Characterization of large and small-plaque variants in the Zika virus clinical isolate ZIKV/Hu/S36/Chiba/2016

Fumihiro Kato,Kenichi Shibasaki,Shigeru Tajima,Chang Kweng Lim,Eri Nakayama,Yasuhiro Kawai,Tomohiko Takasaki,Masayuki Saijo,Satoshi Taniguchi,Masakatsu Taira,Takahiro Maeki

doi:10.1038/s41598-017-16475-2

Abstract

An Asian/American lineage Zika virus (ZIKV) strain ZIKV/Hu/S36/Chiba/2016 formed 2 types in plaque size, large and small. Genomic analysis of the plaque-forming clones obtained from the isolate indicated that the clones forming small plaques commonly had an adenine nucleotide at position 796 (230Gln in the amino acid sequence), while clones forming large plaques had a guanine nucleotide (230Arg) at the same position, suggesting that this position was associated with the difference in plaque size. Growth kinetics of a large-plaque clone was faster than that of a small-plaque clone in Vero cells. Recombinant ZIKV G796A/rZIKV-MR766, which carries a missense G796A mutation, was produced using an infectious molecular clone of the ZIKV MR766 strain rZIKV-MR766/pMW119-CMVP. The plaque size of the G796A mutant was significantly smaller than that of the parental strain. The G796A mutation clearly reduced the growth rate of the parental virus in Vero cells. Furthermore, the G796A mutation also decreased the virulence of the MR766 strain in IFNAR1 knockout mice. These results indicate that the amino acid variation at position 230 in the viral polyprotein, which is located in the M protein sequence, is a molecular determinant for plaque morphology, growth property, and virulence in mice of ZIKV.

Highlights

Zika virus (ZIKV) in the genus Flavivirus and the family Flaviviridae was first isolated from a sentinel rhesus monkey in Uganda and from mosquito in 1947 and 1948, respectively[1,2]
We demonstrated that the viral solution of the ZIKV/Hu/S36/Chiba/2016-Vero[2] isolate contained at least two phenotypes of ZIKV with different plaque size formation and growth kinetics in vitro and that a nucleotide at position 796, which locates in M protein, was responsible for the differences
We proved that the G796A mutation decreased the virulence of ZIKV MR766 strain in IFNAR1-KO mice

Summary

Introduction

Zika virus (ZIKV) in the genus Flavivirus and the family Flaviviridae was first isolated from a sentinel rhesus monkey in Uganda and from mosquito in 1947 and 1948, respectively[1,2]. ZIKV infection epidemics spread to the other Pacific regions, as well as the Americas in 2014–2015. The recent epidemics in Pacific regions and the Americas were caused by the introduction of Asian/American lineage ZIKV18,19. Mouse models of ZIKV infection were established using engineered mice such as interferon receptor knockout (KO) mice, interferon regulatory factor-KO mice, STAT2-KO mice, and immune competent SJL and Balb/c strains[27,28,29,30,31,32,33,34,35,36,37]. During the characterization process of the ZIKV/Hu/S36/ Chiba/2016 isolate in vitro, it was revealed that the isolate included at least two variants with different phenotypes. The genetic determinant that caused the difference in phenotypes was determined using the reverse genetics system

Methods

Results

Conclusion