Abstract
Novel members of the subfamily Gammaherpesvirinae, hosted by eight mammalian species from six orders (Primates, Artiodactyla, Perissodactyla, Carnivora, Scandentia, and Eulipotyphla), were discovered using PCR with pan-herpesvirus DNA polymerase (DPOL) gene primers and genus-specific glycoprotein B (gB) gene primers. The gB and DPOL sequences of each virus species were connected by long-distance PCR, and contiguous sequences of approximately 3.4 kbp were compiled. Six additional gammaherpesviruses from four mammalian host orders (Artiodactyla, Perissodactyla, Primates, and Proboscidea), for which only short DPOL sequences were known, were analyzed in the same manner. Together with available corresponding sequences for 31 other gammaherpesviruses, alignments of encoded amino acid sequences were made and used for phylogenetic analyses by maximum-likelihood and Bayesian Monte Carlo Markov chain methods to derive a tree which contained two major loci of unresolved branching details. The tree was rooted by parallel analyses that included alpha- and betaherpesvirus sequences. This gammaherpesvirus tree contains 11 major lineages and presents the widest view to date of phylogenetic relationships in any subfamily of the Herpesviridae, as well as the most complex in the number of deep lineages. The tree's branching pattern can be interpreted only in part in terms of the cospeciation of virus and host lineages, and a substantial incidence of the interspecies transfer of viruses must also be invoked.
PCR assays with degenerate primers have been used for over a decade for the amplification of unknown herpesvirus DNA polymerase (DPOL) gene sequences. These methods have the potential to detect virtually every mammalian, avian, or reptilian herpesvirus (7, 31). In fact, more than 100 novel herpesviruses have been discovered with the help of such universal PCR methods (4, 5, 8-11, 14, 17, 19, 27, 28, 33), and new phylogenetic herpesvirus lineages within the Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae subfamilies of the Herpesviridae have emerged (21-24). In addition, previous studies with great apes revealed evidence for two lymphocryptovirus (LCV) lineages and two rhadinovirus (RHV) lineages in the Lymphocryptovirus and Rhadinovirus genera of the Gammaherpesvirinae, leading to speculations that a second human LCV related to Epstein-Barr virus (9) and a second human RHV related to human herpesvirus 8 (HHV-8) (14) may exist.
Despite the tremendous accumulation of knowledge on the existence of hitherto unknown herpesviruses, only limited sequence information (i.e., a few hundred base pairs) became available in most of the cases. This information is sufficient to assess whether a virus is already known or novel and allows for assignment to a herpesvirus subfamily. However, a more precise phylogenetic analysis is often not possible, and more extensive sequence data are therefore desirable.
In the present study, we wanted to further extend insight into gammaherpesvirus (GHV) evolution by analyzing mammalian hosts from different orders and various geographic sites, either living in the wild or held in captivity. Members of mammalian orders for which no GHV was known were of particular interest. For the detection of unknown herpesviruses, we envisaged a bigenic PCR approach targeting two conserved genes, the DPOL and the glycoprotein B (gB) genes, separately. The advantage was that these genes are adjacent in GHV genomes, and long-distance PCR (LD-PCR) could be used to close the gap between the generated gB and DPOL sequence information, resulting in a final sequence of approximately 3.4 kbp. This bigenic approach had been used already for the amplification of novel betaherpesviruses and GHVs of primates, artiodactyls, bats, and rodents (3, 11, 25, 33). Here, we used it for a broad analysis of mammalian Gammaherpesvirinae and discovered novel GHVs in eight different host species from six mammalian orders.
MATERIALS AND METHODS
Sample collection and processing, PCR methods, and sequence analysis.
Tissue samples were collected from deceased animals which either had been housed in captivity or had been free ranging (Table 1). DNA was prepared with the QiaAmp tissue kit according to the manufacturer's instructions.
TABLE 1.
Mammalian species and novel GHVs
| Host order and species | Status and origin of host | Specimen(s) | Novel virus (abbreviation) | Most similar GHVa on the basis of:
|
|
|---|---|---|---|---|---|
| gB (% identity over 370 aab) | DPOL (% identity over 760 aab) | ||||
| Primates | |||||
| Gorilla (Gorilla gorilla) | Free ranging; Congo Zoological garden, Germany | Blood, lung tissue | Gorilla gorilla rhadinovirus 1 (GgorRHV-1) | PtroRHV-1 (94) | PtroRHV-1 (88) |
| Common squirrel monkey (Saimiri sciureus) | Free ranging; French Guiana German Primate Centre, Germany Zoological garden, Germany | Spleen, lung, brain, heart, liver, kidney, duodenum, colon, and lymph node tissues; blood | Saimiri sciureus gammaherpesvirus 2 (SsciGHV-2)c | TterGHV-1 (74) | TterGHV-1 (73) |
| Proboscidea Asian elephant (Elephas maximus) | Zoological garden, Germany | Oral swabs | Elephas maximus gammaherpesvirus 1 (EmaxGHV-1) | PLHV-3 (48) | PLHV-2 (53) |
| Carnivora | |||||
| Spotted hyena (Crocuta crocuta) | Free ranging; Tanzania | Lung tissue, blood | Crocuta crocuta gammaherpesvirus 1 (CcroGHV-1) | EzebGHV-1 (76) | EzebGHV-1 (80) |
| Lion (Panthera leo) | Free ranging; Tanzania | Lung tissue, blood | Panthera leo gammaherpesvirus 1 (PleoGHV-1) | RRV (60) | BoHV-4 (61) |
| Artiodactyla | |||||
| Pygmy hippopotamus (Hexaprotodon liberiensis) | Zoological garden, Germany | Blood | Hexaprotodon liberiensis gammaherpesvirus 1 (HlibGHV-1) | AlHV-1 (60) | PLHV-3 (65) |
| Alpine chamois (Rupicapra rupicapra) | Free ranging; Switzerland | Lung, liver, heart, and brain tissues | Rupicapra rupicapra gammaherpesvirus 1 (RrupGHV-1) | OvHV-2 (91) | OvHV-2 (88) |
| Bearded pig (Sus barbatus) | Zoological garden, Singapore | Blood | Sus barbatus rhadinovirus 1 (SbarRHV-1) | BbabGHV-1 (71) | BbabGHV-1 (77) |
| Babirussa (Babyrousa babyrussa) | Zoological garden, Indonesia | Blood | Babyrousa babyrussa rhadinovirus 1 (BbabRHV-1) | SbarRHV-1 (71) | SbarRHV-1 (77) |
| Perissodactyla | |||||
| Mountain zebra (Equus zebra) | Free ranging; Namibia | Blood | Equus zebra gammaherpesvirus 1 (EzebGHV-1) | CcroGHV-1 (76) | CcroGHV-1 (80) |
| Black rhinoceros (Diceros bicornis) | Zoological garden, Germany | Blood | Diceros bicornis gammaherpesvirus 1 (DbicGHV-1) | BoHV-4 (79) | BoHV-4 (87) |
| South American tapir (Tapirus terrestris) | Zoological garden, Germany | Blood | Tapirus terrestris gammaherpesvirus 1 (TterGHV-1) | SsciGHV-2 (74) | SsciGHV-2 (73) |
| Scandentia | |||||
| Tree shrew (Tupaia belangeri) | German Primate Centre, Germany | Lymph node tissue | Tupaia belangeri gammaherpesvirus 1 (TbelGHV-1) | HVA (51) | HVS (63) |
| Eulipotyphla | |||||
| Common shrew (Sorex araneus) | Free ranging; Germany | Spleen tissue | Sorex araneus gammaherpesvirus 1 (SaraGHV-1) | EmaxGHV-1 (47) | TbelGHV-1 (53) |
PtroRHV-1, Pan troglodytes rhadinovirus 1; PLHV-3, porcine lymphotropic herpesvirus 3; RRV, rhesus monkey rhadinovirus; BoHV-4, bovine herpesvirus 4; AlHV-1, alcelaphine herpesvirus 1; OvHV-2, ovine herpesvirus 2; BbabGHV-1, Babyrousa babyrussa gammaherpesvirus 1; HVA, herpesvirus ateles. Other abbreviations are as given in the table.
Approximate length. aa, amino acids.
Designated gammaherpesvirus 2 because one GHV of Saimiri sciureus is already known (HVS; GenBank accession no. X64346).
Panherpesvirus consensus PCR for the amplification of 160 to 181 bp (excluding primer-binding sites) of the DPOL gene was carried out as described previously (3). Samples that did not yield an amplification product were rerun 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 the polymerase was increased twofold.
For the amplification of the gB genes of as-yet-unknown GHVs, two degenerate, deoxyinosine-containing primer sets (GH1 and GH2) (Table 2) were used. The primer sequences were deduced from the gB gene sequences of equine herpesvirus 2 and rhesus monkey rhadinovirus (Table 3), respectively. PCR was carried out at an annealing temperature of 46°C under the conditions described for the DPOL gene.
TABLE 2.
Primers for amplification of the gB gene
| Primer set abbreviation | Name of primera | Sequenceb (5′ to 3′) | Approximate product length (bp) |
|---|---|---|---|
| GH1 | 2759s | CCTCCCAGGTTCARTWYGCMTAYGA | 700 |
| 2762as | CCGTTGAGGTTCTGAGTGTARTARTTRTAYTC | ||
| 2760s | AAGATCAACCCCAC(N/I)AG(N/I)GT(N/I)ATG | 500 | |
| 2761as | GTGTAGTAGTTGTACTCCCTRAACAT(N/I)GTYTC | ||
| GH2 | 3029s | CCCAGTTGCARTWYGC(N/I)TAYGA | 420 |
| 3033as | GCCAGGCGTTGCGT(N/I)TARTARTTRTA | ||
| 3031s | CAAGATTAACCCCAC(N/I)AG(N/I)GT(N/I)ATG | 350 | |
| 3032as | TTGCGTGTAGTAGTTGTAYTC(N/I)CTRAACAT |
s, sense; as, antisense.
I, inosine.
TABLE 3.
Viruses, abbreviations, and accession numbers
| Virus name (sequences analyzed) | Abbreviation | GenBank accession no. |
|---|---|---|
| Viruses from this study (gB and DPOL sequences) | ||
| Babyrousa babyrussa rhadinovirus 1 | BbabRHV-1 | AY177146 |
| Crocuta crocuta gammaherpesvirus 1 | CcroGHV-1 | DQ789371 |
| Diceros bicornis gammaherpesvirus 1 | DbicGHV-1 | AY197560 |
| Elephas maximus gammaherpesvirus 1 | EmaxGHV-1 | EU085379 |
| Equus zebra gammaherpesvirus 1 | EzebGHV-1 | AY495965 |
| Gorilla gorilla rhadinovirus 1 | GgorRHV-1 | AY177144 |
| Hexaprotodon liberiensis gammaherpesvirus 1 | HlibGHV-1 | AY197559 |
| Panthera leo gammaherpesvirus 1 | PleoGHV-1 | DQ789370 |
| Rupicapra rupicapra gammaherpesvirus 1 | RrupGHV-1 | DQ789369 |
| Saimiri sciureus gammaherpesvirus 2 | SsciGHV-2 | AY138584 |
| Sorex araneus gammaherpesvirus 1 | SaraGHV-1 | EU085380 |
| Sus barbatus rhadinovirus 1 | SbarRHV-1 | AY177147 |
| Tapirus terrestris gammaherpesvirus 1 | TterGHV-1 | AF141887 |
| Tupaia belangeri gammaherpesvirus 1 | TbelGHV-1 | AY197561 |
| Previously described viruses (complete and partial genome sequences) | ||
| Alcelaphine herpesvirus 1 | AlHV-1 | AF005370 |
| Bovine herpesvirus 4 | BoHV-4 | AF318573 |
| Callitrichine herpesvirus 3 | CalHV-3 | AF319782 |
| Epstein-Barr virus | EBV | AY037858 |
| Equine herpesvirus 2 | EHV-2 | NC 001650 |
| Herpesvirus ateles | HVA | AF083424 |
| Herpesvirus saimiri | HVS | X64346 |
| Human herpesvirus 8 | HHV-8 | U75698 |
| Macaca fuscata rhadinovirus | MfusRHV | NC_007016 |
| Murid herpesvirus 4, or murine gammaherpesvirus 68 | MuHV-4 | U97553 |
| Ovine herpesvirus 2 | OvHV-2 | NC_007646 |
| Rhesus monkey lymphocryptovirus | RLV | X00784 |
| Rhesus monkey rhadinovirus | RRV | AF029302 |
| Porcine lymphotropic herpesvirus 1 | PLHV-1 | AF478169 |
| Porcine lymphotropic herpesvirus 2 | PLHV-2 | AY170317 |
| Porcine lymphotropic herpesvirus 3 | PLHV-3 | AY170316 |
| Previously described viruses (gB and DPOL sequences) | ||
| Apodemus flavicollis rhadinovirus 1 | AflaRHV-1 | DQ821580 |
| Bandicota indica rhadinovirus 4 | BindRHV-4 | EF128043 |
| Bandicota savilei rhadinovirus 1 | BsavRHV-1 | DQ821581 |
| Bat gammaherpesvirus 1 | BatGHV-1 | DQ788623 |
| Bat gammaherpesvirus 4 | BatGHV-4 | DQ788626 |
| Bat gammaherpesvirus 5 | BatGHV-5 | DQ788629 |
| Caprine herpesvirus 2 | CpRHV-2 | AF283477 |
| Clethrionomys glareolus rhadinovirus 1 | CglaRHV-1 | AY854169 |
| Herpesvirus of badger | BadHV | AF376034 |
| Macaca fascicularis rhadinovirus 1 | MfasRHV-1 | AY138583 |
| Macaca fascicularis rhadinovirus 2 | MfasRHV-2 | EU085377 |
| Mus cervicolor rhadinovirus 1 | McerRHV-1 | DQ821582 |
| Mus musculus rhadinovirus 1 | MmusRHV-1 | AY854167 |
| Pan troglodytes rhadinovirus 1 | PtroRHV-1 | AY138585 |
| Pan troglodytes rhadinovirus 2 | PtroRHV-2 | EU085378 |
Nested LD-PCR was performed with the TaKaRa-Ex PCR system according to the instructions of the manufacturer (Takara Bio Inc., Japan) by using virus-specific primers (not listed). For the second round, 1 μl of the first-round product was used as a template. Cytochrome b gene (cytB) sequences were amplified as described previously (11). PCR product purification and direct sequencing with dye terminator chemistry, as well as nucleotide sequence analysis and amino acid sequence prediction, were performed as described previously (13).
Phylogenetic analyses.
Analyses were carried out at the level of encoded amino acid sequences. Sets of amino acid sequences for DPOL and gB from partial gene sequences were aligned using ClustalW (30). Positions in alignments that had missing characters in any sequence and regions considered too divergent for justified aligning were removed before the alignments were used for phylogenetic analyses.
Measures of amino acid sequence divergence used the matrix of Jones et al. (15). The PHYLIP package (12) was applied in a preliminary evaluation of trees by the neighbor-joining (NJ) method with bootstrap values (1,000 replicates). The PAML package (version 3.15) (34) was employed in making maximum-likelihood (ML) evaluations of amino acid sequence alignments for sets of candidate trees to examine aspects of branching patterns, with a discrete gamma distribution of five classes of substitution rates across sites and with the scoring of output trees by the RELL method (16).
Trees were also derived from alignments by Bayesian analysis using Monte Carlo Markov chains (BMCMC) with MrBayes version 3.1 (26). DPOL and gB sequences were treated in separate partitions with independent parameters, the rate model specified a discrete gamma distribution of four classes of substitution rates plus a class of invariant sites, and runs were for one million generations with sampling every 100th generation. The program's defaults were used for other parameters, including the specification of priors and two independent runs of one cold and three heated chains. At least 5,001 (out of 10,001) samples were discarded as burn-in.
Provisional nomenclature and abbreviations.
The viruses from which the de novo detected sequences originated were named after the host species and the herpesvirus subfamily to which the virus was tentatively assigned, for example, Sorex araneus gammaherpesvirus 1. They are listed with their abbreviations and corresponding GenBank accession numbers in Table 3, together with the names of the previously described viruses and the accession numbers of the sequences that were analyzed for comparison.
Nucleotide sequence accession numbers.
The nucleotide sequences of the novel herpesviruses have been deposited in GenBank under the following accession numbers: Crocuta crocuta gammaherpesvirus 1, DQ789371; Diceros bicornis gammaherpesvirus 1, AY197560; Hexaprotodon liberiensis gammaherpesvirus 1, AY197559; Panthera leo gammaherpesvirus 1, DQ789370; Rupicapra rupicapra gammaherpesvirus 1, DQ789369; Saimiri sciureus gammaherpesvirus 2, AY138584; Sorex araneus gammaherpesvirus 1, EU085380; and Tupaia belangeri gammaherpesvirus 1, AY197561.
RESULTS AND DISCUSSION
Bigenic de novo detection of novel GHVs.
Mammals from seven different orders from Africa, Asia, Europe, and South America, either living in the wild or held in captivity, were investigated for the presence of GHVs. Blood and tissue specimens from deceased individuals were collected, and species comprised the Asian elephant (Elephas maximus; order Proboscidea), the gorilla (Gorilla gorilla; order Primates), the squirrel monkey (Saimiri sciureus; order Primates), the pygmy hippopotamus (Hexaprotodon liberiensis; order Artiodactyla), the black rhinoceros (Diceros bicornis; order Perissodactyla), the Alpine chamois (Rupicapra rupicapra; order Artiodactyla), the spotted hyena (Crocuta crocuta; order Carnivora), the lion (Panthera leo; order Carnivora), the tree shrew (Tupaia belangeri; order Scandentia), and the common shrew (Sorex araneus; order Eulipotyphla [formerly Insectivora]) (Table 1).
Before assays for herpesviruses, the authenticity of the specimens was checked by the amplification and sequencing of roughly 0.3 kbp of the cytB gene. Species identification by means of cytB gene sequencing is a commonly used technique (20). Here, the morphological species determination was confirmed for all animals (data not shown).
To detect herpesvirus sequences, we first performed a pan-herpesvirus DPOL PCR. In blood and/or tissue samples of each mammalian species (except the gorilla and Asian elephant), a novel DPOL sequence was found (data not shown), and by comparison with known herpesvirus sequences, each sequence was determined to originate from an unknown GHV (Table 1). In the gorilla and elephant samples, sequences were found which had been published recently by others (18, 32).
For the detection of the gB gene of each novel GHV, two degenerate PCR sets were used (sets GH1 and GH2) (Table 2). With either the GH1 set or the GH2 set, gB sequences from all GHV-positive specimens were amplified (data not shown). The primer sets GH1 and GH2 were also used to amplify gB sequences of four GHVs for which we had already published short DPOL sequences (Table 1). These GHVs originated from the following hosts: Equus zebra and Tapirus terrestris (order Perissodactyla) (7) and Sus barbatus and Babyrousa babyrussa (order Artiodactyla) (10).
In total, partial gB and DPOL sequences for 14 GHVs were now available. To confirm that the two members of each of the 14 gB-DPOL sequence pairs did in fact originate from the same virus genome, we chose specific nested primers for each virus, with the sense primers located in the gB gene and the antisense primers located in the DPOL gene. By using the TaKaRa-Ex PCR kit, fragments of 2.7 to 3 kbp from all specimens were obtained and sequenced by primer walking (primers are not listed). The sequences of these fragments overlapped the initial gB and DPOL sequences without mismatches. Finally, contiguous sequences of about 3.4 kbp for all 14 GHVs were successfully compiled. The sequences were subjected to open reading frame and subsequent BLAST analyses. Partial open reading frames of gB and DPOL genes in each sequence were identified (Table 1).
The 14 GHVs were detected in specimens from hosts from seven orders (Primates, Proboscidea, Artiodactyla, Perissodactyla, Carnivora, Scandentia, and Eulipotyphla), and 8 GHVs were previously unknown. The gB genes of five novel GHVs (EmaxGHV-1, Tupaia belangeri gammaherpesvirus 1, Sorex araneus gammaherpesvirus 1, Panthera leo gammaherpesvirus 1, and Hexaprotodon liberiensis gammaherpesvirus 1) were only roughly 60% or less identical to those GHV genes which were used for the design of the degenerate gB primers. These findings demonstrate for the first time that a limited number of degenerate gB primers is sufficient for the universal detection of mammalian GHVs and that the primers can be designed on the basis of a small number of GHV sequences. In particular, the primer set GH1 (primers 2759 to 2762) was a powerful tool in the present study, as well as in several earlier investigations (10, 25, 33).
In previous studies using the same techniques of gB and DPOL gene amplification, LD-PCR was sometimes not successful (11, 33). This failure may have resulted from the reduced sensitivity of LD-PCR compared to that of a short-fragment standard PCR, sometimes causing a negative LD-PCR result for specimens with low virus copy numbers. In addition, a negative LD-PCR result can be attributed to the fact that a gB sequence and a DPOL sequence, although amplified from the same specimen, may not originate from the same virus genome, because individuals are often (latently) infected with more than one herpesvirus. Such complex situations, i.e., infections of single animals with similar GHVs, were observed during a previous analysis of multivirus-infected chimpanzees and macaques. In that study, oligonucleotides with locked nucleic acid substitutions were used to specifically inhibit the PCR amplification of a GHV sequence, thereby enabling the amplification of a second, different GHV sequence from the same specimen with the same degenerate primers (25). In the present study, the nested LD-PCR was successful for all gB-DPOL sequence pairs analyzed. This may indicate that additional GHVs were not present in these samples.
Phylogenetic relationships in the Gammaherpesvirinae.
Novel sequence data were available for the 14 viruses described above plus 31 other GHVs for which information has been reported previously in the literature. Alignments of partial sequences of DPOL and gB, which contained 664 and 285 amino acid residues, respectively, after the removal of loci with gaps and unalignable regions, were made for these 45 species. Based on previous experience (21, 22), we regarded this data set to be of adequate size to resolve most details of GHV phylogeny. In order to locate the GHV root position, a second pair of alignments was made that additionally contained data for 17 alphaherpesviruses and 12 betaherpesviruses. Final DPOL and gB lengths for this pair were 523 and 238 residues, respectively. DPOL and gB alignments were concatenated for phylogenetic analyses. Phylogenetic trees were derived by two separate routes: first by the NJ method, followed by ML analysis to examine particular aspects, and second by BMCMC.
The NJ method with bootstrap values was applied to define a preliminary tree. In conjunction with previous work (23), this approach served to identify 11 clades with good confidence (Table 4). Three of these (clades 1, 3, and 6) correspond to genera currently defined (Lymphocryptovirus) or awaiting approval by the International Committee on Taxonomy of Viruses (Macavirus and Percavirus), and four additional clades (7 to 10) contain representatives of the genus Rhadinovirus. The remaining four clades (2, 4, 5, and 11) apparently do not belong to any of the currently defined genera. Specific aspects of branching patterns, concerning mostly deep branches, were then pursued by ML analysis as described previously (23). This process gave a top-scoring tree, together with runner-up variants that indicated uncertainty about certain branching details. Separately, the BMCMC method was employed and gave a single tree with high posterior probabilities associated with all branches. The NJ-plus-ML approach and the BMCMC approach both yielded the same top-scoring tree, and this tree is shown in Fig. 1A. It is known that BMCMC can yield overpositive results (1, 6, 29), and we have dealt with this possibility by producing an alternative tree (shown in Fig. 1B) in which four loci where the ML analyses gave uncertain branching assignments have been collapsed into multifurcations. We regard the latter tree as the more conservative, secure interpretation of the analyses. Two of the unresolved branching loci, in the Macavirus (alcelaphine herpesvirus 1)-like clade (clade 3) and in the HHV8-like clade (clade 8), we consider to reflect simply the relatively short alignment length available plus the closeness of the species involved and to be likely to be fully resolvable with more input data. The other two are deeper in the tree and may represent fundamental properties of the phylogeny.
TABLE 4.
Major clades of GHVs
| Clade no. | Clade name(s)a | Member(s) of clade |
|---|---|---|
| 1 | Lymphocryptovirus (genus) | EBV, RLV, CalHV-3 |
| 2 | EmaxGHV-1 | EmaxGHV-1 |
| 3 | Macavirus (genus) | OvHV-2, RrupGHV-1, CpRHV-2, AlHV-1, PLHV-1, PLHV-2, PLHV-3, HlibGHV-1 |
| 4 | MmusRHV-1 like | MmusRHV-1, McerRHV-1, BsavRHV-1 |
| 5 | BatGHV-1 like | BatGHV-1, BatGHV-4, BatGHV-5 |
| 6 | Percavirus (genus) | CcroGHV-1, EzebGHV-1, EHV-2, BadHV, SaraGHV-1 |
| 7 | Rhadinovirus (genus), HVS like | HVS, HVA, TbelGHV-1 |
| 8 | Rhadinovirus, HHV-8 like | HHV-8, PtroRHV-1, GgorRHV-1, MfasRHV-1, PtroRHV-2, RRV2695, MfusRHV-1, MfasRHV-2 |
| 9 | Rhadinovirus, BoHV-4 like | BoHV-4, DbicGHV-1, BbabRHV-1, SbarRHV-1, PleoGHV-1 |
| 10 | Rhadinovirus, MuHV-4 like | MuHV-4, AflaRHV-1, BindRHV-4, CglaRHV-1 |
| 11 | TterGHV-1 like | TterGHV-1, SsciGHV-2 |
FIG. 1.
Phylogenetic trees for the Gammaherpesvirinae. Panel A shows the phylogenetic tree as obtained by BMCMC (equivalent to the top-scoring tree from the NJ-ML analysis). The root position was determined by analyses using alpha- and betaherpesvirus sequences to provide an out-group. Three closely related viruses (rhesus monkey rhadinovirus 2695, Macaca fascicularis rhadinovirus 1 [MfasRHV-1], and MfasRHV-2) are shown together as rhesus monkey rhadinovirus (RRV) isolates. The major lineages are indicated with numbers 1 to 11. Panel B shows the same tree collapsed to form multifurcations at four locations of uncertainty in the NJ-ML analysis. The divergence scale at the bottom, indicating the number of substitutions per site, applies to both panels. EBV, Epstein-Barr virus; RLV, rhesus monkey lymphocryptovirus; CalHV-3, callitrichine herpesvirus 3; OvHV-2, ovine herpesvirus 2; RrupGHV-1, Rupicapra rupicapra gammaherpesvirus 1; CpRHV-2, caprine rhadinovirus 2; AlHV-1, alcelaphine herpesvirus 1; PLHV-1, porcine lymphotropic herpesvirus 1; HlibGHV-1, Hexaprotodon liberiensis gammaherpesvirus 1; McerRHV-1, Mus cervicolor rhadinovirus 1; BsavRHV-1, Bandicota savilei rhadinovirus 1; CcroGHV-1, Crocuta crocuta gammaherpesvirus 1; EzebGHV-1, Equus zebra gammaherpesvirus 1; EHV-2, equine herpesvirus 2; BadHV, badger herpesvirus; SaraGHV-1, Sorex araneus gammaherpesvirus 1; HVA, herpesvirus ateles; TbelGHV-1, Tupaia belangeri gammaherpesvirus 1; PtroRHV-1, Pan troglodytes rhadinovirus 1; GgorRHV-1, Gorilla gorilla rhadinovirus 1; BoHV-4, bovine herpesvirus 4; DbicGHV-1, Diceros bicornis gammaherpesvirus 1; BbabRHV-1, Babyrousa babyrussa rhadinovirus 1; SbarRHV-1, Sus barbatus rhadinovirus 1; PleoGHV-1, Panthera leo gammaherpesvirus 1; AflaRHV-1, Apodemus flavicollis rhadinovirus 1; BindRHV-4, Bandicota indica rhadinovirus 4; CglaRHV-1, Clethrionomys glareolus rhadinovirus 1; SsciGHV-2, Saimiri sciureus gammaherpesvirus 2.
The location of the root of the GHV clade was investigated by both BMCMC and ML methods using input alignments for a total of 74 viruses from all three subfamilies and utilizing the alpha- and betaherpesvirus subtree as the out-group for the GHV subtree. BMCMC placed the GHV root on the branch between the LCV clade (clade 1) and the rest. For ML analysis, the input set of trees comprised the multifurcated GHV tree described above with the subtree of the alpha- and betaherpesviruses (as previously determined) (24) inserted successively into every branch, giving 79 input trees in all. ML evaluation of this set yielded the same root position as BMCMC. The root locus determined in this way was then transferred to the 45 GHV trees and is the basis of the root shown in Fig. 1A and B.
A detailed examination of phylogenetic relationships in the Gammaherpesvirinae was previously carried out using alignments of amino acid sequences corresponding to up to 28 genes from 12 virus species (23), that is, far fewer species than were included in the present study but much longer input alignments. Figure 2A shows a summary tree from that investigation, with seven major lineages, of which four were placed in a clade whose branching details could not be confidently resolved (this clade was termed the multifurcated clade [MF clade]). At the time of that analysis, additional species for which smaller but usable sets of sequence data were available all fell into one of these seven major lineages. The present analysis assigned more species to all of these seven lineages, except for the LCV lineage (clade 1). Figure 2B shows the equivalent tree for the present analysis, with four additional major lineages (EmaxGHV-1, Mus musculus rhadinovirus 1 [MmusRHV-1] like, bat gammaherpesvirus 1 [BatGHV-1] like, and TterGHV-1 like). The branching pattern of the older tree remains unchanged, except that we now assign additional structure to what was the MF clade, with the herpesvirus saimiri (HVS)-like lineage branching first. Also in this region, we note that the previously uniquely extended terminal branch for murid herpesvirus 4 (MuHV-4) (Fig. 2A) is now unremarkable within a heterogeneity of branch end points (Fig. 1, clade 10).
FIG. 2.
Major lineages in the Gammaherpesvirinae. Panel A shows major features of the GHV tree derived by McGeoch et al. (23), with seven lineages. Panel B reduces the tree in Fig. 1B to 11 major lineages (equivalent to the clades in Table 4), with the mammalian orders to which the hosts of each lineage belong listed. In both panels, the extents of regions of multiple branching near tips are shown as heavy lines. Clades whose branching details could not be confidently resolved are termed MF. EHV-2, equine herpesvirus 2; BoHV-4, bovine herpesvirus 4; Perisso., Perissodactyla; Artio., Artiodactyla; Carni., Carnivora; Eulipo., Eulipotyphla.
As an aid to discussion, we designate the successor of the MF clade (i.e., excluding the HVS-like lineage and including the novel TterGHV-1-like lineage) MF1 (Fig. 2B) and we designate as MF2 the novel, deeper, unresolved clade composed of the MmusRHV-1-like, BatGHV-1-like, Percavirus, and HVS-like clades plus the MF1 clade (Fig. 2B). The four new major lineages spring from origins across the tree. The EmaxGHV-1 line originates deep in the tree, with the second deepest branch point after the LCV; MmusRHV-1 like and BatGHV-1 like are lineages in the large MF2 clade, while the TterGHV-1-like lineage is a new line within MF1. In total, 11 major lineages are now defined and numbered in Table 4. Seven lineages contain multiple viruses with hosts all falling within a single mammalian order, and another four contain viruses with hosts falling into two, three, or four orders, as indicated in Fig. 2B. In addition, lineage 11 contains GHVs (TterGHV-1 and Saimiri sciureus gammaherpesvirus 2) of two hosts (Tapirus terrestris and Saimiri sciureus) belonging to different mammalian superorders (Laurasiatheria and Euarchontoglires). This finding may suggest that for viruses in the latter four clades, distant interspecies transfer has been an important part of their evolutionary history. In accord with this assumption, a herpesvirus previously isolated from a shrew (2) has turned out to be a MuHV-4-like GHV (A. J. Davison, personal communication).
In earlier analyses of Herpesviridae phylogeny, it was possible to discern within each of the subfamilies a substantial degree of congruence between the tree branching pattern and the corresponding pattern for lineages of the mammalian hosts, and this congruence was taken to indicate extensive cospeciation of herpesviruses and hosts (see reference 24). In these interpretations, the Gammaherpesvirinae presented the most complicated (and, thus, the least satisfactory) case, and the applicability of cospeciational interpretation declines further with the extensive detail now available for the GHV tree. First, among the 11 lineages defined, only those for which the host species come solely or predominantly from a single mammalian order can be of primary utility. Second, the deeper branching details of the tree prove rather unproductive for constructing any unified coevolutionary correspondence across host lineages. In particular, the two deepest distinct lineages, i.e., those of LCV and EmaxGHV-1, are not simultaneously compatible with a single cospeciational scheme, and in the MF2 clade, the unresolved nodes for major lineages do not enable any compelling interpretation. On the other hand, clear dispersed examples of cospeciation can be seen in the terminal branchings within major lineages. Most notably, these include the LCVs (clade 1), the macaviruses (clade 3), two lineages of rodent GHVs (MmusRHV-1 like and MuHV-4 like; clades 4 and 10, respectively), and the two distinct sublineages of HHV-8-like viruses (clade 8) in the MF1 clade. The Macavirus clade was previously used as a primary calibration point in importing a time scale from host divergence dates (23), with the divergence of swine and ruminant viruses dated as 63.8 million years before the present, and we regard that application as remaining valid. In summary, there are substantial indications in the GHV tree of evolution both by cospeciation with host lineages and by transfer between widely distinct hosts.
Overall, our analysis makes Gammaherpesvirinae phylogeny the most extensively characterized among those of the three subfamilies in terms of the number of viruses included and also the most complex, considering the number of distinct deep lineages and the details of relationships among them.
Acknowledgments
We thank Sonja Liebmann, Ute Buwitt, and Julia Tesch for excellent technical assistance. The supply of samples from Primates, Artiodactyla, Perissodactyla, Scandentia, and Eulipotyphla by Kerstin Mätz-Rensing (Deutsches Primatenzentrum, Göttingen, Germany), Fabian Leendertz (Robert Koch-Insitut, Berlin, Germany), Andreas Ochs (Berlin Zoological Gardens, Berlin, Germany), Werner Schempp (Institut für Humangenetik und Anthropologie, Universität Freiburg, Freiburg, Germany), and Ingolf Stodian (Nationalparkamt Vorpommersche Boddenlandschaft, Schaprode, Germany) is gratefully acknowledged. We are indebted to Tanzania National Parks, the Tanzania Wildlife Research Institute, the Ngorongoro Conservation Area Authority, the Tanzania Commission for Science and Technology, and Tanzania government ministries for permission to undertake research. The Tanzania National Parks and Tanzania Wildlife Research Institute veterinary units and all members of the Serengeti Viral Transmission Dynamics and Lion projects are especially thanked for assistance with sample collection.
T.L. was supported by the Royal (Dick) School of Veterinary Studies, University of Edinburgh, and the joint National Institutes of Health/National Science Foundation Ecology of Infectious Diseases Program under grant no. NSF/DEB0225453 (field work).
Footnotes
Published ahead of print on 23 January 2008.
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