Study On The Biology Characteristics Of Neurovirulent H5N1 Avian Influenza Virus

Since the emergence in Italy in 1878, the avian influenza virus (AIV) broke out in the world endlessly. And since the first case of the H5N1 AIV infected people and caused mortality in 1997, there were 553 cases of H5N1 AIV infection,323 of them died from then on. This posed great threat on the poultry industry and the human public healthy security.Waterfowl, as the natural host of AIV, did not display any symptoms when infected. But the long-time evolution equilibrium was disrupted when AIV broke out in two parks of Hong Kong. The majority of exotic migratory birds was killed and developed system infection, including the dysfunction of the severe central nervous system (CNS). The viruses harbored highly pathogenicity to waterfowl were isolated in Japan, Thailand and south China.AIV broke out in a duck farm in Hubei province in 2006. The ducks developed severe CNS dysfunction including violent tremors, marked loss of balance and uncontrollable shaking of heads. A H5N1 AIV was isolated from the brain tissue of the infected ducks. The whole genome of the virus was sequenced and the phylogenisis of the virus was performed. We also analyzed the pathogenicity of the virus to the different host. And comparative proteomics was performed to study the protein expression change patten when chicken brain tissue and SK-N-SH cells were infected by hm/06, trying to reveal the mechanism of the interaction between virus and host cell as well as the neuropathologenesis of the host. Meanwhile, the Solexa high-through sequencing was formed to investigate the mouse lung when infected by neurovirulent H5N1 virus and H1N1/2009, building a foundation of the mechanism of pathogenesis when host mix-infected by different kinds of influenza virus. The principal contents were described as following:1 Whole genome sequencing and phylogenesis analysis of the neuvirulent AIV A/duck/Hubei/hangmei01/06(hm/06)A H5N1AIV was isolated from the brain tissue of the infected ducks developed central neural systerm dysfunction. And the whole genome of the virus was sequenced. Sequences analysis revealed that the HA cleavage site of the virus was "SPQRERRRKR". There was a K deletion when compared with H5N1 viruses isolated in Vietnam, Thailand and south China in 2004. The sites 222 and 224 of the HA protein were Gln and Gly at the receptor binding site, demonstrating that the isolate still possessed an avian-specific receptor binding property. Meanwhile, the NA protein displayed a 20-amino acid deletion at positions 49 to 68, and NS1 also had a 5-amino acid deletion at position 80-84.Phylogenetic analyses revealed that this virus was closely clustered with those avian influenza viruses defined as Fujian-like viruses isolated after 2005 in south China. The HA gene clade of the virus is 2.3.4, which was one of the most four epidemic subleaneage. The PB1?PA?HA?NP?M and NS genes contained the highest homology with the virus, A/China/GDO 1/2006, isolated in human in 2006. And the viruses of highest homology with NA and PB2 were A/duck/Fujian/1734/2005 and A/chicken/zhejiang/24/2005, respectively.2 Pathogenicity to embryo eggs, chicken, mouse and duck of the neurovirulect H5N1 virusThe EID50?IVCP and LD50 were determined to evaluate the pathogenicity of the neurovirulect H5N1 virus to embryo eggs, chicken and mouse. The results displayed that virus was highly pathogenic to embryo eggs, chicken and mouse. The duck regression experiment was also performed and the ducks developed the same CNS dysfunction as that of the field infected ducks. The virus distribution displayed that the virus titer in brain tissue was high and virus was isolated in most infected ducks brain. With difference to the traditional target organs of the AIV were intestine and lung, the virus was nearly isolated in the lung of the ducks infected by neurovirulent virus. This demonstrated the main target organ of the neurovirulent virus was brain tissue, and this may be associated with the host neurotropism of the virus.3 The pathology model of the chicken brain tissue infected by the neurovirulect H5N1 virusThe brain pathology analysis was performed to further investigate the pathogenicity of the neurovirulent virus to host. Most chickens developed the neural symptoms when infected by the virus, and virus was also isolated in the chicken brain. Infected chicken brain tissues developed classic non-suppurative encephalitis. The cerebral pia mater was severely edema with the turgescent of the meningeal vessels. Different degrees of neuronal degeneration, inflammatory reactions such as neutrophil infiltrates, considerable satellitosis, and neuronophagia could be observed. Severely perivascular cuffing and degeneration of myelin was accompanied with virus infection. And immunohistochemical staining displayed that viral antigens were detected mainly in the glial cells in the cerebrum and cerebellum. Meanwhile, TUNEL staining displayed that the glial cells of the cerebrum and cerebellum were also displayed apoptosis when the brain infected by the virus.4 Comparative proteomics analysis of the chicken brain tissue and neural cells infected by the neurovirulect H5N1 virus hm/06Proteomics was applied to investigate the interaction of the virus and host as well as the protein differential expression when chicken brain tissue and SK-N-SH (SK) cells were infected by the neurovirulent virus. Meanwhile, proteomics was performed on the SK cell infected by a non-neurovirulent H5N1 AIV as a control. By using 2-DE, coupled with MALDI-TOF MS/MS, a set of differentially expressed cellular proteins were identified. But the differentially expressed proteins of the infected tissue and SK cell were not identical. The differential proteins caused by the non-neurovirulent virus were different with that of the hm/06 virus did. The significant changed protein participated in regulation of cytoskeleton proteins, proteins associated with ubiquitin-proteasome pathway, neural signal transduction, molecular synthesis and metabolism, protein transporting and modification. Notablely, some proteins took part in neural signal transduction and axon morphology stabilization. And some proteins participated in the pathogenesis progress of Huntington's diseases.5 Inhibition of protease inhibitor and siRNA on the cell apoptosis raised by hm/06Protease inhibitor and siRNA of protein c-Abl and Cdk5 were used to inhibit the expression of the two proteins. TUNEL staining and western blot assay were applied to determine the change of SK cell apoptosis and the expression of the apoptosis marker after infected by hm/06. Results showed that the expression of Cdk5 was obviously inhibited by the protease inhibitor and siRNA. The SK cell apoptosis and the expression of the marker were also inhibited. This might indicated that protein Cdk5 had participated in the progress of the tissue and cell apoptosis caused by hm/06.6 Transcriptome analysis of the mouse lung infected by H5N1 AIV and A/HIN1/2009Solexa high-through sequencing was applied to analyse the transcriptome of the mouse lung infected by H5N1 AIV and A/HIN1/2009. A series of differential genes were identified associated with the mixed infection. And we also observed lots of genes developed different forms of the alternative splicing events. The differentially expressed genes and the splicing transcripts might associate with the high virulence and pathogenisis mechanism of H5N1 and H1N1 influenza viruses mixed infection.