Characterization of a novel wood mouse virus related to murid herpesvirus 4

Abstract

Two novel gammaherpesviruses were isolated, one from a field vole (Microtus agrestis) and the other from wood mice (Apodemus sylvaticus). The genome of the latter, designated wood mouse herpesvirus (WMHV), was completely sequenced. WMHV had the same genome structure and predicted gene content as murid herpesvirus 4 (MuHV4; murine gammaherpesvirus 68). Overall nucleotide sequence identity between WMHV and MuHV4 was 85 % and most of the 10 kb region at the left end of the unique region was particularly highly conserved, especially the viral tRNA-like sequences and the coding regions of genes M1 and M4. The partial sequence (71 913 bp) of another gammaherpesvirus, Brest herpesvirus (BRHV), which was isolated ostensibly from a white-toothed shrew (Crocidura russula), was also determined. The BRHV sequence was 99.2 % identical to the corresponding portion of the WMHV genome. Thus, WMHV and BRHV appeared to be strains of a new virus species. Biological characterization of WMHV indicated that it grew with similar kinetics to MuHV4 in cell culture. The pathogenesis of WMHV in wood mice was also extremely similar to that of MuHV4, except for the absence of inducible bronchus-associated lymphoid tissue at day 14 post-infection and a higher load of latently infected cells at 21 days post-infection.

Fig. 1.

PCR amplification of the coding regions of the genes M1 (a), M2 (b) and M3 (c) from viruses isolated from FV1, WM1, WM2, WM7 and WM8, in comparison with MuHV4. TG, Trigeminal ganglia; S, spleen.

Fig. 2.

Variation between the genome sequences of WMHV and MuHV4. The lower part of the panels represents the genome, commencing in the first panel at the start of U and ending in the last panel with one copy of TR, which is shown in a thicker format. Protein-coding regions are depicted by shaded arrows, with connecting introns indicated by white horizontal bars and genes encoding the tRNA-like genes (1–8) shown as arrowheads. Internal tandem repeats are represented by black horizontal bars. The upper part of each panel shows the nucleotide divergence (nd) calculated for a 100 nt window, shifted by increments of 3 nt. A nucleotide position was counted as divergent if it differed between the two sequences; insertions and deletions were not scored.

Fig. 3.

Divergence between the amino acid sequences of predicted protein-coding regions in WMHV, BRHV and MuHV4. The histogram illustrates sequence divergence (% non-identity) between the amino acid sequence of predicted protein-coding regions in WMHV and MuHV4 (grey bars, all coding regions) and WMHV and BRHV (black bars, coding regions up to ORF52).

Fig. 4.

Alignment of the predicted nucleotide sequences of the tRNA-like genes and miRNAs from MuHV4 and WMHV. A diagrammatic representation of the genomic region showing the relative positions of these non-coding RNAs is shown at the top. The positions of the M1–M3 ORFs and viral tRNA-like transcripts (t1–t8) are indicated by arrows. The positions of the miRNAs (miR-M1-1 to miR-M1-9) derived from primary transcripts of the tRNA-like RNAs are shown by vertical lines. Sequence alignments of the tRNA/miRNA transcripts are shown below. The sections processed to generate the tRNA-like molecules are shaded grey and pre-miRNAs are shaded blue. The positions of the A and B boxes of the RNA polymerase III promoters are shown by boxes, as are the positions of the processed miRNAs. The positions of the anti-codons in the tRNAs are shown in blue type. Differences between MuHV4 and WMHV are shown in red type. Data for MuHV4 are from Bowden et al. (1997) and Pfeffer et al. (2004).

Fig. 5.

Alignments of the predicted amino acid sequences of M1, M3 and M4 (a), M2 (b), ORF51 (c) and ORF73 (d). Each individual alignment consists of the sequences from MuHV4, WMHV and BRHV, with residues that differ from the consensus (or from each other in the case of ORF73) shaded grey. In (a), the alignments for M1, M3 and M4 are aligned with each other because these three proteins are related via the residues in bold type; each sequence contains a predicted signal peptide (lower case). In (b), the positions of PXXP motifs (P1–P9), tyrosine residues 120 and 129 and the CD8 CTL epitope (CTL) are indicated above the sequence. In (c), the bold residues indicate potential N-linked glycosylation sites in gp150 encoded by ORF51. In (d), the positions of the Brd4- and Brd2-interacting domains of the ORF73 protein are shown above the sequences.

Fig. 6.

Virological analyses of WMHV infection of wood mice. Wood mice (three per time point) were infected intranasally with 4×10⁵ p.f.u. MuHV4 or WMHV. Results are shown as means±sd; the asterisk represents a statistically significant difference between species (P<0.05). (a) Infectious virus recovered from the lung at 7 and 14 days p.i. Titres were measured by plaque assay on NIH3T3 cells. (b) Mean leukocyte numbers per spleen. (c) Infective centre (i.c.) assay of the level of latency in splenocytes. Infectious virus titres in the samples were analysed in parallel and were subtracted from the number of total infectious centres.

Fig. 7.

Cellular response to WMHV and MuHV4 infection in the lungs of wood mice at 14 days p.i. (a, b) Infection with MuHV4. (a) Intense peribronchial focal lymphocyte infiltration with evidence of lymphatic follicle formation (F). B, Bronchiole. (b) Focal perivascular B-cell infiltration with lymphatic follicle formation (F). A, Artery. (c, d) Infection with WMHV. (c) Moderate peribronchiolar focal lymphocyte infiltration (arrows). B, Bronchiole. (d) Artery with focal B-cell-dominated (CD45R-positive) perivascular lymphocyte infiltration (arrows). There is evidence of B-cell rolling and emigration (arrowheads). Sections were stained with haematoxylin and eosin (a, c), or for the B-cell marker CD45R, using the avidin–biotin–peroxidase complex method and Papanicolaou's haematoxylin counterstain (b, d). Bars, 50 μm (a); 20 μm (b–d).