【摘要】 为了解2023年分离自我国广东省家鸭H1N4亚型禽流感病毒（AIV）GD/S1385株的生物学特性，本研究利用一步法RT-PCR扩增GD/S1385株全基因组，并利用二代测序平台对其全基因组测序。在GISAID数据库中经BLAST比对，并下载所有H1N4亚型AIV的全基因组序列。利用MegAlign软件分析GD/S1385株与其他H1N4亚型AIV各基因节段的同源性，采用MEGA7.0软件构建GD/S1385株与上述AIV各基因节段的进化树，采用GISAID数据库中的FluSurver程序分析GD/S1385株各基因节段关键氨基酸位点的变异。BLAST比对结果显示，H1N4亚型AIV GD/S1385株与鸡源H5N1亚型AIV NP基因序列的同源性最高为99.2%，其他基因节段分别与H1N1、H4N2、H6N2、H8N4和H10N4等野鸟源AIV相应基因序列的同源性最高达98.2%～99.8%。同源性分析结果显示，GD/S1385株与GISAID数据库中12株H1N4 AIV HA和NA基因序列的同源性分别为84.3%～91.8%和83.2%～97.5%，内部基因PB2、PB1、PA、NP、M和NS基因序列的同源性分别为86.6%～94.8%、89%～95.6%、88.4%～95.1%、88%～94.4%、92.5%～96.2%和71.1%～93.4%。进化树结果显示，GD/S1385株部分基因节段与野鸟源H1N1、H4N2、H5N1、H6N2、H8N4和H10N4等亚型AIV处于同一进化分支，但与H1N4亚型AIV的遗传距离均较远。序列分析结果显示，GD/S1385株HA蛋白裂解位点氨基酸序列为³²³PSIQSRGL³³⁰，符合低致病性禽流感病毒（LPAIV）的分子特征；NA蛋白未发生茎部缺失，也未出现神经氨酸酶抑制剂类药物的耐药性突变；PB2蛋白出现³⁸⁹R、⁵⁹⁸T,PB1蛋白出现³V、⁶²²G,PA蛋白出现³⁷A、⁴⁰⁹S,NP蛋白出现¹⁰⁵V,NS1蛋白出现¹⁰⁶M等多个哺乳动物适应性的氨基酸突变位点。将GD/S1385株以10⁶EID₅₀/50μL经鼻腔感染BALB/c小鼠，评估病毒对哺乳动物的感染性。结果显示，GD/S1385株感染组小鼠的肺脏和鼻甲内均可检测到高水平的病毒，病毒滴度分别为6.1log₁₀EID₅₀/mL和3.6log₁₀EID₅₀/mL；在1只小鼠的肾脏和脾脏内还检测到低水平的病毒，脑内未检测到病毒；且可引起感染组小鼠体质量下降8.2%。对照组小鼠各脏器均未检测到病毒，且体质量呈一直上升趋势。上述结果表明，H1N4 AIV GD/S1385株是一种新型重组病毒，具有明显的遗传多样性，并可以在小鼠的呼吸道内高效复制，具有感染哺乳动物和人类的潜在风险。本研究阐明了H1N4亚型AIV的遗传进化特点和对哺乳动物的感染风险，为禽流感的监测和综合防控提供了数据支持。更多还原

【Abstract】 To investigate the biological characteristics of the H1N4 subtype avian influenza virus （AIV） isolated from duck in the live poultry market of Guangdong Province in 2023,the whole genome of the virus was amplified by one-step RT-PCR,and sequenced by the next-generation sequencing platform.The strains with the highest homology were searched by BLAST in the GISAID database,and download the whole genome sequence of all H1N4 subtype AIVs.MegAlign software was used to analyze the homology between GD/S1385 and the other H1N4 subtype AIVs,and MEGA7.0 software was used to construct the phylogenetic trees of GD/S1385 strain and the above AIVs.The variation of key amino acid substitutions in each gene segment of GD/S1385 strain was analyzed by using the FluSurver program in GISAID database.BLAST results indicated that the NP gene of GD/S1385 shares the highest homology with the chicken H5N1 virus,and the homology was 99.2%.The other genes of GD/S1385 strain share the highest homology with some wild bird AIVs,such as H1N1,H4N2,H6N2,H8N4,and H10N4,with the highest homology between98.2%and 99.8%.Homology analysis between GD/S1385 and the other twelve H1N4 viruses in the GISAID database showed that the homology of HA and NA genes was 84.3%-91.8%and 83.2%-97.5%,respecti-vely.The homology of PB2,PB1,PA,NP,M and NS was 86.6%-94.8%,89%-95.6%,88.4%-95.1%,88%-94.4%,92.5%-96.2%and 71.1%-93.4%,respectively.The phylogenetic tree results further showed that the genes of GD/S1385 were clustered in the same group with those wild birds AIVs,such as H1N1H4N2,H5N1,H6N2,H8N4 and H10N4.However,the genetic distance between GD/S1385 and the other H1N4 subtype AIVs was far.The sequence analysis results indicated that the amino acid motif at the cleavage site of the HA protein of the isolate was³²³PSIQSRGL³³⁰,containing only one basic amino acid,a characteristic of low pathogenicity avian influenza virus.There was no stem deletion and mutation that was resistant to the neuraminidase inhibitors in the NA protein.The internal genes contain multiple mammalian adaptive amino acid mutation sites,such as³⁸⁹R and⁵⁹⁸T in PB2,³V,⁶²²G in PB1,³⁷A and⁴⁰⁹S in PA,¹⁰⁵V in NP,and¹⁰⁶M in NS1.Mice were inoculated intranasally with 10⁶EID₅₀of the virus GD/S1385 in a volume of 50μL to evaluate the pathogenicity to mammals.The results indicated that GD/S1385 strain replicated well in the lung and turbinate of mice,and the virus titers were6.1log₁₀EID₅₀/mL and 3.6log₁₀EID₅₀/mL,respectively.Furthermore,low titer viruses were also detected in the kidney and spleen of one mouse,and no virus was detected in the brain of any mice,and caused 8.2%body weight loss in mice.On the contrary,no virus was detected in the organs of the control group,and the body weight of the control group showed an increasing trend.Collectively,the H1N4 AIV GD/S1385 strain is a novel reassorted virus with obvious genetic diversity,which replicated efficiently in the respiratory tract of mice,bearing the potential risk to infect mammals and humans.This study elucidates the genetic evolution characteristics of H1N4 subtype AIV and the risk of infection inmammals and provides data support for avian influenza surveillance and comprehensive prevention and control.更多还原