Li Yanfang, Chen Sujuan, Zhang Xiaojian, Fu Qiang, Zhang Zhiye, Shi Shaohua, Zhu Yinbiao, Gu Min, Peng Daxin, Liu Xiufan
College of Veterinary Medicine, Yangzhou University, Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, P.R. China.
PLoS One. 2014 Apr 17;9(4):e95539. doi: 10.1371/journal.pone.0095539. eCollection 2014.
Since 2003, H5N1-subtype avian influenza viruses (AIVs) with both a deletion of 20 amino acids in the stalk of the neuraminidase (NA) glycoprotein (A-) and a deletion of five amino acids at positions 80 to 84 in the non-structural protein NS1 (S-) have become predominant. To understand the influence of these double deletions in the NA and NS1 proteins on the pathogenicity of H5N1-subtype AIVs, we selected A/mallard/Huadong/S/2005 as a parental strain to generate rescued wild-type A-S- and three variants (A-S+ with a five-amino-acid insertion in the NS1 protein, A+S- with a 20-amino-acid insertion in the NA stalk, and A+S+ with insertions in both NA and NS1 proteins) and evaluated their biological characteristics and virulence. The titers of the AIVs with A- and/or S- replicated in DEF cells were higher than that of A+S+, and the A-S- virus exhibited a replication predominance when co-infected with the other variants in DEF cells. In addition, A-S- induced a more significant increase in the expression of immune-related genes in peripheral blood mononuclear cells of mallard ducks in vitro compared with the other variants. Furthermore, an insertion in the NA and/or NS1 proteins of AIVs resulted in a notable decrease in virulence in ducks, as determined by intravenous pathogenicity index, and the two insertions exerted a synergistic effect on the attenuation of pathogenicity in ducks. In addition, compared with A+S+ and A+S-, the A-S+ and A-S- viruses that were introduced via the intranasal inoculation route exhibited a faster replication ability in the lungs of ducks. These data indicate that both the deletions in the NA stalk and the NS1 protein contribute to the high pathogenicity of H5N1 AIVs in ducks.
自2003年以来，神经氨酸酶（NA）糖蛋白茎部缺失20个氨基酸（A-）且非结构蛋白NS1第80至84位缺失5个氨基酸（S-）的H5N1亚型禽流感病毒（AIVs）已成为优势毒株。为了解NA和NS1蛋白中的这些双重缺失对H5N1亚型AIVs致病性的影响，我们选择A/野鸭/华东/S/2005作为亲本毒株，以产生拯救的野生型A-S-和三个变体（NS1蛋白中有5个氨基酸插入的A-S+、NA茎部有20个氨基酸插入的A+S-以及NA和NS1蛋白中均有插入的A+S+），并评估它们的生物学特性和毒力。在DEF细胞中复制的带有A-和/或S-的AIVs滴度高于A+S+，并且当与DEF细胞中的其他变体共同感染时，A-S-病毒表现出复制优势。此外，与其他变体相比，A-S-在体外诱导野鸭外周血单核细胞中免疫相关基因的表达有更显著的增加。此外，通过静脉致病性指数测定，AIVs的NA和/或NS1蛋白中的插入导致鸭的毒力显著降低，并且这两个插入对鸭致病性的减弱发挥协同作用。此外，与A+S+和A+S-相比，通过鼻内接种途径引入的A-S+和A-S-病毒在鸭肺中表现出更快的复制能力。这些数据表明，NA茎部和NS1蛋白中的缺失均有助于H5N1 AIVs在鸭中的高致病性。
