Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentuckygrid.266539.d, Lexington, Kentucky, USA.
Zuckerman Mind Brain Behavior Institute, Columbia Universitygrid.21729.3f, New York, New York, USA.
J Virol. 2021 Aug 25;95(18):e0097121. doi: 10.1128/JVI.00971-21.
The newly identified influenza D virus (IDV) of the family has a wide host range with a broad geographical distribution. Despite the first appearance in U.S. pig herds in 2011, subsequent studies demonstrated that IDV is widespread in global cattle populations, supporting a theory that IDV utilizes bovines as a primary reservoir. Our investigation of the two reference influenza D viruses, D/swine/Oklahoma/1334/2011 (OK/11), isolated from swine, and D/Bovine/Oklahoma/660/2013 (660/13), isolated from cattle, revealed that 660/13 replicated to titers approximately 100-fold higher than those for OK/11 in multiple cell lines. By using a recently developed IDV reverse-genetics system derived from low-titer OK/11, we generated recombinant chimeric OK/11 viruses in which one of the seven genome segments was replaced with its counterpart from high-titer 660/13 virus. Further characterization demonstrated that the replication level of the chimeric OK/11 virus was significantly increased only when harboring the 660/13 nucleoprotein (NP) segment. Finally, through both gain-of-function and loss-of-function experiments, we identified that one amino acid residue at position 381, located in the body domain of NP protein, was a key determinant for the replication difference between the low-titer OK/11 virus and the high-titer 660/13 virus. Taken together, our findings provide important insight into IDV replication fitness mediated by the NP protein, which should facilitate future study of the infectious virus particle production mechanism of IDV. Little is known about the virus infection and production mechanism for newly discovered influenza D virus (IDV), which utilizes bovines as a primary reservoir, with frequent spillover to new hosts, including swine. In this study, we showed that of two well-characterized IDVs, 660/13 replicated more efficiently (approximately 100-fold higher) than OK/11. Using a recently developed IDV reverse-genetics system, we identified viral nucleoprotein (NP) as a primary determinant of the different replication capacities observed between these two nearly identical viruses. Mechanistic investigation further revealed that a mutation at NP position 381 evidently modulated virus fitness. Taken together, these observations indicate that IDV NP protein performs a critical role in infectious virus particle production. Our study thus illustrates an NP-based mechanism for efficient IDV infection and production .
新鉴定的 家族流感 D 病毒（IDV）具有广泛的宿主范围和广泛的地理分布。尽管它于 2011 年首次出现在美国猪群中，但随后的研究表明，IDV 在全球牛群中广泛存在，这支持了 IDV 利用牛作为主要宿主的理论。我们对从猪中分离到的两种参考流感 D 病毒，即 D/猪/俄克拉荷马州/1334/2011（OK/11）和从牛中分离到的 D/牛/俄克拉荷马州/660/2013（660/13）进行了研究，结果表明，660/13 在多种细胞系中的复制滴度比 OK/11 高约 100 倍。通过使用最近开发的源自低滴度 OK/11 的 IDV 反向遗传学系统，我们生成了重组嵌合 OK/11 病毒，其中 7 个基因组片段之一被高滴度 660/13 病毒的对应片段替换。进一步的特征分析表明，只有当携带 660/13 核蛋白（NP）片段时，嵌合 OK/11 病毒的复制水平才会显著增加。最后，通过功能获得和功能丧失实验，我们确定位于 NP 蛋白体域中的 381 位的一个氨基酸残基是低滴度 OK/11 病毒和高滴度 660/13 病毒之间复制差异的关键决定因素。综上所述，我们的研究结果提供了关于 NP 蛋白介导的 IDV 复制适应性的重要见解，这将有助于进一步研究 IDV 感染性病毒颗粒产生的机制。
对于新发现的流感 D 病毒（IDV），人们对其病毒感染和产生机制知之甚少，IDV 利用牛作为主要宿主，经常溢出到包括猪在内的新宿主。在这项研究中，我们表明，在两种特征良好的 IDV 中，660/13 的复制效率更高（约高 100 倍）。使用最近开发的 IDV 反向遗传学系统，我们确定病毒核蛋白（NP）是这两种几乎相同的病毒之间观察到的不同复制能力的主要决定因素。机制研究进一步表明，NP 位置 381 的突变明显调节了病毒适应性。总之，这些观察结果表明，IDV NP 蛋白在感染性病毒颗粒的产生中起着关键作用。因此，我们的研究表明了一种基于 NP 的 IDV 感染和生产的机制。
