruses revealed a slightly higher percentage of identity to HHV-8 than to EBV. Therefore, all were tentatively designated rhadinoviruses.

Phylogenetic analysis was performed with multiple alignments of (i) the short partial DPOL sequences of all novel gammaherpesviruses (n = 21) or (ii) concatenated gB-DPOL amino acid sequences (n = 8), with gaps removed. In both trees, two distantly related clades resulted. Clade I comprised MHV-68 and the other viruses of group I. Clade II included the novel virus of the house mouse (MmusRHV1) and the other group II viruses (Fig. (Fig.33).

FIG. 3.
Phylogenetic analysis of novel rodent gammaherpesviruses. A phylogenetic tree was constructed by using the amino acid sequences encoded by the gB-DPOL fragments of the novel rodent gammaherpesviruses (Table (Table4)4) and of known gammaherpesviruses, ...

MmusRHV1 and all other group II viruses clearly differed from MHV-68, because they revealed percentages of identity between 45% and 55% in comparison to MHV-68 on the nucleotide and amino acid levels. Beyond this, the phylogenetic placement of the group I and group II clades within the Gammaherpesvirinae was uncertain, even in the 1,100-aa tree (Fig. (Fig.33).

For a more meaningful phylogenetic analysis of MmusRHV1, we further extended the MmusRHV1 sequence from open reading frame 8 (ORF8), gB, through ORF7 and into ORF6, encoding the major DNA-binding protein. This was achieved with degenerate PCR targeting ORF6 and subsequent LD-PCR between ORF6 and ORF8. A final contiguous sequence of approximately 8 kbp was generated, extending from ORF6, MDBP, to ORF9, DPOL. A phylogenetic tree was constructed with >3,000 aa, including the EBV-like lymphocryptoviruses, the HHV-8-like rhadinoviruses, and the AlHV-1-like members of the proposed genus Macavirus. Again, MmusRHV1 branched distantly from MHV-68. A multifurcated clade was obtained, comprising MmusRHV1 and three subclades (Rhadinovirus, Percavirus, and Macavirus species) (Fig. (Fig.44).

FIG. 4.
Extended phylogenetic analysis of the novel virus MmusRHV1. A phylogenetic tree was constructed by using the amino acid sequences encoded by the MDBP-DPOL fragment of MmusRHV1 and of known gammaherpesviruses, available in the GenBank database. MCMV was ...

Finally, the occurrence of MmusRHV1 in the blood of free-living rodents was analyzed by specific PCR. For this purpose, 104 house mice were trapped in the United Kingdom, and samples were tested with nested PCR specific for the MmusRHV1 gB gene. Five samples (about 5%) were positive for MmusRHV1 (Table (Table4).4). In contrast, 26 wood mice (A. sylvaticus), 11 bank voles (M. glareolus), and 77 field voles (M. agrestis) from the United Kingdom were negative for MmusRHV1 in spleen samples (blood samples were not available).

DISCUSSION

Here, we present results of the first comprehensive search for alpha-, beta-, and gammaherpesviruses in M. musculus, R. norvegicus, and other rodents, using PCR approaches that target different conserved genes. The study was based on >1,000 samples, including 289 brain and ganglion samples. It revealed a plethora of novel beta- and gammaherpesviruses.

Amplification of the gB gene and subsequent LD-PCR, spanning the gB-DPOL segment, was successful for one-third (n = 14) of these novel viruses. The most likely reason for the missing amplification of the gB gene in the remaining viruses is that gB is not conserved enough to allow for the amplification of all vertebrate herpesvirus gB genes with a limited number of degenerate primer sets (as is possible with the DPOL gene). Therefore, binding with low specificity of one or more gB primers or a complete absence of binding may have occurred. In addition, several samples probably contained an insufficient genome copy number.

Seventeen novel rodent betaherpesviruses were identified. Sixteen of these clustered with MCMV and the RCMV strains RCMV-M and RCMV-E, forming a large, separate clade with four subclades (Fig. (Fig.2).2). This analysis also showed that RCMV-M and RCMV-E are separate species, in line with the remarkable differences in their individual gene contents, as reported previously (27, 2). In Peromyscus maniculatus (deer mouse), as well as in R. norvegicus and R. rattus, several cytomegaloviruses have been detected recently. It remains to be determined how these relate to the rodent viruses described here, because only short partial sequences of the ORFs of UL33 (P. maniculatus) and R87 (Rattus species) have been published (22, 18).

Among the novel betaherpesviruses, AflaCMV3 was unique because it did not cluster closely with the MCMV- and RCMV-related cytomegaloviruses, including AflaCMV1 and AflaCMV2, which originated from the same host (A. flavicollis). As soon as the isolation and propagation of AflaCMV3 in tissue culture succeeds, its repertoire of nonconserved genes can be compared with those of the human betaherpesviruses and assessed for its suitability in model studies.

No evidence of alphaherpesviruses in rodents was obtained. Several reasons may account for this failure. Their prevalence may be quite low, preventing detection in a limited number of samples, and, as their loci of latency are unknown, we may have missed them through our choice of specimens. In addition, the primers used for universal detection of herpesviruses may sufficiently bind to and amplify rodent alphaherpesvirus DPOL genes only in samples with high viral loads. It is possible that such samples might be obtained only under certain rare disease conditions. However, it is also possible that rodent alphaherpesviruses either never developed or became extinct earlier during herpesvirus evolution. Further studies are needed to clarify this issue.

We identified 21 novel rodent gammaherpesviruses. They clearly clustered into two different groups (I and II), as revealed by the differences in (i) their sequences, (ii) their G+C contents, and (iii) the presence of CpG suppression (Table (Table4),4), as well as (iv) their phylogeny (Fig. (Fig.3).3). The most interesting member of group II is MmusRHV1. It is the first gammaherpesvirus of M. musculus and was detected in 75 samples (31% of all tested samples) from 33 individuals of M. musculus in Germany and the United Kingdom but not in organs of the other rodent species. These data firmly indicate that MmusRHV1 is the first gammaherpesvirus that naturally infects M. musculus. The comparatively low frequency of MmusRHV1 detection in blood samples (roughly 5%) most likely reflects the biology of the virus. Virus may reside (and be detected) in the spleens of all infected animals but may be detected in only a small proportion of blood samples, as is the case for mice experimentally infected with MHV-68 (24).

Rowe and Capps (19) discovered a mouse virus that causes thymic necrosis in newborn mice (M. musculus). This virus exhibits T-cell tropism, persists in salivary glands, and could not be propagated in cell culture. It was named mouse thymic virus (MTV) and classified as MuHV-3. Our recent seroepidemiological studies have shown that MuHV-3 (like MCMV) has ubiquitous presence in free-living European house mice (1). We were therefore concerned that MmusRHV1 might in fact be MuHV-3. No sequences of MuHV-3 are available in public databases, but recently a partial DPOL sequence of MuHV-3 was amplified and found to be betaherpesvirus-like (R. S. Livingston, University of Missouri—Columbia, personal communication). Therefore, MmusRHV1 and MuHV-3 are completely different herpesvirus species.

MHV-68 infection of laboratory mice is extensively studied in the pursuit of insight into gammaherpesvirus pathogenesis. However, its natural hosts are M. glareolus (4) and several Apodemus species (3; also this study), the latter being in accordance with the close phylogenetic relationship of MHV-68 to AflaRHV1 and AsylRHV1 (Fig. (Fig.3).3). In addition, MHV-68 infection fails to reproduce all aspects of human gammaherpesvirus disease. Since laboratory mice are originally derived from M. musculus, MHV-68 has not been studied in its natural host. The investigation of MmusRHV1 (from M. musculus) or its close relative McerRHV1 (from M. cervicolor) in laboratory mice may result in data that reflect more reliably gammaherpesvirus infection in nature. In addition, it may facilitate the study of aspects of human gammaherpesvirus disease that are not visible in the MHV-68 model. MmusRHV1 and McerRHV1 branch distantly from MHV-68 within the Gammaherpesvirinae. Therefore, their content of individual (nonconserved) genes may differ considerably from that of MHV-68. Such genes represent the individual makeup of each herpesvirus and are the basis for their unique pathogenic properties. To characterize their genomes and study their biology, MmusRHV1 (and/or McerRHV1) will have to be isolated and propagated in tissue culture.

The rodent herpesviruses described here for the first time add significant data to herpesvirus phylogeny, and their further characterization will amend our understanding of herpesvirus biology. In particular, MmusRHV1, the first gammaherpesvirus to naturally infect M. musculus, and the related virus in M. cervicolor (McerRHV1), as well as the novel betaherpesvirus AflaCMV3, will be explored for potential as experimental tools for the study of beta- and gammaherpesvirus pathogenesis.

Acknowledgments

Rodents from Germany were kindly provided by Hans-Joachim Pelz (Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Nematologie und Wirbeltierkunde, Münster, Germany), Matthias Wenk (Landesforstanstalt, Eberswalde, Germany), Ronny Wolf (Institut für Zoologie, Universität Leipzig, Leipzig, Germany), Ralf-Udo Mühle (Universität Potsdam, Gülpe, Germany), the late Rolf Gattermann (Institut für Zoologie, Halle/Saale, Germany), Martin Hornung (Veterinäramt Radolfzell, Germany), Egon Splisteser (Rathenow, Germany), Paul Frank (Nauen, Germany), Georg Wolf, and Hans-Joachim Haupt (Alt-Ruppin, Germany). We kindly acknowledge the help of Sonja Liebmann (Robert Koch-Institut, Berlin, Germany), Martina Steffen, Heike Kubitza, Roswitha Mattis, Kirsten Tackmann, Silviana Schilling, Claudia Dettmer, and Robert Friedrich (Friedrich Loeffler-Institut, Wusterhausen, Germany), as well as the support of Franz J. Conraths (Friedrich Loeffler-Institut, Wusterhausen, Germany), Sonja Hartnack (Universität Bern, Switzerland), and Walter Bäumler (TU München, Germany). Rodent trapping and necropsy in Thailand was supported by Prasartthong Promkerd and Yuvaluk Khoprasert (Bangkok, Thailand).

Work in the United Kingdom was funded by a Royal Society (London) fellowship to J.P.S. and by the Veterinary Training and Research Initiative program, funded by the Department of Environment, Fisheries and Rural Affairs and the Higher Education Funding Council of England.

Footnotes

[down-pointing small open triangle]Published ahead of print on 16 May 2007.

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