貂源H9N2亚型流感病毒的分离鉴定和血清学调查及其传播特性研究

发布时间：2018-03-21 14:58

本文选题：H9N2亚型　切入点：水貂　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：H9N2亚型流感病毒可引起人以及各种动物低致病性呼吸道疾病。水貂对其易感,由于我国水貂养殖特点为狐狸、貉子与其混合饲养,从而加大了狐狸、貉子感染H9N2亚型流感病毒的机会。为研究H9N2流感病毒在水貂群中的流行及进化情况,以及是否在狐狸和貉子种群中造成感染。2015年本实验室从山东省中东部地区收集到105份水貂、32份狐狸和20份貉子的肺组织,并从水貂肺组织中分离到六株H9N2亚型流感病毒,A/Mink/Shandong/Z1/2015,A/Mink/Shandong/Z2/2015,A/Mink/Shandong/Z3/2015,A/Mink/Shandong/Z4/2015,A/Mink/Shandong/Z5/2015和A/Mink/Shandong/Z6/2015。而在狐狸和貉子的肺组织中没有分离到流感病毒。将六株分离毒株的核苷酸序列与GenBank中收录的H9N2亚型流感病毒的核苷酸序列进行比对发现,该六株分离毒株的八个基因片段同源性为99.2%~100%;其PB1、PA、HA、NP、NA、M和NS基因与A/mink/Shangdong/F10/2013(H9N2)(Mk/SD/F10/13)毒株的相应片段同源性最高,都在97.0%以上,而PB2基因与A/environment/Suzhou/14/2013(H7N9)同源性最高,为99.4%。为进一步揭示六株分离毒株的分子特征,用DNASTAR软件分别推导其各基因片段的氨基酸序列,并与参考毒株进行比较分析。分析结果表明,该六株分离毒株八段氨基酸序列的同源性为98.2-100%。六株分离毒株的HA蛋白第226位氨基酸均为亮氨酸(Leu),具有SAα-2,6-Gal的受体结合特性;其裂解位点处氨基酸序列为RSSR,表明六株病毒均是低致病性的毒株;HA蛋白有8个潜在糖基化位点,与参考毒株相比,在218位氨基酸处丢失了一个潜在的糖基化位点,在313位氨基酸处增加了一个潜在的糖基化位点;六株分离毒株PB2蛋白的氨基酸序列在627位编码谷氨酸Glu,第701位编码天冬酰胺Asn,第627位符合禽源流感的特点,但第701位的天冬酰胺Asn可以补偿第627位突变产生的影响,增强其在哺乳动物体内的复制能力。为了进一步分析六株分离毒株的基因片段的起源,用GeneBank中的H9N2亚型流感病毒的序列作为参考序列,进行系统发生分析。HA基因的系统发生树分析得出:六株分离毒株属于欧亚Ⅲ系的Y280亚系分支,NA基因与内部基因PB1、PA、NP、NS的系统发生树分析表明:六株分离毒株属于欧亚Ⅱ系的Shanghai/F/98亚系分支,M基因的系统发生树表明:六株分离毒株属欧亚Ⅱ系的Hong Kong/G1/97亚系分支。且以上7个片段与Mk/SD/F10/13毒株具有最近的亲缘关系;PB2基因的系统发生树分析表明:六株分离毒株属于欧亚Ⅰ系的Korea/38349-p96323/96亚系分支,且与A/environment/Suzhou/14/2013(H7N9)有很近的亲缘关系。为研究貂源H9N2亚型流感病毒能否在水貂种群中水平传播,是否可以跨种传播给狐狸、貉子。本研究用实验室保存的Mk/SD/F10/13毒株进行动物实验。动物实验结果,发现在接种后的第4-8天,哨兵水貂出现了精神沉郁、食欲减退、流清亮鼻涕、流眼泪、轻微的咳嗽等症状,最后攻毒水貂和哨兵水貂都恢复健康,没有造成死亡。排毒检测发现哨兵水貂在第4~6天内能够通过鼻腔向外界排毒。剖检能发现肺脏出血、气管环出血、脾脏肿大出血等病理学变化。组织病理学观察发现肺脏表现出血性间质性肺炎,且肺泡壁充血增厚。第14天采集水貂血液样本分离血清进行HI抗体检测发现HI抗体均为阳性。哨兵狐狸和貉子没有表现临床症状,也没检测到排毒,但第14天采集狐狸和貉子血清检测发现HI抗体均为阳性。同时进行了狐狸和貉子的致病性实验,攻毒狐狸和貉子没有表现临床症状,也没检测到排毒,而第14天采集狐狸和貉子血清检测发现HI抗体也为阳性。这些结果表明:在实验条件下,貂源H9N2亚型流感病毒可以在水貂种群中水平传播,并可以跨种传播给狐狸、貉子。此外,为进一步研究H9N2亚型流感病毒在水貂、狐狸和貉子种群中的流行情况,进行了血清学调查。调查结果:水貂血清中抗H9N2亚型流感病毒的抗体阳性率达为31%(97/313),高于2013年的20%。狐狸血清中抗H9N2亚型流感病毒的抗体阳性率为59.4%(76/128),貉子血清中抗H9N2亚型流感病毒的抗体阳性率为41.4%(106/256)。该结果表明H9N2亚型流感病毒在水貂群中的流行越来越广泛,并在狐狸和貉子群中流行。本研究证实了貂源H9N2亚型流感病毒可以在水貂种群中水平传播,并可以跨种传播给狐狸、貉子。而且在毛皮动物种群中广泛流行。此外,貂源H9N2亚型流感病毒在水貂体内持续进化。这可能会对毛皮动物养殖业造成危害,而且毛皮动物也可能作为H9N2流感病毒的中间宿主而感染其他动物,甚至可能感染人类。本研究对做好H9N2流感病毒的防制和监测工作提供了重要的分子流行病学资料,因而具有重要的公共卫生学意义。
[Abstract]:H9N2 subtype influenza virus can cause the human and animal low pathogenic respiratory diseases. The mink susceptible, because the characteristics of China's mink breeding fox, raccoon and mixed feeding, thereby increasing the fox, raccoon infected with H9N2 subtype influenza virus epidemic and opportunities. The evolution of the flu virus group of mink H9N2 flow, and whether the fox and raccoon population caused by infection of.2015 years from the middle part of Shandong province to collect 105 copies of 32 copies of mink, Fox and raccoon 20 lung tissue, lung tissue and isolated from mink in six strains of influenza virus subtype H9N2, A/Mink/Shandong/Z1/2015, A/Mink/Shandong/Z2/2015, A/Mink/Shandong/Z3/2015, A/Mink/Shandong/Z4/2015 A/Mink/Shandong/Z5/2015 and A/Mink/Shandong/Z6/2015., and the fox and raccoon in lung tissue was not isolated influenza virus. Six strains of isolated strains Nucleotide sequence and nucleotide sequences of GenBank included in the H9N2 subtype of influenza virus comparison showed that the six strains of isolated strains of eight genes homologous to 99.2%~100%; PB1, PA, HA, NP, NA, M and NS gene and A/mink/Shangdong/F10/2013 (H9N2) (Mk/SD/ F10/13) of the corresponding homologous strains the highest in more than 97%, while the PB2 gene and A/environment/Suzhou/14/2013 (H7N9) the highest homology to 99.4%. for further reveal the molecular characteristics of six strains of isolated strains, the amino acid sequences by DNASTAR software are derived the gene fragments, and the results were compared with reference strains. The results show that the six strains of isolates eight amino acid sequence homology was 98.2-100%. of six strains of isolated strains of HA protein of 226th amino acids were leucine (Leu), with SA alpha -2,6-Gal receptor binding characteristics of the amino acid sequence of the cleavage site; As RSSR, showed that six strains of the virus are low pathogenic strain; HA protein has 8 potential glycosylation sites, compared with the reference strains, 218 amino acids at the loss of a potential glycosylation sites, a potential glycosylation site in the amino acid 313 increase; six strains of isolates of PB2 protein in 627 amino acid sequence encoding glutamic acid Glu, 701st bit Asn 627th bit encoding asparagine, in line with the characteristics of avian influenza, but 701st of the 627th Asn asparagine can compensate the effect produced by mutation, enhance its ability in the complex system of mammalian origin. In order to further analysis of six strains of gene fragment isolated strains, with GeneBank sequence in the H9N2 subtype of influenza virus as a reference sequence for the phylogenetic analysis of phylogenetic tree analysis of.HA gene of six strains of isolates belonging to the Eurasian Y280 sub branch of the Department of, NA gene and PB1 gene of PA, internal, NP, tree NS system analysis showed that six strains of isolates belonging to the Eurasian Shanghai/F/98 sub branch of the Department, the system of M gene phylogenetic tree showed that six strains of isolates belonging to Hong Kong/G1/97, the Eurasian II sub line branches. And more than 7 pieces with Mk/SD/F10/13 strains had the closest relationship; phylogenetic tree analysis of PB2 gene showed that six strains of isolates belonging to the Korea/38349-p96323/96 sub line I Eurasian branch, and A/environment/Suzhou/14/2013 (H7N9) are closely related. For the study of mink H9N2 subtypes of influenza viruses can spread in the mink population level, whether can cross species spread to the fox, raccoon. This study used Mk/SD/F10/13 strains preserved in the laboratory of animal experiment. The results of animal experiment, found in the first 4-8 days after inoculation, the spirit of Shen Yu sentinel mink, reduced appetite Back flow clear nose, tears, slight cough and other symptoms, finally challenged mink and mink are sentinel health recovery, caused no death. Detection of sentinel mink can detox detoxification to the outside through the nasal cavity in the first 4~6 days. Autopsy found lung hemorrhage, tracheal bleeding and pathological changes of spleen hemorrhage the pathological observation found. Pulmonary manifestations of hemorrhagic interstitial pneumonia, histopathology, and alveolar wall congestion thickening. Blood samples collected fourteenth days of mink separation of serum HI antibody detection of HI antibody was positive. The fox and raccoon does not show clinical symptoms, also not detected detoxification, but fourteenth days to collect the fox and raccoon serum detection HI antibody was positive. At the same time tested pathogenic Fox and raccoon, Fox and raccoon attack showed no clinical symptoms, no detectable detoxification, and fourteenth days to collect the fox and raccoon dog Sub serum detection HI antibody was also positive. These results show that under the experimental conditions, the source of mink H9N2 subtype influenza virus can spread in mink population level, and can spread to the fox, raccoon. In addition, for the further study of H9N2 subtype influenza virus in mink, Fox and raccoon population prevalence the serological investigation has been conducted. Results: the positive rate of antibody of mink serum anti influenza virus subtype H9N2 was 31% (97/313), the antibody positive rate higher than the 2013 fox 20%. serum anti influenza virus subtype H9N2 was 59.4% (76/ 128), the antibody positive rate of serum anti H9N2 raccoon influenza virus type 41.4% (106/256). The results show that the H9N2 subtype of influenza virus in mink in popular more and more widely, and popular in the fox and raccoon populations. This study confirmed the mink H9N2 influenza virus from mink populations in water Flat transmission, and can spread to the fox, and raccoon. Popular in fur animal populations. In addition, the source of mink H9N2 subtype influenza virus continued evolution in mink body. This may cause harm to the fur animal breeding, animal fur and may also serve as intermediate hosts of H9N2 influenza virus infection and other animal. May even infect humans. This study for the H9N2 influenza virus prevention and monitoring work provides important molecular epidemiological data, so it has important significance in public health.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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