Molecular evidence for distinct genotypes of monkey B virus (herpesvirus simiae) which are related to the macaque host species

Abstract

Although monkey B virus (herpesvirus simiae; BV) is common in all macaque species, fatal human infections appear to be associated with exposure to rhesus macaques (Macaca mulatta), suggesting that BV isolates from rhesus monkeys may be more lethal to nonmacaques than are BV strains indigenous to other macaque species. To determine if significant differences that would support this supposition exist among BV isolates, we compared multiple BV strains isolated from rhesus, cynomolgus, pigtail, and Japanese macaques. Antigenic analyses indicated that while the isolates were very closely related to one another, there are some antigenic determinants that are specific to BV isolates from different macaque species. Restriction enzyme digest patterns of viral DNA revealed marked similarities between rhesus and Japanese macaque isolates, while pigtail and cynomolgus macaque isolates had distinctive cleavage patterns. To further compare genetic diversity among BV isolates, DNA sequences from two regions of the viral genome containing genes that are conserved (UL27 and US6) and variable (US4 and US5) among primate alphaherpesviruses, as well as from two noncoding intergenic regions, were determined. From these sequence data and a phylogenetic analysis of them it was evident that while all isolates were closely related strains of BV, there were three distinct genotypes. The three BV genotypes were directly related to the macaque species of origin and were composed of (i) isolates from rhesus and Japanese macaques, (ii) cynomolgus monkey isolates, and (iii) isolates from pigtail macaques. This study demonstrates the existence of different BV genotypes which are related to the macaque host species and thus provides a molecular basis for the possible existence of BV isolates which vary in their levels of pathogenicity for nonmacaque species.

FIG. 1

SDS-PAGE analysis of BV-infected cell proteins and glycoproteins. Vero cells were infected with BV strains and labeled from 4 to 24 h postinfection with [³⁵S]methionine (A) or [¹⁴C]glucosamine (B). Polypeptides were separated on SDS–8% polyacrylamide gels. The monkey species from which isolates were made are identified above the lane numbers. Virus isolates shown in lanes 1 to 10 are E2490, 16293, 12930, 20620, 9400371, E90-136, 1504-11, Kumquat, 7709642, and 7709609, respectively. MCP, major capsid protein.

FIG. 2

Antigenic reactivities of macaque sera and BV isolates. In a standard ELISA (A) sera from cynomolgus monkeys (352-84, 267-76, and 410-94), pigtail macaques (91099, 92209, and 90153), and rhesus monkeys (MM339-95, MM336-95, and MM329-95) were tested at a 1:4,000 dilution with antigens prepared from rhesus (E2490), cynomolgus (E90-136), pigtail (Kumquat), and Japanese (7709609) macaques. cELISA (B) was also performed with homologous serum-viral antigen combinations from rhesus (left), pigtail (center), and cynomolgus (right) monkeys. Soluble antigens used to compete these reactions were from rhesus (⧫), cynomolgus (▴), and pigtail (■) macaque BV isolates.

FIG. 3

RFLP profiles of BV isolates. Purified viral DNA from BV isolates was digested with BamHI, PstI, or SalI, and fragments were separated on 0.8% agarose gels. Isolates from rhesus macaques are in lanes 2 to 5 (strains E2490, 16293, 12930, and 20620, respectively); cynomolgus macaque isolates are in lanes 6 and 7 (strains 9400371 and E90-136, respectively); pigtail macaque isolates are in lanes 8 and 9 (1504-11 and Kumquat, respectively); and Japanese macaque isolates are in lanes 10 and 11 (7709642 and 7709609, respectively). HVP2 strain OU1-76 is shown in lane 1 for comparison. Phage λ DNA cut with EcoRI and EcoRI plus HindIII was used as a size marker. Fragment sizes in kilobase pairs are indicated.

FIG. 4

Comparison of sequence from US4 (gG) through US6 (gD). DNA sequences were amplified by PCR, and products were sequenced directly. Numbering above the aligned sequences represents the positions in the alignment rather than nucleotide numbers of the E2490 sequence. All sequences are shown referenced to the E2490 strain sequence, with identical residues indicated by dots. Termination codons of US4 and US5 and start codons of US5 and US6 in the E2490 sequence are shaded; mRNA poly(A) and transcriptional termination motifs in the US5-to-US6 intergenic region are underlined. The US4 ORF runs from positions 1 to 60 of the aligned sequences; the US5 ORF runs from positions 293 to 688; and the US6 ORF runs from positions 1186 to 1318 of the alignment. The sequence for isolate SMHV is taken from Bennett et al. (1) and Slomka et al. (26).

FIG. 4

Comparison of sequence from US4 (gG) through US6 (gD). DNA sequences were amplified by PCR, and products were sequenced directly. Numbering above the aligned sequences represents the positions in the alignment rather than nucleotide numbers of the E2490 sequence. All sequences are shown referenced to the E2490 strain sequence, with identical residues indicated by dots. Termination codons of US4 and US5 and start codons of US5 and US6 in the E2490 sequence are shaded; mRNA poly(A) and transcriptional termination motifs in the US5-to-US6 intergenic region are underlined. The US4 ORF runs from positions 1 to 60 of the aligned sequences; the US5 ORF runs from positions 293 to 688; and the US6 ORF runs from positions 1186 to 1318 of the alignment. The sequence for isolate SMHV is taken from Bennett et al. (1) and Slomka et al. (26).

FIG. 5

Phylogenetic relationships of BV isolates. Three data sets were used to produce the trees shown. (A) The total aligned sequence shown in Fig. 4; (B) the combined coding sequences of the US4, US5, and US6 ORFs shown in Fig. 4; (C) 437 bp of coding sequence spanning the D2 region of the gB gene (UL27 ORF) (3). Tamura-Nei distances were used to construct trees by the neighbor-joining method. Numbers along branches represent bootstrap confidence levels generated with 500 replications.