RESUMO
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.
Assuntos
Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H3N2/classificação , Virus da Influenza A Subtipo H5N1/classificação , Vírus da Influenza A/classificação , Neuraminidase/genética , Deleção de Sequência , Animais , Aves , China , DNA Viral/análise , Evolução Molecular , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Virus da Influenza A Subtipo H5N1/enzimologia , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A/enzimologia , Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Filogenia , Doenças das Aves Domésticas/virologia , Alinhamento de SequênciaRESUMO
To date, nine neuraminidase (NA) subtypes of avian influenza viruses have been identified. In order to differentiate the NA of avian influenza viruses rapidly, a reverse transcription PCR (RT-PCR) was developed. Nine pairs of NA-specific primers for the RT-PCR were designed based on the analysis of 509 complete NA sequences in GenBank. The primers were designed to amplify partial NA genes and each pair is unique to a single NA subtype (N1-N9). By nine RT-PCRs simultaneously in a set of separate tubes, the subtype of NA was determined by subsequent agarose gel electrophoresis and ethidium bromide staining, since only one of the nine RT-PCRs would give a product of expected size for each virus strain. In comparison with the established method of sequence analysis of 101 reference strains or isolates of avian influenza viruses, the RT-PCR method had a sensitivity of 97.3% and a specificity of 91.1% in subtyping avian influenza viruses. These results indicate that the RT-PCR method described below provides a specific and sensitive alternative to conventional NA-subtyping methods.
Assuntos
Patos/virologia , Vírus da Influenza A/classificação , Vírus da Influenza A/enzimologia , Neuraminidase/classificação , Neuraminidase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Primers do DNA , Vírus da Influenza A/genética , RNA Viral/genética , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
The hemagglutinin (HA) and neuraminidase(NA)gene from subtype H5N1 avian influenza virus were directly inserted into the transferring vector pllS, resulting in the recombinant transferring vector p11SH5ANA. Then p11SH5ANA was transfected into the chicken embryo fibroblasts (CEF), which was pre-infected with wild type fowlpox virus, to generate the recombinant fowlpox virus coexpressing H5A and NA (rFPV-11SH5ANA). By selection of blue plaques on the CEF, rFPV-11SH5ANA was obtained and purified. Experiments on SPF chickens demonstrated that the HI antibody titers in chickens vaccinated with HA-NA coexpressed vaccine was higher than those with HA expressed monovalent vaccines, and all chickens receiving either rFPV-11SH5ANA or rFPV-11SH5A were completely protected from the virulent AIV(H5N1) challenge, while those receiving wt-FPV experienced 100% mortality. The results showed that the rFPV-11SH5ANA was a safe and highly efficient gene engineering vaccine candidate for preventing HPAI.
Assuntos
Galinhas/virologia , Vírus da Varíola das Aves Domésticas/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Animais , Embrião de Galinha , Vírus da Varíola das Aves Domésticas/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/imunologia , Neuraminidase/genética , Neuraminidase/imunologia , TransfecçãoRESUMO
Because of the interference of maternal antibodies, the recombinant fowlpox virus (rFPV) vaccine has not been used widely. The selection of a well-defined FPV nonessential region might be a desirable way to solve this problem. Two pairs of primers were designed according to the genome of a pathogenic FPV to amplify two flanking regions (FPV1 and FPV2) of the supposed nonessential region by PCR, and then a series of plasmid vectors were constructed to generate the expression vector pP12LS, which containing FPV1, FPV2, the expression cassette of P11-LacZ reporter gene and the promoter Ps. To abtain the vector pP12LSF, the F gene of ZJ1 strain Newcastle Disease Virus (NDV) was inserted into pP12LS, in which the F gene was located downstream of the promoter Ps. pP12LSF was transfected into chicken embryo fibroblast (CEF) pre-nfected with 282E4 strain FPV. The recombinant FPV, rFPV-FSC, was purified by blue plaque selection. The LacZ and F genes could be expressed by rFPV-FSC after 20 successive passages in CEF. The FPV nonessential region was the only difference between rFPV-FSC and rFPV-FSB. These two rFPVs could induce completely protection in SPF chickens against lethal challenge with F48E8 strain NDV. However, the protective efficacy showed a significant difference in commercial chickens with maternal antibodies. The protective efficacy of rFPV-FSC was 100% and rFPV-FSB was 61.54%. The results indicate that the selection of a well-defined FPV nonessencial region is an effective way to increase the protective efficacy of rFPVs, especially in chickens with maternal antibodies.
