In order to understand the prevalence and variation of influenza B viruses, the antigenic and genetic characteristics of influenza B viruses circulating in Mainland China during April, 2011 to March, 2012 were analyzed. The results showed the B Victoria lineage viruses were much more prevalent than B Yamagata lineage during this period, phylogenetic analysis showed vast majority of Victoria lineage viruses belong to genetic group 1, intra-clade reassortant between HA1 and NA gene was identified in a minor proportion of the viruses. 72.8% of the B/Victoria-lineage viruses were antigenically closely related to the vaccine strain B/Brisbane/60/2008. B Yamagata component was not included in the trivalent influenza vaccine in China during the study period, however vast majority of B Yamagata lineage viruses were antigenically and genetically closely related to the representative virus B/Hubei-Wujiagang/158/2009(97.8%) and B/Sichuan-Anyue/139/2011(85.2%) in China, reassortant between HA1 and NA was not identified in B Yamagata lineage viruses. Overall, the predominant circulating influenza B viruses in 2011-2012 season in China were matched by current influenza vaccine and the selected representative viruses were proved to represent the antigenic and genetic characteristics of the circulating viruses.
China ; Humans ; Influenza B virus ; classification ; genetics ; immunology ; Influenza Vaccines ; genetics ; immunology ; Phylogeny ; Time Factors

Animals ; Humans ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; administration & dosage ; genetics ; immunology ; Influenza, Human ; prevention & control ; virology ; Vaccines, Virus-Like Particle ; administration & dosage ; genetics ; immunology

A/Puerto Rico/8/34 (PR8)-derived recombinant viruses have been used for seasonal flu vaccines; however, they are insufficient for vaccines against some human-fatal H5N1 highly pathogenic avian influenza (HPAI) viruses (HPAIV) due to low productivity. Additionally, the polymerase basic 2 (PB2) protein, an important mammalian-pathogenicity determinant, of PR8 possesses several mammalian-pathogenic mutations. We previously reported two avian PB2 genes (01310 and 0028) related to efficient replication in embryonated chicken eggs (ECEs) and nonpathogenicity in BALB/c mice. In this study, we generated PR8-derived H5N1 recombinant viruses harboring hemagglutinin (attenuated) and neuraminidase genes of a clade 2.3.2.1c H5N1 HPAIV (K10-483), as well as the 01310 or 0028 PB2 genes, and investigated their replication and immunogenicity. Compared with a control virus harboring six internal PR8 genes (rK10-483), the recombinant viruses possessing the 01310 and 0028 PB2 genes showed significantly higher replication efficiency in ECEs and higher antibody titers in chickens. In contrast to rK10-483, none of the viruses replicated in BALB/c mice, and all showed low titers in Madin-Darby canine kidney cells. Additionally, the recombinant viruses did not induce a neutralization antibody but elicited decreased protective immune responses against K10-483 in mice. Thus, the highly replicative and mammalian nonpathogenic recombinant H5N1 strains might be promising vaccine candidates against HPAI in poultry.
Animals ; Chickens ; Efficiency ; Eggs ; Hemagglutinins ; Influenza in Birds* ; Influenza Vaccines ; Kidney ; Mice ; Neuraminidase ; Ovum ; Poultry ; Reverse Genetics ; Seasons ; Vaccines ; Virulence

Despite recent innovative advances in molecular virology and the developments of vaccines, influenza virus remains a serious burden for human health. Vaccination has been considered a primary countermeasure for prevention of influenza infection. Live attenuated influenza vaccines (LAIVs) are particularly attracting attention as an effective strategy due to several advantages over inactivated vaccines. Cold-adaptation, as a classical means for attenuating viral virulence, has been successfully used for generating safe and effective donor strains of LAIVs against seasonal epidemics and occasional pandemics. Recently, the advent of reverse genetics technique expedited a variety of rational strategies to broaden the pool of LAIVs. Considering the breadth of antigenic diversity of influenza virus, the pool of LAIVs is likely to equip us with better options for controlling influenza pandemics. With a brief reflection on classical attenuating strategies used at the initial stage of development of LAIVs, especially on the principles underlying the development of cold-adapted LAIVs, we further discuss and outline other attenuation strategies especially with respect to the rationales for attenuation, and their practicality for mass production. Finally, we propose important considerations for a rational vaccine design, which will provide us with practical guidelines for improving the safety and effectiveness of LAIVs.
Antigenic Variation ; Cross Protection ; Humans ; Influenza Vaccines ; Influenza, Human ; Orthomyxoviridae ; Pandemics ; Reverse Genetics ; Seasons ; Tissue Donors ; Vaccination ; Vaccines, Inactivated

Humans ; Influenza A Virus, H5N1 Subtype ; classification ; genetics ; pathogenicity ; Influenza Vaccines ; immunology ; Influenza, Human ; drug therapy ; prevention & control ; transmission ; Vaccination

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

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.
Animals ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Hemagglutinins/genetics* ; Influenza B virus/metabolism* ; Influenza Vaccines/genetics* ; Mammals/metabolism* ; Mice ; Mice, Inbred BALB C

This study aimed to develop an effective experimental vaccine against highly pathogenic H5N1 Avian Influenza (HPAI) virus and to optimize their immunization programs. As reported previously, various DNA-based or recombinant vaccinia viral(Tiantan)-based H5N1 vaccine candidates, which containing a single cistronic construct (HAop, or NAop) or a bicistronic construct (HAop/M2 or NAop/M1) of H5N1 influenza virus (Anhui strain) were constructed and characterized in our lab. In this study, we further analysed the immunogenicity in mice of these vaccine candidates by various protocols (single or combined immunization). Our results showed that: comparing with immunization with DNA-based or rTTV-based H5N1 vaccine only, combined DNA-based with rTTV-based H5N1 vaccine immunization via prime-boost strategy enhanced immune response significantly against multi-H5N1 antigens detected by hemagglutination inhibition (HI) assay, NA- or M1- or M2-specific antibody detection, and micro-neutralizing antibody test and IFN-gamma ELISpot assay. Priming with DNA-based vaccine induced higher level of humoral response against HA or NA antigen than priming with rTTV-based vaccine; In contract, M1 and M2-specific antibody levels were higher among that of priming with rTTV -based vaccine. These findings provide a basis for further development of novel H5N1 vaccines and for the optimization of the immunization programs of combined multi-antigens vaccine candidates.
Animals ; Antigens, Viral ; genetics ; immunology ; Female ; Immunization ; methods ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza Vaccines ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccination ; methods ; Vaccines, DNA ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology ; Vaccinia ; genetics ; immunology

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

High-yield H3N2 subtype swine influenza virus for large-scale vaccine production in cell culture was generated by reverse genetics. The rescued H3N2 (rH3N2) candidate virus contained hemagglutinin (HA) and neuraminidase (NA) genes derived from a field isolate A/Swine/Henan/S4/01 (H3N2), PB2 gene from A/PR/8/34, and the other five internal genes from A/Goose/Dalian/3/01 (H9N2). The rH3N2 virus titer in MDCK cell culture were measured by hemagglutination assay and the maximum virus titre of 1:512 hemagglutination unit was obtained after infection of MDCK cell for 60 h. The results of the present study indicated that rH3N2 virus was suitable for growth in MDCK cell culture and is feasible to be used for the production of cell grown influenza vaccine.
Animals ; Cell Line ; Dogs ; Hemagglutination Tests ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; growth & development ; Influenza Vaccines ; Plasmids ; Virus Cultivation