Molecular epidemiology of herpes simplex virus (HSV) by restriction fragment length polymorphism (RFLP)

Abstract

Herpes simplex virus (HSV) is divided into HSV-1 and HSV-2 which are responsible for several clinically important infections. Although HSV-1 and HSV-2 have 50% related in their DNA sequence but there are distinct differences in the biological properties. The severity of the disease depends on infection site of the virus. HSV-1 is more frequently associated with nongenital infection, while HSV-2 is associated with genital infection, however, both type can cause similar disease. Although the typing of HSV isolates brings no benefit for treatment the patient but it is a useful test for study the pathogenesis and epidemiology of the virus. Beside typing, intratypic variation of the viruses studied by molecular biology may be help in understanding the pathogenesis of disease and development of the viruses. Attempt to propagate the virus from 121 HSV clinical isolates was done. 86 isolates were successfully propagated, which were 16 (18.60%) from male and 70 (81.40%) from female. Most of the samples (74.42%) were collected from genital lesions. The results of PCR typing among 86 clinical isolates, 20 of 22 nongenital isolates were HSV-1 (90.90%), two isolates were HSV-2 (9.09%). In genital lesions, 34 of 64 were HSV-1 (53.12%), 28 (43.75%) were HSV-2, and two isolates were mix-infection of HSV-1 and HSV-2 (3.13%). In this study, nongenital lesions were found HSV-1 infection higher than HSV-2 infection, whereas in genital lesions both HSV-1 and HSV- 2 were equally detection. Interestingly, nowadays an increasing prevalence of genital HSV-1 infection has been reported. To study molecular epidemiology of genetic variation of HSV DNAs by RFLP using four restriction endonucleases (RE), BamHI, Kpnl, Hindlll, and EcoR], the patterns were compared to standard HSV-1 (KOS) and HSV-2 (Baylor 186). 20 clinical isolates were randomly selected from each HSV-1 and HSV-2 isolates. They were from both nongenital lesions and genital lesions. Genomic variations are reflected as differences in cleavage patterns and identified based on the gain or loss of restriction maps. The results showed that the great majority of each 20 clinical isolates of HSV-1 distinguished by digestion with BamHI, Kpnl, Hindlll, and EcoRI, were 70%, 50%, 75%, and 70%, respectively, found to be the same pattern as standard HSV-1 (KOS). Among those 20 clinical isolates of HSV-2, 85% were similar to standard HSV-2 (Baylor 186) after digestion with BamHI, Hindlll, and EcoRI but no difference was observed with Kpnl (100%) digestion. To study diversity of RE cleavage patterns among HSV isolates by combination with four enzymes, the patterns of B₁K₁H₁E₁ (35%) which is the same as standard HSV-1 (KOS) showed the combination frequency higher than other patterns. In HSV-2 clinical isolates, the pattern of B₁K₁H₁E₁ (70%) showed highly frequency expressed when compared to standard HSV-2 (Baylor 186). The diversity of patterns was found in HSV-1 isolates (10 patterns) higher than that of HSV-2 isolates (7 patterns). To study restriction enzyme cleavage patterns related to viral gene products, our study indicated that the variation sites were observed on L and ร component of HSV-1 whereas few variations were found in L component and S-L junction of HSV-2. Analysis of HSV patterns after mapping back to gene products, the predicted proteins were found to be mainly structural proteins in both HSV-1 and HSV-2. Some regulatory protein (ICP22) was also demonstrated. HSV strains are differentiated by analysis of RFLP and HSV strains can also be classified into genotype, which the number of enzyme can extend the diversity of genetic variation. The intratypic variation on RE sites can predict HSV gene products and functions. This genotypic difference may possible be one factor involving in differences of pathogenesis and disease development in each person.