| H5N1 subtype avian influenza crossed avian-human species barrier and transmitted from poultry to human directedly in 1997, leading to 6 deaths of 18 infected humans. So far it has been reported 39 confirmed human cases of avian influenza A/(H5N1) .The cases that H5N1 subtype highly pathogenic avian influenza infected human occurred continuously since 2004. It was paid close attention by the people all over the world because the mortality is up to 60%. Reverse genetics is one of the most useful methods to investigate the pathogenic mechanisms of influenza viruses. However, it has not seen that reverse genetics system of the human H5N1 influenza virus isolated from the mainland China was established yet.The human avian influenza virus A/Anhui/2/2005(H5N1) isolated in early period was preserved in our laboratory .It belongs to sublineage Clade2.3.4 and has a close relationship with the avian influenza viruses isolated from the waterfowl in South China.The viruses belonging to Clade2.3.4 exist generally in poultry in South China, threatening the health of the animals and humans.So it is necessary to elucidate pathogenic mechanisms of human avian influenza virus A/Anhui/2/2005(H5N1).Eight plasmids system of the human H5N1 influenza virus isolate A/Anhui/2/2005 was established and it was rescued successfully by reverse genetics in this study.The full genome of the rescued virus R-AH05 had no nucleic acid change compared with the wildtype virus W-AH05 through sequence analysis. R-AH05 also kept highly pathogenic trait of W-AH05 in mice. The 50% mouse lethal dose (MLD50) was 1.5 log10EID50 and 1.2 log10EID50, respectively. The tissue tropism was highly resembled and the mean virus titers were almost equal isolated from respective viscera of the mice infected intranasally with 106EID50 dose of R-AH05 and W-AH05. In conclusion, the rescued virus R-AH05 has congruously biological characteristic with the wildtype virus W-AH05.The results could provide a technique for further research on the pathogenic and crossing-host-barrier mechanisms of the human H5N1 influenza virus isolate. Egypt is the country that has the more serious avian influenza outbreaks in Africa. It has announced 109 confirmed cases of human infection with the H5N1 avian influenza virus by 6 May, 2010, 34 of whom are dead. Most of infected humans had exposure to sick and dead poultry.It happened 2166 highly pathogenic avian influenza outbreaks in poultry from 2006 to 2009. Not only can vaccination in poultry make the poultry industry avert the severe loss, but it can also decrease human cases of avian influenza A/(H5N1).Bird flu vaccines used in Egypt depends much on importing from other countries, commercial bird flu vaccines in China can not prevent completely against H5N1 avian influenza viruses circulating in Egypt. In this study we generated a avian influenza vaccine candidate H5N1(EG) /PR8 by twelve plasmids system.We used the circulating strain A/Chicken /Egypt/A-18-H/2009(H5N1) as the donor of the surface gene HA and NA ,which made the vaccine candidate keep the consensus antigenicity with the circulating viruses. Meanwhile the polybasic cleavage motif -PQGEGRRKR*GLF- was deleted and mutated to low pathogenic molecular characteristics -PQRETR*GLF-.That made the vaccine more safe. PB2,PB1,PA,NP,M and NS genes derived from egg-adapted vaccine virus A/Puerto Rico/8/34(H1N1) kept the vaccine candidate characteristic of well-adapted growth in embryonated chicken eggs, contributing to mass production .Eight genes of this reassortment vaccine candidate strain is correct through sequencing identification.It is well adapted to growth in embryonated chicken eggs, the highest titers up to 210. Its HA gene carried the classic molecular characteristics -PQRETR*GLF- of low pathogenic H5N1 subtype avian influenza viruses, which made it safer. So this vaccine candidate strain is promising to be seed virus for producing avian influenza vaccines exported to Egypt.
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