Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/genetics/*immunology ; Body Weight ; Cross Protection/*immunology ; Disease Models, Animal ; Epitopes, T-Lymphocyte/genetics/immunology ; Female ; Influenza A Virus, H3N2 Subtype/genetics/*immunology ; Influenza Vaccines/*immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/*immunology/mortality/pathology/prevention & control ; Peptides/genetics/*immunology ; Random Allocation ; Survival Analysis ; Vaccines, Synthetic/immunology ; Virus Replication

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

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China.
Animals ; Female ; Influenza A Virus, H3N2 Subtype/*genetics/immunology ; Influenza Vaccines/genetics/immunology/*therapeutic use ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/immunology/*prevention & control/virology ; Reassortant Viruses/genetics/immunology ; Reverse Genetics/methods/*veterinary ; Swine ; Swine Diseases/immunology/*prevention & control/virology ; Vaccines, Inactivated ; Virus Replication

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

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

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

BACKGROUNDH3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics.

METHODSIn order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A).

RESULTSIn this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found.

CONCLUSIONThe results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.

Animals ; COS Cells ; Cercopithecus aethiops ; Dogs ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Influenza A Virus, H3N2 Subtype ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Neuraminidase ; genetics ; Plasmids ; Reassortant Viruses ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Attenuated ; immunology ; Viral Proteins ; genetics

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

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