Molecular method development and establishment of a database for clinical and epidemiological herpes simplex virus 1...

Previous methods of herpes simplex virus 1 (HSV-1) genotype analysis have lacked sufficient discriminatory power for strain analysis within genotypes. The hypervariable reiterative repeat regions in the US1 and US12 introns, known as ReIV, were targeted for strain comparison. PCR methods for these extremely GC-rich target regions were optimized to give reproducible amplicons that were visualized by capillary electrophoresis relative to size standards. Analysis of the size, shape, and pattern of the resulting signatures enabled strain discrimination. Primary clinical specimens were used to develop the assay and the analysis algorithm. A blinded clinical study of 147 in-state and 51 out-of-state samples, including matched specimen-isolate pairs, was then performed. All primary clinical samples had been collected between 2004 and 2011 for viral diagnosis and previously found to be positive for HSV-1 by real-time PCR. The combined database contained patterns from 264 samples collected from 199 patients with a total of 176 unique signatures, none of which were dominant in the population. Matches between the signatures of the more than 50 specimen-isolate pairs were always seen. Signatures also matched across multiple samples collected from individual patients (six such cases), as well as some additional signature matches where epidemiological links were likely. Results were reproducible on repeat testing of individual specimens, even after months in frozen storage. The protocol has multiple potential clinical and public health uses.