Genetic diversity and host adaptation of avian H5N1 influenza viruses during human infection

The continuing pandemic threat posed by avian influenza A/H5N1 viruses calls for improved insights into their evolution during human infection. We performed whole genome deep sequencing of respiratory specimens from 44 H5N1-infected individuals from Indonesia and found substantial within-host viral diversity. At nearly 30% of genome positions multiple amino acids were observed within or across samples, including positions implicated in aerosol transmission between ferrets. Amino acid variants detected our cohort were often found more frequently in available H5N1 sequences of human than avian isolates. We additionally identified previously unreported amino acid variants and multiple variants that increased in proportion over time in available sequential samples. Given the importance of the polymerase complex for host adaptation, we tested 121 amino acid variants found in the PB2, PB1 and PA subunits for their effects on polymerase activity in human cells. We identified multiple single amino acid variants in all three polymerase subunits that substantially increase polymerase activity including some with effects comparable to that of the widely recognized adaption and virulence marker PB2-E627 K. These results indicate highly dynamic evolutionary processes during human H5N1 virus infection and the potential existence of previously undocumented adaptive pathways.

禽流感A/H5N1病毒持续构成的疫情威胁，迫切需要深入了解其在人类感染过程中的演化。本研究对来自印度尼西亚的44名H5N1感染者的呼吸道样本进行了全基因组深度测序，发现病毒在宿主内部存在显著的多样性。在基因组近30%的位置上，观察到多个氨基酸存在于样本内部或跨样本之间，包括与貂之间气溶胶传播相关的位置。在我们队列中检测到的氨基酸变异，在可用的H5N1人类分离株中比禽类分离株更为常见。此外，我们还发现了之前未报告的氨基酸变异以及多个随时间在可用序列样本中比例增加的变异。鉴于聚合酶复合体对宿主适应的重要性，我们对PB2、PB1和PA亚基中发现的121个氨基酸变异进行了测试，以评估其对人类细胞中聚合酶活性的影响。我们在所有三个聚合酶亚单位中识别出多个单氨基酸变异，这些变异显著增加了聚合酶活性，其中一些变异的效果与广泛认可的适应性和致病性标记PB2-E627 K相当。这些结果表明，在人类H5N1病毒感染过程中存在着高度动态的演化过程，以及可能存在未被记录的适应性途径。