H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV...H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.展开更多
The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10...The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150 μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50 μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5 μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.展开更多
Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ）...Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ） carrying hemagglutinin （HA） gene of AIV H9 subtype. After cell fusion, one positive hybridoma cell strain was screened out by hemagglutination inhibition assay （ HI ）, and another positive hybddoma call strain was screened out by ELISA. After subcloning 3 times, the two cell strains could still secret antibodies against the HA of AIV H9 subtype. The mono- clonal antibodies did not react with Newcastle disease virus, AIV H5 subtype and duck adenovirus A. Their subtypes were IgG2b with kappa light chain. These two hybridoma cell strains may play an important role in rapid diagnosis and early-warning surveillance of AIV H9 subtype.展开更多
In the present study,we used nucleotide and protein sequences of avian influenza virus H5N1,which were obtained in Asia and Africa,analyzed HA proteins using ClustalX1.83 and MEGA4.0,and built a genetic evolutionary t...In the present study,we used nucleotide and protein sequences of avian influenza virus H5N1,which were obtained in Asia and Africa,analyzed HA proteins using ClustalX1.83 and MEGA4.0,and built a genetic evolutionary tree of HA nucleotides.The analysis revealed that the receptor specificity amino acid of A/HK/213/2003,A/Turkey/65596/2006 and etc mutated into QNG,which could bind withá-2,3 galactose andá-2,6 galactose.A mutation might thus take place and lead to an outbreak of human infections of avian influenza virus.The mutations of HA protein amino acids from 2004 to 2009 coincided with human infections provided by the World Health Organization,indicating a“low–high–highest–high–low”pattern.We also found out that virus strains in Asia are from different origins:strains from Southeast Asia and East Asia are of the same origin,whereas those from West Asia,South Asia and Africa descend from one ancestor.The composition of the phylogenetic tree and mutations of key site amino acids in HA proteins reflected the fact that the majority of strains are regional and long term,and virus diffusions exist between China,Laos,Malaysia,Indonesia,Azerbaijan,Turkey and Iraq.We would advise that pertinent vaccines be developed and due attention be paid to the spread of viruses between neighboring countries and the dangers of virus mutation and evolution.展开更多
Several universal influenza virus vaccine candidates based on eliciting antibodies against the hemagglutinin stalk domain are in development. Typically, these vaccines induce responses that target group 1 or group 2 h...Several universal influenza virus vaccine candidates based on eliciting antibodies against the hemagglutinin stalk domain are in development. Typically, these vaccines induce responses that target group 1 or group 2 hemagglutinins with Httle to no cross-group reactivity and protection. Similarly, the majority of human anti-stalk monoclonal antibodies that have been isolated are directed against group 1 or group 2 hemagglutinins with very few that bind to hemagglutinins of both groups. Here we review what is known about the human humoral immune response to vaccination and infection with H7 subtype influenza viruses on a polyclonal and monoclonal level. It seems that unlike vaccination with H5 hemagglutinin, which induces antibody responses mostly restricted to the group 1 stalk domain, H7 exposure induces both group 2 and cross-group antibody responses. A better understanding of this phenomenon and the underlying mechanisms might help to develop future universal influenza virus vaccine candidates.展开更多
As a leading cause of respiratory disease, influenza A virus(IAV) presents a pandemic threat in annual seasonal outbreaks. Given the limitation of existing anti-influenza therapies, there remains to be a requirement f...As a leading cause of respiratory disease, influenza A virus(IAV) presents a pandemic threat in annual seasonal outbreaks. Given the limitation of existing anti-influenza therapies, there remains to be a requirement for new drugs. Compound Yi-Zhi-Hao pellet(CYZH) is a famous traditional Chinese medicine(TCM) used in the clinic, whose formula has been recorded in Complication of National Standard for Traditional Chinese Medicine to treat common cold. In this study, we found that CYZH exhibited a broad-spectrum anti-influenza activity and inhibited the expression of viral RNA and proteins in vitro. Mechanistically, CYZH had no inhibitory activities against viral protein hemagglutinin and IAV RNA-dependent RNA polymerase. Instead, it induced activation of erythroid 2-related factor 2(Nrf2) and nuclear factor kappa B(NF-κB), which subsequently upregulated heme oxygenase-1(HO-1) expression.Also, CYZH protected cells from oxidative damage induced by reactive oxygen series. In conclusions,CYZH inhibits IAV replication in vitro, at least partly by activating expression of the Nrf2/HO-1 pathway.展开更多
Even with implementation of current influenza vaccines,influenza still claims up to 500,000 lives worldwide annually,indicating a need for a better vaccine strategy.We have developed a technology to generate unique S_...Even with implementation of current influenza vaccines,influenza still claims up to 500,000 lives worldwide annually,indicating a need for a better vaccine strategy.We have developed a technology to generate unique S_(60)-HA1 pseudovirus nanoparticles(PVNPs)that display the receptor-binding HA1 domains of influenza viruses.Each self-assembled S_(60)-HA1 PVNP consists of a T=1 icosahedral S_(60) nanoparticle that resembles the inner shell of norovirus capsid and 60 surface-displayed HA1 antigens that are excellent vaccine targets.Soluble S_(60)-HA1 PVNPs presenting HA1 antigens of H7N9 influenza virus subtypes have been produced efficiently in large amount.Their three-dimensional(3D)structures have been solved by cryogenic electron microscopy.The PVNP-displayed HA1 antigens react with HA-specific antibody,and retain authentic sialic acid binding specificity and hemagglutinate human erythrocytes.The PVNPs are highly immunogenic,eliciting high titers of HA1-specific antibodies in mice and the mouse sera strongly inhibited hemagglutinations of homologous and heterologous influenza virus HA proteins.Therefore,the S_(60)-HA1 PVNPs may provide useful reagents to study influenza viruses and offer a potential new vaccine tactic to fight the deadly influenza disease.展开更多
The H5N1 avian influenza virus (AIV) has widely spread in Asia, Europe and Africa, making a large amount of economic loss. Recently, our research group has screened a common neutralizing mono- clonal antibody named 8H...The H5N1 avian influenza virus (AIV) has widely spread in Asia, Europe and Africa, making a large amount of economic loss. Recently, our research group has screened a common neutralizing mono- clonal antibody named 8H5, which can neutralize almost all H5 subtype AIV ever isolated so far. Obvi- ously, this monoclonal antibody would benefit for research and development of the universal AIV vac- cine and design of the drug against H5N1 AIV in high mutation rate. In this study, the homology mod- eling was applied to generate the 3D structure of 8H5 Fab fragment, and "canonical structure" method was used to define the specified loop conformation of CDR regions. The model was subjected to en- ergy minimization in cvff force field with Discovery module in Insight II program. The resulting model has correct stereochemistry as gauged from the Ramachandran plot calculation and good 3D-structure compatibility as assessed by interaction energy analysis, solvent accessible surface (SAS) analysis, and Profiles-3D approach. Furthermore, the 8H5 Fab model was subjected to docking with three H5 subtype hemagglutinin (HA) structures deposited in PDB (ID No: 1jsm, 2ibx and 2fk0) respectively. The result indicates that the three docked complexes share a common binding interface, but differ in bind- ing angle related with HA structure similarity between viral subtypes. In the light of the three HA inter- faces with structural homology analysis, the common neutralizing epitope on HA recognized by 8H5 consists of 9 incontinuous amino acid residues: Asp68, Asn72, Glu112, Lys113, Ile114, Pro118, Ser120, Tyr137, Tyr252 (numbered as for 1jsm sequence). The primary purpose of the present work is to provide some insight into structure and binding details of a common neutralizing epitope of H5N1 AIV, thereby aiding in the structure-based design of universal AIV vaccines and anti-virus therapeutic drugs.展开更多
Interferon-inducible transmembrane protein 3(IFITM3)inhibits influenza virus infection by blocking viral membrane fusion,but the exact mechanism remains elusive.Here,we investigated the function and key region of IFIT...Interferon-inducible transmembrane protein 3(IFITM3)inhibits influenza virus infection by blocking viral membrane fusion,but the exact mechanism remains elusive.Here,we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin(HA).The restriction of IFITM3 on HAmediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses.Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA_(2) subunit but not HA_(1) subunit of H5 and H7 virus.Truncated analyses showed that the transmembrane domain of the IFITM3 and HA_(2) subunit might play an important role in their interaction.Finally,this interaction of IFITM3 was also verified with HA_(2) subunits from other subtypes of influenza A virus and influenza B virus.Overall,our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain,providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.展开更多
OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonate...OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonated hen eggs, and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03) and Editseg (version 3.69) softwares. RESULTS: The amino acid sequences at the cleavage site between HA1 and HA2 domains of H9N2 viruses isolated in China are R-S-S-R. One pigeon strain contains seven potential glycosylation sites on the HA protein molecule, while all others have eight. There are 2 to 15 differences of amino acid sequences distributed at 24 different positions on the HA protein molecules among six H9N2 viruses. The H9N2 viruses with multiple lineages of HA genes were co-circulating in China recently. CONCLUSION: The highest possibility is that human influenza A (H9N2) virus was derived from Chicken H9N2 virus, and not derived from pigeon H9N2 virus. However, it is still unknown whether the H9N2 virus could transmit from person to person. The H9N2 viruses with multiple lineages of HA genes are co-circulating in China.展开更多
Avian influenza (AI) virology surveillance is the most important method to monitor AI virus (AIV) in poultry so as to effectively prevent and control AI outbreaks. Monoclonal antibodies (MAb)-based assays are highly s...Avian influenza (AI) virology surveillance is the most important method to monitor AI virus (AIV) in poultry so as to effectively prevent and control AI outbreaks. Monoclonal antibodies (MAb)-based assays are highly sensitive and specific for AIV detection, and much practical and economic for test-in-field or onsite. Many such assays have been developed and are still in developing since the H5N1 highly pathogenic AI (HPAI) outbreaks occurred in South East Asia in 2003. A MAb-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed in our lab during late 1990s and early 2000s. Meanwhile, AIV H7 and H5 subtype specific-MAbs have been successfully developed in our laboratory to enhance the Dot-ELISA and other MAb-based assays for AIV detection. Production and purification of the H7 and H5 MAbs were made to provide essential reagents for Dot-ELISA and other immunoassays, and the current development of a novel Biosensor technique for rapid detection of AIV from clinical and field specimens.展开更多
This research reveals the phylogenetic history and structural information of the hemagglutinin（HA） and neuraminidase（NA） from novel avian influenza virus A/Hangzhou/1/2013（HTN9_2013） strain from human infected i...This research reveals the phylogenetic history and structural information of the hemagglutinin（HA） and neuraminidase（NA） from novel avian influenza virus A/Hangzhou/1/2013（HTN9_2013） strain from human infected in China. Strains closely related to the HTN9 2013 strain were obtained from Nation Center for Biotechnology Information（USA）-basic local alignment search tool（NCBI-BLAST） searching, and the phylogenetic trees were con- structed. The 3D structures of HA and NA from H7N9 2013 strain were built by homology modeling technology, and molecular dynamics（MD） simulations were performed on the high-performance computer cluster. Characteristic amino acid sites were then screened from multiple sequence alignment（MSA） via home-made Python script and mapped onto the 3D structures. The thermodynamic characteristic root-mean-square-fluctuation （RMSF） of these sites in the structure was also analyzed with MD trajectories. The HA of HTN9_2013 strain is closely related to the A/duck/Zhejiang/12/2011 strain isolated in China, while the NA of HTN9 2013 strain is mostly related to the A/mallard/Czech Republic/13438-29K/2010 strain isolated in Europe. The 3D structures of HA and NA from H7N9 2013 stain are mostly identical to the existing structure of H7 and N9. A total of 11 and 14 characteristic ami- no acid sites were identified in HA and NA, respectively, in HTN9_2013. Structural analysis indicates that certain sites in the top region of HA are important, at which the mutation of some amino acids can impact the receptor bin- ding that may be related to its infection of human beings.展开更多
Influenza virus is the causative agent of the seasonal and occasional pandemic flu.The current H1N1 influenza pandemic,announced by the WHO in June 2009,is highly contagious and responsible for global economic losses ...Influenza virus is the causative agent of the seasonal and occasional pandemic flu.The current H1N1 influenza pandemic,announced by the WHO in June 2009,is highly contagious and responsible for global economic losses and fatalities.Although the H1N1 gene segments have three origins in terms of host species,the virus has been named swine-origin influenza virus(S-OIV)due to a predominant swine origin.2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects.Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection.Primary sequence analysis of the 2009 S-OIV hemagglutinin(HA)reveals its closest relationship to that of the 1918 pandemic influenza virus,however,analysis at the structural level is necessary to critically assess the functional significance.In this report,we report the crystal structure of soluble hemagglutinin H1(09H1)at 2.9Å,illustrating that the 09H1 is very similar to the 1918 pandemic HA(18H1)in overall structure and the structural modules,including the five defined antiboby(Ab)-binding epitopes.Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV,and people born around the 1918 are resistant to the current pandemic,yet younger generations are more susceptible to the 2009 pandemic.展开更多
Seasonal influenza epidemics and influenza pandemics caused by influenza A virus(IAV)has resulted in millions of deaths in the world.The development of anti-IAV vaccines and therapeutics is urgently needed for prevent...Seasonal influenza epidemics and influenza pandemics caused by influenza A virus(IAV)has resulted in millions of deaths in the world.The development of anti-IAV vaccines and therapeutics is urgently needed for prevention and treatment of IAV infection and for controlling future influenza pandemics.Hemagglutinin(HA)of IAV plays a critical role in viral binding,fusion and entry,and contains the major neutralizing epitopes.Therefore,HA is an attractive target for developing anti-IAV drugs and vaccines.Here we have reviewed the recent progress in study of conformational changes of HA during viral fusion process and development of HA-based antiviral therapeutics and vaccines.展开更多
There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs.The main component of the current split influe...There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs.The main component of the current split influenza A virus vaccine is viral hemagglutinin(HA)which induces a strong antibody-mediated immune response.To develop a modern vaccine against influenza A viruses,the current research has been focused on the universal vaccines targeting viral M2,NP and HA proteins.Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface,and each monomer consists of a globular head(HA1)and a“rod-like”stalk region(HA2),the latter being more conserved among different HA subtypes and being the primary target for universal vaccines.In this study,we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A(H1N1)virus HA as a model,tested its immunogenicity in mice,solved its crystal structure and further examined its immunological characteristics.The results show that the HA globular head can be easily prepared by in vitro refolding in an E.coli expression system,which maintains its intact structure and allows for the stimulation of a strong immune response.Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.展开更多
A mutation network for the hemagglutinin gene(HA) of the novel type A(H1N1) influenza virus was constructed.Sequence homology analysis indicated that one HA sequence type from the viruses mainly isolated from Mexico w...A mutation network for the hemagglutinin gene(HA) of the novel type A(H1N1) influenza virus was constructed.Sequence homology analysis indicated that one HA sequence type from the viruses mainly isolated from Mexico was likely the original type in this epidemic.Based on the 658A and 1408T mutations in HA,the viruses evolving into this epidemic were divided into three categories,the Mexico,the transitional and the New York type.The three groups of viruses presented distinctive clustering features in their geographic distributions.展开更多
Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines.In this study,we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic...Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines.In this study,we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen(HA) from the strains A/New Caledonia/20/99(H1N1)(pV1A5) and A/California/04/2009(H1N1)(pVEH1) ,respectively.After verifying antigen expression,the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model.Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies.HA-specific T-cells were also measured in IFN-γELISpot assays.The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD50 of mouse-adapted A/New Caledonia/20/99(H1N1) .Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses(P<0.05) than the pVAX1 control group.Additionally,the pV1A5 vaccine effectively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100%compared with a 40%survival rate in the pVEH1 vaccinated group(P<0.05) .Our study indicates that the seasonal influenza DNA vaccine completely protects against the homologous A/New Caledonia/20/99 virus(H1N1) ,while the pandemic influenza DNA vaccine only partially protects against this virus.展开更多
基金supported by the National Modern Agricultural Industry Technology System Project of China(CARS-41)the Science and Technology Plan Project of Guangdong Province,China(2012B020306002 and 2012B091100078)
文摘H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.
文摘The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150 μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50 μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5 μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.
基金funded by the National Key Technology R&D Program(2006BAK20A29)the Shenzhen Entry-Exit Inspection and Quarantine Project(sz2008102)
文摘Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ） carrying hemagglutinin （HA） gene of AIV H9 subtype. After cell fusion, one positive hybridoma cell strain was screened out by hemagglutination inhibition assay （ HI ）, and another positive hybddoma call strain was screened out by ELISA. After subcloning 3 times, the two cell strains could still secret antibodies against the HA of AIV H9 subtype. The mono- clonal antibodies did not react with Newcastle disease virus, AIV H5 subtype and duck adenovirus A. Their subtypes were IgG2b with kappa light chain. These two hybridoma cell strains may play an important role in rapid diagnosis and early-warning surveillance of AIV H9 subtype.
基金supported by CAS Innovation Program(KSCX2-YW-N-063)China MOST(2006BAD06A01),IDRC,USDA and NIH.
文摘In the present study,we used nucleotide and protein sequences of avian influenza virus H5N1,which were obtained in Asia and Africa,analyzed HA proteins using ClustalX1.83 and MEGA4.0,and built a genetic evolutionary tree of HA nucleotides.The analysis revealed that the receptor specificity amino acid of A/HK/213/2003,A/Turkey/65596/2006 and etc mutated into QNG,which could bind withá-2,3 galactose andá-2,6 galactose.A mutation might thus take place and lead to an outbreak of human infections of avian influenza virus.The mutations of HA protein amino acids from 2004 to 2009 coincided with human infections provided by the World Health Organization,indicating a“low–high–highest–high–low”pattern.We also found out that virus strains in Asia are from different origins:strains from Southeast Asia and East Asia are of the same origin,whereas those from West Asia,South Asia and Africa descend from one ancestor.The composition of the phylogenetic tree and mutations of key site amino acids in HA proteins reflected the fact that the majority of strains are regional and long term,and virus diffusions exist between China,Laos,Malaysia,Indonesia,Azerbaijan,Turkey and Iraq.We would advise that pertinent vaccines be developed and due attention be paid to the spread of viruses between neighboring countries and the dangers of virus mutation and evolution.
文摘Several universal influenza virus vaccine candidates based on eliciting antibodies against the hemagglutinin stalk domain are in development. Typically, these vaccines induce responses that target group 1 or group 2 hemagglutinins with Httle to no cross-group reactivity and protection. Similarly, the majority of human anti-stalk monoclonal antibodies that have been isolated are directed against group 1 or group 2 hemagglutinins with very few that bind to hemagglutinins of both groups. Here we review what is known about the human humoral immune response to vaccination and infection with H7 subtype influenza viruses on a polyclonal and monoclonal level. It seems that unlike vaccination with H5 hemagglutinin, which induces antibody responses mostly restricted to the group 1 stalk domain, H7 exposure induces both group 2 and cross-group antibody responses. A better understanding of this phenomenon and the underlying mechanisms might help to develop future universal influenza virus vaccine candidates.
基金supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China (2012ZX10004501-004-001) The Science & Technology project of Urumqi of Xinjiang Uygur Autonomous Region (Y151310010): Study on the mechanism of anti-influenza virus effect of compound Yi-Zhi-Hao Pellets
文摘As a leading cause of respiratory disease, influenza A virus(IAV) presents a pandemic threat in annual seasonal outbreaks. Given the limitation of existing anti-influenza therapies, there remains to be a requirement for new drugs. Compound Yi-Zhi-Hao pellet(CYZH) is a famous traditional Chinese medicine(TCM) used in the clinic, whose formula has been recorded in Complication of National Standard for Traditional Chinese Medicine to treat common cold. In this study, we found that CYZH exhibited a broad-spectrum anti-influenza activity and inhibited the expression of viral RNA and proteins in vitro. Mechanistically, CYZH had no inhibitory activities against viral protein hemagglutinin and IAV RNA-dependent RNA polymerase. Instead, it induced activation of erythroid 2-related factor 2(Nrf2) and nuclear factor kappa B(NF-κB), which subsequently upregulated heme oxygenase-1(HO-1) expression.Also, CYZH protected cells from oxidative damage induced by reactive oxygen series. In conclusions,CYZH inhibits IAV replication in vitro, at least partly by activating expression of the Nrf2/HO-1 pathway.
基金The research described in this study was supported by the National Institute of Allergy and Infectious Diseases(NIAID,No.R56 AI148426-01A1 to M.T.)Cincinnati Children Hospital Medical Center(CCHMC,Innovation Funds 2018-2020,GAP Fund 2020-2021,and Research Innovation and Pilot Grant 2020-2021 to M.T.)+1 种基金the Center for Clinical and Translational Science and Training(CCTST)of the University of Cincinnati College of Medicine(Pilot Collaborative Studies Grant 2018-2019 to M.T.)that was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health(No.UL1TR001425).
文摘Even with implementation of current influenza vaccines,influenza still claims up to 500,000 lives worldwide annually,indicating a need for a better vaccine strategy.We have developed a technology to generate unique S_(60)-HA1 pseudovirus nanoparticles(PVNPs)that display the receptor-binding HA1 domains of influenza viruses.Each self-assembled S_(60)-HA1 PVNP consists of a T=1 icosahedral S_(60) nanoparticle that resembles the inner shell of norovirus capsid and 60 surface-displayed HA1 antigens that are excellent vaccine targets.Soluble S_(60)-HA1 PVNPs presenting HA1 antigens of H7N9 influenza virus subtypes have been produced efficiently in large amount.Their three-dimensional(3D)structures have been solved by cryogenic electron microscopy.The PVNP-displayed HA1 antigens react with HA-specific antibody,and retain authentic sialic acid binding specificity and hemagglutinate human erythrocytes.The PVNPs are highly immunogenic,eliciting high titers of HA1-specific antibodies in mice and the mouse sera strongly inhibited hemagglutinations of homologous and heterologous influenza virus HA proteins.Therefore,the S_(60)-HA1 PVNPs may provide useful reagents to study influenza viruses and offer a potential new vaccine tactic to fight the deadly influenza disease.
基金the National Natural Science Foundation of China (Grant Nos. 30500092, 30640017 and 30600106)National Science and Technology Project in the 10th Five-Year Period (Grant No. 2004BA519A73)+2 种基金Key Projects in the National Science & Technology Pillar Program (Grant No. 2006BAI01B06)Major Science and Technology Project of Fujian Province (Grant No. 2004YZ01)Key Science and Technology Project of Fujian Province (Grant No. 2005Y020)
文摘The H5N1 avian influenza virus (AIV) has widely spread in Asia, Europe and Africa, making a large amount of economic loss. Recently, our research group has screened a common neutralizing mono- clonal antibody named 8H5, which can neutralize almost all H5 subtype AIV ever isolated so far. Obvi- ously, this monoclonal antibody would benefit for research and development of the universal AIV vac- cine and design of the drug against H5N1 AIV in high mutation rate. In this study, the homology mod- eling was applied to generate the 3D structure of 8H5 Fab fragment, and "canonical structure" method was used to define the specified loop conformation of CDR regions. The model was subjected to en- ergy minimization in cvff force field with Discovery module in Insight II program. The resulting model has correct stereochemistry as gauged from the Ramachandran plot calculation and good 3D-structure compatibility as assessed by interaction energy analysis, solvent accessible surface (SAS) analysis, and Profiles-3D approach. Furthermore, the 8H5 Fab model was subjected to docking with three H5 subtype hemagglutinin (HA) structures deposited in PDB (ID No: 1jsm, 2ibx and 2fk0) respectively. The result indicates that the three docked complexes share a common binding interface, but differ in bind- ing angle related with HA structure similarity between viral subtypes. In the light of the three HA inter- faces with structural homology analysis, the common neutralizing epitope on HA recognized by 8H5 consists of 9 incontinuous amino acid residues: Asp68, Asn72, Glu112, Lys113, Ile114, Pro118, Ser120, Tyr137, Tyr252 (numbered as for 1jsm sequence). The primary purpose of the present work is to provide some insight into structure and binding details of a common neutralizing epitope of H5N1 AIV, thereby aiding in the structure-based design of universal AIV vaccines and anti-virus therapeutic drugs.
基金supported by the National Natural Science Foundation of China (31702210, 31972719)the CAMS Innovation Fund for Medical Sciences (2020–12M-5-001)
文摘Interferon-inducible transmembrane protein 3(IFITM3)inhibits influenza virus infection by blocking viral membrane fusion,but the exact mechanism remains elusive.Here,we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin(HA).The restriction of IFITM3 on HAmediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses.Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA_(2) subunit but not HA_(1) subunit of H5 and H7 virus.Truncated analyses showed that the transmembrane domain of the IFITM3 and HA_(2) subunit might play an important role in their interaction.Finally,this interaction of IFITM3 was also verified with HA_(2) subunits from other subtypes of influenza A virus and influenza B virus.Overall,our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain,providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.
文摘OBJECTIVE: To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. METHODS: Viruses were passed in embryonated hen eggs, and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03) and Editseg (version 3.69) softwares. RESULTS: The amino acid sequences at the cleavage site between HA1 and HA2 domains of H9N2 viruses isolated in China are R-S-S-R. One pigeon strain contains seven potential glycosylation sites on the HA protein molecule, while all others have eight. There are 2 to 15 differences of amino acid sequences distributed at 24 different positions on the HA protein molecules among six H9N2 viruses. The H9N2 viruses with multiple lineages of HA genes were co-circulating in China recently. CONCLUSION: The highest possibility is that human influenza A (H9N2) virus was derived from Chicken H9N2 virus, and not derived from pigeon H9N2 virus. However, it is still unknown whether the H9N2 virus could transmit from person to person. The H9N2 viruses with multiple lineages of HA genes are co-circulating in China.
文摘Avian influenza (AI) virology surveillance is the most important method to monitor AI virus (AIV) in poultry so as to effectively prevent and control AI outbreaks. Monoclonal antibodies (MAb)-based assays are highly sensitive and specific for AIV detection, and much practical and economic for test-in-field or onsite. Many such assays have been developed and are still in developing since the H5N1 highly pathogenic AI (HPAI) outbreaks occurred in South East Asia in 2003. A MAb-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed in our lab during late 1990s and early 2000s. Meanwhile, AIV H7 and H5 subtype specific-MAbs have been successfully developed in our laboratory to enhance the Dot-ELISA and other MAb-based assays for AIV detection. Production and purification of the H7 and H5 MAbs were made to provide essential reagents for Dot-ELISA and other immunoassays, and the current development of a novel Biosensor technique for rapid detection of AIV from clinical and field specimens.
基金Supported by the National Natural Science Foundation of China（Nos.81172725, 30271175）.
文摘This research reveals the phylogenetic history and structural information of the hemagglutinin（HA） and neuraminidase（NA） from novel avian influenza virus A/Hangzhou/1/2013（HTN9_2013） strain from human infected in China. Strains closely related to the HTN9 2013 strain were obtained from Nation Center for Biotechnology Information（USA）-basic local alignment search tool（NCBI-BLAST） searching, and the phylogenetic trees were con- structed. The 3D structures of HA and NA from H7N9 2013 strain were built by homology modeling technology, and molecular dynamics（MD） simulations were performed on the high-performance computer cluster. Characteristic amino acid sites were then screened from multiple sequence alignment（MSA） via home-made Python script and mapped onto the 3D structures. The thermodynamic characteristic root-mean-square-fluctuation （RMSF） of these sites in the structure was also analyzed with MD trajectories. The HA of HTN9_2013 strain is closely related to the A/duck/Zhejiang/12/2011 strain isolated in China, while the NA of HTN9 2013 strain is mostly related to the A/mallard/Czech Republic/13438-29K/2010 strain isolated in Europe. The 3D structures of HA and NA from H7N9 2013 stain are mostly identical to the existing structure of H7 and N9. A total of 11 and 14 characteristic ami- no acid sites were identified in HA and NA, respectively, in HTN9_2013. Structural analysis indicates that certain sites in the top region of HA are important, at which the mutation of some amino acids can impact the receptor bin- ding that may be related to its infection of human beings.
基金This work is supported by the intramural grant of the Chinese Academy of Sciences(Grant No.KSCX2-YW-R-158)the National Basic Research Program(973 Program)(Grant Nos.2010CB534004 and 2005CB523001)+1 种基金G.F.G.is a distinguished young investigator of the NSFC(Grant No.30525010)Dr.Christopher Vavricka is,partly,supported by the Fellowship for Young International Scientists of the Chinese Academy of Sciences(Grant No.2009Y2BS2).
文摘Influenza virus is the causative agent of the seasonal and occasional pandemic flu.The current H1N1 influenza pandemic,announced by the WHO in June 2009,is highly contagious and responsible for global economic losses and fatalities.Although the H1N1 gene segments have three origins in terms of host species,the virus has been named swine-origin influenza virus(S-OIV)due to a predominant swine origin.2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects.Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection.Primary sequence analysis of the 2009 S-OIV hemagglutinin(HA)reveals its closest relationship to that of the 1918 pandemic influenza virus,however,analysis at the structural level is necessary to critically assess the functional significance.In this report,we report the crystal structure of soluble hemagglutinin H1(09H1)at 2.9Å,illustrating that the 09H1 is very similar to the 1918 pandemic HA(18H1)in overall structure and the structural modules,including the five defined antiboby(Ab)-binding epitopes.Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV,and people born around the 1918 are resistant to the current pandemic,yet younger generations are more susceptible to the 2009 pandemic.
基金We thank Chungen Pan for assistance in preparing the Fig.4B.SJ and LD were supported by the US NIH grants(Nos.AI68002,AI46221)SL and RL were supported by the Natural Science Foundation of China(Grant No.30772602)the Ministry of Education’s New Century Talent Program(NCET-06-0753).
文摘Seasonal influenza epidemics and influenza pandemics caused by influenza A virus(IAV)has resulted in millions of deaths in the world.The development of anti-IAV vaccines and therapeutics is urgently needed for prevention and treatment of IAV infection and for controlling future influenza pandemics.Hemagglutinin(HA)of IAV plays a critical role in viral binding,fusion and entry,and contains the major neutralizing epitopes.Therefore,HA is an attractive target for developing anti-IAV drugs and vaccines.Here we have reviewed the recent progress in study of conformational changes of HA during viral fusion process and development of HA-based antiviral therapeutics and vaccines.
基金by the National Basic Research Program(973 Program)(Grant No.2011CB504703)We are grateful to Dr Christopher Vavricka and Dr Guangwen Lu for their help.GFG is a leading principal investigator of the Innovative Research Group of the National Natural Science Foundation of China(Grant No.81021003).
文摘There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs.The main component of the current split influenza A virus vaccine is viral hemagglutinin(HA)which induces a strong antibody-mediated immune response.To develop a modern vaccine against influenza A viruses,the current research has been focused on the universal vaccines targeting viral M2,NP and HA proteins.Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface,and each monomer consists of a globular head(HA1)and a“rod-like”stalk region(HA2),the latter being more conserved among different HA subtypes and being the primary target for universal vaccines.In this study,we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A(H1N1)virus HA as a model,tested its immunogenicity in mice,solved its crystal structure and further examined its immunological characteristics.The results show that the HA globular head can be easily prepared by in vitro refolding in an E.coli expression system,which maintains its intact structure and allows for the stimulation of a strong immune response.Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.
文摘A mutation network for the hemagglutinin gene(HA) of the novel type A(H1N1) influenza virus was constructed.Sequence homology analysis indicated that one HA sequence type from the viruses mainly isolated from Mexico was likely the original type in this epidemic.Based on the 658A and 1408T mutations in HA,the viruses evolving into this epidemic were divided into three categories,the Mexico,the transitional and the New York type.The three groups of viruses presented distinctive clustering features in their geographic distributions.
基金supported by the National High Technology Research and Development Program of China(Grant No.2006AA10A205)the National Key Technology Research and Development Program(Grant No. 2006BAD06A05)the National Key Program for Infectious Diseases of China(Grant No.2009ZX10004-103)
文摘Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines.In this study,we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen(HA) from the strains A/New Caledonia/20/99(H1N1)(pV1A5) and A/California/04/2009(H1N1)(pVEH1) ,respectively.After verifying antigen expression,the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model.Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies.HA-specific T-cells were also measured in IFN-γELISpot assays.The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD50 of mouse-adapted A/New Caledonia/20/99(H1N1) .Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses(P<0.05) than the pVAX1 control group.Additionally,the pV1A5 vaccine effectively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100%compared with a 40%survival rate in the pVEH1 vaccinated group(P<0.05) .Our study indicates that the seasonal influenza DNA vaccine completely protects against the homologous A/New Caledonia/20/99 virus(H1N1) ,while the pandemic influenza DNA vaccine only partially protects against this virus.
