[Cloning of full-length genes of H5N1 subtype Avian influenza virus strain A/duck/Shandong/093/2004 and analysis of the sequences].
The eight full-length genes, including the 5' and 3' ends of H5N1 subtype Avian influenza virus (A/duck/ Shandong/093/2004) were amplified by using the universal primers and H5 specific primers. The method used for the amplification of Avian influenza virus's full-length sequence was more easily and rapidly than that of rapid amplification of cDNA ends assay (RACE). The amplified segments were cloned into the T vector PCR 2.1, respectively. Three to five positive clones of each gene were sequenced and the same two sequencing results of the full-length genes were obtained. The phylogenetic analysis results showed that all the eight segments of the A/duck/Shandong/093/2004 were different from the A/Quail/Hongkong/G1/97 and A/Chicken/Beijing/1/94, but showed highly similarity (99% and above) to that of four H5N1 strains, which were isolated in 2002 in duck. It revealed that this strain was resulted from re-assortment of H5N1 rather than H9N2. The NA sequence of A/D/SD/04 was analyzed and the result demonstrated that there are 20 amino acids missing in 48 - 68 sites, however, there was no residue lost in NS gene in 263 to 277 sites. The motif of HA cleavage site is PQRERRRKKR/G, which is the characteristic of HPAIV. The 226 amino acid residue was Met (M), which can react with both Aalpha-2, 3Gal and SAalpha-2, 6Gal receptor. And the 627 residue of PB2 was Glutamic acid (E). The result mentioned above confirmed that H5N1 subtype AIV has multiple determinants in its virulence. A/D/SD/04 is the mid-strain evolving from HPAIV to a virulent strain of mammal.
Long
Jin-xue
JX
Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture, Yangzhou University, China.
Wang
Qu-zhi
QZ
Lu
Jian-hong
JH
Liu
Yu-liang
YL
Liu
Xiu-fan
XF
D003001:Cloning, Molecular; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
D019267:Hemagglutinin Glycoproteins, Influenza Virus; C057027:PB2 protein, Influenzavirus A; D014763:Viral Matrix Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C109963:hemagglutinin, avian influenza A virus; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase