Biological Characterizations Research And Establishment Of Reverse Genetics System For H5N1 Avian Influenza Virus DKFJ/01/02

Highly pathogenic avian influenza was listed as type A acute infectious disease by Office International Des Epizooties (OIE). H5N1 subtype highly pathogenic avian influenza viruses (HPAIV) always cause disaster damage for the poultry industry, and recently, the subtype avian influenza viruses caused human infections and deaths in several occasions.So the researches about the molecular of biological properties basis of AIV have been constantly emphasized.To understand the molecular evolution of the A/Duck/FuJian/01/02(DKFJ/01/02) viruses in Mainland China, the entire genomes of the virus were completely sequenced. Results shown that the HA and NA genes of the DKFJ/01/02 shared very highly homology with GSGD/1/96, the virus have deletion in the stalk of HA genes and highly pathogenic properties. The internal genes based on the homology with A/DKGX/07/99. Therefore, there is a gene recombination between different genotype influenza viruses.According to the data of molecular evolution DKFJ/01/02, we study the biological properties of the virus. A group of BALB/c mice that is a mammalian model were separately intranasally inoculated with 106EID₅₀ of DKFJ/01/02. These mice were monitored daily for 14 days for weight loss and mortality. The weight of the mice decline markedly, and it cause mice to die in ten days time. Virus titers in lungs were 7.4±0.4 three days later. intranasally inoculated with 101EID₅₀ to 106EID₅₀ of DKFJ/01/02 could cause complete fatal in mice; And the LD₅₀ values of DKFJ/01/02 were 0.5log10EID₅₀/mL. These results suggest that DKFJ/01/02 was a highly pathogenic avian influenza virus. DKFJ/01/02 have the same titers after 100TCID₅₀ infected MDCK that have 1-adamantylamine and no 1-adamantylamine, the titers is between 25 and 27, so DKFJ/01/02 have resistance phenotype to 1-adamantylamine.We established the eight-plasmid reverse genetics system of DKFJ/01/02. The total RNA was extracted from the allantoic fluids of embryonated chicken eggs containing DKFJ/01/02 virus. Using the total RNA extracted as template, the genes encoding PB2, PB1, PA, HA, NP, NA, M and NS were amplified by RT-PCR. Then they were subsequently cloned into the vector pBD. 293T cells were transfected by the 8 recombinant pBD plasmids and rescued the recombinant virus. The recombinant virus was successfully rescued and named as R-DKFJ. Two groups of BALB/c mice were separately intranasally inoculated with 106EID₅₀ of DKFJ/01/02 or R-DKFJ. Virus titers in lungs were 7.4±0.4 logEID₅₀/mL and 7.1±0.1 logEID₅₀/mL respectively. And the LD₅₀ values of R-DKFJ and DKFJ/01/02 were 0.5log10EID₅₀/mL and 0.98log10EID₅₀/mL, respectively. 100TCID₅₀ infected MDCK that have 1-adamantylamine and no 1-adamantylamine, the titers is between 25 and 27. The results of replication kinetics after 100TCID₅₀ infected MDCK suggest the titers is exceed 108TCID₅₀/mL, These results suggest that rescued virus R-DKFJ retained the same biological properties as the wild virus DKFJ/01/02 in pathogenicity resistance to 1-adamantylamin and replication ability.We systemically study the basis biological properties of molecular mechanism of H5N1 AIV and established the eight-plasmid reverse genetics system of DKFJ/01/02. The research is a good base for the molecular evolution, structure, and function and pathogenicity mechanism of H5N1 AIV, it is benefit to further development of new-type AIV gene vaccine.