Preliminary Study On An Inactivated Avian H9N2 Influenza Virus Vaccine

Since it firstly occurred in the Guangdong province in 1992, H9N2 avian influenza virus(H9N2 AIV) has been sporadically isolated and becomes predominant among avian influenza virus isolates. As the most prevalent subtype, H9N2 AIV continued to circulate in the poultry population. It has been difficult to control the H9N2 virus because of its low-pathogenicity, inapparent infection. What’s more, H9N2 AIV can lead to a high mortality associated with the co-infection, resulting serious economic losses for the poultry industry in China.The inactivated vaccines were taken in China as early as 1998 to prevent H9N2 AIV infection in chickens. There were relatively large differences between the vaccine strains and dominant strains in antigenicity after 2006 because of these vaccines strains were isolated before 2005. Our previous study illustrated that H9N2 AIV underwent significant antigenic drift. Virus continues to persist in chicken populations, even in those vaccinated flocks in recent years. Therefore, the commercial vaccines did not provide a complete protection for the endemic H9N2 AIV, reminding us to update the vaccine strains timely.In this study, the reverse genetics technique was used to develop a candidate strain in order to match the recent epidemic strains in antigenicity. Based on the screening of donor strains, the two genes(HA and NA) from H9N2 AIV(A/Chicken/Jilin/DH104/2012) and six genes(PB2, PB1, PA, NP, M, and NS) from H11N6 influenza virus(A/Swan/Jilin/SN8/2009) were amplified and cloned into the p CSA/G plasmid. The recombinant plasmids were co-transfected into 293 T and MDCK cell mixtures for 72 h. The cell supernatant was collected and inoculated into embryonated eggs and MDCK cells, respectively. The rescued virus from the allantoic fluid and cell supernatant were identified by hemagglutinination assay and the cytopathic effect. Moreover, no significant changes in the viral genome and hemagglutinin titer were detected after thirteen passages both in eggs and MDCK cells. The recombinant viruses were inactivated by the addition of 0.1% formalin(v/v) and emulsified with Imject Alum Adjuvanta at a ratio of 3:1(w/w) and used for immunization. The results indicated that the HI titer in the sera reached the protective level at 7 days post first-vaccination and achieved to the peak titer of 12 log2 at 6 days post second-vaccination, remaining at a high level(7 log2) for at least 39 days. Additionally, viral shedding was completely blocked in vaccinated birds after challenge with a homologous H9N2 AIV at 3 days. In contrast, the virus shedding from oropharyngeal and cloacal swabs at 3, 5, 7 dpi and lung and kidney at 3 dpi can be detected in the commercial vaccinated group. The cross-HI test demonstrated that the sera antibody from the commercial vaccine can partially reacted with H9N2 DH104 strain and reached to a cross-HI titer peak of 6 log2 at 36 days post first-vaccination. Taken together, the reassortant H9N2 strain based on a reverse genetics system can induce immune response and has the ability to provide protection against homologous virus infections, which will be a potential vaccine for commercial application.