Study on molecular epidemiology of genital herpes and development of nucleic acid probes for detection of herpes simplex viruses

Abstract

Herpes simplex virus (HSV) isolated from genital herpes cases in Thailand were characterized using the 3 restriction endonucleases (RE), BamHI, KpnI and EcoRI. By making use of presence or absence of cleavage sites and variation in mobility of DNA fragments at the terminal and joint regions of HSV genomes, 120 clinical isolates obtained from the Center for Sexually Transmitted Diseases, Bangrak Hospital, Ministry of Public Health, Bangkok, were analyzed. Results revealed that 114 (95.0%) were HSV-2, 2 (1.7%) were HSV-1, 3 (2.5%) mixed HSV-2 infection and 1 (0.8%) mixed HSV-1 and HSV-2 infection. According to the criteroa used in the present study, the DNAs of Thai HSV-2 isolates wers in highly conserved and could be classified into 25 groups which have distinct RE cleavage patterns and distributions from isolates of other countries. In order to verify a correlation of typing methods between RE analysis and tissue culture-fluorescent antibody staining (TC-FA) technique using type-specific monoclonal antibodies, 94 genital herpes isolates randomly selected from the 120 clinical isolates were used. Restriction endonucleases were apparently superior to monoclonal antibodies since they could also reveal identity of virus in miewd HSV populations. Type-specific monoclonal antibodies failed to distinguish identity of virus in mixed infection; they identified 2 (2.1%) and 92 (97.9%) of isolates as type 1 and type 2, respectively. To rapidly detect and distinguish between the 2 types, a nucleic acid hybridization assay to identify clinical isolates of HSV was developed using in vitro synthesized, radioactively labeled RNA transcripts from HSV specific DNA fragments cloned in transcription vector pGEM3. The RNA probe derived from the HSV-1 BamHI-Q DNA fragment hybridized strongly with HSV-1 and HSV-2, while the RNA probe derived from HSV-1 HindIII - PvuII DNA fragment hybridized with HSV-1 DNA only. The results of HSV typing using both common and type-specific probes correlated well with typing by TC-FA and RE analysis. Neither of these probes bound to cytomegalovirus, Epstein - Barr virus, varicella zoster virus, nor pseudorabies virus DNA. In order to increase the sensitivity of HSV detection and to detect the viral gene at a single cell level without use of radioisotope material, an in situ hybridization with Photobiotin-labeled double-stranded (ds) DNA probes prepared from HSV DNA was utilized. This assay provided an earlier detection of virus in cell culture by producing a signal at the site of virus replication, i.e., in the nucleus with an ease of preparation of Photobiotinlabeled ds DNA compared to biotinylated ds DNA using conventional nick translation.