Animals ; Birds ; China ; epidemiology ; Humans ; Influenza A Virus, H5N1 Subtype ; physiology ; Influenza A Virus, H7N7 Subtype ; physiology ; Influenza A Virus, H9N2 Subtype ; physiology ; Influenza A virus ; physiology ; Influenza, Human ; epidemiology ; prevention & control

The expression of the hemagglutinins of Avian influenza virus H5 H7and H9 subtypes was studied in this article by Pichia pastoris, one of the eukaryotis expression systems. Three reconstructed expression plasmids and engineering strains, named pPIC9K-H5HA, pPIC9K-H7HA, pPIC9K-H9HA and GS115/pPIC9K-H5HA, GS115/pPIC9K-H7HA, GS115/pPIC9K-H9HA repectively, were obtained. The reconstructed yeast engineering strains were identified by MD and MM plate selecting and PCR. The induced interests proteins were examined by SDS-PAGE and Western-bloting,the results showed that the interest genes were expressed exactly. And this will be helpful in the future study of antigen detection and antibody detection kit, as well in the subunit vaccines developing.
Animals ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; Influenza A Virus, H7N7 Subtype ; genetics ; Influenza A Virus, H9N2 Subtype ; genetics ; Pichia ; genetics ; metabolism

Specific primers and TaqMan MGB probes were designed with Primer Express 2.0 software according to the conserved region of the H5, H9, H7 subtype AIV hemagglutinin gene to make research of real-time fluorescent one-step PCR in the differential detection of H5, H9, H7 subtype avian influenza inactivated vaccines. The result showed that the method was specific and reproducible. No cross-reaction was discovered with other avian disease vaccines. Real-time fluorescent PCR provided a specific, sensitive, rapid and convenient method for the subtype identification of avian influenza inactivated vaccines.
Animals ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Humans ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza A Virus, H7N7 Subtype ; immunology ; Influenza A Virus, H9N2 Subtype ; immunology ; Influenza A virus ; classification ; immunology ; Influenza Vaccines ; analysis ; classification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Vaccines, Inactivated ; analysis

OBJECTIVEIn March 2012, an H7N7 subtype avian influenza virus (AIV) named A/wild goose/Dongting/PC0360/2012 (H7N7) (DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization.

METHODSRNA was extracted from environment samples (including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed.

RESULTSOur results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in Mainland China in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus.

CONCLUSIONStrengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

Amino Acid Sequence ; Animals ; China ; Embryo, Nonmammalian ; virology ; Feces ; virology ; Geese ; virology ; Genome, Viral ; Influenza A Virus, H7N7 Subtype ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Lakes ; virology ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; RNA, Viral ; genetics ; Real-Time Polymerase Chain Reaction ; veterinary