ABSTRACT
To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Subject(s)
Animals , Antibodies, Viral , Blood , Bacteriophage T7 , Genetics , Allergy and Immunology , Metabolism , Chickens , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Viral , Immunization , Influenza A virus , Genetics , Allergy and Immunology , Influenza Vaccines , Allergy and Immunology , Influenza in Birds , Allergy and Immunology , Metabolism , Peptides , Genetics , Allergy and Immunology , Metabolism , Polymerase Chain Reaction , Recombinant Fusion Proteins , Specific Pathogen-Free Organisms , Viral Matrix Proteins , Genetics , Allergy and Immunology , MetabolismABSTRACT
To examine the phylogenetic information regarding the gene pool of AIV in domestic ducks in eastern China, the NA genes of twenty-six viruses isolated during 2002-2006, including two H1N1 strains, tenH3N1 strains and fourteen HSN1 strains, which reflected the predominant N1 subtype viruses were subjected to phylogenetic analysis. The results indicated that AIVs of N1 subtype circulating in domestic ducks in eastern China were undergoing a gradual evolution. Analysis of the deduced amino acid sequences revealed that NAs from all isolated H5N1 viruses had a 20-aa deletion in the stalk region (residues 49-68), whereas no deletion was seen in the NAs from other HA subtype viruses. The viruses of H3N1 and H1N1 might have a propensity for reassortment of NA genes, whereas no direct evidence of reassortment of NA gene was obtained in H5N1 viruses.
Subject(s)
Animals , Humans , Birds , China , DNA, Viral , Evolution, Molecular , Influenza A Virus, H1N1 Subtype , Classification , Genetics , Influenza A Virus, H3N2 Subtype , Classification , Genetics , Influenza A Virus, H5N1 Subtype , Classification , Genetics , Influenza A virus , Classification , Genetics , Influenza in Birds , Virology , Influenza, Human , Virology , Neuraminidase , Genetics , Phylogeny , Poultry Diseases , Virology , Sequence Alignment , Sequence DeletionABSTRACT
Two H5N1 avian influenza viruses (AIV), A/mallard/Huadong/S/2005 (S, IVPI = 2.65, in mallard) and A/mallard/Huadong/Y/2003 (Y, IVPI = 0, in mallard), were capable of distinct in pathogenicity to non-immunized mallards (Anas platyrhynchos). There were two amino acid residues difference in the HA cleavage site between two viruses, 322 (S, Leu; Y, Gln) and 329 (S, deletion; Y, Lys). Based on the variation, a series of recombinant viruses carrying HA gene either from S or Y virus with mutation at 322 and/or 329 were constructed via reverse genetics system to explore the influence of the two amino acid residues on viral pathogenicity in mallards. Recombinant viruses with S virus backbone were completely attenuated in terms of their virulence to ducks when position 322 (L322Q) and/or position 329 (-329K) of HA gene had been mutated. The critical role that L322 and -329 of HA protein from S virus play in the high virulence to ducks were influenced by the entire background of that protein because the recombinant virus with HA gene from Y and other seven genes from S were completely attenuated even if Q322L and K329- mutations of HA gene had been achieved. Recombinant viruses with Y virus backbone significantly increased their virulence to ducks when position 322 (Q322L) and/or position 329 (K329-) of HA gene had been mutated. All recombinant viruses carrying HA gene from Y with Q322L and/or K329-mutations and other seven genes from S were completely attenuated in terms of virulence to ducks whereas all recombinant viruses carrying HA gene from Y with same mutations and other seven genes from Y gained significant virulence. It seems that the compatibility among eight genes might be an important factor for HA to exert its functions. Results indicated that the mutation at amino acid position 322 and deletion at 329 in HA cleavage site significantly influence the pathogenicity of S and Y viruses in mallard, the compatibility among eight genes also contribute to the pathogenicity of both viruses in mallard.