H9亚型禽流感病毒HA基因的进化分析及鹅源H9N2亚型禽流感病毒对鹅的致病性研究

发布时间：2018-09-12 17:51

【摘要】：禽流感病毒(Avian Influenza Virus,AIV)能够感染多种禽类和包括人类在内的哺乳类动物。自1994年我国首次报道从广东鸡群中分离到H9N2亚型AIV以来,该病毒一直在我国家禽中广泛流行。虽然该病毒致病力较低,但是能引起家禽的呼吸道症状以及产蛋家禽明显的产蛋下降给我国家禽养殖业带来巨大的经济损失。水禽尤其是鸭、鹅是AIV天然的贮存库,在AIV的传播过程中起到重要的作用。在流感病毒的基因组的八个片段中,HA基因具有决定致病力、抗原性和宿主范围等方面的重要作用,而且HA基因最容易发生变异。因此研究H9N2亚型AIV对鹅的致病性以及监测其HA基因的变异情况,有非常重要的意义。1.H9亚型AIV的分离鉴定以及HA基因的变异分析本研究汇集了2012~2013年山东不同地区的疑似感染H9亚型AIV的病例,收集肺脏样品,分离到25株病毒,经鉴定为AIV。利用PCR技术扩增25株AIV分离株的HA基因,测序,分析其变异情况。结果显示:(1)25株AIV分离株的HA基因核苷酸同源性和氨基酸同源性分别在94.5%~100.0%、93.6%~100.0%之间;(2)遗传进化分析结果表明,25株AIV分离株均属于欧亚分支中的Y280-like亚分支;(3)AIV的HA蛋白有7~9个潜在糖基化位点,其中218位糖基化位点缺失,145位新增糖基化位点;(4)25株AIV分离株HA蛋白裂解位点均为RSSR↓GIF,符合低致病力AIV的特征;(5)25株AIV分离株的受体结合位点较保守,234位受体结合位点均为L(亮氨酸),仅198位受体结合位点存在变异,具有与哺乳动物唾液酸α,2-6受体结合的特征。2.H9N2亚型AIV对鹅的致病性研究本研究选用本实验室分离、鉴定并保存的一株鹅源H9N2亚型AIV(以下称为g AIV-H9N2)进行人工感染试验。将60只2周龄健康鹅随机分为静脉注射组、点眼滴鼻组和对照组,每组20只,其中静脉注射组和点眼滴鼻组每只人工接种1 m L g AIV-H9N2增殖尿囊液(ELD50=10-7.5/0.2 m L),对照组接种等量的无菌生理盐水。试验组与对照组分开隔离饲养,每天观察各组试验鹅的发病情况和临床表现。在攻毒后第3、6、9、12、15、21、28d采集血液一次,分离血清,用HA-HI方法测定血清中H9亚型AIV抗体水平,用细胞因子ELISA方法检测试剂盒检测血清中IL-2、IL-6、IFN-β、IFN-γ等细胞因子的表达水平,同时测定血液生化指标。在攻毒后第3、6、9、12、15d剖杀每组的鹅3只,观察剖检变化;并采集脑、肝脏、肺脏、胰脏、脾脏等组织器官,用4%福尔马林溶液固定,按常规方法制备石蜡切片、HE染色,进行病理组织学观察。结果显示:(1)人工感染后静脉注射组和点眼滴鼻组鹅表现为精神萎顿,采食量下降,饮水减少,排白色稀便,生长缓慢,呼吸系统症状。静脉注射组较点眼滴鼻组的鹅临床感染症状明显,试验过程中无死亡。人工感染后第9 d开始,所有鹅均表现耐过,精神状态、采食量、饮水均恢复正常。对照组鹅均无异常症状。(2)人工感染后,第3d,可见静脉注射组和点眼滴鼻组肺脏出血,心外膜出血。第6d,可见静脉注射组和点眼滴鼻组腺胃潮红,肺脏出血。第9d,可见静脉注射组和点眼滴鼻组均肝脏淤血、出血,脾脏肿大、有坏死点,肺脏出血。第12d,可见静脉注射组和点眼滴鼻组肺脏出血、脑膜出血点。第15d,可见脾脏肿大,肺脏出血。比较发现,静脉注射组的各种病变均比点眼滴鼻组严重。对照组雏鸭未见肉眼可见病变。(3)人工感染后,各组鹅的病理组织学变化如下。肺脏:攻毒组肺脏出血明显。气管:攻毒组气管杯状细胞增多,分泌功能增强。肠道:静脉注射组肠绒毛杯状细胞坏死,绒毛断裂、脱落,点眼滴鼻组病变不明显。脑:静脉注射组脑小胶质细胞增生。肝脏:静脉注射组肝细胞水肿、坏死,点眼滴鼻组炎性细胞浸润,淤血。胰脏:局部有典型的炎性细胞聚集。(4)抗体水平测定:静脉注射组和点眼滴鼻组的抗体水平都在攻毒后逐渐升高,到达高峰后降低,可见静脉注射组抗体水平升高较快,且前期抗体水平值较高。静脉注射组和点眼滴鼻组最高抗体滴度都是7log2。(5)细胞因子测定:各攻毒组的IL-2、IL-6、IFN-β、IFN-γ水平明显高于对照组,在攻毒15天后基本恢复至与对照组持平。(6)血液生化指标变化如下。总蛋白变化趋势:在攻毒后,静脉注射组和点眼滴鼻组的总蛋白量不断升高,至攻毒后第6d,水平达到最高值,之后逐渐下降;可见静脉注射组的总蛋白水平高于点眼滴鼻组。球蛋白变化趋势:基本与总蛋白的变化趋势一致。AST/ALT变化趋势:人工感染后,水平整体下降,静脉注射组和点眼滴鼻组下降趋势一致。肌酐的变化趋势:人工感染后,静脉注射组和点眼滴鼻组肌酐水平先升高后降低,在第6d达到最高值,而后下降;可见静脉注射组和点眼滴鼻组的变化幅度基本一致,无太大区别。
[Abstract]:Avian Influenza Virus (AIV) can infect a variety of poultry and mammals including humans. Since the first report of H9N2 subtype AIV isolated from Guangdong chicken flocks in 1994, the virus has been widespread in poultry in China. Although the virulence of the virus is low, it can cause respiratory symptoms in poultry. Waterfowl, especially ducks and geese, are the natural reservoirs of AIV and play an important role in the transmission of AIV. Among the eight genomic fragments of influenza viruses, HA gene is the major determinant of pathogenicity, antigenicity and host range. Therefore, it is very important to study the pathogenicity of H9N2 subtype AIV in geese and to monitor the variation of its HA gene. 1. Isolation and identification of H9 subtype AIV and analysis of variation of HA gene. This study collected suspected cases of H9 subtype AIV from different areas of Shandong Province from 2012 to 2013. The results showed that: (1) The nucleotide homology and amino acid homology of HA gene of 25 AIV isolates were 94.5%-100.0% and 93.6%-100.0% respectively; (2) Genetic evolution analysis showed that 25 AIV isolates had the same nucleotide homology and amino acid homology. The isolates belong to the Y280-like subbranch of Eurasian branch; (3) There are 7-9 potential glycosylation sites in the HA protein of AIV, of which 218 are deleted and 145 are newly added glycosylation sites; (4) The HA protein cleavage sites of 25 AIV isolates are RSSR_GIF, which conform to the characteristics of low pathogenicity AIV; (5) The receptor binding sites of 25 AIV isolates are more than those of 25 AIV isolates. Conservative, 234 receptor binding sites are L (leucine), only 198 receptor binding sites have mutations, with mammalian sialic acid alpha, 2-6 receptor binding characteristics. 2. H9N2 subtype of AIV pathogenicity to goose This study selected our laboratory isolation, identification and preservation of a goose-derived H9N2 subtype of AIV (hereinafter referred to as G AIV-H9N2) for human. Sixty two-week-old healthy geese were randomly divided into intravenous injection group, intraocular drip nose group and control group, with 20 geese in each group. The intravenous injection group and intraocular drip nose group were artificially inoculated with 1 m L AIV-H9N2 proliferative allantoic fluid (ELD50 = 10-7.5/0.2 m L) and the control group was inoculated with the same amount of sterile saline. Serum samples were collected at 3, 6, 9, 12, 15, 21 and 28 days after treatment. The levels of antibodies against H9 subtype AIV in serum were determined by HA-HI method. The levels of cytokines such as IL-2, IL-6, IFN-beta and IFN-gamma in serum were detected by cytokine ELISA kit. Blood biochemical indices were determined. Three geese in each group were killed on the 3rd, 6th, 9th, 12th and 15th day after poisoning to observe the anatomical changes. Brain, liver, lung, pancreas, spleen and other tissues were collected and fixed with 4% formalin solution. Paraffin sections were prepared by routine method and stained with HE for histopathological observation. The intravenous injection group showed more obvious symptoms of clinical infection than the intravenous drip group, and no death occurred during the experiment. No abnormal symptoms were found in the control group. (2) On the 3rd day after artificial infection, pulmonary hemorrhage, epicardial hemorrhage were observed in the intravenous injection group and the intraocular drip nose group. On the 6th day, glandular stomach flushing and pulmonary hemorrhage were observed in the intravenous injection group and the intraocular drip nose group. On the 12th day, pulmonary hemorrhage and meningeal hemorrhage were observed in the intravenous injection group and the eye dropping nose group. On the 15th day, splenomegaly and pulmonary hemorrhage were observed. Comparing with the eye dropping nose group, all kinds of pathological changes in the intravenous injection group were more serious than those in the eye dropping nose group. Trachea: The goblet cells increased and secretory function was enhanced in the treatment group. Intestinal tract: The goblet cells of intestinal villi necrosis, villi rupture, exfoliation were not obvious in the intravenous injection group. Cerebral microglia proliferation was observed in the intravenous injection group. Pancreas: There were typical inflammatory cell aggregation. (4) Antibody level: The antibody level of intravenous injection group and nose dropping group increased gradually after poisoning, and decreased after reaching the peak. The antibody level of intravenous injection group increased faster, and the pre-antibody level was higher. The levels of IL-2, IL-6, IFN-beta and IFN-gamma in each treatment group were significantly higher than those in the control group, and recovered to the same level after 15 days of treatment. (6) The changes of blood biochemical indexes were as follows. The total protein level of the intravenous injection group was higher than that of the intraocular drip group. The change trend of globulin was basically consistent with that of the total protein. Change trend: After artificial infection, the creatinine level of intravenous injection group and intraocular drip nose group increased first, then decreased, reached the highest value on the 6th day, and then decreased; it can be seen that the changes of intravenous injection group and intraocular drip nose group were basically the same, without much difference.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.33

【共引文献】