| Avian influenza (Al) is an infection and/or disease syndrome caused by type A influenza virus. It is widespread in many countries and areas throughout the world, and of great economic losses in poultry industry. The great characteristic of the highly pathogenic avian intluenza (HPAI) is high mortality, and it is classified A virulent infectious disease by O.l.E. In China, several HPAIV isolates were isolated from goose in 1996 in Guangdong province. And a year later, H5NI subtype of AIV infected human directly, especially caused them dead, which was the first time that AIV overacrossed interspecies barrier and intected human.To elucidate the relationships between A/Goose/Guangdong/3/96 (H5N I) [GD3/961 and the other strains isolated in 1997 in Hongkong, NA gene of GD3/96 was amplified by RT-PCR, then cloned into pUCI8 and sequenced. The result of sequencing showed that l4lObp of NA cDNA covered the complete open reading frame, encoding 469 amino acid residues. When compared with A/Hongkong/I 56/97(H5N 1), A/Chicken/HongKong/220/97(H5N 1), A/Teal/Hongkong/W3 12 /97(H6NI) and A/Goose/Guangdong/l/96(l-15N1), there was identity of 89.1%, 89.1%, 99% and 88.4% respectively at nucleotide acid level, and that of 90.8%, 90.8%, 99.2% and 89.8% separately at deduced amino acid level. Deduced amino acid sequence of GD/3/96 had no 19-amino-acid deletion in the stalk region. These results demonstrated that NA gene of I-longkong isolates were not directly derived from Guangdong avian influenza virus.It is very difficult to develop ideal vaccine providing protection against all subtypes of AIV, for the highly frequent antigenic variation of HA and NA gene of AIV. In this study, to produce a kind of vaccine inducing cross-protection in chickens, recombinant viruses expressing NP gene and co-expressing HA-NA or I-IANP genes of AIV were constructed. To construct transfer plasmids, NP gene of A/Goose/Guangdong/1/96(H5NI)IGDI/96j and LacZ gene were cloned into the vector of fowlpox virus (FPV), NA gene of GD3/96 or NP gene of GDI/96 was cloned into transfer plasmid containing HA gene of GD3/96 respectively, which was constructed previously. Then these recombinant vectors were transferred on CEF3cells infected with parent fowlpox virus S-FPV-0l7. Based on the expression ofreporter gene, fOwlpox virus recombinants (rFPV) were selected by blue plaque,purified by several cycles cloning of blue plaque, and identified by PCR andWestern-blot. rFPV-HA-NA, rFPV-HA-NP and rFPV-NP with stable geneticproperties could efficiently express foreign genes of A IV.Eight weeks old SPF chickens were immunized with rFPV-HA-NA, rFPV-HA-NP. rFPV-HA and rFPV-NP respectively by wing web puncture, chickens vaccinatedWith S-FPV-017 and unvaccinated were used as control. Four weeks aftervaccination, all chickens were chaIlenged with a lethal dose of H5N l and H7N lsubtypes of highJy pathogenic avian influenza (HPAIV) by intramuscular injection.Serum antibodies (Hl, AGP and ELISA), distribution of T lymphocytessubpopulation in peripheral blood, re-isolation of challenge virus in cIoacal,morbidity and morta1ity were used to evaluate the efficacy of rFPVs. High titers ofAIV specific HI and ELlSA antibodies were detected in aIl serum species collectedfrom ch ickens two weeks post-vaccination, but the resutts of AGP antibody were al lnegative. The resuIt of dynarn ics of T lymphocytes indicated that vaccination ofrFPV could effectively stimuIate ceIl-mediated immune response of chickens andactivate T cell. The percentages of CD3+, CD4+, CD8+ and y8TCR+ T cells inperipheral blood of chickens immunized with rFPV were increased. Theimmunization of rFPV could efficiently prevent the percentage of T lympocytes fromsharp dropping observed in control chickens, after challenge with HPAIV. Chickensvaccinated with rFPV-HA-NA, rFPV-HA-NP, rFPV-HA were all protected fromdeath when chaIlenged with H5N l virus, while the protection rates of against H7Nlvirus were 100%, 40% and 0, respectively.
|
PDF Full Text Request