Identification of novel rodent herpesviruses, including the first gammaherpesvirus of Mus musculus

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.

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 sites. (B) Long-distance nested PCR was performed with specific primers (open triangles). The amplified fragments are represented by dashed lines between the primer-binding sites. (C) The number of novel rodent herpesvirus contiguous sequences is specified. Their locations are depicted with thick solid lines. βHV, betaherpesviruses; γHV, gammaherpesviruses. At the top of the figure, the genomic locus spanning ORF6 (MDBP) to ORF9 (DPOL) is depicted with black arrows, indicating the direction of transcription. ORF numbering (ORF6 to ORF9) is adapted from the ORF nomenclature of herpesvirus saimiri (accession no. X64346). The start of the ruler corresponds with the first base of ORF6.

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 3) and of known betaherpesviruses, available in the GenBank database. A multiple alignment of 1,100 aa (concatenated) was analyzed by the neighbor-joining method of the program MacVector (version 8.0). A midpoint-rooted phylogram is shown. The branch length is proportional to evolutionary distance (scale bar). Results of bootstrap analysis (100-fold) are indicated at the nodes of the tree to the left of the first vertical divider. In addition, the alignment was analyzed with the program TREE-PUZZLE (version 5.0). The tree topology was the same (not shown). Support values, estimated by the quartet puzzling (QP) tree search and expressing QP reliability (in percentages), are indicated to the left of the second vertical divider. In addition, the alignment was analyzed by the maximum-likelihood method of the PHYLIP program package (version 3.65). The results of bootstrap analysis (100-fold) are presented to the right of the second vertical divider. Nodes that did not appear consistently in all analyses or that had bootstrap values mainly below 70% are masked with a thick black bar. The novel viruses are marked with black oval symbols. Herpesvirus genera and groups I to IV of the members of the genus Muromegalovirus, as well as the host orders, are indicated. Abbreviations of viruses are listed with full virus names in Materials and Methods and in Table 3.

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 4) and of known gammaherpesviruses, available in the GenBank database. MCMV was used as the outgroup. A multiple alignment of 1,100 aa (concatenated) was analyzed as described in legend to Fig. 2. The novel viruses are marked with black oval symbols. Herpesvirus genera and groups I to II of the rodent rhadinoviruses, as well as the host orders, are indicated. Abbreviations of viruses are listed with full virus names in Materials and Methods and in Table 4.

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 used as the outgroup. A multiple alignment of >3,000 aa (concatenated) was analyzed as described in the legend to Fig. 2. MmusRHV1 is indicated in boldface type. Herpesvirus genera are indicated.