Sequence-based methods for identifying epidemiologically linked herpes simplex virus type 2 strains

Abstract

Traditional methods for confirming the identity of herpes simplex virus (HSV) isolates use restriction fragment length polymorphism (RFLP). However, RFLP is less amenable to high-throughput analyses of many samples, and the extent to which small differences in RFLP patterns distinguish between different viral strains remains unclear. Viral HSV type 2 (HSV-2) DNA isolates from 14 persons experiencing a primary HSV-2 infection and from their sexual partners were analyzed by RFLP and heteroduplex mobility assays. We also compared the HSV-2 sequences from seven regions, including noncoding regions between UL19 and UL20, UL24 and UL25, UL37 and UL38, and UL41 and UL42 and coding segments of the gC, gB, and gG genes. Although the resulting RFLP patterns of the couples were almost identical, minor banding differences existed between the source and susceptible partners in five couples. Heteroduplex mobility assays were unable to distinguish between unrelated strains. Overall, 22 sites of sequence variation were found in 1,482 bp of analyzed sequence. The DNA sequences differentiated between all unrelated infections, and epidemiologically related isolates had identical sequences in all but two pairs. Our results suggest that a multilocus assay based on several DNA sequences has the potential to be an informative tool for identifying epidemiologically related HSV-2 strains.

FIG. 1.

Results of RFLP for six selected pairs. HSV-2 genomic DNA was digested using the SalI enzyme and electrophoresed on a 0.6% agarose gel overnight with a 1-kb molecular weight marker. RFLP pattern differences between partners can be seen with pairs 13 and 4.

FIG. 2.

HMA results for HSV-2 region NC1 amplicons by pair and partner. A radiolabeled amplicon from HSV-2 strain 333 was hybridized to each sample and then electrophoresed on a 0.6% polyacrylamide gel overnight. Slight mobility shifts are visible for pair 2, pair 8, partner B of pair 1, and partner A of pair 3.

FIG. 3.

Individual genotypes at each polymorphism. The consensus alleles (blue) and minor alleles (yellow) determined by the population studied are listed for each individual at each locus where a sequence polymorphism was observed by DNA sequence analysis. Missing data are indicated by gray squares or the letter N. The number of C repeats at locus 48802 and the number of GT repeats at locus 84796 are listed for each individual. Asterisks indicate regions where no sequence variation was found. (A) Related pairs with matching sequences; (B) related pairs with differing sequences.