Preparation Of Hemagglutinin And Neuraminidase Subtype-specific Reference Antisera Against Avian Influenza Virus

Avian Influenza, or "bird flu", is a contagious disease of animals caused by avian influenza viruses(AIVs). The AIVs continue to circulate among poultryin Asia and remain a threat to both veterinaryandhuman public health. The virus can be divided to 16 hemagglutinin and 9 neuraminidase subtypes.Some subtypes have been evolved to different clades with spatio-temporal pattern. AIVs strain changesor mutates easily and gradually in two ways: antigenic drift and antigenic shift. Mutation in virusesresults in huge and highly flexible pool of diversity and cause changes in characteristics, such asinfectious ability, pathogenicity, and surface protein shape. Therefore, it is critical to detect the virus andidentify the subtype to diagnosis the case of the diseases, the classical and gold rule of subtypeidentification were hemagglutinin inhibitation (HA-HI) and neuraminidase inhibitation (NA-NI) tests.Reference antigens and sera of HA-HI and NA-NI were donated friendly by foreign scientists as an aidassistance almost 20 years ago, but some of the reagents made by foreign isolates couldn't diagnosis theunknown local virus due to the huge mutation. So some of diagnosis reagents were produced from thedomestic inactivated viruses, but the inactivated whole virus contained unnecessary proteins which leadto the lower specificity and interfere to the clinical diagnosis. Therefore, HA and NA subtype-specificreference sera should to be prepared for diagnosis accuratelyand rapidly.The subtype-specific reference antiseras were prepared by eukaryotic expression recombinantplasmid of HA or NA in SPF chiken. 16 HA subtypes of a total 29 representative strains and 9 NAsubtypes of a total 10 representative strains were selected and synthesized. The genes were optimizedwith chickens'codon bias and the Kozak sequence (-GCCGCC-) were inserted into upstream of ORF.Then these genes were cloned into the eukaryotic expression vector pCAGGS. The recombinantplasmids were identified by enzyme digest and indirect immunofluorescence assay, respectively.SPF chickens were immunized intramuscularly with the recombinant plasmid with the dose of 200μg, 200μL and the sera were harvested post 4weeks immunization.HA-HI test demonstrated that the titer of HA subtype-specific reference antisera can reach at least 6log2 and with high sensitivity. Theresults of cross HA-HI test manifested there is no interaction with other subtypes serums, the antiserasare strong specificity. H5 and H9 HA subtype-specific reference antiseras were mainly analysised. TheHAsubtype-specific reference antiseras which against 4 strains H5N1 HPAIVs were prepared fromWB,ST and SX clades, and it can identify difference virus which the specificity were better than commercialH5 subtype serums. The subtype-specific reference antiseras against 6 representative H9N2 strainswhich existed in China from 1996 to 2008 were prepared; the results indicated that the subtype-specificreference antiseras have more specificityand sensitivity than whole seras.The serumcontained NAantibody ideantified through macro and micro NA-NI tests. The NAsubtype-specific reference antiseras have more specificity and no cross reaction compared to serumsprepared byvirus.29 HAand 10 NArecombinant plasmid constructs in this studyexpressed accurately, which can be used for DNAvaccine research, and subtype-specific reference sera can be used in clinical diagnosisand other researchs.