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

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 (230^Gln in the amino acid sequence), while clones forming large plaques had a guanine nucleotide (230^Arg) 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.

Figure 1

Plaque phenotypes of the ZIKV/Hu/S36/Chiba/2016 isolate in Vero cells (a,b). Plaque morphology of ZIKV/Hu/S36/Chiba/2016-Vero2 (a) and ZIKV/Hu/S36/Chiba/2016-Vero3 (b) in Vero cells cultured with the supernatants amplified two and three times, respectively (c and d). Sequencing electropherograms at nucleotide position 796 in ZIKV/Hu/S36/Chiba/2016-Vero2 (c) and -Vero3 (d). Two different primers (upper and lower electropherograms) were used for the sequencing of the region.

Figure 2

Growth properties of the large-plaque ChibaS36#3LP and small-plaque ChibaS36#8SP clones of ZIKV/Hu/S36/Chiba/2016. (a) Plaque morphology of the clones in Vero cells. (b,c) Growth kinetics of each clone in Vero cells (b) and in C6/36 cells (c). Cells were plated into 6-well culture plates and infected with the ZIKV clones at a multiplicity of infection of 0.01 PFU/cell. Values represent the mean and standard deviation from 3 independent experiments. P values were calculated by using two-way ANOVA test.

Figure 3

(a) Schematic representation of the construction of ZIKV infectious molecular clone rZIKV-MR766/pMW119-CMVP and mutant clone G796A/rZIKV-MR766/pMW119-CMVP. (a–h) RT-PCR fragments and nucleotide regions on the MR766 genome. A detailed explanation of the strategy is described in the Methods section. (b) Plaque morphology of the recombinant rZIKV-MR766 virus and its parental strain MR766 in Vero cells.

Figure 4

Growth properties of the recombinant G796A/rZIKV-MR766 virus. (a) Plaque morphology of the recombinant and parental MR766 strains in Vero cells. (b and c) Growth kinetics of the recombinant and parental viruses in Vero cells (b) and in C6/36 cells (c). Cells were plated into 6-well culture plates and infected with the viruses at a multiplicity of infection of 0.01 PFU/cell. Values represent the mean and standard deviation from three independent experiments. P values were calculated by using two-way ANOVA test.

Figure 5

Survival rate of mice infected with 1 × 10² PFU of MR766 or G796A/rZIKV-MR766. Mice in the two groups were monitored for 14 days after virus challenge.

Figure 6

Schematic representation of prM protein of ZIKV. Numbers indicate amino acid positions on the polyprotein. TM, transmembrane.