H7N9亚型流感病毒遗传演化分析及H7N9弱毒疫苗在哺乳动物模型上的免疫效力评价

发布时间：2018-01-15 03:14

本文关键词：H7N9亚型流感病毒遗传演化分析及H7N9弱毒疫苗在哺乳动物模型上的免疫效力评价　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：H7N9亚型流感病毒目前在我国呈散发流行态势,且在不断的进化重组,不仅给我国的养禽业带来了巨大的经济损失,也给人类健康造成了巨大的威胁。截止到2016年2月28日,已造成747人感染,297人死亡。本研究对2013年来本实验室分离到的210株H7N9病毒株进行了系统的遗传演化分析和关键分子特征分析。根据时间划分,可将2013年至2016年2月间的H7N9疫情分为四个时期。比较各个基因同源性发现,HA和NA基因的核苷酸同源性分别处在96.9-100%和97.3-100%之间;另外6个内部基因片段PB2、PB1、PA、NP、M和NS的核苷酸同源性分别为95.1-100%、95.3-100%、94.7-100%、93.6-100%、95.2-100%和93.7-100%。根据各基因进化树划分结果,可以将66株全基因组测序的2013分支病毒分为12个基因型。第一波疫情疫情背景较为简单,只有3个基因型;第二波和第三波疫情,背景较为复杂,基因型分别为8个和7个;但主要基因型不变,仍为基因1型。以上结果表明,H7N9病毒表面基因在进化上相对稳定,内部基因的进化率要高于表面基因,这可能是因为H7N9在我国蔓延的过程中不断的与H9N2亚型禽流感病毒发生重组所致。进一步分析发现,所有本实验室2013分支H7N9病毒分离株M2蛋白都有S31N突变,表明对金刚烷胺类药物耐药,所有H7N9病毒仍对神经氨酸酶抑制剂敏感;所有分离到的H7N9病毒裂解位点均只有一个碱性氨基酸,所有PB2蛋白627位为E,均为低致病性特征。耐药毒株的出现以及人传人的可能性,促使人们研发一种有效的疫苗来应对H7N9病毒。本研究利用反向遗传操作技术,将人分离株A/Anhui/1/2013(H7N9)(AH/1)的表面基因HA和NA与冷适应流感病毒株A/AnnArbor/6/60(H2N2)(AAca)的6个内部基因片段重组,构建出一株重组弱毒疫苗株H7N9/AAca。利用多种动物模型评价了该疫苗的安全性、免疫原性和保护效力。在小鼠和雪貂模型上,该疫苗株的毒力显著致弱,小鼠和雪貂感染后,均不表现出临床症状,且病毒的复制局限于上呼吸道并呈低水平复制。在小鼠、豚鼠和雪貂上,H7N9/AAca一免之后能诱导产生一定程度的血凝抑制和中和抗体水平,二免之后诱导产生的血凝抑制和中和抗体水平显著升高并达到了理想的水平。在小鼠、豚鼠和雪貂上,H7N9/AAca一免之后并不能完全阻止病毒复制,但是能够显著降低病毒复制的水平,而且动物不表现出临床症状;二免之后,该疫苗能完全阻止病毒在动物体内的复制。另外,H7N9/AAca一免之后能够完全阻止病毒经飞沫和直接接触感染豚鼠。综上所述,我国流行的H7N9亚型流感病毒目前相对稳定,主要流行基因型为G1型。H7N9/AAca具有良好的安全性和免疫原性,并且具有良好的免疫保护效力。H7N9/AAca弱毒疫苗可以作为储备疫苗以应对目前的H7N9疫情。动物实验结果表明,该疫苗具有在人上进行下一步临床试验的价值。
[Abstract]:The H7N9 subtype influenza virus is spreading and recombination in China, which not only brings great economic losses to poultry industry in China. It also poses a huge threat to human health. As of February 28th 2016, 747 people had been infected. In this study, 210 strains of H7N9 virus isolated in our laboratory since 2013 were systematically analyzed for genetic evolution and key molecular characteristics. The H7N9 outbreak from 2013 to February 2016 can be divided into four stages. The nucleotide homology of HA gene and na gene was 96.9-100% and 97.3-100%, respectively. The nucleotide homology of the other six internal gene fragments PB2, PB1, PANPM and NS were 95.1-100, 95.3-100 and 94.7-100%, respectively. 93.6-100%, 95.2-100% and 93.7-1000.The results were divided according to the phylogenetic tree of each gene. There were 12 genotypes of the 66 strains of the virus which were sequenced in the whole genome. The background of the first wave of the virus was relatively simple, with only 3 genotypes. In the second and third waves, the background was complicated, and the genotypes were 8 and 7, respectively. The results showed that the surface gene of H7N9 virus was relatively stable in evolution, and the evolution rate of internal gene was higher than that of surface gene. This may be due to the continuous recombination of H7N9 with H9N2 subtype avian influenza virus during the spread of H7N9 in China. All strains of H7N9 virus in our laboratory had S31N mutation, indicating that they were resistant to amantadine, and all H7N9 viruses were still sensitive to neuraminidase inhibitors. All the isolated H7N9 virus cleavage sites had only one basic amino acid, and all PB2 proteins at position 627 were characterized by low pathogenicity, the emergence of drug-resistant strains and the possibility of human-to-human transmission. In order to develop an effective vaccine against H7N9 virus, this study uses reverse genetic manipulation technology. The surface genes HA and na of the human isolate A / Anhui / 1 / 2013 H7N9 / AH / 1 were compared with the cold adapted influenza strain A / R Ann Arborr / 6 / 60. Six internal gene fragments of H2N2AAAca) were recombined. A recombinant attenuated vaccine strain H7N9 / AAca. was constructed to evaluate the safety, immunogenicity and protective efficacy of the vaccine in mice and ferrets using various animal models. The virulence of the vaccine strain was significantly weakened, neither mice nor ferrets showed any clinical symptoms after infection, and the replication of the virus was limited to the upper respiratory tract and was replicated at a low level in mice, guinea pigs and ferrets. After H7N9 / AAca immunization, a certain degree of hemagglutination inhibition and neutralizing antibody level were induced. The level of hemagglutination inhibition and neutralizing antibody induced after the second immunization was significantly increased and reached the desired level. In mice, guinea pigs and ferrets, H7N9 / AAca immunization did not completely prevent the replication of the virus after the administration of H7N9 / AAca in mice, guinea pigs and ferrets. But it can significantly reduce the level of viral replication, and animals do not show clinical symptoms; After the second immunization, the vaccine could completely stop the replication of the virus in animals. In addition, H7N9 / AAca could completely prevent the virus from infecting guinea pigs through droplets and direct contact. The H7N9 subtype influenza virus in China is relatively stable at present. The main genotype is G1. H7N9 / AAca has good safety and immunogenicity. And has a good immune protection. H7N9 / AAca attenuated vaccine can be used as a reserve vaccine to respond to the current H7N9 epidemic. The vaccine has the value of further clinical trials in humans.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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