Influence Of Antibody-mediated Immune Pressure On Neuraminidase Gene Mutations Of Avian Influenza Virus H9N2

The LG1strain which was isolated in our lab was proliferated in the SPF embryos. Then,the cDNA of NA and HA gene of H9N2avian influenza isolate was amplified, cloned andsequenced. The result of the sequence analysis shows that cDNA of NA gene is composed of1401bp nucleotides, encoding for466amino acids, and HA gene is composed of1683bpnucleotides, encoding for560amino acids.The first round of passage is started with LG1strain. The leghorn chickens sourced SPFchicken were inoculated inactivated elai-vaccine (LG1strain of H9N2subtype) at thebeginning of egg laid, the serum for the hemagglutination inhibition H9antibody titer was29.Fertilized eggs are collected and hatched after laid, as the embryo with maternal antibodies.At the same time, SPF chicken eggs are got directly from the same strain as the embryoswithout maternal antibodies. The LG1strain of H9N2-AIV is passed continuously inembryos in4separate serials with antibodies against LG1(A1-4) and in2separate serialswithout antibodies (B1-2) for40passages. The viruses in each passage are preserved in ourlab to determine the sequences and be compared to the original strain.Viruses with stable mutation of the40thpassage (A1-40and A3-40) are chosen for thesecond round of passage as in the first round. The serials with antibodies are A1-40-A andA3-40-A, and serials without antibodies are A1-40-B and A3-40-B. At the10th,20th,30th,and40th passages in each serial, HA and NA gene sequences were determined and comparedto the original strain.The result shows that, in the first round of LG1strain,3serials have a stable mutation of“G” to “A” at#99of the neuraminidase gene（NA）from the20th passage resulting in achange of Met to Ile and2have a stable mutation of “A” to “G” at#473of the NA gene fromthe30th passage resulting in a change of Asn to Ser which occurred in the50th passage ofanother experiment. Eighty continuous passages in chicken embryos without antibody do nothave the same mutation, indicating that the mutations of the2positions are associated withselective pressure of antibodies. Analysis of the ratios of nonsynonium (NS) vs synonium (S)mutations of nucleic acids demonstrates that NS/S of4parallel experiments with antibodiesis4.6(32/7) compared with2.0(16/8) of the2experiments without antibodies and this significant discrepancy implies the selective pressure of antibodies.In the second round, HAdid not form new stable mutations.The results suggested that the antibody selective pressure influenced mutations of H9N2AIV NA gene during the passages in embryos with maternal antibody, but less than the HAgene. Embryos with maternal antibody to certain viruses could be used as experimentalmodels to study the immune selective pressures on viral mutations.