某些动物病毒在宿主细胞内的繁殖特点

共研究了9种动物病毒在宿主细胞内的繁殖特点。 一、无囊膜的病毒粒子在宿主细胞内常呈晶格排列。经常可观察到A型口蹄疫病毒在胞质内呈晶格排列,但0型口蹄疫病毒却观察不到晶格排列。 二、观察到猪传染性水泡瘸病毒的特异装配形式。 三、病毒繁殖时,诸如线粒体、粗面内质网与溶酶体等重要细胞器超徽结构的变化,似与病毒的分类地位无关。 四、大多数RNA病毒繁殖时,宿主细胞核的超微结构变化明显,而DNA病毒繁殖时,宿主细胞核早期超微结构的变化不显著。 五、动物病毒感染时能引起宿主细胞内出现一些新的结构,除毒浆结构(Viropla”)外,还有层板膜结构,微管与徽纤维。     

FMDV非结构蛋白2C主要表位区与3AB基因的组合表达与活性分析

近年的研究表明, 口蹄疫病毒(FMDV)非结构蛋白(NSP)2C在区分灭活疫苗免疫动物与自然感染动物方面具有潜在的价值, 为了建立敏感性更高的鉴别诊断方法, 将截取了2C蛋白N-端和C-端的主要B细胞表位区和完整3AB蛋白基因组合后, 进行了原核表达, 得到了分子量约为47.6 kD的目的蛋白2C￠3AB。通过Western blotting分析证明, 表达产物能被FMDV感染动物阳性血清识别, 具有很好的反应性。以通过电泳纯化的目的蛋白作抗原进行间接ELISA检测不同来源的动物血清, 结果表明, 该抗原仅与感染动物血清具有很好的反应活性, 而与健康动物与免疫动物血清无反应, 说明重组蛋白2C￠3AB可作为区分灭活疫苗免疫动物与感染动物的鉴别诊断抗原。用2C￠3AB和3ABC为抗原进行间接ELISA, 对比检测田间血清样品, 结果显示2C￠3AB-ELISA敏感性比3ABC-ELISA高。说明以重组蛋白2C￠3AB作为鉴别诊断抗原, 能进一步提高对临界值血清的检出率, 这对区分灭活疫苗免疫动物与FMDV隐性感染动物与带毒动物具有非常重要的意义。     

轮状病毒肠毒素NSP4:一个多功能糖蛋白

轮状病毒(rotavirus,RV)是在世界范围内造成婴幼儿和幼年动物病毒性腹泻的最主要病原。A组RV的NSP4蛋白被认为是病毒的肠毒素,近年来的研究发现,这个非结构蛋白NSP4在RV致病过程中起着重要的作用。本文简要综述了多功能肠毒素NSP4的研究进展。     

口蹄疫病毒非结构蛋白3A、3B和2C基因的表达及产物纯化与活性检测

[目的]口蹄疫病毒(FMDV)非结构蛋白(NSP)3A、3B和2C基因的表达及产物纯化与活性检测.[方法]利用原核表达系统表达了FMDV NSP 3A、3B和富含B细胞抗原位点序列的2C蛋白.利用高浓度尿素裂解包涵体,采用稀释法和氧化型、还原型谷胱甘肽系统相结合方法对2C蛋白进行复性.用金属鳌合亲合层析的方法对表达的FMDV NSP 3A、3B和2C进行纯化.采用ELISA方法对比检测了3种纯化蛋白在检测羊血清NSP抗体的效果.[结果]检测得知3A和3B为可溶性表达蛋白,2C以包涵体形式表达.通过Western-blot分析,表明纯化后蛋白能与FMDV感染动物血清发生特异性反应.纯化的3A、3B和复性后的2C融合蛋白与3ABC抗原的检测结果具有很高的符合性.[结论]该研究为建立鉴别FMDV自然感染动物和灭活疫苗免疫动物的酶联免疫电转移印迹技术(EITB)提供了所需的材料.     

口蹄疫病毒单克隆抗体的制备及应用研究进展

口蹄疫是严重影响全球政治经济的烈性动物传染病,快速诊断及有效防治对口蹄疫的防控具有重要意义。单克隆抗体具有高特异性、均质、活性单一等优点,在生物医学领域中有广泛用途。目前,国内外学者制备了多种抗口蹄疫病毒的单克隆抗体,并应用于口蹄疫病毒抗原定型、疫苗量化、抗体水平监测、自然感染与疫苗免疫动物的鉴别诊断,以及口蹄疫病毒抗原表位分析等方面。我们简要综述口蹄疫病毒单克隆抗体的制备及应用进展。     

3A蛋白104–115位氨基酸缺失口蹄疫A型标记病毒的构建

【目的】构建一株含3A非结构蛋白104–115位氨基酸缺失的口蹄疫A型标记病毒,分析其生物学特性和发展标记疫苗的潜力。【方法】采用融合PCR技术,在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆p QAHN中引入3A104–115位氨基酸的缺失,构建全长重组质粒。全长质粒经NotI线化后转染表达T7RNA聚合酶的稳定细胞系,拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性,并用实验室开发的针对3A优势表位(AEKNPLE)的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。【结果】成功拯救到一株含3A 104–115位氨基酸缺失的口蹄疫A型标记病毒,3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。【结论】本研究构建的3A蛋白104–115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株,用于我国未来口蹄疫A型的有效防控。     

小RNA病毒——新型的基因工程疫苗载体

小RNA病毒(Picornaviruses)是最小的RNA动物病毒,这类病毒的研究具有重要的意义,①小RNA病毒可以引起人和动物的许多严重疾病,如脊髓灰质炎、甲型肝炎、口蹄疫、水泡病等;②口蹄疫病毒(FMDV)是最早发现的动物病毒,脊髓灰质炎病毒(Poliovirus,PV)是最早被纯化并获得结晶的病毒。近来的研究发现,小RNA病毒尤其是PV有可能成为新的疫苗载体,可以插入某些已知或推测的病毒中和抗体决定簇的基因区域。本文仅就这方     

口蹄疫病毒非结构蛋白3abc基因的克隆与表达

口蹄疫病毒(Foot-and-mouth disease virus,FMDV)非结构蛋白(NSP)—3ABC可用于注苗与感染动物的鉴别诊断,合成该基因的PCR引物,并在引物5’端和3'端分别加入含BamHⅠ和HindⅢ限制性酶切位点序列。以FMDV毒株基因组RNA为模板,利用RT-PCR技术扩增3abc基因,得到的基因片段与T载体连接,转化DH5α。提取重组载体pT-3ABC,经BamHⅠ/HindⅢ双酶切后与载体pET32a连接,转化宿主菌BL21(DE3)plysS,IPTG诱导表达目的蛋白。SDS—PAGE及Western Blotting检测和鉴定结果表明,在大肠杆菌中成功表达了NSP—3ABC蛋白,分子量约56kDa,且该表达产物可与FMDV感染的动物血清产生免疫反应。ELISA试验结果显示,表达蛋白可用于FMDV注苗与感染动物的鉴别诊断。     

一种实用的筛选病毒抗原表位方法的建立(英)

采用基因分段克隆、表达结合蛋白质印迹, 筛选到了口蹄疫病毒非结构蛋白3ABC上高结合力、保守的感染相关线性表位,分别位于3ABC蛋白上第106～155和156～190位氨基酸.这两个表位可与感染不同血清型口蹄疫病毒动物康复血清反应,但不与来自健康免疫动物和未接触病毒动物的血清发生反应.实验表明,用基因工程表达的多肽筛选抗原表位的方法是可行的.     

口蹄疫病毒非结构蛋白3AB双抗体夹心ELISA方法的建立

抗原纯净度是口蹄疫 (Foot-and-mouth disease,FMD) 灭活疫苗质量检验的一项重要内容,一般采用疫苗2–3次免疫动物后,检测非结构蛋白 (Non-structural protein,NSP) 抗体是否阳转,判断疫苗抗原的纯净度。文中旨在建立定量检测FMD灭活疫苗抗原中NSP 3AB含量的ELISA方法,为疫苗质量控制提供参考方法。利用口蹄疫病毒 (Foot-and-mouth disease virus,FMDV) NSP 3A单克隆抗体和辣根过氧化物酶 (Horseradish peroxidase,HRP) 标记的3B单克隆抗体,建立定量检测NSP 3AB含量的双抗体夹心ELISA检测方法。采用原核表达并纯化的3AB蛋白作为标准品,标准品系列稀释,绘制标准曲线,以标准品与未加抗原的阴性对照吸光值 (OD) 的比值大于2.0的标准品最低浓度为最低检测限。标准品浓度介于4.7–600.0 ng/mL之间时,测得的OD值与浓度呈线性相关,回归曲线呈直线,相关系数R2=0.99,确定最低检测限为4.7 ng/mL。检测12份未纯化灭活抗原中3AB蛋白含量介于9.3–200.0 ng/mL之间;而纯化后的病毒抗原中3AB蛋白残留量低于最低检测限;33份来自不同厂家的成品疫苗抗原中9份疫苗抗原3AB蛋白含量在9.0–74.0 ng/mL之间,其余24份疫苗抗原中3AB蛋白残留量低于最低检测限。检测3AB蛋白含量的双抗体夹心ELISA方法能够特异、敏感地检测疫苗抗原中的3AB蛋白含量,为疫苗质量控制与纯净度检验提供了一种可供选择的检测方法。     

Pathogenesis of Primary Foot-and-Mouth Disease Virus Infection in the Nasopharynx of Vaccinated and Non-Vaccinated Cattle

A time-course pathogenesis study was performed to compare and contrast primary foot-and-mouth disease virus (FMDV) infection following simulated-natural (intra-nasopharyngeal) virus exposure of cattle that were non-vaccinated or vaccinated using a recombinant adenovirus-vectored FMDV vaccine. FMDV genome and infectious virus were detected during the initial phase of infection in both categories of animals with consistent predilection for the nasopharyngeal mucosa. A rapid progression of infection with viremia and widespread dissemination of virus occurred in non-vaccinated animals whilst vaccinated cattle were protected from viremia and clinical FMD. Analysis of micro-anatomic distribution of virus during early infection by lasercapture microdissection localized FMDV RNA to follicle-associated epithelium of the nasopharyngeal mucosa in both groups of animals, with concurrent detection of viral genome in nasopharyngeal MALT follicles in vaccinated cattle only. FMDV structural and non-structural proteins were detected in epithelial cells of the nasopharyngeal mucosa by immunomicroscopy 24 hours after inoculation in both non-vaccinated and vaccinated steers. Co-localization of CD11c⁺/MHC II⁺ cells with viral protein occurred early at primary infection sites in vaccinated steers while similar host-virus interactions were observed at later time points in non-vaccinated steers. Additionally, numerous CD8⁺/CD3^- host cells, representing presumptive natural killer cells, were observed in association with foci of primary FMDV infection in the nasopharyngeal mucosa of vaccinated steers but were absent in non-vaccinated steers. Immunomicroscopic evidence of an activated antiviral response at primary infection sites of vaccinated cattle was corroborated by a relative induction of interferon -α, -β, -γ and -λ mRNA in micro-dissected samples of nasopharyngeal mucosa. Although vaccination protected cattle from viremia and clinical FMD, there was subclinical infection of epithelial cells of the nasopharyngeal mucosa that could enable shedding and long-term persistence of infectious virus. Additionally, these data indicate different mechanisms within the immediate host response to infection between non-vaccinated and vaccinated cattle.     

口蹄疫病毒与宿主细胞的相互作用研究进展

口蹄疫病毒（FMDV）导致了偶蹄动物口蹄疫的发生,它是一类有着自身特点的RNA病毒。首先,FMDV衣壳蛋白VP1识别结合宿主细胞膜上的整联蛋白等受体,以内吞的方式进入细胞,利用宿主细胞成分完成病毒蛋白的合成。这些新合成的L^pro、2C和3C^pro等病毒致病因子进一步抑制宿主基因的转录和翻译,诱导细胞凋亡和白噬,并抑制干扰素介导的一系列先天性和获得性免疫反应。宿主则在病毒侵染细胞的初期,利用病毒识别受体等来识别病毒并诱导合成干扰素等细胞因子,介导多种免疫反应以清除病毒。病毒和宿主两者在持续的利用和较量中完成疾病的发生和痊愈等。其次,不断发现的病毒受体、结合基序、致病因子及宿主细胞的多种免疫调节因子将成为相关领域新的研究内容。综上,开发高效安全疫苗、增强自身免疫力及利用RNAi直接抑制病毒RNA等便成为现代FMDV防治的主要内容。     

A Safe Foot-and-Mouth Disease Vaccine Platform with Two Negative Markers for Differentiating Infected from Vaccinated Animals

Vaccination of domestic animals with chemically inactivated foot-and-mouth disease virus (FMDV) is widely practiced to control FMD. Currently, FMD vaccine manufacturing requires the growth of large volumes of virulent FMDV in biocontainment-level facilities. Here, two marker FMDV vaccine candidates (A₂₄LL3D_YR and A₂₄LL3B_PVKV3D_YR) featuring the deletion of the leader coding region (L^pro) and one of the 3B proteins were constructed and evaluated. These vaccine candidates also contain either one or two sets of mutations to create negative antigenic markers in the 3D polymerase (3D^pol) and 3B nonstructural proteins. Two mutations in 3D^pol, H₂₇Y and N₃₁R, as well as RQKP_9-12→PVKV substitutions, in 3B₂ abolish reactivity with monoclonal antibodies targeting the respective sequences in 3D^pol and 3B. Infectious cDNA clones encoding the marker viruses also contain unique restriction endonuclease sites flanking the capsid-coding region that allow for easy derivation of custom designed vaccine candidates. In contrast to the parental A₂₄WT virus, single A₂₄LL3D_YR and double A₂₄LL3B_PVKV3D_YR mutant viruses were markedly attenuated upon inoculation of cattle using the natural aerosol or direct tongue inoculation. Likewise, pigs inoculated with live A₂₄LL3D_YR virus in the heel bulbs showed no clinical signs of disease, no fever, and no FMD transmission to in-contact animals. Immunization of cattle with chemically inactivated A₂₄LL3D_YR and A₂₄LL3B_PVKV3D_YR vaccines provided 100% protection from challenge with parental wild-type virus. These attenuated, antigenically marked viruses provide a safe alternative to virulent strains for FMD vaccine manufacturing. In addition, a competitive enzyme-linked immunosorbent assay targeted to the negative markers provides a suitable companion test for differentiating infected from vaccinated animals.     

Advances in novel vaccines for foot and mouth disease: focus on recombinant empty capsids

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.     

Development of a Foot-and-Mouth Disease Virus Serotype A Empty Capsid Subunit Vaccine Using Silkworm (Bombyx mori) Pupae

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae (Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID(50)). Furthermore, the 50% bovine protective dose (PD(50)) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD(50) per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases.     

口蹄疫新型疫苗及其免疫特性研究进展

口蹄疫是世界性重大动物疫病,接种疫苗是预防该病的重要策略之一。随着现代分子生物学技术的发展,对一些新型口蹄疫疫苗如亚单位疫苗、活载体疫苗、核酸疫苗、可饲疫苗、多表位疫苗等的研究和探索已全面展开。我们简要介绍了近年涌现的口蹄疫新型疫苗,以为口蹄疫分子疫苗的设计提供参考。