title: Analysis of adaptive mutations selected during the consecutive passages of hepatitis E virus produced from an infectious cDNA clone.
abstract: 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.
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Nagashima
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan.
authors.forename: Tominari
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan.
authors.forename: Toshinori
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan.
authors.lastname: Tanggis
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan.
authors.forename: Suljid
authors.initials: S
authors.lastname: Jirintai
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nishizawa
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi 329-0498, Japan. Electronic address: hokamoto@jichi.ac.jp.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Okamoto
mesh_terms: D000220:Adaptation, Biological / Q000235:genetics*; D019943:Amino Acid Substitution; D002460:Cell Line; D018076:DNA, Complementary; D016679:Genome, Viral; D016752:Hepatitis E virus / Q000145:classification / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010802:Phylogeny; D012367:RNA, Viral; D016865:Reassortant Viruses / Q000235:genetics; D011995:Recombination, Genetic; D012641:Selection, Genetic; D019562:Viral Load; D014776:Virus Cultivation; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral
keywords: Adaptation; Cell culture; Hepatitis E virus; Infectious cDNA clone; Reverse genetics system; Synonymous mutation