To understand how human cytomegalovirus (HCMV) might change and evolve after
reactivation, it is very important to understand how the nucleotide sequence of cultured
HCMV changes after in vitro passaging in cell culture, and how these changes affect the
genome of HCMV and the consequent variation in amino acid sequence. Strain JHC of HCMV
was propagated in vitro for more than 40 passages and its biological and genetic changes were
monitored. For each passage, real-time PCR was performed in order to determine the genome
copy number, and a plaque assay was employed to get virus infection titers. The infectious
virus titers gradually increased with passaging in cell culture, whereas the number of virus
genome copies remained relatively unchanged. A linear correlation was observed between the
passage number and the log10 infectious virus titer per virus genome copy number. To
understand the genetic basis underlying the increase in HCMV infectivity with increasing
passage, the whole-genome DNA sequence of the high-passage strain was determined and
compared with the genome sequence of the low-passage strain. Out of 100 mutations found in
the high-passage strain, only two were located in an open reading frame. A G-T substitution in
the RL13 gene resulted in a nonsense mutation and caused an early stop. A G-A substitution in
the UL122 gene generated an S-F nonsynonymous mutation. The mutations in the RL13 and
UL122 genes might be related to the increase in virus infectivity, although the role of the
mutations found in noncoding regions could not be excluded.

Keywords: Human cytomgalovirus, Mutations, RL13, UL122