Biological Characteristics Of An H5N1 Avian Influenza Virus And Its Infectious Clone

An H5N1 avian influenza virus (AIV) isolated from China was studied its molecular evolution and biological characteristics and established an eight-plasmid reverse genetics system for this virus in this paper.Firstly, this virus named as DSH/8/01(H5N1) was sequenced and molecular evolutionarily analyzed, which compared with fifteen H5N1 AIV isolated from different regions in East Asia during the period of 1996 to 2004.DNA sequence analysis of these viruses genes showed that this virus excluded from NA gene in this study was closely related with GGD/3/97and GGD/1/96,which isolated from gooses in Guangdong, China in 1996 and 1997.But, it was comparatively far related with CHK/220/97,HK/483/97 and HK/486/97,which isolated from chickens and human in HongKong during the 1997 outbreak of AIV. Therefore, I suppose that DSH/8/01 in this study possibly came from early H5N1 AIV in Southern China, which is a GGD/1/96-like virus.Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. Fifty percent of Egg Infective Doses (EID50), Intravenous Pathogenecity index (IVPI) to chicken of this virus, Mean Death Time to chicken of intranasal and intravenous inoculated virus were 10-8.33/0.1ml, 3.00,3.82days and 1.00 day respectively. This virus titer of chicken's lung was most high, which was 6.98(log10EID50/0.1ml). The replicated virus also was isolated from the heart muscle, bursa of Fabricius, kidney, brain, spleen, liver in chickens, whereas only a few chickens' intestine can isolated this virus and chickens' pancreas cannot. Accordingly, this virus is a H5N1 Highly Pathogenic Avian Influenza Virus.This virus intranasal inoculated BALB/c mice experiment showed that it had had evident pathogenicity to mice. The majority of mice infected intranasally with 106 to 105EID50 of this virus showed clinical signs, more significant weight loss, even led to death. Whenas, mice infected intranasally with 104 to 101EID50 of this virus had no significant weight loss. 50% Lethal Doses to BALB/c mice (MLD50) of this virus was 104.16EID50.The highest virus titers in the mice's tissue and organs were lung and brain, but This virus titer in the brain was higher on day 6 than on day 4.The replicated virus also was recovered from kidney, spleen, liver, the heart muscle and blood, whereas intestine and pancreas nearly can not isolated this virus. Thus, pathogenicity of this virus to chickens and mice was obviously different, and these test also proved that the virulence mechanism responsible for the lethality of influenza virus in birds also operates in mammalian host. I established an eight-plasmid reverse genetics system for this virus, and succeeded to rescue infectious AIV in order to research its structure and function and its pathogenicity mechanism for the future.In conclusion, the research is a good base for development of new-type AIV vaccine, the further study for the molecular evolution, structure, and function and pathogenicity mechanism of H5N1 AIV.