Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institutgrid.417834.d, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institutgrid.417834.d, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
J Virol. 2021 Aug 25;95(18):e0044521. doi: 10.1128/JVI.00445-21.
Highly pathogenic avian influenza virus H5N8 clade 2.3.4.4 caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade 2.3.4.4A in 2014/2015 and clade 2.3.4.4B from 2016 up to today. Both clades were highly virulent in chickens, but only clade B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 2.3.4.4 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and experiments indicated that clade 2.3.4.4A and clade 2.3.4.4B viruses have a preference for NS1 of 237 aa and 217 aa, respectively, over NS1 of 230 aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C terminus (CTE) of clade B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade A virus was observed. Remarkably, the NS1 of clade A and clade B, regardless of length, was efficient in blocking interferon (IFN) induction in infected chickens, and changes in the NS1 C terminus reduced the efficiency for interferon antagonism. Together, the NS1 C terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks. The panzootic H5N8 highly pathogenic avian influenza viruses of clade 2.3.4.4A and 2.3.4.4B devastated the poultry industry globally. Clade 2.3.4.4A was predominant in 2014/2015 while clade 2.3.4.4B was widely spread in 2016/2017. The two clades exhibited different pathotypes in ducks. Virus factors contributing to virulence and transmission are largely unknown. The NS1 protein is typically composed of 230 amino acids (aa) and is an essential interferon (IFN) antagonist. Here, we found that the NS1 protein of clade 2.3.4.4A preferentially evolved toward long NS1 with 237 aa, while clade 2.3.4.4B evolved toward shorter NS1 with 217 aa (exclusively found in Europe) due to stop codons in the C terminus (CTE). We showed that the NS1 CTE of H5N8 is required for efficient virus replication, transmission, and endotheliotropism in ducks. In chickens, H5N8 NS1 evolved toward higher efficiency to block IFN response. These findings may explain the preferential pattern for short NS1 and high fitness of the panzootic H5N8 in birds.
高致病性禽流感病毒 H5N8 属 2.3.4.4 分支在全球范围内引发了家禽疫情，规模空前。该病毒由亚洲野生鸟类分两波传播：2014/2015 年的 2.3.4.4A 分支和 2016 年至今的 2.3.4.4B 分支。这两个分支在鸡中均具有高度致病性，但只有 B 分支病毒在鸭中表现出高致病性。导致这些大流行的 H5N8 2.3.4.4 病毒具有致病性和传播性的病毒因素在很大程度上尚不清楚。NS1 蛋白通常由 230 个氨基酸（aa）组成，是一种多功能蛋白，也是一种致病性因素。在这里，我们研究了 2013 年至 2019 年 H5N8 NS1 蛋白的进化轨迹及其在 H5N8 病毒在鸡和鸭中的适应性中的作用。序列分析和实验表明，2.3.4.4A 分支和 2.3.4.4B 分支病毒对 237 aa 和 217 aa 的 NS1 有偏好，而不是 230 aa 的 NS1。217 aa 的 NS1 仅见于欧洲的家养和野生鸟类中。B 分支病毒的 NS1 C 端（CTE）延长降低了病毒在鸡和鸭中的传播和复制能力，并部分削弱了鸭内皮的全身趋向性。相反，对 A 分支病毒适应性的影响较小。值得注意的是，A 分支和 B 分支的 NS1，无论长度如何，都能有效地阻断感染鸡中的干扰素（IFN）诱导，而 NS1 C 端的变化降低了干扰素拮抗的效率。总之，NS1 C 端有助于 H5N8 病毒在鸡和鸭中的有效传播和高适应性。属于 2.3.4.4A 和 2.3.4.4B 分支的大流行高致病性禽流感 H5N8 病毒在全球范围内摧毁了家禽业。2014/2015 年以 2.3.4.4A 分支为主，2016/2017 年广泛传播 2.3.4.4B 分支。这两个分支在鸭中表现出不同的病原型。导致致病性和传播性的病毒因素在很大程度上尚不清楚。NS1 蛋白通常由 230 个氨基酸（aa）组成，是一种必需的干扰素（IFN）拮抗剂。在这里，我们发现 2.3.4.4A 分支的 NS1 蛋白优先向 237 aa 的长 NS1 进化，而 2.3.4.4B 分支则由于 C 端（CTE）中的终止密码子而向 217 aa 的短 NS1 进化（仅在欧洲发现）。我们表明，H5N8 的 NS1 CTE 是病毒复制、传播和在鸭中内皮趋向性所必需的。在鸡中，H5N8 NS1 进化为更高效率地阻断 IFN 反应。这些发现可以解释短 NS1 和大流行的 H5N8 在鸟类中高适应性的偏好模式。
