Herpes simplex virus 2 (HSV-2) evolves faster in cell culture than HSV-1 by generating greater genetic diversity

Abstract

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.

High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9

“…Viral factors participating in the replication phase, together with the intrinsic high recombination rate exhibited by poxviruses and herpesviruses, could also reduce Cas9 precision. Moreover, the fact that herpesviruses can generate a great degree of genetic diversity when replicating in vitro also calls into question the off-target-free accuracy of Cas9 when engineering dsDNA viruses [23, 28]. In order to address this matter, we deep sequenced two isolates of every recombinant virus generated by the two-progenies method, as well as the parental viruses (Table S4).…”

“…We consistently detected a higher number of de novo MVs among HSV-2 recombinants than among HSV-1 counterparts (, Table S8). Nonetheless, we previously reported that HSV-2 evolves faster in vitro than HSV-1 [23]. The fact that these recombinants were plaque-isolated five times in cell culture might explain the appearance of these de novo MVs.…”

“…MVs were then additionally filtered by coverage >200. An additional filter was implemented to detect those MVs with high frequency but low coverage, where read depth at the given position had to be greater than the product obtained from dividing 200 (coverage threshold) by the variant frequency (0–100) at the given position [23]. MVs were considered to be de novo when, after coverage filtering, their frequency in the previous parental viral population was <0.01.…”

“…Bowtie 2 alignments were visualized and screened using Integrative Genomics Viewer v2.8.2 [22] to detect large gaps and rearrangements. Minor variants (MVs) were detected as previously described [23]. Briefly, VarScan v2.4.3 [24] was used with settings intended to minimize sequencing-induced errors from the raw calling of MVs.…”

Structural variation turnovers and defective genomes: key drivers for the in vitro evolution of the large double-stranded DNA koi herpesvirus (KHV)

“…Serial passages of viruses in cell culture may lead to the accumulation of mutations and gene disruptions (Spatz 2010;Colgrove et al, 2014). These mutations can modify viral adaptation and increase or decrease virulence (Boutier et al, 2017;López-Muñoz et al, 2021;Vancsok et al, 2017). In the case of KHV, a previous work using short-read sequencing showed that 99 consecutive in vitro passages onto CCB cells resulted in the accumulation of less than 60 small variations (<100 nt) (Klafack et al, 2019).…”