Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture

Abstract

Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13−UL128L+) or both RL13 and UL128L (RL13−UL128L−). RL13−UL128L− viruses produced greater yields of infectious progeny than RL13−UL128L+ viruses, and RL13−UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks

“…Passage of EHV‐1 (Allen, Yeargan, et al., 1983; Bonass et al., 1994) and other herpesviruses (Dargan et al., 2010; Tyler et al., 2007) in vitro is known to select mutants that have a growth advantage over wild‐type virus. The majority of viruses analysed in the present study were sequenced at passage 3 or 4 after their original isolation from the animals, to minimize the extent of adaptation, but it is possible that mutations occurred in vitro .…”

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

“…These findings led to the conclusion that the lack of HCMV transmission to leukocytes and HUVEC infection are markers of HCMV attenuation [47]. Interestingly, sequential mutations associated with the adaptation of human cytomegalovirus to growth in HELF cultures have been shown to be associated with greater virus yields [48].…”

“…The genetic determinants of EC-and leuko-tropism remained elusive until 2004, when a 2-year collaborative investigation by researchers from Pavia (Italy) and Munich (Germany) provided evidence that the UL131-128 locus (UL128L) [48] within the ULb¢ region of the HCMV genome is indispensable for virus replication in ECs and virus transmission to leukocytes [6]. At the beginning of this study, the following findings were critical in directing interest to the ULb¢ region as a prime candidate: (i) the loss of EC-and leuko-tropism in the laboratory strains, Towne and AD169, was associated with the loss of the great majority of ULb¢ [52,53]; (ii) the low-passaged strain Toledo, which was shown to be tropism-deficient [51], was found to display an inversion of ULb¢ [53]; and (iii) extensive propagation of EC-and leuko-tropic clinical isolates in HELFs was found to be consistently associated with the selection of tropism-deficient variants [37,45,[54][55][56] in the presence of only minor variations in the relevant viral genomes.…”

Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication

“…Spontaneous defects clearly arise elsewhere in the HCMV genome, yet their significance is only occasionally defined functionally (13,18,26,27). Indeed, whole-genome sequencing of multiple clinical strains following growth in vitro reveals that clinical HCMV genomes invariably mutate whether passaged in fibroblasts, epithelial cells, or endothelial cells (28).…”