四株分离自同一猪场的H3N2亚型猪流感病毒的进化分析

猪作为流感病毒异源毒株间发生基因重组的"混合容器"，其呼吸道上皮细胞上同时存在着能够感染人（SA α-2，6-Gal）和禽（SA α-2，3-Gal）两种流感病毒的受体，具备产生新型流感病毒的潜力。在我们的前期研究中，连续两年（2013年和2014年）从南宁地区某个规模化养猪场当中分离获得了2株新型甲型流感病毒重配的H3N2亚型猪流感病毒（swine influenza viruses，SIVs）。为了解SIVs在同一地方的遗传进化规律，我们在2018年至2019年间对该猪场进行了持续的监测，并于2019年再次成功分离获得了2株H3N2亚型的三源重组毒株，命名为A/swine/Guangxi/JG13/2019（简称JG13/2019）和A/swine/Guangxi/JG20/2019（简称JG20/2019）。遗传进化分析表明其基因重配形式与2013年分离株A/swine/Guangxi/JGB4/2013（简称JGB4/2013）和2014年分离株A/swine/Guangxi/JG1/2014（简称JG1/2014）相同，表面基因HA和NA来源于类人H3N2谱系，内部基因NP、M、PA、PB1和PB2来源于2009年甲型H1N1大流感谱系（pdm/09H1N1），NS基因来源于古典型H1N1谱系。此外，新分离株JG13/2019和JG20/2019同早期分离株JGB4/2013和JG1/2014 HA和NA基因的核苷酸相似性分别为95.3%~97.4%和93.9%~97.0%，内部基因（NP、M、PA、PB1和PB2）的核苷酸相似性为96.2%~98.1%，NS基因的核苷酸相似性为97.1%~97.6%。通过分析比较这些年代不同毒株之间的关键氨基酸位点差异，结果发现JG20/2019和JG13/2019的HA蛋白仍旧保持了与人型受体结合的分子特征（190D、226I和228S），却也出现了V223I或P227S的新变化，JG13/2019的PA蛋白（R356K）和PB2蛋白（I588T）也与之前的毒株有所不同。这些位点上的氨基酸改变是否影响到病毒的致病能力、复制能力以及跨种间传播能力，有待今后进一步研究。历经6年，携带有pdm/09 H1N1多种内部基因片段（PB2、PB1、PA、M、NP）和类人表面基因（HA和NA）的H3N2亚型SIVs依旧在同一个猪场的猪群中流行，虽然其关键的功能区域出现了基因突变，但是仍然保持着能够感染人的受体结合特性。因此，加强对SIVs流行情况的监测，将为今后防控人类流感大暴发提供预警。

Phylogenetic Analysis of Four Strains of H3N2 Swine Influenza Virus Isolated from the Same Pig Farm

Pigs are regarded as a "mixing vessel" of influenza viruses, who could reassort with heterologous strains. They pose a potential threat to human health, due to the existence of the human-type (SA α-2,6-Gal) and the avian-type (SA α-2,3-Gal) receptors on the respiratory epithelial cells. Our previous study showed that two novel reassortants H3N2 swine influenza viruses(SIVs) with 2009 pandemic H1N1 internal genes were isolated in 2013 and 2014 from a large-scale pig farm of Nanning. To understand the genetic evolution of SIVs in the same pig farm, the consecutive surveillance was performed from 2018 to 2019. Two triple-reassortant H3N2 SIVs named as A/swine/Guangxi/JG13/2019 (JG13/2019) and A/swine/Guangxi/JG20/2019 (JG20/2019) were isolated once more in the pig farm mentioned above in 2019. The analysis of phylogenetic tree demonstrated that their reassortant form were similar to previous strains, A/swine/Guangxi/JGB4/2013 (JGB4/2013) and A/swine/Guangxi/JG1/2014 (JG1/2014), containing the surface genes HA and NA derived from the human-like H3N2 lineage, the internal genes NP, M, PA, PB1 and PB2 derived from the pdm09/H1N1 lineage and the NS gene derived from the classical swine H1N1 lineage. Besides, the homologies of HA and NA genes between the new isolates and previous isolates were 95.3%-97.4% and 93.9%-97.0%, respectively at the nucleotide level. The internal genes (NP, M, PA, PB1 and PB2) were 96.2%-98.1%, NS genes were 97.1%-97.6%. It was found that the protein HA still remained 190D, 226I and 228S which bind to human receptor. However, there were still two mutations at the position 223 (V→I) and 227 (P→S). Furthermore, there were two sites different from previous strains on PA protein and PB2 protein, which were R→K at the position 356 and I→T at the position 588. Whether these changes could affect the pathogenicity, replication and the ability of cross-species transmission needs to be further studied in future. After six years, H3N2 SIVs carrying internal gene fragments (PB2, PB1, PA, M and NP) of pdm/09 H1N1 and human-like surface genes (HA and NA) were still epidemic in pigs from the same farm. Although gene mutations have occurred in key functional domains, they still maintained the receptor binding characteristics to infect human. Therefore, the strength of surveillance for SIVs will provide the pre-alarm for the possible outbreak of human pandemic.