Genome-wide characterization of the seasonal H3N2 virus in Shanghai reveals natural temperature-sensitive strains conferred by the I668V mutation in the PA subunit

Seasonal H3N2 influenza viruses are recognized as major epidemic viruses, exhibiting complex seasonal patterns in regions with temperate climates. To investigate the influence of viral evolution and mutations on the seasonality of influenza, we performed a genome-wide analysis of samples collected from 62 influenza A/H3N2-infected patients in Shanghai during 2016–2017. Phylogenetic analysis of all eight segments of the influenza A virus revealed that there were two epidemic influenza virus strains circulating in the 2016–2017 winter season (2016–2017win) and 2017 summer season (2017sum). Replication of the two epidemic viral strains at different temperatures (33, 35, 37, and 39 °C) was measured, and the correlation of the mutations in the two epidemic viral strains with temperature sensitivity and viral replication was analyzed. Analysis of the replication kinetics showed that replication of the 2016–2017win strains was significantly restricted at 39 °C compared with that of the 2017sum strains. A polymerase activity assay and mutational analysis demonstrated that the PA I668V mutation of the 2016–2017win viruses suppressed polymerase activity in vitro at high temperatures. Taken together, these data suggest that the I668V mutation in the PA subunit of the 2016–2017win strains may confer temperature sensitivity and attenuate viral replication and polymerase activity; meanwhile, the 2017sum strains maintained virulence at high temperatures. These findings highlight the importance of certain mutations in viral adaptation and persistence in subsequent seasons.

季节性H3N2流感病毒（seasonal H3N2 influenza viruses）是一类公认的主要流行性病毒，在温带气候区域呈现复杂的季节流行特征。为探究病毒演化与突变对流感季节性的影响，本研究对2016-2017年间上海地区62例甲型H3N2流感确诊患者的样本开展全基因组分析。对甲型流感病毒（influenza A virus）全部8个基因节段的系统发育分析显示，2016-2017年冬季（2016–2017win）与2017年夏季（2017sum）分别流行着两种不同的流行性病毒毒株。本研究检测了这两种流行性毒株在33、35、37与39℃不同温度下的复制能力，并分析了两种毒株所携带的突变与温度敏感性及病毒复制能力的相关性。复制动力学分析结果表明，与2017sum毒株相比，2016–2017win毒株在39℃条件下的复制受到显著抑制。聚合酶活性检测与突变分析结果证实，2016–2017win毒株所携带的PA（polymerase acidic protein）I668V突变在高温环境下可抑制病毒聚合酶活性。综合以上实验数据，本研究表明：2016–2017win毒株PA亚基上的I668V突变可能赋予病毒温度敏感性，并减弱其复制能力与聚合酶活性；而2017sum毒株在高温条件下仍可维持毒力。本研究结果凸显了部分突变在病毒适应宿主环境并于后续流行季持续传播过程中的关键作用。