Genetic evolution and pathogenicity analysis of a canine-origin H9N2 subtype influenza virus
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摘要: 【目的】明确犬源H9N2亚型流感病毒的分子变异特征及致病性,为今后探究流感病毒的禽—犬跨种传播机制提供科学依据。【方法】从流浪犬中分离H9N2亚型流感病毒,利用RT-PCR扩增其8个基因节段进行BLAST同源比对,然后基于国内外的22株参考毒株进行遗传进化分析,并通过小鼠滴鼻感染试验评估分离毒株的致病性。【结果】从采集的393份犬鼻拭子样品中分离获得1株H9N2亚型流感病毒,命名为A/canine/Guangxi/LZ11/2018(简写为LZ11)。分离毒株LZ11为三重组毒株,其中,HA、NA和NS基因属于BJ/94类谱系,PB2和M基因属于G1类谱系,PB1、PA和NP基因属于F/98类谱系;病毒基因组成属于近年我国广泛流行的S基因型。分离毒株LZ11的HA蛋白在第324~331位存在1个裂解位点(PSRSSR↓GL),符合低致病性流感病毒的特征;HA蛋白还出现226Q→L突变,具有优先结合人类α-2,6唾液酸受体的能力;NA蛋白在119E、151D、276E、292R和294N未发生突变,但M2蛋白出现31S→N突变,说明分离毒株LZ11仍对神经氨酸酶抑制剂敏感,但已对金刚烷胺类药物产生耐药性;聚合酶蛋白出现多个哺乳动物适应性相关的氨基酸位点突变,包括PB2蛋白的292I→V、PB1蛋白的368I→V及PA蛋白的409S→N和356K→R。分离毒株LZ11虽然未引起小鼠出现典型的临床症状,但可在肺脏和上鼻窦有效复制,病毒滴度分别为3.82和5.98 lg PFU/mL。【结论】分离获得的犬源H9N2亚型流感病毒属于近年流行的S基因型,其8个基因节段分属于3种谱系(BJ/94类谱系、G1类谱系和F/98类谱系),且存在多个哺乳动物适应性相关氨基酸位点突变,具备感染哺乳动物的分子特征,可在小鼠脏器内有效复制。鉴于人类与宠物犬的密切关系,今后应加强对犬源H9N2亚型流感病毒的监测与防控。Abstract: 【Objective】To understand the characteristics of molecular variation of canine-origin H9N2 subtype influenza virus and its pathogenicity in mice. It would provide the scientific evidence for the cross-species transmission of aviancanine.【Method】An H9N2 subtype influenza virus was isolated from stray dogs,eight of its gene segments were amplified by RT-PCR for BLAST homology comparison,and then genetic evolution analysis was conducted based on 22 reference strains at home and abroad,and pathogenicity of the isolated strains was evaluated by mouse nasal drip infection test.【Result】From collected 393 samples of dog nose swabs,one subtype H9N2 influenza virus strain was isolated,named A/canine/Guangxi/LZ11/2018(abbreviated as LZ11). Genetic evolution analysis showed that LZ11 was a triple-ressortant strain,which contained HA,NA and NS genes from BJ/94-like lineage,PB2 and M genes from G1-like lineage,PB1,PA and NP genes from F/98-like lineage,whose genetic composition belonged to S genotype circulating widely in China recently. Sequence analysis showed that there was a cleavage site(PSRSSR↓GL) in the HA protein at position 324-331 of LZ11 isolate,which was consistent with the characteristics of low pathogenic influenza virus. HA protein had 226 Q→L mutation,which preferentially bound human alpha-2,6 sialic acid receptors. There were no mutations in 119 E,151 D,276 E,292 R and 294 N in NA protein,while there was an 31 S→N mutation occurred in M2 protein,indicating that the LZ11 isolate was sensitive to neuraminidase inhibitors but resistant to amantadine drugs. Multiple amino acid site mutations related to mammalian adaptation were found in polymerase proteins,including 292 I→V in PB2 protein,368 I→V in PB1 protein,and 409 S→N and 356 K→R in PA protein. The results showed that although this isolate did not cause any typical clinical symptoms in mice,it could replicate effectively in the lungs and turbinates,that viral titer could reach 3.82 and 5.98 lg PFU/mL respectively.【Conclusion】The isolated canine-origin H9N2 subtype influenza virus belongs to to S genotype circulating widely in China recently,whose eight gene segments belong to three lineages,including BJ/94-like,G1-like and F/98-like lineage.It contains several adaptive amino acid mutations in mammals and can replicate well in the organs of mice with the molecular characteristics to infect mammals. Due to the closer relationship between human and pet dogs,the surveillance and prevention and control for canine-origin H9N2 subtype influenza virus should be strengthened in future.
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Keywords:
- influenza virus /
- H9N2 subtype /
- dogs /
- genetic evolution /
- pathogenicity
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