表达O型口蹄疫病毒衣壳蛋白的重组腺病毒构建及其免疫原性分析

为构建表达O型口蹄疫病毒（foot-and-mouth disease virus, FMDV）衣壳蛋白的复制缺陷型人5型腺病毒（Ad5）为载体的重组腺病毒,本研究设计、合成特异性引物并扩增出FMDV-OZK93的P12A、3B3C基因,通过融合PCR方法连接2个片段,获得P12A3B3C基因后插入到pDC316-mCMV-EGFP质粒,构建了能够表达FMDV-OZK93株衣壳蛋白前体P12A和3B3C蛋白酶的重组穿梭质粒pDC316-mCMV-EGFP-P12A3B3C。利用AdMaxTM腺病毒包装系统进行重组腺病毒rAdv-P12A3B3C-OZK93的包装、鉴定及扩增;并感染人胚胎肾细胞HEK-293进行表达验证。将鉴定正确且高度纯化后的重组腺病毒肌肉免疫小鼠进行免疫原性分析。结果显示,rAdvP12A3B3C-OZK93在病毒传代过程中目的基因稳定存在,且病毒滴度可达1×10^9.1 TCID₅₀/mL。间接免疫荧光和Western blotting结果均表明rAdv-P12A3B3C-OZK93在HEK-293细胞中表达了FMDV特异...     

口蹄疫病毒3D基因的克隆表达及功能分析

利用RT-PCR技术扩增了口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,并将其克隆到原核表达质粒载体pET-28a(+)中.3D基因经测序确认后在大肠杆菌BL-21中表达,表达产物纯化的目的蛋白进行Western-blotting检测,获得分子量约55KDa的单一3D基因表达产物.利用RNA体外复制体系和荧光定量PCR技术,证明纯化的3D基因表达产物RNA依赖的RNA聚合酶具有较高的酶活性,可以在体外从头合成FMDV RNA,且主要以引物依赖的方式合成病毒基因组.     

小RNA病毒蛋白翻译调控元件研究进展

真核生物的起始复合物并不是在起始AUG处形成 ,而是在mRNA的 5′末端形成 ,其识别信号就是 5′末端的帽子结构。小RNA病毒科成员RNA 5′末端没有帽子结构 ,而有一个病毒编码的小蛋白质与基因组共价相连。小RNA病毒的蛋白翻译起始于 5′非翻译区中的内部顺式调控元件 ,称为内部核糖体进入位点 (IRES)。口蹄疫病毒 (foot and mouthdiseasevirus,FMDV)是该科病毒的典型代表 ,引起偶蹄动物的急性接触性传染病。完整FMDV含有单链正股RNA、衣壳蛋白及少量装配过程中夹带的非结构蛋白和宿主细胞肌动蛋白 ,其基因组RNA全长约 8 5kb ,可直接作为信使RNA。对IRES的一、二级结构进行了比较 ,对IRES与翻译起始因子的相互作用以及对病毒毒力的影响作了综述。     

口蹄疫重组鸡痘病毒诱导猪特异性细胞毒性T淋巴细胞(CTL)杀伤活性检测

目的:评价口蹄疫重组鸡痘病毒vUTAL3CP1诱导猪产生特异性细胞毒性T淋巴细胞(CTL)杀伤活性的能力。方法:用PCR方法亚克隆O型FMDVVP1基因C末端部分片段(第130～213AA)。将其插入真核表达载体pDisplay中,构建质粒pDisplay-mVP1。将pDisplay-mVP1转染PK15细胞,经3次G418加压筛选,并用RT-PCR和IFA鉴定,证明获得表达目的基因的PK15/pDisplay-mVP1细胞。最后,利用该细胞作为靶细胞,用LDH法检测口蹄疫重组鸡痘病毒vUTAL3CP1免疫猪的特异性CTL杀伤活性。结果:vUTAL3CP1免疫组在效靶比25∶1和50∶1时,CTL裂解活性分别达到42.84%±32.1%和61.94%±42.8%,显著高于灭活疫苗组与其它对照组(p<0.01)。结论:vUTAL3CP1可以诱导猪产生高水平的特异性细胞毒性T淋巴细胞(CTL)杀伤活性,为进一步的FMDV基因工程疫苗的研究奠定了基础。     

Asia1型口蹄疫病毒第V群猪源和牛源毒株全基因组比较分析

为了查明Asia1型FMDV第Ⅴ群猪源和牛源毒株的序列差异, 采用RT-PCR方法, 对Asia1型FMDV第Ⅴ群猪源分离毒株Asia1/HN/06的基因组全序列进行了扩增和测序, 并与第Ⅴ群牛源和猪源参考毒株基因组进行比较分析。结果表明, Asia1/HN/06毒株全基因组序列长约8236 nt [含38个A的poly(A)尾], 其中5'NCR长1116 nt, 前导蛋白(L)编码区长603 nt, 结构蛋白与非结构蛋白编码区的核苷酸序列为6990 nt, 3'NCR长93 nt, 3￠端是至少含有38个A的poly(A)尾巴。猪源毒株和牛源毒株的全基因比较分析表明, 属于第Ⅴ群, 全基因编码区核苷酸和氨基酸的同源性均为98.0%, 主要差别是猪源毒株Asia1/HN/06在细胞受体结合位点变为RDD和155位置的N变为S或D, 该群毒株3A更具有猪源毒株特征, 有4个特异性氨基酸变异。明确了Asia1型FMDV第Ⅴ群猪源和牛源毒株的序列差异, 为进一步利用反向遗传技术研究猪源和牛源毒株差异位点或基因在病毒表型变异中的作用奠定基础。     

Characteristics of codon usage bias in two regions downstream of the initiation codons of foot-and-mouth disease virus

The mechanism of utilization of alternative two AUGs in foot-and-mouth disease virus (FMDV) is still unknown to date. In this study, the characteristics of codon usage bias (CUB) of the region between the two AUGs (the region-La) and of the same-sized region behind the second AUG (the region-Lb) in 94 different FMDV RNA sequences were analyzed using relative synonymous codon usage (RSCU) values. The results indicated that many codons with negative CUB were preferentially used in the region-La. There were two conserved residues (Thr and Cys) on the 4th and 6th residue positions of the region-La. The conserved residues had a general tendency to choose synonymous codons with negative CUB. Although most positions in the region-La did not contain conserved residues, many positions tended to use codons with negative CUB in this region. Among these codons, the majority belonged to the amino acids containing synonymous codons with clearly positive and negative CUB, including Asp, Val, Ile, Leu, Thr, Ala, Ser, Asn and Arg. The presence of many codons with negative CUB in the region-La might impair the efficiency of the first AUG selection. The phylogenetic incongruence of the region-La and the region-Lb implied that intertypic recombination played an important role in the evolution of FMDV. Furthermore, due to the existence of more positions with positive CUB and more widespread phylogenetic incongruence in the region-Lb than the region-La, a probable relationship between the degree of CUB and the evolution of the two target regions was revealed.     

Viral fitness can influence the repertoire of virus variants selected by antibodies

Minority genomes in the mutant spectra of viral quasispecies may differ in relative fitness. Here, we report experiments designed to evaluate the contribution of relative fitness to selection by a neutralizing monoclonal antibody (mAb). We have reconstructed a foot-and-mouth disease virus (FMDV) quasispecies, with two matched pairs of distinguishable mAb-escape mutants as minority genomes of the mutant spectrum. Each mutant of a pair differs from the other by 11-fold or 33-fold in relative fitness. Analysis of the mutant spectra of virus populations selected with different concentrations of antibody in infections in liquid culture medium has documented a dominance of the high fitness counterpart in the selected population. Plaque development as a function of increasing concentration of the antibody has shown that each mutant of a matched pair yielded the same number of plaques, although the high fitness mutant required less time for plaque formation, and attained a larger plaque size at any given time-point. This result documents equal intrinsic resistance to the antibody of each mutant of a matched pair, confirming previous biochemical, structural, and genetic studies, which indicated that the epitopes of each mutant pair were indistinguishable regarding reactivity with the monoclonal antibody. Thus, relative viral fitness can influence in a significant way the repertoire of viral mutants selected from a viral quasispecies by a neutralizing antibody. We discuss the significance of these results in relation to antibody selection, and to other selective forces likely encountered by viral quasispecies in vivo.     

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

重组鸡痘病毒和DNA疫苗对口蹄疫病毒的豚鼠免疫保护

将构建的携带FMDV衣壳蛋白P1-2A和蛋白酶3C编码基因的重组鸡痘病毒活载体疫苗vUTAL3CP1以及编码FMDVP1-2A基因和猪IL-18基因的重组DNA疫苗pVIRIL18P1,分别以单独和混合的方式给豚鼠进行2次免疫,然后测定FMDV特异性结合抗体、中和抗体和T淋巴细胞增殖反应,并用250ID50的FMDV进行攻击,观察其保护效果。结果表明这2种基因工程疫苗均能诱导豚鼠产生特异性的体液免疫及细胞免疫应答。其中以vUTAL3CP1两次免疫组的效果最好,其诱导的抗体水平已接近于常规灭活疫苗,而细胞免疫水平则比后者高得多。攻击保护结果表明该组完全保护率可达3/4,而另外两组也具有一定保护效果。上述研究结果为进一步进行大动物免疫攻毒试验,并最终筛选出最佳疫苗和免疫程序奠定了基础。