Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.
Animals ; Base Sequence ; Birds ; China/epidemiology* ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus/immunology* ; Hemagglutinins/genetics* ; Humans ; Influenza A Virus, H7N9 Subtype/isolation & purification* ; Influenza in Birds ; Influenza, Human/virology* ; Neuraminidase/genetics* ; Phylogeny ; RNA, Viral/genetics* ; Sequence Analysis, DNA

Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection.
Antibodies, Monoclonal ; immunology ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; immunology ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Influenza A Virus, H10N8 Subtype ; Neutralization Tests

Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China.
Animals ; Antibodies, Viral ; immunology ; Electrophoresis, Polyacrylamide Gel ; Female ; Hemagglutination Inhibition Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; analysis ; immunology ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Rabbits ; Reassortant Viruses ; genetics ; immunology

To analyze the antigenic and genetic characteristics of the influenza A (H3N2) virus in mainland China during the surveillance year of 2013-2014, the antigenic characteristics of H3N2 virus were analyzed using reference ferret anti-sera. The nucleotide sequences of the viruses were determined by Sanger dideoxy sequencing, phylogenetic trees were constructed with the neighbor-joining method, and the genetic characteristics of the viruses were determined in comparison to current vaccine strains. The results showed that most of the H3N2 viruses were antigenically closely related to the A/Victoria/361/2011 vaccine strain cell-propagated prototype virus (99.6%). Using the A/Texas/50/2012 egg isolate as the reference antigen, 15.1% of the viruses were found to be closely antigenically related to it, while 11.9% of strains were closely antigenically related to the egg-propagated epidemic strain, A/Shanghai-Changning/1507/2012. Phylogenetic analysis of HA genes indicated that the A(H3N2) viruses in this surveillance year were in the same clade, but no drug resistant mutation was identified in the NA genes. During the 2013-2014 influenza surveillance year, no significant genetic change was detected in either the HA or NA genes of the A(H3N2) viruses, while significant mutations were found in egg isolates resulting from their adaptation during propagation in eggs. The antigenic and genetic changes should be investigated in a timely manner to enable the selection of an appropriate vaccine strain in China.
Animals ; Antigenic Variation ; Base Sequence ; Chick Embryo ; China ; Genetic Variation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; virology ; Molecular Sequence Data ; Mutation ; Phylogeny

Hemagglutinin (HA) contains a head domain with a high degree of variability and a relatively conserved stem region. HA is the major viral antigen on the surface of the influenza virus. To define the biologic activities of chimeric HA bearing different head domains and stem regions or their potential use, a HA chimeric gene containing the head domain of the H7 subtype virus and stem region of the H3 subtype virus was modified and expressed using a baculovirus expression vector. Then, the secreted protein was purified and its biologic activities characterized. Approximately 1.4 mg/mL cH7/3 HA could be obtained, and its molecular weight was ≈ 70 kD. The trimer form of cH7/3 protein had hemagglutination activity and could be recognized by specific antibodies. The method described here can be used for further studies on the screening of HA stem-reactive antibodies or the development of vaccines with conserved epitopes.
Antibodies, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hemagglutination ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza Vaccines ; genetics ; immunology ; Influenza, Human ; prevention & control ; virology

Adult ; Antibodies, Viral ; blood ; immunology ; Cross Reactions ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Humans ; Influenza A Virus, H1N1 Subtype ; immunology ; Influenza A Virus, H3N2 Subtype ; immunology ; Influenza B virus ; immunology ; Influenza Vaccines ; immunology ; Orthomyxoviridae ; immunology ; Seasons ; Vaccination

Fragments encoding amino acids 76-130 in the linear conserved region (LCR) of A/Hubei/1/2010 (H5N1) HA2 was fused to hepatitis B core antigen (HBc) to generate a LCR-HBe virus-like particle (VLP). Results showed that the fusion protein of LCR-HBc was highly expressed in this prokaryotic expression system. The purified LCR-HBc particle stimulated high levels of IgG production in mice with a titer of > 1:12 800, and provided 50% cross-protection against lethal challenge by H1N1 viruses.
Amino Acid Sequence ; Animals ; Female ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Interferon-gamma ; biosynthesis ; Lung ; pathology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data

OBJECTIVETo prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.

METHODSRecombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.

RESULTSThe 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.

CONCLUSIONThe 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Animals ; Chick Embryo ; Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; prevention & control ; virology ; Neuraminidase ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Synthetic ; immunology

Influenza, caused by influenza virus, is a serious respiratory illness which poses a global public health threat. Vaccination is the primary strategy for the prevention and control of influenza. Although both inactivated vaccines and the live attenuated vaccines are effective in preventing influenza, the current vaccines have poor efficacy in the elderly and fail to provide protection against heterosubtype viruses. Development of a safer and more effective influenza vaccine that provides broad cross protection, overcoming the intrinsic limitation of the current vaccines, has been a scientific challenge. During the past decades, structural biology, reverse genetic and other virological technologies developed quickly and sped the progress of influenza vaccinology. Some new strategies for developing influenza vaccine have been generated, produced encouraging results, which showed great prospect as next-generation of influenza vaccines.
Disease Outbreaks ; prevention & control ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Humans ; Influenza Vaccines ; biosynthesis ; immunology ; Influenza, Human ; immunology ; prevention & control ; virology ; Orthomyxoviridae ; immunology ; Vaccines, Attenuated ; immunology ; Vaccines, Inactivated ; immunology

Since March 2009, pandemic A/H1N1/2009 influenza virus has been spreading throughout many countries including China. The emerged virus caused great harm to human health and social economy. Hemagglutinin (HA) is the most important viral surface glycoprotein, mainly possessing three kinds of functions: (1) binding to host cell receptor, (2) triggering the fusion between viral envelop and target cell membrane, (3) stimulating the body to generate the neutralizing antibody. Advances in the structure, primary function, evolution and antigenicity of pandemic A/H1N1/2009 influenza virus HA protein are reviewed in this paper.
Animals ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; pathogenicity ; physiology ; Influenza, Human ; epidemiology ; virology ; Pandemics