24Herpes simplex viruses (HSV) are difficult to sequence due to their large DNA genome, 25 high GC content, and the presence of repeats. To date, most HSV genomes have been 26 recovered from culture isolates, raising concern that these genomes may not accurately 27 represent circulating clinical strains. We report the development and validation of a DNA Herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) are 61 alphaherpesviruses causing over 4 billion human infections that can manifest as oral and 62 genital ulcerations, neonatal disease, herpetic keratitis, and encephalitis (1, 2). While
63HSV-2 has traditionally been associated with genital herpes, HSV-1 comprises the 64 majority of first episode genital herpes infections in high income countries (3). HSV 65 genome evolution is notable for extensive HSV-1 recombination within HSV-2 genomes, 66 with no detectable HSV-2 recombination into HSV-1 genomes (4, 5).
67To date, most human herpes simplex virus genome sequencing has been 68 performed on culture isolates (6-10). Culture is a pragmatic method to enrich for viral 69 sequences and many clinical virology labs have rich banks of cultured HSV isolates.
70However, without the ability to compare these sequences to sequence recovered directly 71 from clinical samples, interpretation of sequence results has been tempered by the 72 concern that culture isolates might not accurately represent viral sequence in vivo.
73Other viruses such as influenza and parainfluenza viruses have shown that culture 74 adaptation results in radically different viral sequence and receptor binding properties 75 that do not accurately reflect selection pressures in vivo (11-13). Culture of the 76 polyomaviruses BK and JC viruses is often performed in SV40 large T-antigen 77 immortalized cell lines, allowing near complete loss of the BKV and JCV large T-antigen 78 via transcomplementation, representing loss of one-third of the viral genome (14, 15).79 Culture adaptation of human herpesvirus 6A/B results in large tandem repeats in the 80 origin of replication and other regions that are not found in low-passage clinical isolates 81 and likely help accelerate viral replication in vitro (16-18). Similarly, laboratory passage 82 of human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus can result in 83 surprisingly large deletions comprising multiple genes and kilobases (19-22).
84Many clinical studies of HSV conducted at our institution and throughout the 85 world have utilized swabs to obtain DNA, which have the advantages of being easily 86 collected, stable at room temperature, and can be sequenced directly from the patient.
87To fully take advantage of the rapidly growing field of genomics to understand HSV 88 pathogenesis and diversity, we created a high-throughput method for sequencing HSV 89 from DNA swab and culture material. Capture sequencing has become commonly used 90 in human exome sequencing, oncology panels, and for other herpes viruses (23-25).
91We report here the development of wet-lab and dry-la...