RESUMO
This study aimed to establish a method for the detection and identification of H7N9 avian influenza viruses based on the NA gene by pyrosequencing. According to the published NA gene sequences of the avian influenza A (H7N9) virus, a 15-nt deletion was found in the NA gene of H7N9 avian influenza viruses. The 15-nt deletion of the NA gene was targeted as the molecular marker for the rapid detection and identification of H7N9 avian influenza viruses by pyrosequencing. Three H7N9 avian influenza virus isolates underwent pyrosequencing using the same assay, and were proven to have the same 15-nt deletion. Pyrosequencing technology based on the NA gene molecular marker can be used to identify H7N9 avian influenza viruses.
Assuntos
Animais , Sequência de Bases , Aves , Galinhas , Sequenciamento de Nucleotídeos em Larga Escala , Métodos , Subtipo H7N9 do Vírus da Influenza A , Classificação , Influenza Aviária , Virologia , Dados de Sequência Molecular , Neuraminidase , Genética , Filogenia , Doenças das Aves Domésticas , Virologia , Proteínas Virais , GenéticaRESUMO
Based on the genomic sequence of NDV08-004 strain (GenBank accession number FJ794269), seven pairs of primers were designed to amplify the genomic fragments by RT-PCR and cloned into pGEM-Teasy vector. The fragments (named A to G) were sub-cloned into transcription vector pOLTV5 according to the universal RE site and the plasmid named NDV08-004-pO which contained the full length cDNA of NDV08-004 strain was constructed. Three helper plasmids (pCI-NP, pCI-P and pCI-L) together with NDV08-004-pO were co-transfected into BSR T7/5 cells, and the transfection supernatant was inoculated into SPF embryonated eggs to rescue the virus. The virus was rescued successfully and identified by HA and RT-PCR and sequencing. The rescue system constructed in this study provided a good foundation for the further related research.
Assuntos
Animais , Embrião de Galinha , Sequência de Bases , Vetores Genéticos , Genética , Dados de Sequência Molecular , Doença de Newcastle , Virologia , Vírus da Doença de Newcastle , Genética , Plasmídeos , Genética Reversa , MétodosRESUMO
Mutation in any of five key amino acid residues (at positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses leads to resistance against the amantodine class of anti-influenza drugs. In this study, a pyrosequencing method was described to rapidly detect established five molecular markers of resistance to M2 blockers, amantadine. The residues L26, V27, A30, S31 and G34 in the M2 protein were targeted for pyrosequencing, and 94 avian influenza viruses were used to perform the amantadine resistance analysis. Our results showed that most of avian influenza viruses were amantadine resistant, Mutations V27I and S31N were founded in these isolates.
Assuntos
Animais , Amantadina , Usos Terapêuticos , Antivirais , Usos Terapêuticos , Galinhas , Farmacorresistência Viral , Genética , Vírus da Influenza A , Genética , Influenza Aviária , Tratamento Farmacológico , Virologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Thirteen isolates of Class I Newcastle disease virus obtained from healthy poultry in China during 2008 were characterized genotypically in this study. All the isolates were proved to be lentogenic strains based on the deduced amino acid sequence of the Fusion protein gene. Molecular epidemiological analysis showed that 13 isolates could be subdivided into 2 distinct genotypes, 11 isolates belonged to genotype 2, and other 2 isolates belonged to genotype 3. Results indicated two genotypes of Class I Newcastle disease virus might widely exist in domestic poultry in China.