In vitro evolution of herpes simplex virus 1 (HSV-1) reveals selection for syncytia and other minor variants in cell culture

Abstract

The large dsDNA virus herpes simplex virus 1 (HSV-1) is considered to be genetically stable, yet it can rapidly evolve in response to strong selective pressures such as antiviral treatment. Deep sequencing has revealed that clinical and laboratory isolates of this virus exist as populations that contain a mixture of minor alleles or variants, similar to many RNA viruses. The classic virology approach of plaque purifying virus creates a genetically homogenous population, but it is not clear how closely this represents the mixed virus populations found in nature. We sought to study the evolution of mixed versus highly purified HSV-1 populations in controlled cell culture conditions, to examine the impact of this genetic diversity on evolution. We found that a mixed population of HSV-1 acquired more genetic diversity and underwent a more dramatic phenotypic shift than a plaque-purified population, producing a viral population that was almost entirely syncytial after just ten passages. At the genomic level, adaptation and genetic diversification occurred at the level of minor alleles or variants in the viral population. Certain genetic variants in the mixed viral population appeared to be positively selected in cell culture, and this shift was also observed in clinical samples during their first passages in vitro. In contrast, the plaque-purified viral population did not appear to change substantially in phenotype or overall quantity of minor allele diversity. These data indicate that HSV-1 is capable of evolving rapidly in a given environment, and that this evolution is facilitated by diversity in the viral population.

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

“…(B) Four representative pictures are showing the plaque morphology phenotype of HSV-1 clone 3 in Vero cells (48 hpi), before (passage 0, P0), and after ten passages (P10) in Vero cells. This syncytial plaque phenotype is due to the previously well-described syncytia-inducing mutation in UL27 CDS (see Sheet I in S1 Table , variant #38: R858H) [ 44 ]. Tiled images (4 x 4) were taken using a Leica DM IL LED inverted microscope equipped with a Leica DFC3000-G digital camera.…”

“…Kuny et al [ 44 ] did not find any changes in plaque phenotype in an HSV-1 purified population passaged ten times in Vero cells. However, we observed dramatic changes in plaque morphology in the purified HSV-1 clone 3 after ten passages in Vero cells ( Fig 1B ).…”

“…In contrast with HSV-1 clone 3, the frequency of this variant did not increase at P10 for HSV-2 clone 1, not acquiring the syncytial plaque phenotype. Neither this variant nor other syncytia-inducing MVs in gB, such as L817P [ 44 ], were detected when purified clones were passaged in HaCaT cells, for both HSV subtypes. These results suggested that even purified viral populations with low diversity are able to quickly change or evolve just in a few passages in cell culture, being more prone to happen in some cell lines than in others.…”

“…On the other hand, in vitro studies of viral evolution can bring an ideal scenario with more controlled and reduced selective pressures, where gaining insights into the mechanisms governing the generation of genetic variability can be easier. Kuny et al [ 44 ] have recently reported that a heterogeneous population of HSV-1 increased its genetic diversity and changed its phenotype more dramatically than a plaque-purified population of the same strain, after ten passages in Vero cells. Certain genetic MVs already present in the heterogeneous population appeared to be positively selected, whereas the purified population did not appear to increase its MVs repertoire.…”

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin

“…We hypothesise that the previous HSV-1 gC mutant, which was extensively passaged but not sequenced, had other mutations that diminished skin pathogenesis. HSV-1 genomes accumulate genetic changes that become apparent with serial passage, for example SNPs that lead to increase in syncytia formation [ 66 ]. Using the HSV-1 BAC for generating new mutant viruses is a practical method that minimises undesired genetic changes, although sequencing of the derived viruses remains important because some mutations may occur even at low passage numbers, as we noticed with the 17-37 and m2kb-S viruses.…”