Primary Study On The Pathogenicity And HA Gene Of Avian Influenza Viruses Isolated From Muscovies

In the late 2002 year, several muscovy avian influenza happened in some districts of Zhejiang and Fujian provinces, where apparent nervous symptoms were found on the infected poultry, and necrosis in the brain demonstrated on the dead poultry. To study pathogen characteristics of these viruses, the viral HA genes were analyzed and the viruses were inoculated into the animals to observe their pathogenicities.The four H5N1 subtype avian influenza viruses isolated from muscovies died with necrosis in brain, named LH, ZA, LSD and LSM strain, were inoculated into 12-day-old embryonated chicken eggs, the allantoic fluids with virus were concentrated and purified in the super-centrifuge. The viral genomic RNA was extracted and complementary DNA of HA gene was amplified with one-tube RT-PCR. The product was cloned into the pGEM-T vector and transformed into DH5a, then positive recombinant plasmid was sent to sequence. The result shows that HA gene of the four isolates has a 1732bp-long nucleotide, containing 1707bp of ORF, which encodes 568 amino acids. The proteolytic cleavage sites were all hexylbasic amino acids RRRKKR, with exception of LH (GKRKKR), which were the characteristic sequence of the highly pathogenic strains; The sequence of potential glycosylation sites and receptor binding sites are similar to those of the other reference HPAI isolates, besides the additional N-glycosylation sites NNT and NST residues in 170aa-172aa of LH and LSD respectively, and a NGS appeared in 260aa-262aa of LSM; While with the potent O-glycosylation sites in LH, a SAYP disappeared in 171aa-174aa but added a PS in 157aa-158aa, and in LSD, 171aa-174aa changed from SAYP to STYP; Analysis of their relationship with Megalin indicates that the homology reaches 96.1-98.6%. Thecomparison of the test strains with the others also shows the high homology of 95.1-98.6% with national isolates, but the homology level decreased among the international strains (85.6-94.3%). The phylogenetic relationships divided the tested strains into two subgroups, all falling within the Eurasian lineage with the other six national strains and have a distinct branch with the North-American lineage.Two test strains, LH and LSD, were selected to test their pathogenecity with the other two duck HPAIV strains, NJ and SH, as the controls. The laboratory animals included chickens, geese, quails, broiler ducks, layer ducks and pigeons. After challenging with the viruses, clinical symptoms appeared and some animals died. The results suggest that both test and control viruses have a high pathogenicity for chicken, goose and quail (the mortality may reaches 100%); The mortality of ducks varied from 16.7% to 83.3%, and only one pigeon died. The common clinical signs before dead included depression, decreased feed consumption and cyanosis of unfeathered skins. Congestion, hemorrhage and necrosis in subcutaneous, internal organs and digestive tracts were the common gross lesions on the dead poultry. The histologic lesions on ducks showed the same with cellular degeneration, necrosis and organizational hemorrhage and congestion. The serious damages in the immune organs were particularly apparent. The survived ones remained clinically healthy, but milder lesions could be observed in some organs. Through the pathogenicity test, it may be concluded that the four isolates are highly pathogenic for chickens, geese and quails, low pathogenic for ducks and non-pathogenic for pigeons. The pathogenicity of the different or the same isolates for different animals were different, test strains were more virulent than the controls, for the mortality were higher and the lesions more serious.In one word, the four test isolates were all highly pathogenic avian influenza viruses with higher pathogenicity than the earlier ones, the HA genes varied in some extent and belong to the Eurasian lineage, closer to the national isolates than the international ones.