Establishment Of Reverse Genetics System Of Avian Influenza Virus

Waterfowl are regarded as the natural reservior of influenza viruses, in order to study the pathogenic molecular basis of highly pathogenic avian influenza virus to waterfowl and establish a suitable animal model, this paper made the following experiment.First, we established the eight-plasmid reverse genetics system of DKFJ/49/05. The total RNA was extracted from the allantoic fluids of embryonated chicken eggs containing DKFJ/49/05 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.Second, a group of ducks that made as waterfowl animals model were separately inoculated with different density R-DKHB and DKHB/49/05. We observed that if were there vital and dead ducks and detected the virus titers in the different organ and secretion. Three ducks were killed to obtain the organ to make the pathological picture. The result show that the rest group all died except 102EID50R-DKHB and 102EID50DKHB/49/05 group; the virus titers in throat were 3.3±0.8logEID50 and cloacal were 2.5±0.5logEID50 in 106EID50DKHB/49/05 group and the virus titers in throat were 3.9±0.6ogEID50 and cloacal titers were 2.9±1.2logEID50 in 106EID50 R-DKHB group; all organic virus titers were high in 106EID50R-DKHB and 106EID50DKHB/49/05 group, the virus titers in brain were the highest 6.63±0.17logEID50 and 6.59±0.28logEID50; the pathological picture result show that it show up edema and nerve cell death in the brain and nothing changed in the lung and thrombus in the small blood vessel of kidney and glandular cell death.