| To assess the pathogenicity of influenza A virus in ducks,two viruses A/goose/Guangdong/1/96(H5N1)(GS/GD/1/96) and A/duck/GuangdongJiedong/23/2004(H5N1)(DK/GDJD/23/04) isolated from Guangdong province were selected in the study,20 SPF ducks with 4 weeks old in each group were inoculated nasally with 106 EID50 to evaluate the pathogenicity of two viruses.The content of virues,the distribution of virues and the pathological changes in multiorgans in SPF ducks were caused by the H5N1 subtype avian influenza virus,so the virus titration of multiorgans and swabs,the real time PCR,and also pathological and immunochemistry analysis at different time point were done.All ducks survived post infection(p.i.) with GS/GD/1/96 and didn't show any clinical signs,no virus was detected from the organs on days p.i,whereas DK/GDJD/23/04 caused 100%mortality in the ducks,furthermore,DK/GDJD/23/04 caused ducks with severe neurological dysfunction,viruses were re-isolated from many tissues.Histological lesions were observed in multiple parenchyma organs and were characterized by congestion, hemorrhage,degeneration to necrosis or'heterophilis infliltration.This virus could replicate very well in the brains,and the titers reach to the highest.In this study,Enzyme-Linked ImmunoSorbent Assay(ELISA),CCK assay were used to research the dynamic changes of the content of IL-2,IFN-γ in serum and T lymphocytes to ConA in spleen of 4-week-old duck infected with AⅣ.The content of IL-2,IFN-γ in serum were increased compared with control groups and statistically significant(p＜0.05), But the proliferative function of T lymphocytes to ConA in spleen of 4-week-old ducks infected with GS/GD/1/96 were no significant different(p＞0.05).The content of IL-2,IFN-γ in serum were increased compared with control groups and statistically significant(p＜0.05,p＜0.01),But the proliferative function of T lymphocytes to ConA in spleen of 4-week-old ducks infected with DK/GDJD/23/04 were decreased compared with control ducks and statistically significant(p＜0.01).The results showed that cellular immune in ducks infected with DK/GDJD/23/04 was critically inhibited,which is the primary element in immune subordinate ducks.Genome sequencing and analysis showed that the two viruses are highly pathogenic strains,in the molecular structure,both the HA protein glycosylation sites exactly the same, the difference between the two HA protein has two:first,DK/GDJD/23/04 is the cleavage site for the five consecutive basic amino acids(-RRRKK-),GSGD/1/96 for six consecutive basic amino acids(-RRRKKR-),Second,Receptor binding sites of 224-229 have changed. The amino acids of DK/GDJD/23/04 HA protein for RLVPKI and the sequence of GSGD/1/96 HA protein for RLVPEI,which has E to K changed in 228 amino acid site.The difference between the two NA proteins have two:first,DK/GDJD/23/04 has a 20-amino acid delection in NA stalk(residues49-68),the corresponding loss of the four potential glycosylation sites.There was no delection in NA stalk in GSGD/1/96.The second is DK/GDJD/23/04 NA protein in the 386 has a potential glycosylation site,and GSGD/1/96 NA protein in the 386 did not have.A 20-amino acid delection in NA stalk(residues49-68)and a 5-amino acid delection in NS1 stalk(residues80-84)which were in the most of influenza A viruses have been the co-genetic marker of the majority isolates circulating during recent years.Phylogenetic analysis revealed that both HA and NA genes of DK/GDJD/23/04 had genetically close relationship with DK/China/E319-2/03,which suggested that they came from the same origin.
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