Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events

H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.

H6N6亚型禽流感病毒（avian influenza viruses, AIVs）已在野生鸟类、家禽乃至哺乳动物中被广泛检出。近期有研究报道，H6N6病毒参与了H5、H7亚型禽流感病毒的产生过程。为探究H6N6病毒的出现机制、进化模式及流行潜力，本研究对198株H6N6病毒的全基因组进行了分析，其中包含截至2019年1月收录于NCBI与GISAID数据库的168株H6N6病毒，以及2014年11月至2019年1月期间于我国分离获得的30株病毒分离株。通过系统发育分析，本研究将198株H6N6病毒划分为98个基因型。分子钟分析结果显示，我国境内H6N6病毒的进化过程保持稳定，未受到选择压力的干扰。值得注意的是，本研究的实验室分离株与H6N2、H5N6、H7N9、H5N2、H4N2及H6N8共6种亚型病毒发生了基因重配，共产生9例新型H6N6重配事件。上述结果表明，H6N6病毒可充当H5N6、H6N6及H7N9病毒进化过程中的中间媒介。动物实验结果显示，10株代表性H6N6病毒对鸡仅表现出低致病性，且无需预先适应性改变即可感染小鼠。本研究结果提示，H6N6病毒在禽流感病毒的整体进化过程中发挥着重要作用，因此需持续监测并评估H6N6亚型病毒的流行潜力。