Department of Medical Microbiology, Academic Medical Center, Amsterdam, the Netherlands
Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands.
J Virol. 2020 Jun 16;94(13). doi: 10.1128/JVI.00195-20.
Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans. H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.
高致病性禽流感 (HPAI) 病毒在野生鸟类和家禽中持续存在，并继续导致人类感染，死亡率很高。迄今为止，已报告超过 850 例确诊的 H5N1 病毒感染病例，其中约 60%为致命病例。人们一直担心这些或类似的禽流感病毒会演变成能够在人与人之间有效传播的病毒，从而引发严重的流感大流行。此前的研究表明，受体特异性的改变是适应人类和向可传播病毒进化的标志。通过下一代测序检测到，在 HPAI A/H5N1 病毒血凝素的受体结合部位内检测到大量遗传多样性，这些多样性是在人类感染过程中进化的。在这里，我们研究了在这些人类感染过程中检测到的取代对功能的影响。在拯救了 21 种突变病毒后，受体结合部位（RBS）中的大多数取代都产生了有活力的病毒，但病毒复制、进入和稳定性常常受到阻碍。在经过修饰的红细胞和聚糖阵列的测量中，没有一种测试取代能够单独导致受体偏好的明显转变。尽管几种取代的组合可以导致人类型受体特异性，但在人类感染期间，单个血凝素中单一氨基酸取代的积累很少，从而降低了病毒适应人类的风险。H5 病毒继续对公共卫生构成威胁。由于这些病毒对人类来说是免疫上的新病毒，因此当它们适应在人与人之间传播时，可能会引发大流行。禽流感病毒需要多个适应性突变才能结合人类型受体、增加血凝素 (HA) 的稳定性并在人类细胞中复制。然而，关于人类感染期间适应性突变的知识有限。之前的一项研究表明，在人类感染期间，H5N1 的受体结合部位存在大量多样性。因此，我们在多种不同的检测中分析了观察到的氨基酸变化，包括病毒复制、稳定性和受体特异性。在测试的取代中，没有一种导致向能够气溶胶传播的适应人类的病毒明显转变。值得注意的是，获得人类型受体特异性需要多个氨基酸突变，并且关键位置 226 的变异性是不可容忍的，从而降低了自然获得它们的风险。
