Analysis of enzymatic digestion pattern of two open reading frames of Varciella-Zoster genome from Kuwaiti patients using the RFLP technique

Abstract

Background and objectives: Varicella-Zoster virus (VZV) is a human herpes virus that usually attacks young children and commonly causes chicken pox (Varicella). Following primary infection, a lifelong latent infection is established. The virus often reactivates during adulthood or senesces to cause shingles (Zoster). Little is known regarding the genotypes of Varicella in Kuwait. The aim of this study was to genotype Varicella samples collected from patients in Kuwait.

Materials and methods: Samples from 60 cases of chicken pox were typed. The DNA extraction was performed using the commercially available DNA extraction kit. Two sets of oligonucleotide primers were used to amplify the intervening sequences with polymerase chain reaction to identify VZV DNA in clinical samples. The BglI and PstI endonucleases were used to digest. The PCR amplicons for PCR-RFLP typing.

Results: Relatively consistent restriction enzyme digestion profiles for different VZV strains were observed. Limited genetic differences between VZV samples were found. Three VZV strains were identified (A, B and C) with type B representing 86.6%, type A 11.7% and type C being 1.7%. We found that distinct restriction fragment length polymorphism isolates from the same origin or nationality were very similar.

Conclusion: Varicella strains with cutting sites for both enzyme PstI and BglI (typeB) were more prevalent. Molecular amplification of viral DNA by PCR and restriction digestion could be used for VZV typing as an alternative method to serological assays.

Keywords: Genotyping; Kuwait; RFLP; Restriction enzymes; Varicella.

Fig. 1

A schematic diagram indicating the PCR amplification of two ORf's fragments using two primersset. Ethidum bromide agarose gel indicating different genotypes corresponding to restriction enzyme (PstI and BglI) digestion. The 350 and 225 base pair amplicons were incubated with PstI and BglI restriction enzymes for digestion. The product of digestion is represented by agarosegel showing sizes of digestion products. The capital letters (A, B, C, D) in the lower portion of the figure represent the genotypes.

Fig. 2

Map showing geographic distribution of the resident locations of patients coming to Infectious Diseases Hospital, Kuwait, as prepared from the information provided by Civil ID of each patient. North (N = 10/B), North Central (NC = 11/B), Central (20/B), South Central (SC = 5/B), South (S = 7/A; 6/B; 1/C).