The Protection Efficacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus

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.

Pathogenicity and amino acid sequences of hemagglutinin cleavage site and neuraminidase stalk of differently passaged H9N2-avian influenza virus in broilers

Low pathogenic Avian Influenza (AI) virus has the ability to evolve to high pathogenic viruses resulting in significant economic losses in the poultry sector. This study aims at assessing the impact of H9N2 viral passaging in broilers and its relatedness to pathogenicity and amino acid (a.a) sequences of the hemagglutinin (HA) cleavage site and neuraminidase (NA) stalk. The original H9N2 AI virus (P0) was used to challenge ten-21 days old broilers. Individual recovery of H9N2 virus from homogenates of trachea, lungs and airsacs was attempted in 9 days old chicken embryos, as a conclusion of the first passage (P1). Tracheal isolates of H9N2 were passaged for a second (P2) and a third (P3) time in broilers, followed by a similar embryonic recovery procedure. The a.a. sequence of a part of HA1 cleavage site and Neuraminidase stalk were compared among the differently passaged viruses;an assessement of the relatedness of the determined a.a. sequences to the pathogenicity in broilers, based on frequency of mortality, morbidity signs, gross and microscopic lesions at 3 days post challenge with the P1, P2, and P3-H9N2, is concluded. An increase in certain morbidity signs and specific lesions was observed in P2- and P3-H9N2 challenged broilers compared to birds challenged with P1-H9N2. A conserved R-S-S-R amino acid sequence at the HA1 cleavage site was observed in the differently passaged H9N2, associated with a variability in the NA stalk-a.a sequences. The passaging of the low pathogenic H9N2 virus in broilers leads to a trend of increase in pathogenicity, manifested in higher frequency of morbidity signs, and of specific gross and microscopic lesions of the examined organs. This passaging was associated with a conserved a.a. sequence of the hemaglutinin cleavage site and a variability in the sequence of the neuraminidase stalk. A detailed study of the potential of the detected variability in the neuraminidase stalk of H9N2 in induction of a higher pathogenicity in broilers will be the subject of future investigations.

Preparation of Monoclonal Antibodies against Hemagglutinin of Avian Influenza Virus H9 Subtype by Plasmid Immunization

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.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

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.

Development of H5 subtype-specific monoclonal antibodies (MAb) and MAb-based assays for rapid detection of H5 avian influenza

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.

禽流感病毒样颗粒疫苗研究进展

禽流感是由禽流感病毒(Avian influenza virus,AIV)感染引起的一种禽类传染病,给家禽业造成了巨大的经济损失。AIV因抗原漂移和抗原转换可能会产生新的变异毒株和亚型,因此禽流感是在世界范围内需要被重点关注的禽类传染病。病毒样颗粒(virus-like particles,VLPs)能自组装成类似天然病毒结构的蛋白颗粒,易被免疫系统识别,但无遗传物质,安全性较高,是疫苗制备的新方向。禽流感VLPs是基于AIV蛋白模拟禽流感病毒外表面而构成的蛋白颗粒,具有很强的免疫原性,用其制成的疫苗具有无传染性、稳定和不易失活等优势,是一种安全性较高的疫苗类型。笔者就禽流感VLPs疫苗的研发基础、与疫苗有关的结构蛋白及疫苗的通用性和优缺点进行综述,以期对未来禽流感疫苗的研发和防控提供理论参考。

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin(HA)cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes.We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process.Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons,and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops.Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses,that is,to inhibit the gene expression of avian influenza virus H5N1 subtypes,we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.

Phylogenetic and Structural Analysis of Major Surface Proteins Hemagglutinin and Neuraminidase of Novel Avian Influenza Virus A H7N9 from Chinese Patient

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.

H5亚型禽流感重组鸡痘病毒活载体疫苗的构建及其遗传稳定性与免疫效力

以鹅源H5亚型禽流感病毒 (AIV)基因组为模板 ,用RT PCR扩增血凝素 (Hemaggluti nin ,HA)基因 ,克隆入鸡痘病毒表达载体pFG1 1 75 ,转染鸡痘病毒感染的鸡胚成纤维细胞 ,通过蓝斑筛选和间接免疫荧光检测 ,获得表达HA基因的重组鸡痘病毒 (Recombinantfowlpoxvir us,rFPV HA)。rFPV HA经鸡胚成纤维细胞连续传 1 5代后 ,报告基因LacZ和HA基因可稳定表达。用 1 0 3PFU和 1 0 5PFU的rFPV HA免疫无特定病原体的 (Specificpathogenfree,SPF)鸡 ,免疫后 2 2d血凝抑制 (Hemagglutinininhibition ,HI)抗体监测阳性率分别为 0 %和 2 0 % ,但均抵御了H5亚型毒株的致死性攻击 ,保护率为 1 0 0 %。结果表明 ,构建了表达HA基因的重组鸡痘病毒 ,该重组病毒具有良好遗传稳定性 ,免疫鸡可提供完全保护 ,显示出了一定的应用前景。

禽流感HA基因疫苗脂质体的制备及其理化性质

用改良的逆相蒸发法制备了Ｈ７亚型禽流感保护性抗原血凝素（ＨＡ）基因重组质粒ｐＳＶＨ７脂质体。电镜观察表明，该脂质体多呈球形的单层或多层结构，脂质体粒径分布均匀，粒径范围２０～７００ｎｍ，平均粒径１６３ｎｍ，主要粒径分布位于５０～２００ｎｍ（８４．８０％）。重组质粒ＤＮＡ在脂质体的制备过程中保持了稳定性，并能抵抗ＤＮＡ酶的水解破坏作用。吸收光谱表明，在２４８ｎｍ及２６８ｎｍ处脂质体有２个特征吸收峰，ＤＮＡ的存在不改变吸收峰的位置，但能增加吸收峰的强度。

表达H5亚型禽流感病毒HA基因的DNA疫苗免疫保护效力研究

表达高致病力禽流感病毒分离株A/Goose/Guangdong/1/96(H5N1) [GD/1/96(H5N1)]HA基因的DNA疫苗质粒pCIHA5具有良好的免疫保护性,为了将其开发并应用于实际,对其免疫保护效力进行了进一步的研究。结果表明,用10、40、70、100和150g pCIHA5和油苗1次免疫后,其免疫保护率分别为12.5%(1/8)、58.3%(7/12)、72.7%(8/11)、50.0%(6/12)、66.7%(8/12)和100%(12/12),其中70和100g剂量组的病毒分离结果为1/8和2/6;用剂量为5、20、35和50g pCIHA5分别免疫2次,免疫保护率分别为45.5%(5/11)、58.3%(7/12)、58.3%(7/12)和91.7%(11/12),5g的病毒分离结果为1/5,其余组未分离出病毒;pCIHA5免疫后AGP抗体均未检出,油苗免疫后其HI抗体虽高,但AGP抗体也呈阳性。结果显示,加强免疫应为DNA疫苗质粒pCIHA5的安全免疫方式,DNA疫苗质粒pCIHA5虽具有较为稳定的免疫原性和免疫保护性,但有必要进一步提高其在肌肉组织中的有效表达水平和抗体反应水平。

百脉根表达H5N1亚型禽流感血凝素的研究

【目的】将H5N1亚型禽流感病毒血凝素(AIVHA)基因转入牧草植物中,利用豆科牧草作为植物生物反应器来生产禽流感抗原蛋白,探索研制禽流感转基因植物可饲用疫苗的可行性。【方法】以百脉根子叶柄外植体作为转化受体,通过农杆菌介导法将AIVHA基因导入百脉根,子叶柄外植体经过共培养、筛选分化、再生,得到抗性植株。对抗性植株进行了PCR、RT-PCR检测和Western blot分析。【结果】证明AIVHA基因已经导入到百脉根基因组中,在核酸水平和蛋白水平都得到了表达。【结论】利用百脉根表达禽流感抗原蛋白是可行的。

H5亚型禽流感重组鸡痘病毒活载体疫苗的免疫效力

利用表达H5亚型禽流感病毒血凝素基因的重组鸡痘病毒疫苗免疫SPF鸡和无母源抗体的商品鸡,通过比较免疫后血凝抑制(HI)抗体应答水平、攻毒后发病率和死亡率等指标评价其免疫保护作用。免疫后21d,重组鸡痘病毒免疫组仅有13%～20%鸡的HI抗体检测呈阳性。在同亚型禽流感病毒攻击后,重组鸡痘病毒疫苗免疫组产生了100%的保护率,而未免疫组全部死亡。结果表明:重组鸡痘病毒疫苗不能激发高滴度HI抗体应答,但可抵御同亚型禽流感病毒致死性攻击,保护效果达到或优于目前应用的灭活苗,显示出良好的应用前景。

鸡源株与人源株H9N2流感病毒血凝素受体结合位点氨基酸比较与分析

H9N2病毒可以感染多种禽类和哺乳动物 ,包括人类。流感病毒血凝素受体结合位点的氨基酸可以影响受体结合特性。为了解鸡源株与人源株H9N2病毒受体结合部氨基酸的差异 ,作者对二者进行了比较与分析 ,结果表明 ,鸡源株与人源株血凝素受体结合部位氨基酸的第 137、183、190、2 2 6位点存在差异 ,鸡源株在这些位点分别为K、N、AorVorT、LorQ ,而人源株则分别为R、H、E、L。虽然尚不清楚这些位点的改变对病毒与细胞亲和力及宿主范围的影响 ,但由于H9N2在我国养鸡业的普遍存在 ,加强H9N2病毒的分子流行病学监测有重要意义。

封闭式饲养鸡场H9N2亚型禽流感病毒HA基因在5年内的遗传变异

选择一个于 1 998年开始发生H9亚型禽流感的封闭式大型养鸡场 ,连续 5年内分离到 2 2株H9N2亚型病毒 ,对其中 9株与 1 998年分离株进行HA基因序列和病毒抗原性的比较结果表明 ,这些分离株均与 1 998年的具有较高的序列同源性 ,且在本研究期内HA基因的这些变化尚未产生引起交叉保护性改变。初步推断这些分离株均系 1 998年分离株在场内循环传播变化得来 ,其HA基因的变异可能与频繁的疫苗免疫选择压力有关。这为进一步研究禽流感病毒变异的规律和制定正确的禽流感防治对策具有重要意义。

禽流感血凝素基因的原核表达及其在H9亚型诊断中的应用

根据H9N2亚型禽流感病毒血凝素基因序列设计并合成引物 ,从本室分离并保存的H9N2亚型禽流感病毒中扩增了预计约 16 83bp的血凝素基因 ,将此扩增产物克隆进pMD18_T载体 ,限制性酶切及序列测定后 ,进一步将其亚克隆到pGEX_KG中 ,与GST蛋白融合表达。SDS_PAGE和Western印迹表明缺失信号肽后的HA基因在大肠杆菌中获得了表达 ,表达产物具有免疫学活性 ,融合蛋白的分子量约为 90kD ,位于包涵体中。包涵体经变性、复性处理 ,利用复性产物作为抗原包被酶标板建立了检测H9亚型禽流感抗体ELISA方法。结果表明应用HA重组蛋白作为诊断H9亚型禽流感抗原具有特异性强、敏感性高、重复性好的特点 ,可用于H9亚型禽流感抗体的检测。

禽流感病毒A/Chicken/Guangdong/SS/94(H_9N_2)HA基因的克隆及序列分析

RT-PCR was employed to amplify the cDNA of HA gene of influenza A/Chicken/Guangdong/SS/94(H 9N 2)with a pair of degenerate primers and the cDNA were cloned into the T-T windows of plasmid pMD18-T.The inserts were sequenced,at first time,and the results revealed that the HA gene had a long complete open reading frame and composed of 1,683 nucleotides,coding for 560 amino acids.The amino acid sequences of the HA connecting peptide revealed that A/Chicken/Guangdong/SS/94(H 9N 2)had X-X-X-R(X,not basic amino acid)at the proteolytic cleavage site.The molecular basis of the HA gene was compatible with not highly pathogenicity.Comparison of the degree of homology of HA gene showed 82%-98% nucleotide sequence and amino acid sequence homology among the isolate and the other H 9N 2 subtype AIV in GenBank. Thus, the HA gene of influenza A/Chicken/Guangdong/SS/94 belonged to H 9 subtype.

我国与韩国禽流感H9N2病毒血凝素分子特性的区别

中国与韩国都发生了H9N2亚型禽流感,且都对养禽业造成了严重损失。国际上有不少学者认为韩国的H9N2亚型流感是由中国通过禽肉及其相关产品的输入而引起。本研究针对这一情况对从中国大陆分离的H9N2病毒和自GeneBank读取的韩国H9N2病毒的血凝素核苷酸序列进行了比较分析,结果表明两者属于不同的进化分支。所有中国H9N2病毒的血凝素裂解位点都为-RSSR/G-,而韩国H9N2病毒的血凝素裂解位点为-ASVR/G-、-ASYR/G-或-ASGR/G-。韩国H9N2病毒血凝素的受体结合位点的氨基酸在第183、190和226位点分别H、E和Q;而中国毒株的变异较大,在第183位点全部为N,190位点为A、T或V,第226位点为L或Q。因此,中韩两国的H9N2病毒血凝素分子特性显著不同。

禽流感病毒分离株A/Goose/Guangdong/3/96(H5N1)HA基因序列分析

采用RT_PCR技术,以A/Goose/Guangdong/3/96(H5N1)RNA为模板,扩增了1.73kb 的HA 全基因cDNA。将HAcDNA克隆后进行了序列测定,测序结果表明所扩增的1728 个核苷酸片段包含了完整的HA基因的开放阅读框架和上下游引物序列、蛋白质合成的起始密码子和终止密码子。核苷酸序列比较分析结果表明:A/Goose/Guangdong/3/96(H5N1) 与A/Goose/Guangdong/1/96(H5N1)有11 个核苷酸差异,同源率99.4% ;与A/HongKong/156/97(H5N1) 有25 个核苷酸差异,同源率98.6 % ;与A/Chicken/HongKong/258/97 (H5N1) 有30 个核苷酸差异,同源率98.3% ;它们的氨基酸序列同源率依次分别为99 .2 % 、98.6% 和98.1% 。受体结合位点的氨基酸序列完全一致;HA裂解位点氨基酸序列也完全一致,各有5 个碱性氨基酸插入。这说明上述4 个流感病毒分离株可能来自同一个祖先,具有相同的毒力和相似的生物学特性。

禽流感病毒血凝素分子生物学研究进展

禽流感病毒基因组由 8条单性负链RNA组成 ,血凝素基因是禽流感病毒基因组中变异最大的基因 ,禽流感病毒的抗原性和致病性很大程度上取决于该基因的变异情况。血凝素在病毒吸附及穿膜过程中起关键作用 ,但可刺激机体产生中和抗体来中和病毒的感染力 ,而且血凝素诱导机体产生的体液免疫反应 ,对宿主抵抗禽流感起到了决定性保护作用 ,使目前研制血凝素基因工程疫苗成为禽流感病毒疫苗的研究热点。血凝素发挥功能之前必须裂解为两条肽链 HA1和 HA2 ,其裂解位点的氨基酸残基的组成是决定禽流感病毒致病力高低的主要因素。文章主要从血凝素的基因及其编码的蛋白、裂解位点序列和基因疫苗等角度对禽流感病毒血凝素分子生物学的研究状况进行了综述 。