Preparation of hemagglutinin-specific reference antisera against H4 and H6 subtype avian influenza viruses

In this study,to establish a rapid method to detect H4 and H6avian influenza viruses,hemagglutinin(HA)-specific reference antisera against H4 and H6subtypes of avian influenza virus(AIV)were prepared.HA genes of A/Duck/Hunan/S11090/2012(H4N6)and A/Chicken/Guangdong/S1312/2010(H6N2)were synthesized and optimized,and then inserted into mammalian expression vector pCAGGs,respectively.Recombinant plasmids were constructed and confirmed by double restriction enzyme digestion and sequencing.Then each 4-week-old specific pathogen free chicken was immunized with 200μg HA plasmid three times at a 30-day interval.The sera were collected at 10 days after the third immunization.The results of indirect immunofluorescence and Western-blot indicate that HA genes of recombinant plasmids were successfully expressed.Antibody titers detected by hemagglutination inhibition test were ranging from 64 to 128by using H4 and H6 AIVs as antigens,suggesting high sensitivity of antisera.Meanwhile,the prepared antisera did not cross-react with AIV of other subtypes and Newcastle disease virus,indicating high specificity of prepared antisera.