Prediction of a common neutralizing epitope of H5N1 avian influenza virus by in silico molecular docking

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.     

Prediction of a common neutralizing epitope of H5N1 avian influenza virus by in silico molecular docking

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. Obviously, 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 modeling 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 energy 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： ljsm, 2ibx and 2fk0） respectively. The result indicates that the three docked complexes share a common binding interface, but differ in binding 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： Asp^58, Asn^72, Glu^112, Lys^113, lie^114, Pro^118, Ser^120, Tyr^137, Tyr^252 （numbered as for ljsm 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.     

The mutation network for the hemagglutinin gene from the novel influenza A （H1N1 ） virus

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.     

Genome evolution of novel influenza A （H1N1 ） viruses in humans

The epidemic situation of A H1N1 flu arose in North America in April 2009, which rapidly expanded to three continents of Europe, Asia and Africa, with the risk ranking up to 5. Until May 13th, the flu virus of A H1N1 had spread into 33 countries and regions, with a laboratory confirmed case number of 5728, including 61 deaths. Based on IRV and EpiFluDB database, 425 parts of A H1N1 flu virus sequence were achieved, followed by sequenced comparison and evolution analysis. The results showed that the current predominant A H1N1 flu virus was a kind of triple reassortment A flu virus： （i） HA, NA, MP, NP and NS originated from swine influenza virus; PB2 and PA originated from bird influenza virus; PB1 originated from human influenza virus. （ii） The origin of swine influenza virus could be subdivided as follows： HA, NP and NS originated from classic swine influenza virus of H1N1 subtype; NA and MP originated from bird origin swine influenza virus of H1N1 subtype. （iii） A H1N1 flu virus experienced no significant mutation during the epidemic spread, accompanied with no reassortment of the virus genome. In the paper, the region of the representative strains for sequence analysis （A/California/04/2009 （H1N1） and A/Mexico/4486/2009 （H1N1）） included USA and Mexico and was relatively wide, which suggested that the analysis results were convincing.     

H5N1亚型禽流感病毒血凝素基因的克隆与表达

 根据已知H5N1亚型禽流感病毒血凝素(HA)基因序列设计、合成克隆引物.自灭活的云南地方H5N1亚型病毒阳性临床组织样品中提取总RNA,反转录后采用高可信度DNA聚合酶(Pyobest^TMDNA Polymerase)扩增HA基因,采用Invitrogen定向表达系统(Champion^TMpET directional TOPO expression system)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组HA,分子质量约78ku.采用阳性血清经免疫印迹及ELISA分析重组HA的免疫反应性,结果表明重组HA能与H5N1亚型病毒抗血清发生特异性结合,具有良好的免疫反应性.     

Orisin and future distribution of the new A （H 1N1 ） influenza virus emerging in North America in 2009

The origin of the new A （H1N1） influenza virus recently emerging in North America is a hot controversial topic of significance in disease control and risk assessment. Some experts claimed that it was an unusually mongrelized mix of human, avian and swine influenza viruses, while some others concluded that it was totally a simple re-assortment hybrid of two lineages of swine influenza viruses. Here the phylogenetic diversity of the viral PB1, PA and PB2 gene sequences using online web servers, and the results suggest that all the 8 genetic segments of the new virus were possibly from two lineages of swine influenza viruses, and one of the lineage was a mongrelized mix of human, avian and swine influenza viruses emerging in the world approximately 10 years ago. Considering the recent epidemiological trends of the new virus, we believe it will spread more widely in the world and persist long in human populations. It also could spread among swine populations. The future wide spreading of the new virus may coincide the disappearance of a subtype of previous human influenza A virus.     

无血清微载体培养MDCK细胞和甲型流感病毒H1N1的条件优化

 血清微载体培养MDCK细胞,并接种流感病毒H1N1,优化培养条件,为细胞流感疫苗的工艺研发奠定基础.采用不同的细胞接种量在50mL无血清微载体搅拌瓶中培养MDCK细胞,并接种甲型流感病毒H1N1,检测不同pH值和TPCK-胰酶含量的病毒培养液,不同病毒接种量,补加TPCK-胰酶,以及不同收毒时间对血凝效价的影响.以1.0×10⁵mL^-1的MDCK细胞数量接种到无血清微载体上,病毒培养液pH值为7.2~7.4范围内,TPCK-胰酶质量浓度为1.0μg/mL,接种后不补加胰酶,病毒接种量MOI=1.0,并在72h收获,最高血凝效价达到512.由此获得了在无血清为载体上培养MDCK细胞和甲型流感病毒H1N1的适宜条件.     

H5N1型禽流感病毒HA基因在烟草中的表达

禽流感病毒H5N1是可以直接感染人类的甲型流感病毒，发展植物源口服疫苗是疫苗研究的方向之一．本研究通过农杆菌介导的方法将禽流感病毒H5N1的HA基因转化烟草．共获得38株潮霉素抗性植株，经PCR和Southern-blotting检测，目的基因已整合到转基因植株的基因组中．Western-dotting检测结果表明，目的基因在转基因烟草中得到表达，具有免疫原性，获得了能够表达HA基因的植物口服疫苗候选植株．     

Antigenic epitope peptides of influenza H3N2 virus neuraminidase gene based on experiments

The neuraminidase (NA) in viral surface is one of the main subtype-specific antigen of influenza type A viruses.Neuraminidase is an enzyme to break the bonds between hemagglutinin (HA) and sialic acid to release newly formed viruses from infected cells.In this study,the H3N2 subtype virus NA genes were sequenced and NA proteins were screened for B-cell epitopes and assessed based on immunoinformatics.Based on this results,four peptides DR6,EY7,VG8 and RE8 (covering amino acid residues 151-156,368-374,398-405 and 428-435,respectively) of the NA protein were synthesized artificially.These peptides were used to immunize New Zealand rabbits subcutaneously to raise antisera.Experimental results showed that these four peptides were capable of eliciting antibodies against H3N2 viruses in a specific and sensitive feature,detected in vitro by enzyme-linked immunosorbent assay.Moreover,hemadsorption anti-releasing effects took place in three three-antisera-mixtures at a dilution of 1:40.Alignment using NA gene database showed that amino acid residues in these four epitope peptides were substituted at specific sites in all the NAs sequenced in this study.It was suggested that these NA epitope peptides might be used in combination with HA proteins as vaccine antigens.     

甲型H1N1流感在预防控制措施下的传播数学模型构建

从数学应用的角度,研究甲型H1N1流感的传播规律,将人群分为易感人群、病毒潜伏人群、发病人群、退出者人群四类. 分析了甲型H1N1流感在人群间的转化过程;日接触率和"聚集性突然爆发"事件被数学刻画,尝试性地构建了一个在预防控制阶段的传播数学模型.     

H5N1亚型禽流感毒株A/Duck/HuBei/3/2005的分子特征及致病性研究

采用分子生物学方法,对1株2005年从湖北省某县分离获得的禽流感病毒株(A/Duck/HuBei/3/2005)进行全基因组序列测定.序列分析显示,该分离株为H5N1亚型.HA蛋白在HA1和HA2连接处,含有连续多碱性氨基酸模体(-RRKKR-).根据进化分析结果,分离株A/Duck/HuBei/3/2005的7个基因来源于2004～2005年湖南地区流行株(CK/HN/999/05,DK/HN303/04),但是PA基因片段发生了重排,来源于野禽.动物实验显示DK/HB/3/05对鸡和鸭均具有高致病性;对小鼠有较低致病性.     

Molecular characterization of avian influenza virus (H7N8) isolated from poultry in Central China in the mid-1980s

The molecular and pathogenic properties of avian influenza virus (A/duck/Hubei/216/1985/H7N8) isolated from Hubei Province of China in 1985 were characterized.The hemagglutinin gene (HA) of Dk/Hb/216/85/H7N8 had the multiple amino acid se-quences (-PEIPKGRG-) at the connecting peptide between HA1 and HA2, which is considered to be a distinguishing molecular characteristic of low pathogenicity.The key sites of host markers among the genes (M, NP, NS, PA and PB2) of Dk/Hb/ 216/85/H7N8 were similar to those of...     

禽流感病毒H5N1亚型RNA聚合酶蛋白表达纯化及晶体生长

通过使用原核表达载体大量表达H5N1病毒RNA聚合酶亚基PA-C257,PB1_N25,再经过GST亲和层析和Sephadex G-200层析柱纯化,获得了高纯度的蛋白复合体.采用悬滴气相扩散法筛选蛋白晶体,在1～1.5mol/L乙酸钠和pH7.9条件下获得了理想的晶体,为解析禽流感病毒RNA聚合酶三维结构并进一步认识其生物功能奠定了基础.     

Special features of the 2009 pandemic swine-origin influenza A H1N1 hemagglutinin and neuraminidase

Since the 2009 pandemic H1N1 swine-origin influenza A virus (09 S-OIV) has reminded the world about the global threat of the ever changing influenza virus,many questions regarding the detailed re-assortment of influenza viruses yet remain unanswered.Influenza A virus is the causative agent of the pandemic flu and contains 2 major antigenic glycoproteins on its surface:(i) hemagglutinin (HA);and (ii) neuraminidase (NA).The structures of the 09 S-OIV HA and NA proteins (09H1 and 09N1) have recently been resolved in our laboratory and provide some clues as to why the 09 S-OIV re-assortment virus is highly infectious with severe consequences in humans.For example,the 09H1 is highly similar to the HA of the 1918 influenza A pandemic virus in overall structure and especially in regards to its 5 defined antibody binding epitopes.For 09N1,its most distinctive feature is the lack of a 150-loop active site cavity,which was previously predicted to be present in all N1 NAs,and we hypothesize that the 150-loop may play a important role in the substrate specificity (α2,3 or α2,6 linked sialic acid receptors) and enzymatic mechanism of influenza NA.Combination of the HA and NA with special characteristics for the 09 S-OIV might contribute to its high increased transmissibility in humans.     

Stepwise prediction and statistical screening: B-cell epitopes on neuraminidase of human avian H5N1 virus

The B-cell epitopes of virus are associated with the antiviral drug and the vaccine screening. As the nucleotide sequences of neuraminidase （NA） of stain GD-01-06 were sequenced, we predicted the a-helix and β-fold structure and the indexes of the flexible regions of secondary structure of NA with methods of the Hydrophilicity plot by Kyte-Doolittle, the Surface probability plot by Emini and the An- tigenic index by Jameson-Wolf, and then screened statistically the parameters to predict B-cell epi- topes by the Hierarchical cluster and the Bivariate correlation and the quartiles with SPSS 13.0. The impact of variation of amino acids in NA on its epitopes was analyzed. The predictive results were evaluated by Wu＇s Antigenic Index and SWISS-MODEL. We found that the most possible epitopes on NA were located within or nearby its N-terminal Nos. 120--137, 81--84, 408--415, 273--282, 429--432, 356--368, 46--55, 146--155, 341 --350 and 198--209, which were the dominant regions of NA epitopes. Peptide 120--137 including the glycoprotein domain （NGT126 128） was first chosen as the B-cell epitopes on NA. NA in H5N1 strain isolated after 2003 lacked in No. 53 amino acid （I）, resulting in an increase in the surface flexible region of NA in GD-01-06 and an enlargement to their epitope regions （VEP48-48→ VEPISNTNFL46-55）. Conclusively, prediction of the B-cell epitopes on the NA based on multiple pa- rameters is useful for researches on the molecular immunology and drug screening and immuno-prophylaxis. A deletion of No. 53 amino acid （I） in NA in strain GD-01-06 might increase its anti- genicity.     

H5N1的结构与致病机制

以禽流感病毒H5N1的两种基质蛋白HA、NA的结构为基础,分析了H5N1的基因结构,探索其毒力基础和致病的分子机制.     

Molecular characterization of H1N1 influenza A viruses from human cases in North America

Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A （H1N1） virus in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites （IPSIQSR↓GL）, and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug （i.e. oseltamivir and zanamivir） because no mutations have been observed. Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A （H1N1） virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.     

H5N1型禽流感病毒HA基因在烟草中的表达

禽流感病毒H5N1是可以直接感染人类的甲型流感病毒,发展植物源口服疫苗是疫苗研究的方向之一.本研究通过农杆菌介导的方法将禽流感病毒H5N1的HA基因转化烟草.共获得38株潮霉素抗性植株,经PCR和Southern-blotting检测,目的基因已整合到转基因植株的基因组中.Western-dotting检测结果表明,目的基因在转基因烟草中得到表达,具有免疫原性,获得了能够表达HA基因的植物口服疫苗候选植株.     

A/PR8/34(H1N1)NA基因包装信号对H5N1流感疫苗株NIBRG-14产量的影响

NIBRG-14是采用"6+2"策略制备的一株H5N1灭活疫苗株,其表面抗原HA和NA基因来自于A/Vietnam/1194/2004(H5N1,VN1194),内部基因来自于A/Puerto Rico/8/34(H1N1,PR8),已有研究表明该疫苗株在鸡胚中的产量不佳.本研究发现,在PR8背景下,VN1194NA基因被包装入重组病毒中的效率仅为正常包装量的38%～68%,因此有一部分重组病毒为不含有NAvRNA的缺陷型病毒粒子.本研究通过在VN1194NA基因完整编码区(CDS)的5′和3′两端嵌合PR8NA基因包装信号序列(vRNA3′末端41bp,5′末端67bp)的方法,使重组病毒中NAvRNA的包装效率得到完全恢复,并且病毒在鸡胚的生长滴度提高了10倍,血凝素HA含量提高了约2·7倍,从而为H5N1流感疫苗株的研制提供了新的思考方向.     

Subtyping of type A influenza by sequencing the variable regions of HA gene specifically amplified with RT-PCR

The outbreak of a novel influenza A(H1N1) virus across the globe poses a threat to human health.It is of paramount importance to develop a rapid,reliable and inexpensive diagnostic procedure.Based on the bioinformatic information from public database,primers specific for influenza A virus surface protein haemagglutinin(HA) of several subtypes(including H1,H2,H3,H5,H7 and H9) were designed.Primer-specific PCR products were subjected to sequencing for accurately distinguishing H1 and H3 subtypes from others.T...