The Research Of Two Genotyping Methods For AIV HA Subtype Identification From Wildbirds

Avian Influenza is an acute, highly contagious disease of birds. Not only it is a serious disease in poultry that can cause severe economic losses in many countries, also can directly infect human. Wild birds are considered a potential reservoir and many therefore play a role in the epidemiology of economically important or zoonotic disease. Studing the prevalence of this disease, we may find out the role of wild birds in the virus spreading. Furthermore, it is very important for the disease that we could prevent and control it more effectively. So, it is essential to have an accurate and convenient identification method to identify the influenza virus subtypes for the prevalence and monitor of influenza virus in wild birds.The study refers to the universal primer RT-PCR HA subtype identification methods established by L.P. Phipps and degenerate primers for each HA subtype classification methods designed by Kenji Tsukamoto. A part of influenza virus known and unknown subtypes were confirmed in order to identify their HA subtypes. Amplify all viruses by RT-PCR. The purified CDNAs were inserted into pMD18-T vector, and the recombinant plasmid was transformed into competent DH5a E.Coli, then sequenced when the PCR result was positive for amplifying the inserted gene in recombinant plasmid. And then make homology comparison with corresponding reference sequences of subtypes to determine the HA subtype.BLAST analysis showed that the degenerate primers and universal primers could identify of HA subtypes of avian influenza virus strains exactly, but in practice and on the convenient aspect, making use of universal primers to conduct HA subtype is more superior, more simple. Through validating, making use of universal primers to conduct HA subtype is more suitable for identifying avian influenza virus of wild birds.In order to certify the applicability of universal primers, we also conduct validation experiment, including after the subtype of universal primers, using degenerate primers identified again, making use of universal primers to conduct PCR amplification of AIV-positive strains, and using small fragments to make a BLAST comparation is feasible, and so on.We demonstrate the applicability and feasibility of this method thought many experiments, and then offer a effective and convenient way for identifying avian influenza virus of wild birds of our laboratory.