OBJECTIVETo analyse seasonal influenza epidemic situation in 2006, and to analyse the genetic and antigenic characteristics of viral hemagglutinin (HA) gene.

METHODSThe single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics of these viruses from influenza surveillance network, and the HA1 genes were sequenced based on the antigenic test results according to different isolation times and sites.

RESULTSThe influenza virus types A and B co-circulated in 2006. influenza A H1N1 subtype and Victoria-like B influenza circulated preponderantly during this epidemic season. The HA1 gene sequence of H1N1 viruses showed that 192, 193, 196, 198 positions (located at antigenic site B) have an amino acid substitute, compared with the last circulating strain A/Hubeihongshan/53/2005(H1N1). Two amino acid changes at 142 and 144 positions compared with A/Yunnan/1145/2005 (H3N2). There was no change in influenza B viruses either Victoria-like B or Yamagata-like B virus, i.e . antigenic characteristics is analogous to B/shenzhen/155/2005 and B/tianjin/144/2005, respectively.

CONCLUSIONThe H1N1 and H3N2 influenza viruses had changing antigenic and genetic characteristics in 2006. Influenza virus types B did not change in 2006.

Amino Acids ; analysis ; China ; Hemagglutination Inhibition Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Influenza A Virus, H1N1 Subtype ; immunology ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; immunology ; isolation & purification ; Influenza B virus ; immunology ; isolation & purification ; Time Factors

To construct a recombinant vaccinia virus RVJ1175M2 expressing influenza A3 virus (H3N2) M2 gene, full length gene encoding influenza virus (H3N2) M2 protein was amplified with PCR and cloned into plasmid pJSC1175 which was used for homologous recombination with vaccinia virus Tiantan strain. Along with this, a recombinant vaccinia virus RVJ1175M2 containing the M2 gene was subsequently constructed. It was identified by PCR that the gene of M2 protein was inserted into the TK locus of vaccinia virus Tiantan strain correctly and M2 protein was expressed by recombinant vaccinia virus RVJ1175M2 effectively. Two electrophoretic bands of M2 protein expressed by the infected HeLa cells, one of 15kD and the other of 13kD in accordance with related documents, was deteced by Western-blot. M2 protein distributing on the surface of the infected cells was demonstrated by immunofluorescence and flow cytometry. The results suggested that recombinant vaccinia virus RVJ1175M2 could express M2 protein effectively, this laid a foundation for comparative research on the immune effect of universal vaccine of influenza virus with different kinds of vaccine expressing M2 protein.
HeLa Cells ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Influenza Vaccines ; immunology ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Vaccines, Synthetic ; immunology ; Vaccinia virus ; genetics ; Viral Matrix Proteins ; genetics

The molecular characterization and phylogenetic analysis of hemagglutinin (HA) genes of human influenza H3N2 viruses in Guangdong, China from 2007 to 2010 were studied in this study. By space-time sampling of strains, the HA genes of H3N2 strains from Guangdong were sequenced and searched from Internet, and then the variation and evolution of HA genes were conducted by Lasergene 7.1 and Mega 5.05 and evolutionary rates were analyzed by epidemiological data. The phylogenetic tree was established by alignment of 17 Guangdong strains and 26 global reference strains. Ks rates and Ka rates of HA genes were 2.06 x 10(-3)-2.23 x 10(-3) Nt/Year and 1.05 x 10(-3)-1.21 x 10(3) Nt/Year during 2007-2010, while the velocity of HA1 evolution of Ka was 3. 13 times than that of HA2 evolution. Compared with HA of vaccine strain A/Perth/16/2009, the genetic homologies of Guangdong strains in 2009 reached to 98.8%-99.7% and of Guangdong strains in 2010 reached to 98.0%-98.4%. There were some amino acid substitutions in five epitope regions of HA1 during 2007-2010, especially in B region (N160K) and D region (K174R/N); the K189E/N/Q and T228A in RBS (receptor-binding site) occurred in 2010 as two glycoproteins sites substituted impacted on the HA1 antigenicity. The antigenicity of epidemic H3N2 strains in 2010 was to some degree different that of the vaccine strain A/ Perth/16/2009. According to that there were variations of B and D epitopes and two sites of RBS and two glycoprotein in Guangdong H3N2 HA1 genes, WHO/ CDC should recommend new representative strains during 2011-2012 influenza seasons if H3N2 HA genes further evolve in the near future.
Amino Acid Substitution ; China ; Disulfides ; chemistry ; Epitopes ; genetics ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Mutation ; Phylogeny

OBJECTIVETo identify variations in hemagglutinin genes from influenza viruses (H3N2) isolated from infants and young children with acute respiratory infection (ARI) between March, 2004 and April 2005.

METHODSRNAs from influenza A virus strains (subtype H3) isolated from specimens collected from ARI children were extracted followed by amplification for HA1 fragments from hemagglutinin (HA) genes by RT-PCR. The sequences of the fragments were defined by direct sequencing for the PCR products or the target inserts after the PCR fragments were cloned into the TA-cloning vector pBS-T and analyzed by bioinformatic software.

RESULTSFragments of 987 bps of HA1 (encoding 329 amino acids) from a total of 32 strains of influenza A virus (subtype H3) isolated from the 2004 season and 1 from the 2003 season were amplified and the sequences were compared with vaccine reference strains recommended by WHO which were used in recent years. There were several consistent amino acid variations which involved in both antigenic epitopes A and B and receptor binding site (RBS) for isolated strains in the 2004 influenza season compared with the vaccine strains used during the recent years and the virus strains isolated in March 2004, indicated the antigenic drift of the viruses isolated in 2004 influenza season may lead to variant viruses.

CONCLUSIONThe variations of the HA genes from influenza virus (subtype H3) strains in the 2004-2005 influenza season were confirmed by sequence analysis for the HA1 regions of the hemagglutinin genes, which indicate that the antigenic drift would have been caused by the diversification of the genes and the efficacy of the recently used vaccines should be kept under close watch.

Antigenic Variation ; Child ; China ; epidemiology ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; epidemiology ; immunology ; virology ; RNA, Viral ; genetics ; Sequence Analysis, RNA

To understand the HA1 genetic variation characterization of influenza H3N2 virus isolates in Zhu-hai during 2008-2009, we selected 20 of H3N2 Influenza strains cultured in MDCK cell. Viral RNAs were extracted and amplified by using RT-PCR. The amplified products were purified after identified by gel electrophoresis and then the nucleotide sequences of the amplicons were determined. The results were analyzed by the software ClustalX and MEGA4. 1. When compared with the amino acid sequences of the epitopes of HA1 district of H3N2 influenza vaccine recommended by WHO in 2008, changes were found in those of H3N2 influenza strains in Zhuhai in 2008: K140I in all of H3N2 influenza strains, L157S in 08-0343 and 08-0677, K158R in 08-0466, 08-0620 and 08-0667, K173E in 08-0466 and 08-0620, K173N in 08-0667, and I192T in 08-0667. The epitopes of HA1 district of H3N2 influenza strains in Zhuhai in 2009 are different from that of H3N2 influenza vaccine during the same time: K173Q and P194L occur in all of H3N2 influenza strains, N144K, K158N, and N189K occur in the strains except the strain 09-0056. HA1 domain of H3N2 influenza strains in 2009 has lost a glycosylation site at amino acid position 144 while the glycosylation sites of HA1 domain of H3N2 influenza stains isolated in 2008 remained. This study suggested that H3N2 influenza virus in Zhuhai in 2008 was not evolved a novel variant and H3N2 influenza variant in 2009 was attributed to antigenic drift in HA1 district.
Animals ; Antigens, Viral ; immunology ; Cell Line ; China ; Dogs ; Epitopes ; immunology ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; isolation & purification ; Mutation ; Phylogeny ; Sequence Analysis, DNA

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

OBJECTIVETo understand the antigenic and genetic characteristics of influenza A H3N2 viruses circulated in man in China from 2000 to 2002.

METHODSEmbryonated chicken eggs inoculated with virus for amplification of viral yield. The harvesting egg allantoic fluids with influenza viruses were provided for testing antigen and RNA extraction. Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA amplified by PCR, and the product of PCR was purified. Afterward RNA sequence analysis was performed by the dideoxynucleotide chain termination method using synthetic oligodeoxynucleotide primers. Finally the phylogenetic tree was analyzed with MegAlign software.

RESULTSThe H3N2 viruses isolated during 2000-2002 were different in amino acid sequences on HA1 domain protein molecule from those of A/Wuhan359/1995 H3N2 as well as those of A/Sydney/7/1997 H3N2 strains. There were four different positions of amino acid sequence on HA1 domain protein molecule among the H3N2 viruses isolated in 2000 and during 2001-2002. They located at 83, 186, 202, 222 and 225 position, respectively. Of them 83 and 186 were in antigenic determinant E and B, respectively. The others located at left wall of the receptor binding site (RBS).

CONCLUSIONFrom the end of 2001 to the beginning of 2002, the influenza epidemic in Northern China caused by H3N2 virus was due to occurrence of antigenic and genetic changes of influenza A(H3N2) virus.

Amino Acid Sequence ; Antigenic Variation ; Base Sequence ; China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction

Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Chick Embryo ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; physiology ; Rats ; Rats, Wistar ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Swine ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Virus Replication ; genetics

Several antigen epitopes were designed according to the sequences of Swine influenza virus hemagglutinin (HA) genes and lined with the interval. The gene was amplified by PCR and sub cloned into pET30a (+) vector. The fusion protein was expressed in E. coli BL21 (DE3) by induced with IPTG and purified by affinity chromatography. The molecular weight of the protein was about 20 kD in SDS-PAGE. Immunological activity of the fusion protein was analyzed by Western blot. The results showed that the fusion protein could interact with anti-His antibody and the rabbit antiserum against SIV. The immunized mouse can produced antibodies against the target peptide and HI antibody against SIV H1N1 or H3N2. This study provides a new vaccine candidate for SIV.
Amino Acid Sequence ; Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Base Sequence ; Epitopes ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Hemagglutinins ; genetics ; immunology ; Humans ; Immunization ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

M2 protein of influenza A virus is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in influenza virus replication. It is also a target molecule of anti-virus drugs. We extracted the viral genome RNAs from MDCK cells infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M2 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into a prokaryotic expression vector pET-28a(+) (designated pET-28a(+)-M2) and a eukaryotic expression vector p3xFLAG-CMV-7.1 (designated p3xFLAG-CMV-7.1-M2), respectively. The resulted constructs were confirmed by restriction enzyme digestion and DNA sequencing analysis. We then transformed the plasmid pET-28a(+)-M2 into Escherichia coli BL21 (DE3) strain and expressed it by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). The recombinant M2 protein was purified from the induced bacterial cells using Ni(2+) affinity chromatography. Wistar rats were immunized with the purified M2 protein for producing polyclonal antibodies specific for it. Western blotting analysis and immunofluorescence analysis showed that the produced antibodies were capable of reacting with M2 protein expressed in p3xFLAG-CMV-7.1-M2-transfected cells as well as that synthesized in SIV-infected cells. We also transfected plasmid p3xFLAG-CMV-7.1-M2 into Vero cells and analyzed its subcellular localization by immunofluorescence. The M2 protein expressed in the Vero cells was 20 kDa in size and dominantly localized in the cytoplasm, showing a similar distribution to that in SIV-infected cells. Western blotting analysis of SIV-infected cells suggested that M2 was a late phase protein, which was detectable 12 h post-infection, later than NS1, NP and M1 proteins. It would be a potential molecular indicator of late phases replication of virus. Our results would be useful for studying the biological function of M2 protein in SIV replication.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cercopithecus aethiops ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; RNA ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection ; Vero Cells ; Viral Matrix Proteins ; biosynthesis ; genetics ; Virus Replication ; genetics