【作者】 杨德全；

【导师】 张书霞； 李雁冰；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 本试验利用2株1996年和2004年从我国广东地区分离的H5N1亚型禽流感病毒感染4周龄SPF鸭,针对H5N1亚型禽流感病毒对SPF鸭的致病性进行了系统研究。以10⁶ EID₅0的病毒量鼻腔感染SPF鸭后,于第3、5、7、9 d分别剖杀感染鸭3只,采集喉头拭子、泄殖腔拭子、各组织脏器,进行病毒含量滴定、荧光定量PCR检测、病理组织学和免疫组织化学检验,探讨了H5N1亚型禽流感病毒在鸭体内各脏器的含量、分布、引起的病理变化以及排毒情况。2株禽流感病毒对鸭表现为不同的致病性:GS/GD/1/96毒株对SPF鸭的致死率为0,且无临床症状,剖检后无肉眼可见病理变化,各器官均分离不到病毒,免疫组织化学检测未发现病毒抗原信号;而DK/GDJD/23/04毒株对SPF鸭的致死率为100%,且表现典型的神经症状,剖检可见脑膜点状出血,胸腺出血,脾脏肿大并有坏死灶,肺脏严重充血、出血,腺胃粘膜弥散性出血。病理组织学检查发现全身各脏器以出血、充血、细胞变性坏死和炎性细胞浸润为主要特征的病理变化。各器官均可分离到病毒,脑组织病毒含量最高,免疫组织化学检测发现有病毒抗原信号。应用ELISA、细胞培养技术及CCK测定法,对4周龄SPF鸭感染H5N1 AIV后,其血清中IL-2、IFN-γ的含量,脾脏淋巴细胞对ConA刺激的T淋巴细胞增殖功能动态变化的研究发现,GS/GD/1/96毒株可促使外周血淋巴细胞分泌和释放IL-2和IFN-γ（p＜0.05）,但不能引起外周血T淋巴细胞的转化率的变化;而DK/GDJD/23/04毒株不仅可促使外周血淋巴细胞合成和释:IL-2（p＜0.05）和IFN-γ（p＜0.01）,最主要的是能引起外周血T淋巴细胞的转化率显著降低（P＜0.01）,并引起脾、胸腺及盲肠扁桃体的出血和淋巴细胞减少的变化,说明该病毒具有显著的抑制鸭细胞免疫功能的作用。进一步证实了DK/GDJD/23/04毒株感染后可引起鸭免疫功能抑制。全基因组测序分析表明,2株病毒均为高致病性毒株,在分子结构上,二者HA蛋白上的糖基化位点完全一致,二者HA蛋白的差异有2处,一是DK/GDJD/23/04裂解位点为连续的5个碱性氨基酸（-RRRKK-）,GSGD/1/96为6个连续的碱性氨基酸（-RRRKKR-）;二是在可能的受体结合位点224-229位,DK/GDJD/23/04的氨基酸为RLVPKI,而GSGD/1/96的序列为RLVPEI,即228位氨基酸E到K的变化。二者NA蛋白的差异有2处,一是DK/GDJD/23/04在NA蛋白颈部49-68位缺20个氨基酸,相应的缺失了4个潜在糖基化位点。GSGD/1/96 NA蛋白颈部没有发生缺失。二是DK/GDJD/23/04NA蛋白在386位有糖基化位点,而GSGD/1/96 NA蛋白在386位没有糖基化位点。DK/GDJD/23/04的NA蛋白颈部49-68位20个氨基酸缺失、NS1蛋白在80-84位有5个氨基酸缺失,这些缺失存在于大多数H5N1亚型人源和禽源分离株中,有利于禽流感病毒在家禽中传播,成为2002-2004年流行的优势病毒。上述这些差异可能是2株病毒对鸭致病性差异的重要原因。遗传演化分析表明,二株病毒存在差异,DK/GDJD/23/04与04年及以后的流行株亲缘关系很近,此毒株已在家禽中传播并发生了变异。更多还原

【Abstract】 To assess the pathogenicity of influenza A virus in ducks,two viruses A/goose/Guangdong/1/96（H5N1）（GS/GD/1/96） and A/duck/GuangdongJiedong/23/2004（H5N1）（DK/GDJD/23/04） isolated from Guangdong province were selected in the study,20 SPF ducks with 4 weeks old in each group were inoculated nasally with 10⁶ EID₅₀ to evaluate the pathogenicity of two viruses.The content of virues,the distribution of virues and the pathological changes in multiorgans in SPF ducks were caused by the H5N1 subtype avian influenza virus,so the virus titration of multiorgans and swabs,the real time PCR,and also pathological and immunochemistry analysis at different time point were done.All ducks survived post infection（p.i.） with GS/GD/1/96 and didn’t show any clinical signs,no virus was detected from the organs on days p.i,whereas DK/GDJD/23/04 caused 100%mortality in the ducks,furthermore,DK/GDJD/23/04 caused ducks with severe neurological dysfunction,viruses were re-isolated from many tissues.Histological lesions were observed in multiple parenchyma organs and were characterized by congestion, hemorrhage,degeneration to necrosis or’heterophilis infliltration.This virus could replicate very well in the brains,and the titers reach to the highest.In this study,Enzyme-Linked ImmunoSorbent Assay（ELISA）,CCK assay were used to research the dynamic changes of the content of IL-2,IFN_-γ in serum and T lymphocytes to ConA in spleen of 4-week-old duck infected with AⅣ.The content of IL-2,IFN_-γ in serum were increased compared with control groups and statistically significant（p＜0.05）, But the proliferative function of T lymphocytes to ConA in spleen of 4-week-old ducks infected with GS/GD/1/96 were no significant different（p＞0.05）.The content of IL-2,IFN_-γ in serum were increased compared with control groups and statistically significant（p＜0.05,p＜0.01）,But the proliferative function of T lymphocytes to ConA in spleen of 4-week-old ducks infected with DK/GDJD/23/04 were decreased compared with control ducks and statistically significant（p＜0.01）.The results showed that cellular immune in ducks infected with DK/GDJD/23/04 was critically inhibited,which is the primary element in immune subordinate ducks.Genome sequencing and analysis showed that the two viruses are highly pathogenic strains,in the molecular structure,both the HA protein glycosylation sites exactly the same, the difference between the two HA protein has two:first,DK/GDJD/23/04 is the cleavage site for the five consecutive basic amino acids（-RRRKK-）,GSGD/1/96 for six consecutive basic amino acids（-RRRKKR-）,Second,Receptor binding sites of 224-229 have changed. The amino acids of DK/GDJD/23/04 HA protein for RLVPKI and the sequence of GSGD/1/96 HA protein for RLVPEI,which has E to K changed in 228 amino acid site.The difference between the two NA proteins have two:first,DK/GDJD/23/04 has a 20-amino acid delection in NA stalk（residues49-68）,the corresponding loss of the four potential glycosylation sites.There was no delection in NA stalk in GSGD/1/96.The second is DK/GDJD/23/04 NA protein in the 386 has a potential glycosylation site,and GSGD/1/96 NA protein in the 386 did not have.A 20-amino acid delection in NA stalk（residues49-68）and a 5-amino acid delection in NS1 stalk（residues80-84）which were in the most of influenza A viruses have been the co-genetic marker of the majority isolates circulating during recent years.Phylogenetic analysis revealed that both HA and NA genes of DK/GDJD/23/04 had genetically close relationship with DK/China/E319-2/03,which suggested that they came from the same origin.更多还原