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

LG1 strain of avian influenza virus H9N2 was passaged continuously for 40 generations in chicken embryos with anti-LG1 maternal antibodies in 4 parallel experiments, of which 3 experiments had a stable mutation of "G" to "A" at #99 of the neuraminidase gene(NA)from the 20th passage resulting in a change of Met to Ile and 2 had a stable mutation of "A" to "G" at #473 of the NA gene from the 30th passage resulting in a change of Asn to Ser which occurred in the 50th passage of another experiment. Eighty continuous passages in chicken embryos without antibody did not have the same mutation, indicating that the mutations of the 2 positions were associated with selective pressure of antibodies. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S of 4 parallel experiments with antibodies was 4.6 (32/7) compared with 2.0 (16/8) of the 2 experiments without antibodies and this significant difference implied the selective pressure of antibodies.
Animals ; Antibodies, Viral ; immunology ; Chick Embryo ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; Mutation ; Neuraminidase ; genetics

Abattoirs ; manpower ; Adult ; Antibodies, Viral ; blood ; China ; Humans ; Influenza A Virus, H1N1 Subtype ; immunology ; Influenza A Virus, H3N2 Subtype ; immunology ; Influenza A Virus, H3N8 Subtype ; immunology ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza A Virus, H9N2 Subtype ; immunology ; Influenza A virus ; classification ; immunology ; Influenza, Human ; epidemiology ; prevention & control ; virology ; Middle Aged ; Population Surveillance ; methods ; Seroepidemiologic Studies ; Young Adult

In Korea, several outbreaks of low pathogenic AI (H9N2) viral infections leading to decreased egg production and increased mortality have been reported on commercial farms since 1996, resulting in severe economic losses. To control the H9N2 LPAI endemic, the Korea Veterinary Authority has permitted the use of the inactivated H9N2 LPAI vaccine since 2007. In this study, we developed a killed vaccine using a low pathogenic H9N2 AI virus (A/chicken/Korea/ADL0401) and conducted safety and efficacy tests in commercial layer farms while focusing on analysis of factors that cause losses to farms, including egg production rate, egg abnormality, and feed efficiency. The egg production rate of the control group declined dramatically 5 days after the challenge. There were no changes in feed consumption of all three groups before the challenge, but rates of the control declined afterward. Clinical signs in the vaccinated groups were similar, and a slight decline in feed consumption was observed after challenge; however, this returned to normal more rapidly than the control group and commercial layers. Overall, the results of this study indicate that the safety and efficacy of the vaccine are adequate to provide protection against the AI field infection (H9N2) epidemic in Korea.
Animals ; Chickens ; Emulsions ; Female ; Influenza A Virus, H9N2 Subtype/*immunology ; Influenza Vaccines/*immunology/*standards ; Influenza in Birds/immunology/prevention & control ; Oviparity ; Specific Pathogen-Free Organisms ; Vaccines, Inactivated/immunology

Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Animals ; Antibodies, Viral ; biosynthesis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Genetic Vectors ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H9N2 Subtype ; Plasmids ; Rabbits ; Viral Proteins ; immunology

OBJECTIVETo understand the distribution of influenza A H9N2 virus in chickens and men in Shenzhen area.

METHODSVirus isolation was performed in embryonated hen s eggs with routine method. The antibody to H9N2 virus was detected with micro-hemagglutination inhibition (HI) test, then the results were checked by using the neutralization assay in MDCK cells.

RESULTSTotally 27 strains of influenza A H9N2 virus were isolated from chickens in farm markets in Shenzhen, whereas no H9N2 virus was isolated from men. Approximately 26% of human sera with the HI titers > or =20 to H9N2 virus were detected. However only 7% of chicken sera with the HI titers > or =20 to H9N2 virus were detected. Meanwhile the HI titer and (MGT) of antibody to H9N2 virus in human sera increased with age. It was also found that there was a close relationship between HI antibody titer to H9N2 virus in human sera and occupation.

CONCLUSIONSThe distribution of influenza A H9N2 virus in chicken and men in Shenzhen was rather wide. The human H9N2 virus infection probably derived from chicken H9N2 virus.

Animals ; Antibodies, Viral ; blood ; Chickens ; China ; epidemiology ; Hemagglutination Inhibition Tests ; Humans ; Influenza A Virus, H9N2 Subtype ; Influenza A virus ; classification ; immunology ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Seroepidemiologic Studies

To explore the impact of the history of infection by the influenza A virus subtype H1N1 on secondary infection by the influenza A virus subtype H9N2, pigs non-infected and pre-infected with H1N1 were inoculated with H9N2 in parallel to compare nasal shedding and seroconversion patterns. Unlike pigs without a background of H1N1 infection, nasal shedding was not detected in pigs pre-infected with H1N1. Both groups generated antibodies against H9N2. However, levels of H1N1 antibodies in pigs pre-infected with H1N1 increased quickly and dramatically after challenge with H9N2. Cross-reaction was not observed between H1N1 antibodies and H9N2 viruses. These findings suggest that circulation of the H1N1 virus might be a barrier to the introduction and transmission of the avian H9N2 virus, thereby delaying its adaptation in pigs.
Animals ; Antibodies, Viral ; immunology ; Cross Reactions ; Immune Sera ; immunology ; Influenza A Virus, H1N1 Subtype ; immunology ; physiology ; Influenza A Virus, H9N2 Subtype ; immunology ; Orthomyxoviridae Infections ; blood ; immunology ; Species Specificity ; Swine ; immunology ; virology

OBJECTIVETo understand the epidemic status of avian influenza A virus in chickens and men in Guangzhou area and to prevent men suffering from avian influenza A (H5N1) virus.

METHODSEtiologic and serological surveys were conducted in chickens and men who were working in the poultry farms and slaughter house. Viruses were isolated with both MDCK cells and embryonated chicken eggs. Hemagglutination inhibition tests were performed by routine method.

RESULTSAnti-H9N2 antibody was found in 12.8% of the chickens and 5.1% of the workers.

CONCLUSIONSAvian influenza virus H9N2 subtype existed in chickens and this subtype of influenza A virus might infect men.

Animals ; Antibodies, Viral ; blood ; Chickens ; virology ; China ; Hemagglutination Inhibition Tests ; Humans ; Influenza A Virus, H9N2 Subtype ; immunology ; isolation & purification ; Influenza in Birds ; virology ; Influenza, Human ; virology

OBJECTIVETo determine whether it could protect mice from challenge of lethal influenza virus which group prior infected A(H1N1) pdm09 and H9N2 virus respectively.

METHODS150 BALB/c mice are divided into three groups. Mice are infected A(H1N1) pdm09 virus (pCA07) and poultry H9N2 virus (GZ333) respectively. Infected mice are challenged with 10 times of lethal dose virus (PR8) then compare the viral load, antibody and survival of the two group mice before and after challenged.

RESULTSBoth experimental group mice survived after challenge of lethal influenza virus and lung viral load are lower than that of the first infection. Antibodies derived from the infective virus and challenge virus.

CONCLUSIONPrior infected A(H1N1) pdm09 and H9N2 virus could protect mice from challenge of lethal influenza virus.

Animals ; Antibodies, Viral ; immunology ; Female ; Humans ; Influenza A Virus, H1N1 Subtype ; physiology ; Influenza A Virus, H9N2 Subtype ; physiology ; Influenza, Human ; immunology ; prevention & control ; virology ; Lung ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; Viral Load

The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.
Animals ; Chickens ; virology ; China ; epidemiology ; Humans ; Influenza A Virus, H7N9 Subtype ; genetics ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; physiology ; Influenza in Birds ; epidemiology ; transmission ; virology ; Influenza, Human ; epidemiology ; transmission ; virology ; Vaccination ; Viral Proteins ; classification ; metabolism