Added value of ultra-deep sequencing (UDS) approach for detection of genotypic antiviral resistance of herpes simplex virus (HSV).

Classically, Sanger sequencing is considered the gold standard for detection of HSV drug resistance mutations (DRMs). As a complementary method, ultra-deep sequencing (UDS) has an improved ability to detect minor variants and mixed populations. The aim of this work was to apply UDS performed on MiSeq® Illumina platform to the detection of HSV DRMs and to the evaluation of the subpopulation diversity in clinical samples in comparison with Sanger sequencing. A total of 59 HSV-positive clinical samples (31 HSV-1 and 28 HSV-2) recovered from 50 patients mainly immunocompromised (70%) were retrospectively analyzed. Remarkably, UDS analysis revealed significant differences of relative abundance according to the type of DRMs within TK and Pol: natural polymorphisms and amino acid changes associated with resistance to antivirals were identified as high-abundant mutations (>96%), whereas TK frameshifts conferring resistance to ACV were systematically detected at lower abundance (≈80%). This work also revealed that UDS can detect low-frequency DRMs and provides extensive information on viral population composition.