| H5N1 and H9N2 are two main subtypes of influenza A viruses that circulate in Southeast Asia including China and many other countries worldwide. Avian influenza (AI) associated with these two subtypes has raised great concerns for its economic importance to poultry industry and potential threat to the public health. Highly pathogenic avian influenza (HPAI) caused by some H5N1 strains can lead to high morbidity and mortality, whereas mildly or moderately pathogenic viruses of H9N2 subtype can also induce serious economic consequences in chicken industry. AI is not an eradicable disease, as avian influenza virus (AIV) has a lot of subtypes, undergoes constant antigenic drift and shift, and can infect a variety of birds and mammals. More importantly, there is a large reservoir in wild aquatic birds, therefore prevention and control relying on vaccines and strict biosecurity precautions are realistic approaches. The results from surveillance on live poultry markets in southern China (mainly, Hong Kong) have showed that viruses of these two subtypes coexist and evolve rapidly, and this remind of our lack of preparedness for human influenza pandemic. In this case, it is essential for mainland China to strengthen studies on the distribution, evolution, and pathogenicity of AIVs and development of more effective and safer vaccines by novel approaches. Reverse genetics systems entirely based on cloned cDNAs of influenza virus whose genome is negative-sense and eight-segmented have been established by Neumann, Hoffmann, et al. since 1999, and these developments have implied the perspective future of virus research.In this representation, an AFV isolate of H9N2 subtype, A/Chicken/Shanghai/F/98 (C/SH/F/98), from mainland China was chosen to determine the entire genomic sequence and characterize phylogenetically. The eight cDNAs from C/SH/F/98 were cloned into the bidirectional transcription/expression vector pHW2000, and a panel of reasserted viruses containing HA and NA genes from C/SH/F/98 were generated by reverse genetics technique. Attenuated H5N1 and H5N2 recombinants with modified HA genes were also generated by using this technique.1. Sequence determination and phylogenetic analysis of the eight full-length genes of an H9N2 subtype AIVA previously characterized AIV, C/SH/F/98 (H9N2), was propagated in embryonated chicken eggs and virus in the allantoic fluid was purified by high-speed centrifugation with a sucrose cushion. Viral RNAs were extracted and RT-PCR was performed to amplify the eight gene segments with eight sets of specific primers designed according to published sequences. In order to obtain accurate sequences of 5'-ends and 3'-ends, a modified method of rapid amplification of cDNA ends (5'/3'RACE) was also employed. Sequences of the eight full-length genes were subsequently obtained. The genome of C/SH/F/98 was 13597 nucleotides (nt) in length. Segment one was 234 Int encoding PB2 of 759 amino acid (aa); segment two was also 2341 nt encoding FB1 of 757 aa; segment three consisted of 2233nt encoding PA of 716 aa, and segment four was 1742nt encoding hemagglutinin (HA) of 560 aa. Segment five consisted of 1565nt encoding nucleoprotein (NP) of 498 aa; segment six was made up of 1458nt encoding 466 aa of neuraminidase (NA). Segment seven and segment eight comprised 1027nt and 890nt respectively, and both encode two proteins: matrix 1 (Ml), 252 aa; ion channel M2, 97 aa; nonstructural protein NS1, 217 aa and NS2/NEP (nuclear export protein), 121 aa.The sequences of the eight genes were deposited in GenBank under accession numbers AY250750-AY250756, AF461532. C/SH/F/98 was the first H9N2 AIV with unabridged genomic sequence reported in GenBank.Homology comparison and phylogenetic analysis were carried out with sequence data for the eight genes of C/SH/F/98 and those of other representative H9N2 strains.Phylogenetically, C/SH/F/98 was entirely different from Q/HK/G1/97 which shares internal genes with H5N1 subtype viruses responsible for 1997 Hong Kong Bird flu incident and associated with human... |
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