Analysis of adaptive mutations selected during the consecutive passages of hepatitis E virus produced from an infectious cDNA clone

Virus Res. 2016 Sep 2;223:170-80. doi: 10.1016/j.virusres.2016.07.011. Epub 2016 Jul 30.


To characterize the genomic mutations of hepatitis E virus (HEV) during consecutive passages associated with adaptation to growth in cell culture, a cloned genotype 3 HEV [pJE03-1760F/wt, starting virus (SV)] was passaged 10 times in A549 cells, and the entire genomic sequence of the passage 10 (P10) progeny was determined. Compared to SV, P10 virus possessed two non-synonymous (T2808C and A5054G) and four synonymous mutations (C1213T, T2557C, C3118T and C4435T) in the ORF1. Full-length infectious cDNA clones with a single, double (T2808C and A5054G), or all six mutations, identical to P10, were constructed, and their replication capacity was compared. Four (C1213T, T2557C, T2808C and A5054G) of the six viruses with a single mutation grew more efficiently than SV. The P10 virus propagated more rapidly and grew more efficiently than SV and T2808C+A5054G and reached a higher viral load (95.1- and 8.5-fold, respectively) at 20days post-inoculation. An immunofluorescence analysis revealed that a high percentage (>80%) of cells inoculated with the P10 virus expressed ORF2 proteins, while relatively low percentages (nearly 30% or 5%) inoculated with T2808C+A5054G or SV, respectively, expressed ORF2 proteins. We found that not only non-synonymous but also synonymous HEV mutations are independently associated with increased virus production.

Keywords: Adaptation; Cell culture; Hepatitis E virus; Infectious cDNA clone; Reverse genetics system; Synonymous mutation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptation, Biological / genetics*
  • Amino Acid Substitution
  • Cell Line
  • DNA, Complementary
  • Genome, Viral
  • Hepatitis E virus / classification
  • Hepatitis E virus / genetics*
  • Humans
  • Mutation*
  • Phylogeny
  • RNA, Viral*
  • Reassortant Viruses / genetics
  • Recombination, Genetic
  • Selection, Genetic*
  • Viral Load
  • Virus Cultivation
  • Virus Replication


  • DNA, Complementary
  • RNA, Viral