Molecular Diagnostic And Preventive Techniques Of H5 And H7 Subtypes Influenza Viruses

In the present study, the polymerase chain reaction was used for development of a specific RT-PCR for avian influenza virus, which may be used for diagnostic purposes. A sets of primers were selected from strongly conserved regions of the genome coding for the nucleoprotein (NP) gene and were tested on 26 avian influenza viruses belonging to 14 H subtypes. RNA extracted directly from clinical trachea or cloaca swab and infected tissue samples by Trizol reagent were used as the source RNA for RT-PCR. The amplified products of 236bp were obtained with all these strains after electrophoresis on a 2% agarose gel. The specificity of the reaction was confirmed by hybridization with NP probes labeled by digoxigeniri. When nucleic acids from a variety of avian viruses disease were subjected to the PCR with the sets of primers, no specific amplified products were generated. The sensitivity of the technique was found to he at the level of 10 pictogram level. The RT-PCR were applied to experimental specimens inoculated with 1-15 and 1-17 subtypes AIV. The 34 samples were positive in RT-PCR detection and among them 32 could be confirmed by viruses isolated on embryonated hen抯 eggs. The results of RT-PCR from 6 clinical specimen were confirtned by the virus isolation on embryonated hen抯 egg, either. Apart from diagnosis of influenza virus infection, PCR can also be used for subtyping influenza isolates. Two sets of primers were designed and synthesized to differentiate H5 and H7 subtype avian influenza viruses from other 12 subtypes by RT-PCR. Primers H5F/R or primers of H7F/R were only subtype-specific, cross reactivity with the other 13 subtype influenza viruses were not found, the specificity of the amplified products were confirmed by dot-blot hybridization with digoxigenin labeled 1-15 or H7 HA gene probe. H5 subtype specific amplified products were 242bp DNA fragment and 1-17 subtypes were I 92bp DNA fragment. With the sets of primers H5FS/RS and H7FS/RS which bind to each side of cleavage sites of HA, 998 bp DNA fragment and 712 bp DNA fragment could be amplified by RT-PCR. The purified PCR products sequenced by auto-sequencer. The amino-acid sequence were deduced and the virulence of the virus could be predicted. Technically, the RT-PCR is suited for application for routine diagnostic procedures in a diagnostic laboratory. Based on the sequence of hemagglutinin (HA) and neuraminidase (NA) gene of avian influenza virus A/Goose/Guangdong/1/96(H5N I), two sets of primers were designed and synthesized. The 5? 3 H5 jv H7 k1~31~i 2000 ~- 6 ~ ends of primer抯 adding the sequence of restriction endonuclease BamHI (or HindIII). The complete coding region of HA (or NA) gene was obtained from the viral genome RNA by RT-PCR. The amplified fragments were digested with BamHl (or HindlIl) and then cloned into the ptasmid vector pUCI9 cleaved with BamHl (or HindllI). The recombinant plasmids contain HA (or NA ) genes were designated as pUCH5 or pUCN 1. The HA (or NA) genes were subcloned into the baculovirus transfer vector pBlueBacHisB from pUCHS or pUCN 1. The recombinant baculovirus transfer vectors were screened at random by picking colonies on plates contained ampinicillin. The recombinant transfer vector pBacH5(or pBacNl) was sequenced and used for transfecing Sf~ cells. The recombinant baculovirus transfer vector containing 1-17 HA In correct oriention were designated as pBacH7. After con...