鉴定与 H7N9 病毒株毒力增强相关的血凝素中新型氨基酸取代。

Identifying novel amino acid substitutions of hemagglutinin involved in virulence enhancement in H7N9 virus strains.

机构信息

MHC Key Laboratory of Systems Biology of Pathogen, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

出版信息

Virol J. 2021 Jan 11;18(1):14. doi: 10.1186/s12985-020-01464-1.

DOI:10.1186/s12985-020-01464-1

PMID:33430903

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7798331/

Abstract

BACKGROUND

To identify site-specific features of amino acid substitutions that confer enhanced H7N9 virulence in humans, we independently generated mammalian-adapted variants of A/Anhui/1/2013 (AH-H7N9) and A/Shanghai/2/2013 (SH-H7N9) by serial passaging in Madin-Darby canine kidney (MDCK) cells.

METHODS

Virus was respectively extracted from cell culture supernatant and cells, and was absolutely quantified by using real-time polymerase chain reaction. Viral RNAs were extracted and subjected to sequencing for identifying mutations. Then, site-specific mutations introduced by viral passaging were selected for further constructing HA7 or NA9 mutant plasmids, which were used to generate recombinant viruses. The interaction between the recombinant HA and receptors, H7N9-pseudotyped viruses and receptors were detected.

RESULTS

Both subtypes displayed high variability in replicative capability and virulence during serial passaging. Analysis of viral genomes revealed multiple amino acid mutations in the hemagglutinin 7 (HA7) (A135T [AH-H7N9], T71I [SH-H7N9], T157I [SH-H7N9], T71I-V223I [SH-H7N9], T71I-T157I-V223I [SH-H7N9], and T71I-T157I-V223I-T40I [SH-H7N9]), and NA9 (N171S [AH-H7N9] and G335S [AH-H7N9]) proteins in various strains of the corresponding subtypes. Notably, quite a few amino acid substitutions indeed collectively strengthened the interactions between H7N9 strains and sialic acid receptors. Moreover, some of the amino acid substitutions identified were highly and specifically cytopathogenic to MDCK cells.

CONCLUSIONS

This study demonstrated that AH-H7N9 and SH-H7N9 subtypes can acquire enhanced receptor affinity for sialic receptors through novel amino acid substitutions. Such changes in affinitive interactions are conferred by site-specific mutations of HA7 proteins that affect the virulence and pathology of the virus strain, and/or limited compatibility between the host and the virus strain.

摘要

背景

为了确定导致 H7N9 病毒在人类中增强毒力的氨基酸取代的特定部位特征，我们通过在 Madin-Darby 犬肾（MDCK）细胞中连续传代，分别生成了 A/Anhui/1/2013（AH-H7N9）和 A/Shanghai/2/2013（SH-H7N9）的哺乳动物适应变体。

方法

分别从细胞培养上清液和细胞中提取病毒，并通过实时聚合酶链反应进行绝对定量。提取病毒 RNA 并进行测序以鉴定突变。然后，选择通过病毒传代引入的特定部位突变，进一步构建 HA7 或 NA9 突变质粒，用于生成重组病毒。检测重组 HA 与受体的相互作用、H7N9 假型病毒与受体的相互作用。

结果

两种亚型在连续传代过程中表现出高变异性和不同的复制能力及毒力。病毒基因组分析显示，血凝素 7（HA7）蛋白中存在多种氨基酸突变（A135T[AH-H7N9]、T71I[SH-H7N9]、T157I[SH-H7N9]、T71I-V223I[SH-H7N9]、T71I-T157I-V223I[SH-H7N9]和 T71I-T157I-V223I-T40I[SH-H7N9]）和神经氨酸酶 9（NA9）蛋白中的 N171S[AH-H7N9]和 G335S[AH-H7N9]。值得注意的是，许多氨基酸取代确实共同增强了 H7N9 株与唾液酸受体的相互作用。此外，鉴定出的一些氨基酸取代对 MDCK 细胞具有高度特异性的细胞病变作用。