A peptide of foot-and-mouth disease virus serotype Asia1 generating a neutralizing antibody response, and an immunostimulatory peptide

The epitopes of the capsid of foot-and-mouth disease virus (FMDV) play important roles in the construction of highly immunogenic subunit vaccines. However few epitopes have been found for FMDV serotype Asia1. In this study we screened for epitopes of the VP1 and VP2 proteins of FMDV serotype Asia1 isolate, YNBS/58. Fragments consisting of amino acids 133-163 of VP1 and amino acids 1-33 of VP2 contained epitopes, and both induced lymphoproliferation in guinea pigs. Only the VP1 fragment induced neutralizing antibodies but the VP2 peptide dramatically increased the neutralizing antibody response induced by the VP1 peptide.     

Identification of a novel foot-and-mouth disease virus specific T-cell epitope with immunodominant characteristics in cattle with MHC serotype A31

To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the entire polyprotein sequence of the virus, 442 overlapping pentadecapeptides were tested in proliferation assays using lymphocytes from cattle experimentally infected with FMDV. Four months post-infection cells from all investigated animals (n = 4) responded by proliferation and interferon-gamma production to a peptide located on the structural protein 1D (VP1), amino acid residues 66-80. Major histocompatibility complex (MHC) serotyping of the investigated cattle indicated that all animals shared the MHC serotype A31 which comprises the class II allele DRB3 0701. This may explain the common recognition of this newly discovered epitope. Responses to other peptides could only be observed in one animal and rapidly declined during the time course of the study. These observations point to an immunodominant role of this epitope located on the protein 1D in cattle with MHC serotype A31.     

口蹄疫病毒VP2基因重组表达载体的构建及其稳定表达细胞系的建立

本试验旨在构建Asia 1型口蹄疫病毒(FMDV)的VP2基因重组表达载体,并建立稳定表达VP2基因的BHK-21细胞系。利用RT-PCR方法扩增Asia 1型FMDV的cDNA,获得VP2基因完整的编码区,构建可表达VP2的带有绿色荧光蛋白标记基因的pIRES2-EGFP-VP2重组表达载体。经鉴定正确后,利用LipofectamineTM2000将重组质粒转染293T细胞,通过Western Blot技术检测VP2蛋白的瞬时表达;然后再转染BHK-21细胞,通过G418筛选,形成单克隆细胞系,经荧光筛选到表达EGFP的抗性单克隆细胞,扩繁培养克隆细胞,再通过West-ern Blot技术检测其VP2的表达,最终筛选到稳定表达FMDVVP2基因的BHK-21细胞系。结果表明,VP2基因重组表达载体pIRES2-EGFP-VP2构建正确,能够在293T细胞内瞬时表达;转染BHK-21细胞,经G418筛选到表达VP2基因的BHK-21细胞克隆,经长达60d的传代,获得了稳定表达VP2基因和EGFP的BHK-21细胞系。上述结果表明,我们建立了稳定表达VP2基因的BHK-21细胞系,为进一步探讨VP2基因在FMD...     

南非Ⅱ型口蹄疫病毒抗原表位的筛选及抗原性分析

目前,我国南非Ⅱ型(SATⅡ)口蹄疫病毒(FMDV)的防控形势十分严峻,为了防止SATⅡ型FMD的跨境传入,迫切需要建立其特异的检测方法。本研究以SATⅡ型FMDV结构蛋白氨基酸序列为依据,利用分子生物学软件分析了FMDV结构蛋白VP1~VP3上可能的抗原表位,并人工合成了8条表位多肽。通过采用SATⅡ型FM-DV阳性血清进行ELISA反应,检测其反应原性;通过采用与载体蛋白偶联的合成肽免疫小鼠,测定小鼠血清中抗体效价,检测合成肽的免疫原性。结果表明,合成的8条多肽均能与SATⅡ型FMDV阳性血清结合,其中的6条多肽免疫小鼠后能产生针对多肽的抗体。本研究为利用串联表位为抗原检测SATⅡ型FMDV抗体方法的建立奠定了基础。     

口蹄疫病毒VP1基因研究进展

口蹄疫病毒VP1基因是参与构成病毒粒子的主要中和抗原基因,其表达蛋白可以诱导动物机体产生中和抗体。对VP1基因进行分析,不仅对口蹄疫病毒遗传变异的研究具有指导作用,而且对口蹄疫的流行病学调查、疫源追踪、毒株型和亚型的分析,甚至对新型疫苗的研制也具有重要意义。因此,VP1基因一直是口蹄疫病毒分子生物学研究领域中的热点。文章就口蹄疫病毒VP1基因的特性及其在基因分型、诊断和疫苗研究中的应用进行了综述。     

AsiaⅠ型口蹄疫病毒VP1基因在昆虫细胞/杆状病毒中的表达

利用杆状病毒表达系统对AsiaⅠ型口蹄疫病毒(foot-and-mouth disease virus,FMDV)VP1基因在Sf9昆虫细胞中进行表达,为研究AsiaⅠ型FMDV VP1蛋白功能及建立AsiaⅠ型FMDV血清学诊断方法奠定基础。采用PCR方法从pGEM-T-Easy-AsiaⅠ型VP1质粒中扩增VP1基因,将其插入杆状病毒转座载体pFastBacHTA,构建的重组质粒pFastBacHTA-VP1再转入DH10Bac感受态细胞,经三重抗性与蓝白斑筛选,获得杆状病毒重组质粒Bacmid-VP 1,然后转染Sf9昆虫细胞。PCR鉴定证实VP1基因正确地插入到Bacmid中,成功构建了杆状病毒重组质粒Bacmid-VP1,SDS-PAGE和Western-blotting检测结果表明,VP1基因在Sf9昆虫细胞中表达出约26.5 ku的VP1蛋白。将可溶性表达的融合蛋白用Ni-NTA亲和层析方法进行纯化,通过ELISA分析,能特异性地检测出AsiaⅠ型口蹄疫病毒阳性血清。AsiaⅠ型FMDV VP1基因在杆状病毒表达系统中的成功表达为AsiaⅠ型FMDV VP1蛋白的抗原性及血清学抗体水平检测研究奠定了基础。     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Co-expression of the Bcl-xL antiapoptotic protein enhances the induction of Th1-like immune responses in mice immunized with DNA vaccines encoding FMDV B and T cell epitopes

Foot-and-mouth disease (FMD) is one of the most devastating animal diseases, affecting all cloven-hoofed domestic and wild animal species. Previous studies from our group using DNA vaccines encoding FMD virus (FMDV) B and T cell epitopes targeted to antigen presenting cells, allowed demonstrating total protection from FMDV homologous challenge in those animals efficiently primed for both humoral and cellular specific responses (Borrego et al. Antivir Res 92:359-363, 2011). In this study, a new DNA vaccine prototype expected to induce stronger and cross-reactive immune responses against FMDV which was designed by making two main modifications: i) adding a new B-cell epitope from the O-serotype to the B and T-cell epitopes from the C-serotype and ii) using a dual promoter plasmid that allowed inserting a new cistron encoding the anti-apoptotic Bcl-xL gene under the control of the internal ribosomal entry site (IRES) of encephalomyocarditis virus aiming to increase and optimize the antigen presentation of the encoded FMDV epitopes after in vivo immunization. In vitro studies showed that Bcl-xL significantly prolonged the survival of DNA transfected cells (p?<?0.001). Accordingly, vaccination of Swiss out-bred mice with the dual promoter plasmid increased the total IgG responses induced against each of the FMDV epitopes however no significant differences observed between groups. The humoral immune response was polarized through IgG2a in all vaccination groups (p?<?0.05); except peptide T_3A; in correspondence with the Th1-like response observed, a clear bias towards the induction of specific IFN-γ secreting CD4⁺ and CD8⁺ T cell responses was also observed, being significantly higher (p?<?0.05) in the group of mice immunized with the plasmid co-expressing Bcl-xL and the FMDV B and T cell epitopes.     

Sequence analysis of the non-structural 3A and 3C protein-coding regions of foot-and-mouth disease virus serotype Asia1 field isolates from an endemic country

A total of 18 foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates belonging to two different lineages (including the divergent group) as delineated earlier in VP1-based phylogeny were sequenced in the non-structural 3A and 3C protein-coding regions. The phylogenetic trees representing the regions coding for the non-structural proteins were very similar to that of the structural VP1 protein-coding region. Phylogenetic comparison at 3C region revealed clustering of Asia1 viruses with the isolates of serotypes O, A and C in the previously identified clade. Comparison of amino acid sequences identified lineage-specific signature residues in both the non-structural proteins. Overall analysis of the amino acid substitutions revealed that the 3A coding region was more prone to amino acid alterations than 3C region.     

云南边境O型口蹄疫监测及病毒VP1基因序列分析

口蹄疫是由口蹄疫病毒引起的主要侵袭偶蹄动物的一种急性热性高度接触性传染病。口蹄疫病毒为微RNA病毒科口蹄疫病毒属成员,存在7个不同血清型,病毒VP1蛋白抗原性差异是病毒血清型划分依据,而其编码基因(1D)核苷酸序列差异是同型病毒拓扑型(Topotype)或基因型鉴别依据。采用O/A/C/Asia-1多重RT-PCR技术,对2006年自云南边境地区采集的120份动物组织样品,进行口蹄疫病原监测,检出O型口蹄疫病毒阳性样品15份。对阳性样品中病毒VP1基因全序列进行扩增、纯化后,克隆至pMD18-T载体测序,并与已知代表性毒株进行比对及系统发育分析。结果发现:云南边境O型口蹄疫病毒阳性样品VP1基因核苷酸序列同源性介于77.3%~98.7%,可划分为3个不同的拓扑型或基因型:中东-南亚型(ME-SA)或泛亚型(PAN-Asia)、古典中国型(Cathay)、东南亚型(SEA)。部分样品VP1蛋白表位43位、154位关键性氨基酸位点存在变异。     

猪O型口蹄疫病毒样颗粒的构建及鉴定

为开发猪O型口蹄疫病毒（FMDV）病毒样颗粒（VLPs）基因工程亚单位疫苗,试验参考GenBank中登录的FMDV毒株基因序列（登录号：JN998085）,设计针对VP1、VP2、VP3和VP4 4个基因片段的特异性引物,以O型FMDV O/MYA98/XJ/2010毒株的cDNA序列为模板,对目的基因进行PCR扩增;将获得的VP3、VP1和VP4、VP2基因片段分别插入2个杆状病毒供体质粒（pFastBacDual）的p10和pH双元启动子中,构建pFBD-VP3-VP1和pFBD-VP4-VP2 2个重组转座质粒;将验证正确的2个重组转座质粒分别转化含有穿梭载体（Bacmid）的大肠杆菌DH10Bac感受态细胞,获得2个重组杆粒rBacmid-VP3-VP1和rBacmid-VP4-VP2,经验证正确后,对其进行扩增和提取,将其分别转染Sf9贴壁昆虫细胞,构建2个重组杆状病毒rvAc-VP3-VP1和rvAc-VP4-VP2;2个重组杆状病毒共同感染悬浮培养的Sf9昆虫细胞,利用杆状病毒表达系统在昆虫细胞内对4个基因进行表达,目的蛋白通过间接免疫荧光试验（IFA）、SDS-PAGE、Western blotting及透射电镜（EM）进行检测。结果显示,本研究成功构建2株分别表达FMDV VP1、VP2、VP3和VP4 4个结构蛋白的重组杆状病毒;特异性抗体检测发现,4个蛋白VP1~VP4均成功表达,且具有良好的特异性反应;4个蛋白在Sf9昆虫细胞内能够完成自我组装,形成与天然病毒结构相似的VLPs,直径大小在25~30 nm。本研究利用共感染表达方式在Sf9昆虫细胞内成功制备出FMDV病毒颗粒,为开发高效安全的FMDV基因工程亚单位疫苗开辟了一条新思路。     

O型和Asia1型口蹄疫病毒感染RT-PCR鉴别诊断方法的建立

根据GenBank中O型和Asia1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)的vp3、vp1和2A基因序列,并与其它血清型FMDV的对应基因序列进行比较,设计用于扩增O型和Asia1型FMDV vp1基因的特异性引物,建立O型和Asia1型FMDV RT-PCR鉴别诊断方法。本方法首先用通用型引物进行RT-PCR,确定是否为FMDV感染,然后用特异性引物鉴别O型或Asia1型FMDV的感染。用vp1基因序列分析进行符合性试验,验证了该方法所具有的特异性和敏感性。本方法可用于O型和Asia1型FMD的快速诊断及流行病学调查。     

口蹄疫病毒3C蛋白酶的结构及功能研究进展

口蹄疫病毒能引起牛、羊等偶蹄动物发生高度接触性的传染病口蹄疲,该病常常影响着全球畜牧业的发展.FMDV是小RNA病毒科口蹄疫病毒属的成员,口蹄疫病毒为单股正链RNA病毒,病毒基因组全长约8.5 kb,基因组分为5'非编码区、3'非编码区和一个开放阅读框(ORF).基因组的中部是一大的开放阅读框,编码一多聚蛋白,多聚蛋白在翻译的同时,经二级裂解后,形成3种病毒结构蛋白(VP0,VP3和VP1)和8种非结构蛋白(L,2A,2B,2C,3A,3B,3C和3D).其中3C全长639 bp,编码213个氨基酸.3C蛋白酶是小RNA病毒的共同裂解酶,在多聚蛋白成熟过程中起着极为重要的作用,且在抗病毒药物打靶方面具有一定研究价值.因此,对3C蛋白酶的结构及功能研究进展进行综述很有必要.     

猪感染口蹄疫亚洲Ⅰ型病毒分离及其VP1基因的变异比较

对口蹄疫病毒的分子流行病学的研究通常是以VP1基因序列分析为依据的。本试验分离到1株猪源口蹄疫亚洲Ⅰ型毒株,对其主要抗原基因VP1进行了扩增和测序,并与国内外报道序列进行比对,发现与国内报道珠同源性在83.3%~86.4%之间,与国外报道株的同源性在81.4%~98.4%之间。比较发现本次分离株与国内外报道株的差异的较大,只有2株报道株与其同源性在90%以上。     

猪口蹄疫病毒多抗原表位的原核表达与纯化

利用限制性酶切从重组质粒pShuttle-CMV-VP中得到猪O型口蹄疫病毒VP1(21-60)-(141-160)-(200-213)位氨基酸的基因。将此多抗原表位基因克隆至原核高效表达载体pET43.1 a(+),在E.coliBL21中用IPTG诱导表达了含有猪口蹄疫病毒多抗原表位的融合蛋白,并用镍柱亲和层析法获得了纯化蛋白。W estern-b lot结果表明融合蛋白可被猪O型口蹄疫病毒标准阳性血清所识别,从而为进一步研究FMDV多表位抗原的免疫特性和诊断方法奠定了基础。     

Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs

The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus.In the present study we compared the pathogenicity of different FMDVs in young pigs. In total 32 pigs, 7-weeks-old, were exposed to virus, either by direct inoculation or through contact with inoculated pigs, using cell culture adapted (O1K B64), chimeric (O1K/A-TUR and O1K/O-UKG) or field strain (O-UKG/34/2001) viruses. The O1K B64 virus and the two chimeric viruses are identical to each other except for the capsid coding region.Animals exposed to O1K B64 did not exhibit signs of disease, while pigs exposed to each of the other viruses showed typical clinical signs of foot-and-mouth disease (FMD). All pigs infected with the O1K/O-UKG chimera or the field strain (O-UKG/34/2001) developed fulminant disease. Furthermore, 3 of 4 in-contact pigs exposed to the O1K/O-UKG virus died in the acute phase of infection, likely from myocardial infection. However, in the group exposed to the O1K/A-TUR chimeric virus, only 1 pig showed symptoms of disease within the time frame of the experiment (10 days). All pigs that developed clinical disease showed a high level of viral RNA in serum and infected pigs that survived the acute phase of infection developed a serotype specific antibody response. It is concluded that the capsid coding sequences are determinants of FMDV pathogenicity in pigs.     

Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus

No experimental system to date is available to identify viral T-cell epitopes in swine. In order to reconstruct the system for identification of short antigenic peptides, the swine SLA-2 gene was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence (G4S)3, using splicing overlap extension-PCR (SOE-PCR). The maltose binding protein (MBP)-SLA-2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB1 system. The purified MBP-SLA-2-(G4S)3-beta(2)m protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-beta(2)m protein was used to bind three nonameric peptides derived from the foot-and-mouth disease virus (FMDV) O subtype VP1. The SLA-2-(G4S)3-beta(2)m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and secondary spectra, respectively. The results indicate that the SLA-2-(G4S)3-beta(2)m was 41.6kDa, and its alpha-helix, beta-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-beta(2)m protein was able to bind the nonameric peptides derived from the FMDV VP1 region: 26-34 (RRQHTDVSF) and 157-165 (RTLPTSFNY). The experimental system demonstrated that the reconstructed SLA-2-(G4S)3-beta(2)m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

Identification of a conformational epitope on the VP1 G-H Loop of type Asia1 foot-and-mouth disease virus defined by a protective monoclonal antibody

Although neutralizing antigenic sites of foot-and-mouth disease virus (FMDV) can be defined by selection of monoclonal antibody (MAb) escape mutants, no conformational neutralizing epitope on the major antigenic site located on the G-H loop of type Asia1 FMDV has been precisely mapped. In this study, we generated a potent neutralizing MAb 3E11, which recognized a conformation-dependent epitope and neutralized FMDV Asia1/YS/CHA/05 in vitro. Importantly, a dose of 5.5 NT(50) of the MAb 3E11 completely protected suckling mice from a dose of 10 LD(50) of homologous virus challenge in vivo. Through a 12-mer random peptide phage display, synthetic peptide analysis and constructing a series of FMDV Asia1/YS/CHA/05 mutants using reverse genetic system, we finely mapped the neutralizing epitope as the 12-amino acid peptide (141)SXRGXLXXLXRR(152). These results provide additional insights into the virus-MAb interaction at the amino acid level and may help in the development of an epitope-based Asia1 FMDV vaccine.     

O型口蹄疫病毒结构蛋白VP1的原核表达与抗原性分析

研究分析了O型口蹄疫病毒(FMDV)结构蛋白VP1与当前猪FMDV疫苗血清的免疫反应性.将VP1基因克隆至原核表达载体pET32c,并在大肠埃希菌BL21中得到了表达,Western blot分析表明该重组蛋白与豚鼠O型FMDV标准阳性血清具有良好免疫反应性.目的蛋白经纯化后用ELISA分析其与猪疫苗血清的免疫反应性,结果显示该重组VP1蛋白(rVP1)只能与部分O型FMDV疫苗血清反应.推测当前使用的不同O型FMDV疫苗毒株在VP1重要中和抗原位点G-H环(134 aa～158 aa)与C末端(200 aa～213 aa)存在较大差异.