Establishment Of Reversed Genetic System For H7N9Influenza Virus And Generation Of Candidate Vaccines For H7N9and H5N2Avian Influenza Virus

In our study, the A/Chicken/Shanghai/S1053/2013(CK/53) was selected as the parental strain to establish the eight-plasmid reverse genetics system and to rescue the rCK/53. Meanwhile,186V and226L mutations occurred in HA segment of the2013H7N9AIV. To examine whether they will impact the receptor binding ability, we generated a series of mutant viruses in these sites, and the results suggested that the186V and226L played an important role on the virus binding to human receptors. To further explore its host specificity, we adapted the rCK/53in mammalian cells (MDCK) by6consecutive passages, and E627K mutation in PB2was identified, which has been previously proved to be the molecular marker for the adaptation of avian influenza viruses in mammalian hosts. To prevent and control the wide spreading of H7N9viruses and the great losses it caused, a recombinant virus, rPR8-CK53, was generated as the candidate vaccine seed virus, which contained HA and NA genes of CK/53in the background of internal genes derived from A/Purto Rico/8/34(PR8). Comparison of rCK/53and PR8-CK53in MDCK and A549cells showed that there was no significant difference. These results could provide a promising basis for the further evaluation of the immune protection efficacy of rPR8-CK53in avian species.The co-circulation of multiple subtypes of avian influenza viruses results in the repeatedly emergence of reassortant viruses with novel features. To better control the threat posed by these novel viruses to the animal health, the dominant reassortant viruses must be selected for the development and evaluation of preparatory vaccine. The outbreak of H5N2avian influenza viruses in poultry was detected in China in2013. Phylogenetic analyses of three representative isolates, A/Chicken/Shandong/03/2013(SD03), A/Chicken/Hebei/03/2013(HB03) and A/Chicken/Hebei/2012(HB2012), suggested that their HA was derived from the H5N1viruses of clade7.2but with a low homology to that of the prototype virus (A/Chicken/Shanxi/2/2006). The M gene was derived from H5N1viruses and other six genes originated from H9N2viruses. In this study, we used SD03, HB03and HB2012as the surface gene donor in which the multiple basic amino acids at the HA cleavage site were removed to resemble the characteristics of that of low pathogenic avian influenza viruses. Using PR8as the donor of the six internal genes, we successfully rescued three strains of H5N2candidate vaccine viruses by reverse genetics system, thus, providing a basis for further experimental evaluation of their immune protection efficacy.