Utilization of microsatellite polymorphism for differentiating herpes simplex virus type 1 strains

Abstract

The herpes simplex virus type 1 (HSV-1) genome is a linear double-stranded DNA of 152 kpb. It is divided into long and short regions of unique sequences termed U(L) and U(S), respectively, and these are flanked by regions of inverted internal and terminal repeats. Microsatellites are short tandem repeats of 1- to 6-nucleotide motifs; they are often highly variable and polymorphic within the genome, which raises the question of whether they may be used as molecular markers for the precise differentiation of HSV-1 strains. In this study, 79 different microsatellites (mono-, di-, and trinucleotide repeats) in the HSV-1 complete genome were identified by in silico analysis. Among those microsatellites, 45 were found to be distributed in intergenic or noncoding inverted repeat regions, while 34 were in open reading frames. Length polymorphism analysis of the PCR products was used to investigate a set of 12 distinct HSV-1 strains and allowed the identification of 23 polymorphic and 6 monomorphic microsatellites, including two polymorphic trinucleotide repeats (CGT and GGA) within the UL46 and US4 genes, respectively. A multiplex PCR method that amplified 10 polymorphic microsatellites was then developed for the rapid and accurate genetic characterization of HSV-1 strains. Each HSV-1 strain was characterized by its own microsatellite haplotype, which proved to be stable over time in cell culture. This relevant innovative tool was successfully applied both to confirm the close relationship between sequential HSV-1 isolates collected from patients with multiple recurrent infections and to investigate putative nosocomial infections.

FIG. 1.

Linear map of genomic distribution of HSV-1 microsatellites. The linear double-stranded DNA is represented with the nucleotide scale at the bottom according to the sequence of strain 17 (GenBank accession number X14112). The unique U_L and U_S sequences are shown as heavy solid lines, and the terminal and internal inverted repeats (TR_L, IR_L, IR_S, and TR_S) are shown as hatched boxes. The open reading frames on both the forward and the reverse strands are represented by open arrows. Each vertical line represents one microsatellite locus; the dotted lines and the dashed lines represent monomorphic and polymorphic microsatellites in the samples tested, respectively. This map is restricted to sites with at least four trinucleotide, five dinucleotide, or nine mononucleotide repeats units.

FIG. 2.

Sequences of two polymorphic trinucleotide microsatellites present in HSV-1 genes. (A) Microsatellite analysis of M41(CGT)6, which codes for a stretch of aspartic acid residues (residues 582 to 587) within the UL46 gene, showed two variants, M41(CGT)6 and M41(CGT)5, that led to alleles 366 and 363, respectively. The two variants represented 70% and 30% of the HSV-1 strains tested in this study, respectively. (B) Microsatellite analysis of M69(GGA)4, which codes for a polyglutamyl stretch (residues 79 to 82) within the US4 gene showed two variants, M69(GGA)4 and M69(GGA)5, that led to alleles 242 and 245, respectively. The two variants represented 42% and 58% of the HSV-1 strains tested in this study, respectively.

FIG. 3.

Electropherograms of multiplex PCR products illustrating the haplotype characteristics used to differentiate the HSV-1 strains. The haplotype was defined as the combination of the lengths of 10 amplicons labeled at the 5′ end with 6-carboxyfluorescein and containing HSV-1 microsatellite sequences obtained from the multiplex assay and analyzed by polyacrylamide capillary electrophoresis. The numbers 100 to 500 indicate the sizes (in base pairs) of the PCR amplicons. The haplotypes of strain B (top panel) and strain F (bottom panel) represented here display the length polymorphisms for the 10 loci.