RESUMO
Full-length eight gene segments of avian influenza virus A/duck/Hubei/W1/2004(H9N2) (Dk/Hub/W1/04) were amplified by RT-PCR and completely sequenced. Phylogenetic analysis revealed that Dk/Hub/W1/04 was derived from A/Duck/HongKong/Y280/97, not displaying direct evolutional relationship with A/Quail/HongKong/G1/97 or Hubei H5N1 viruses. Meanwhile, Dk/Hub/W1/04 was found highly related to recent three chicken isolates. The connecting peptide of HA and the deletion in NA stalk were consistent with three chicken isolates, and the number of potential glycosylation site on the HA and NA also was similar or identical to the three chicken isolates. These findings suggested that Dk/Hub/W1/04 is likely to transmit back to ducks from chickens.
Assuntos
Evolução Molecular , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Neuraminidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Aves/virologia , Galinhas/virologia , China/epidemiologia , Patos/virologia , Genoma Viral , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H9N2/classificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Dados de Sequência Molecular , Neuraminidase/química , Filogenia , Alinhamento de SequênciaRESUMO
In order to differently diagnose avian influenza virus (AIV) subtypes, the HA gene of AIV H9 subtype was cloned, expressed and utilized in an enzyme-linked immunoad sorbent assay (ELISA). HA gene (1683bp) of H9N2 AIV was amplified by RT-PCR from a strain of field isolated H9N2 AIV, and its identity was confirmed by sequencing. The HA gene was subcloned into prokaryotic expression vector pGEX-KG with its secretion signal sequence removed. The expressed HA-GST fusion protein in E. coli BL21 was characterized by SDS-PAGE and western blotting analysis as a 90kD protein with immunogenicity. The fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation. The HA-GST fusion protein was used to establish an indirect ELISA for the detection of antibodies to H9 subtypes of AIV. The assay has 91.57% specificity to H9 AIV, 92.31% sensitivity and excellent reduplication. It could be used to differently detect antibodies to H9 AIV.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Humana/diagnóstico , Proteínas Recombinantes , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Humanos , Influenza Humana/virologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
A Latex Agglutination Test (LAT) was developed for quick detection of hemagglutinin serum antibodies of H5 Avian Influenza Virus (AIV) in chicken. Recombinant hemagglutinin protein of H5 AIV were obtained and purified, then HA protein were covalently linked to carboxylated polyethylene latex beads by EDAC. The sensitisation condition such as ionic strength of the diluting mixture pH, concentration, antigen, the times of linking were optimized. 126 fresh serum after vaccination were detected by this LAT and HI simultaneously, the result show that sensitivity for the LAT was 87.03%, the specificity was 88.8%, the coincidence rate of both methods was 87.30%. The result indicated that this LAT method can be used for quick detection of serum antibodies and epidemiological study of H5 AIV.
