RNA干扰抑制口蹄疫病毒复制研究进展

鉴于现有疫苗和抗病毒药物在治疗口蹄疫方面存在的局限性,寻找新型抗病毒策略势在必行,而RNA干扰正是一种有益的尝试。RNA干扰通过沉默哺乳动物细胞中的基因表达,可有效的阻断口蹄疫病毒在宿主体内的复制。论文以口蹄疫病毒引起宿主细胞病变、RNA干扰的机制及RNA干扰抑制FMDV在宿主细胞内复制为内容,探讨如何更加有效地防控口蹄疫。     

Interactions of Bovine Viral Diarrhoea Virus and Foot-and-mouth Disease Virus with Bovine Reproductive Tissues and Embryos

Contents This review will focus upon the interactions of two important viral pathogens in cattle, bovine viral diarrhoea virus (BVDV) and foot-and-mouth disease virus (FMDV), respectively, with bovine reproductive tissues and embryos. Both viruses are widely spread throughout the world and cause serious economic losses due to death, loss of appetite and weight, respiratory disease, reproductive problems (infertility, early embryonic death, abortions) and decreased milk production (Baker, J Am Vet Med Assoc 190, 1449–1458, 1987; Kitching, J Comp Path 118, 89–108, 1998).     

1. Isolation and Characterisation of Equine Rhinopneumontis Virus and Other Equine Herpesviruses from Horses

Equine herpesviruses (EH viruses) were isolated from 9 horses in three separate outbreaks of respiratory disease. The pattern of disease in the three stables is described and evidence is presented that some of the horses were ill, possibly as a result of recurrent infection, and that reactivation of a persistent, latent infection may have occurred. An ulcerative condition of the pharyngeal region was seen in some of the horses with EH virus infection.
The cytopathogenicity for equine foetal kidney cells of the 9 EH viruses varied considerably. One isolate, EH 39 virus, which was recovered from an acute, upper respiratory tract infection, was rapidly cytopathic for equine foetal kidney cell cultures and was shown in neutralisation tests to be identical with, or closely related to equine rhinopneumonitis virus (EH virus type 1) that is associated with acute respiratory disease and abortion in other countries. More slowly cytopathic isolates were recovered from mild to subclinical upper respiratory tract infections. Evidence is presented that the property of slow cytopathogenicity is probably related to the tendency of these viruses to remain cell associated. Slowly cytopathic isolates were recovered from the nasal cavity of horse 89 on two occasions 79 days apart. One of the eight slowly cytopathic isolates, EH 86 virus, was shown to be antigenically distinct from equine rhinopneumonitis virus (EH 39 virus).     

Heterogeneity of Foot-and-mouth Disease Virus: Further Studies on Plaque Formation by Two Plaque-size Variants

Plaque production by a small-plaque (SP) and large-plaque (LP) variant of foot-and-mouth disease virus, type A, strain 119 (FMDV, A119), was influenced by a number of environmental factors. The SP variant produced plaques on cells of the IB-RS-2 cell line from swine kidney and to a lesser degree on primary cultures of swine kidney cells, but plaque formation was inhibited on primary cultures of bovine kidney (BK) cells unless diethylaminoethyl (DEAE) dextran was added to agar overlays. When DEAE dextran-treated agar overlay was used, the LP variant formed larger plaques on BK cells but not on IB-RS-2 cells. Concentrations of DEAE dextran from 0 to 100 µg/ml greatly enhanced the formation of SP virus plaques on BK cells but had little or no effect on the average size of plaques produced by the LP variant. Higher concentrations of polycation enlarged the plaques formed by both variants. Plaque sizes of the SP and LP variants increased as the concentration of agar or agarose in the overlays decreased. Reducing the concentration of agar to 0.75% facilitated the formation of SP virus plaques, but better plaque production occurred under agarose overlays.

The original parent virus consisted predominantly of virus particles that formed small plaques. The rate of neutralization of the parent virus by guinea pig antiserum prepared against the parent virus was faster than antiserum inactivation of a low-passage virus of the same serotype and strain.

     

多重RT-PCR检测猪口蹄疫病毒、猪水泡病病毒和猪水疱性口炎病毒的研究

建立了一种同时检测猪口蹄疫病毒(FMDV)、猪水泡病病毒(SVDV)和猪水疱性口炎病毒(VSV)三种病原体的多重RT-PCR方法。参照文献报道的基因序列,设计合成了三对特异性引物;PCR扩增条件进行优化后,用这三对引物对同一样品中的FMDV、SVDV、VSVRNA模板进行扩增,结果同时得到了三条特异性条带,大小与试验设计相符:FMDV(208bp)、SVDV(862bp)、VSV(638bp),且对猪瘟病毒(CSFV)、猪繁殖与呼吸综合症病毒(PRRSV)和猪传染性胃肠炎病毒(TGEV)核酸扩增结果为阴性;三种病毒RNA模板检出的最小量均为10fg。试验证明,此方法经济、快速、敏感、特异,可用于FMDV、SVDV和VSV这三种猪水泡性疾病的鉴别诊断及流行病学调查。     

口蹄疫病毒抗原胶体金试纸条的研究

利用单克隆抗体技术制备抗口蹄疫病毒的单克隆抗体,特异性试验表明其只与O、A、Asia 1型3种血清型FMDV抗原结合。进而采用胶体金标记技术,以胶体金标记的抗口蹄疫病毒单克隆抗体、多克隆血清抗体和葡萄球菌A蛋白为主要材料,研制口蹄疫快速检测试纸条。该试纸条检测O、A、Asia 1型3种血清型灭活口蹄疫病毒均为阳性,检测水疱性口炎病毒、猪水疱病病毒、蓝舌病病毒、猪蓝耳病病毒4种灭活抗原及小反刍兽疫病毒疫苗株均为阴性,试验结果与口蹄疫实时荧光定量RT-PCR方法的完全一致,表明其具有良好的特异性。敏感性试验结果是,试纸条的检测极限为1∶160稀释的样品,其敏感性相当于实时荧光定量RT-PCR方法的1/64。由于试纸条具有操作方便、检测快速等优点,因此该试纸条可以用于大量临床样品的快速检测和现场检测。     

Susceptibility of Bovine Umbilical Cord Endothelial Cells to Bovine Herpesviruses and Pseudocowpox Virus

The purpose of the study was to determine the susceptibility of bovine umbilical cord endothelial (BUE) cells to bovine herpesvirus (BHV) 1, BHV2, BHV4 and BHV5, and to pseudocowpox virus. The detection limits and growth curves of these viruses in BUE cells were compared with those in Vero, Madin-Darby bovine kidney (MDBK), or bovine fetal diploid lung (BFDL) cells. Detection limits were determined by inoculating cell cultures with serial 10-fold dilutions of these viruses, and growth curves by titration of virus, harvested at various times after infecting cells at a multiplicity of infection of 0.1. The detection limits of BHV2 and BHV4 were lower in BUE cells than in Vero or MDBK cells, and cytopathic effects were observed earlier in BUE cells. In addition, BHV2 and BHV4 grew to higher titres in BUE cells than in Vero or MDBK cells. BUE cells appeared to be equally susceptible to BHV5, but less susceptible to BHV1.1 and BHV1.2 than MDBK cells. The study showed that BUE cells are highly susceptible to BHV2 and BHV4, and that the use of BUE cells can improve the laboratory diagnosis of these viruses. The use of BUE cells could also improve the isolation and growth of pseudocowpox virus.     

Evaluation of cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, and vesicular stomatitis virus in vitro

OBJECTIVE: To evaluate cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against cytopathic and noncytopathic bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), and vesicular stomatitis virus (VSV) in vitro. SAMPLE POPULATION: Primary bovine testicular cells and Mardin Darby bovine kidney cells. PROCEDURES: To evaluate cytotoxicity, cells were added to serial dilutions of each interferon. To evaluate antiviral activity of each interferon, interferons were serially diluted 1:10, and tissue culture cells were added; virus was then added at 3 time points. Prevention of viral infection by interferon was defined as failure to induce cytopathologic effect for VSV, IBRV, and cytopathic BVDV and failure to detect virus immunohistochemically for cytopathic and noncytopathic BVDV. RESULTS: No evidence of cytotoxicity in either cell line was detected after incubation with interferon alfa-2a or interferon alfa-B/D. However, reduced growth rates of tissue culture cells were detected for each interferon when undiluted interferon was tested. Comparable and profound antiviral activities against cytopathic and noncytopathic BVDV were evident for each interferon. Interferon alfa-2a and interferon a-B/D had comparable antiviral activities against VSV. Neither interferon had antiviral activity against IBRV. CONCLUSIONS AND CLINICAL RELEVANCE: The safety and marked in vitro antiviral activity against noncytopathic BVDV, cytopathic BVDV, and VSV suggest that interferons alfa-2a and alfa-B/D may be useful for treatment of natural disease after infection with these viruses.     

Detection of African malignant catarrhal fever virus antigens in cell cultures by immunofluorescence

A fluorescent antibody (FA) test for antigens of African bovine wildebeest-derived malignant catarrhal fever virus was developed. Serum from one of the few survivors of the experimental disease in steers was used to prepare the conjugate. Both a virulent and an attenuated strain of malignant catarrhal fever virus were used to infect bovine thyroid cell cultures. Cells infected with both strains were readily detected by FA staining as early as 24 h post infection, whereas cytopathic effect could be observed by bright-field microscopy only after days 5 or 6 post infection. Controls consisting of normal bovine thyroid cells or infected cells treated with conjugated normal globulins did not show autofluorescence. The reaction was blocked by treatment of infected cells with homologous positive antisera but not by treatment with normal bovine serum or antisera to foot-and-mouth disease, rinderpest, bovine virus diarrhea, Ibaraki, infectious bovine rhinotracheitis, or bovine herpes mammilitis viruses. Treated with African malgnant catarrhal fever virus conjugate did not react.     

Evaluation of techniques to demonstrate foot-and-mouth disease virus in bovine tongue epithelium: comparison of the sensitivity of cattle, mice, primary cell cultures, cryopreserved cell cultures and established cell lines

Tongue epithelia infected with each of the 7 serotypes of foot-and-mouth disease virus (FMDV) were used to evaluate in vivo and in vitro systems for the detection of FMDV. Cattle inoculated by the intradermal route in the tongue (IDL) and suckling mice inoculated intraperitoneally were compared for susceptibility to FMDV with freshly prepared bovine thyroid cell cultures; cultures from cryopreserved bovine thyroid, bone marrow, mammary gland, myocardium, tongue, ovary and kidney cells; cultures from cryopreserved embryonic ovine kidney, newborn ovine kidney, ovine testicle, bone marrow, and chloroid plexus cells; and the continuous porcine kidney cell lines MVPK-1 and S6. The mean titers determined for each serotype in each system were statistically compared. The FMDV titers obtained in freshly prepared bovine thyroid cell cultures and by cattle IDL inoculation were the highest and were statistically indistinguishable. The titers obtained by suckling mouse inoculation were significantly lower than the titers obtained in thyroid cultures for serotypes A, C, Asia 1, and SAT 3. The cattle IDL assay was significantly more sensitive than the mouse assay for serotype A. The cell cultures from the cryopreserved newborn ovine kidney and embryonic ovine kidney were significantly less susceptible to serotype Asia 1 when compared with the fresh bovine thyroid cultures, but not significantly different when compared with the cattle assay for all serotypes. Cryopreservation of bovine thyroid cells directly after trypsinization resulted in the loss of susceptibility to FMDV serotype SAT 2. The other cryopreserved cell culture systems exhibited no or minimal susceptibility to all 7 serotypes, or exhibited considerable inconsistency. The established cell lines MVPK-1 and S6 were not susceptible to serotype A, and were less sensitive to serotype C than other culture systems. Quality control of cell cultures used to evaluate field specimens for FMDV was critical. The cell cultures of cryopreserved ovine kidney cells provided the most practical diagnostic system.     

牛病毒性腹泻-黏膜病病毒分离鉴定及生物学特性研究

为分离鉴定新疆地区牛病毒性腹泻-黏膜病病毒(BVDV)流行毒株,掌握该毒株的生物学特性,本试验在新疆北疆部分地区采集牛病毒性腹泻-黏膜病(BVD/MD)疑似病例的粪便,通过RT-PCR检测、细胞分离培养、间接免疫荧光抗体检测、免疫电镜观察及血清中和试验5种方法对毒株进行分离和鉴定。对毒株的TCID₅₀测定后,再对毒株分别进行乙醚敏感性试验、氯仿敏感性试验、胰蛋白酶敏感性试验、酸碱度敏感性试验、温度敏感性试验及核酸分型试验等理化特性检测。经RT-PCR诊断,病料在286 bp处出现了目的片段。将RT-PCR诊断为阳性的粪便,接种于密度约为80%的单层MDBK细胞出现了细胞病变,盲传5代至出现典型的细胞病变。将F5代细胞培养物采用间接免疫荧光抗体检测,结果产生了与C₂₄V标准毒株相同的特异性黄绿色荧光。免疫电镜观察到了大量呈球形的BVDV粒子,大小20~40 nm。血清中和试验中抗体阳性血清处理组细胞均未出现细胞病变,病毒完全被抗体阳性血清中和。综合以上方法确定分离株为BVDV毒株。对分离株进行毒价和理化特性测定,该毒株TCID₅₀为10^-4.5/0.1 mL,对乙醚和氯仿敏感,对胰蛋白酶敏感,耐碱不耐酸,对温度敏感,经54 ℃ 1 h完全被灭活,属于RNA病毒。本试验成功分离到一株新疆BVDV流行毒株,掌握了该毒株的生物学特性,为今后该病的诊断和防控奠定了基础。     

新城疫、禽流感病毒同胚接种的研究

根据新城疫病毒、禽流感病毒两种病毒均能在鸡胚中繁殖的特点,将两种病毒以最适稀释比例稀释后,分别接种于9—11日龄的鸡胚尿囊腔,同时进行两种病毒同胚培养,并利用血凝试验进行病毒效价的测定。结果表明,在同一鸡胚中两种病毒均能较好地增殖,病毒间干扰现象不明显。本试验在增强疫苗的免疫原性和进一步阐明病毒的增殖机理方面具有重要意义。     

口蹄疫病毒非结构蛋白基因3ABC在大肠杆菌中的高效表达

将牛源O型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)China／99株非结构蛋白基因3ABC从pGEM-T-3ABC重组质粒中亚克隆到原核表达载体pTriEx-4Neo上,成功构建重组表达质粒pTriEx-3ABC。将其转化大肠杆菌Rosetta(DE3)感受态细胞中表达,表达产物经SDS-PAGE可检测到分子量约为59．1ku的目的蛋白带,经薄层扫描分析,目的蛋白占菌体总蛋白的31．4％。Western blotting分析证明表达产物能被FMDv阳性血清所识别。表达产物纯化后作抗原进行ELISA检测,结果表明,表达的蛋白显示特异的免疫学活性,并能区分自然感染动物和注苗动物。研究为以FMDV非结构蛋白为抗原,建立自然感染动物和注苗动物的鉴别诊断方法提供了材料。     

利用RNAi抑制口蹄疫病毒的复制

根据口蹄疫病毒 IRES和 L 串联序列两侧的保守区域设计了 2个引物 ,利用 RT- PCR和 PCR方法扩增出该串联序列 ,并进行了测序。测序结果表明 ,扩增产物与 Gen Bank上相应的序列具有很高的序列同源性 (大于 99% )。在测序的基础上 ,选择了 L 基因上的 1个靶位点 (位于启始密码子下游第 2 2 9nt后 2 1 nt长的序列 ) ,合成了 si RNA表达盒SEC- L2 2 9。细胞单层长成 5 0 %～ 70 %时 ,将纯化的 SEC- L2 2 9转染到 BHK细胞中 ,转染 4 h后用高感染复数 FMDV接种 ,2 4 h后用间接免疫荧光方法对口蹄疫病毒在 BHK细胞中的复制进行检测。研究结果表明 ,SEC- L 2 2 9极大地抑制了口蹄疫病毒在 BHK细胞中的复制 ,且该抑制作用具有序列特异性 ,并降低了 BHK细胞的死亡率。另外 ,2 5 ng和5 0 ng SEC- L2 2 9处理组间对病毒复制的抑制作用差异不明显 ,可能是病毒基因组发生了突变。本试验表明 ,利用 PCR方法合成的 SEC在 BHK细胞中能特异性地抑制 FMDV的复制 ,RNAi技术可能为防治口蹄疫提供一个新的途径     

Bovine viral diarrhea virus: biotypes and disease.

Bovine viral diarrhea virus continues to produce significant economic losses for the cattle industry and challenges investigators with the complexity of diseases it produces and the mechanisms by which it causes disease. This paper updates and attempts to clarify information regarding the roles of noncytopathic and cytopathic bovine viral diarrhea viruses in persistent infections and mucosal disease. It also covers, in brief, what is known of the new diseases: thrombocytopenia and hemorrhagic disease, and a disease resembling mucosal disease that is apparently caused solely by noncytopathic virus. Although a good understanding of the roles of the 2 biotypes in the production of persistent infections and the precipitation of mucosal disease has been obtained, there are still unanswered questions regarding the origin of cytopathic viruses and the mechanism by which they cause pathological changes in cells. It is apparent, however, that cytopathic bovine viral diarrhea viruses arise by mutation of noncytopathic viruses, and it is known that p80 is the marker protein for cytopathic viruses. The previous distinction between mild bovine viral diarrhea and fatal mucosal disease has been eroded with the emergence of new virulent bovine viral diarrhea viruses. The new diseases pose a threat to the cattle industry and present a new challenge for investigators. Index Veterinarius (1984-1994) and Medline (1985-1994) databases and personal files updated since 1987 from BIOSIS Previews and Biosciences Information Services were used to search the literature.     

ELISpot技术检测猪抗口蹄疫γ干扰素方法的建立

猪口蹄疫免疫防御是控制猪口蹄疫疫情的重要手段,免疫水平指标通常用免疫抗体水平判定。对于其细胞免疫水平检测尚缺乏成熟可靠的技术,本试验利用市售ELISpot试剂盒建立猪口蹄疫特异γ干扰素检测方法,刺激物分别采用疫苗全病毒颗粒（O/Mya98/XJ/2010）、口蹄疫病毒T细胞表位多肽池,以植物血凝素（PHA）为阳性对照,对细胞浓度、刺激物浓度、孵育时间等进行优化。优化条件为：外周血PBMC新鲜提取或冻存细胞成活率90%以上,最佳细胞数为2×10⁵个/孔,最佳反应时间是16 h,病毒粒子146 S含量为100 ng/mL,多肽最佳浓度10 μg/mL,PHA的最佳浓度是20 μg/mL。ELISpot技术检测口蹄疫病毒感染猪γ干扰素方法的建立,为口蹄疫免疫力评价及口蹄疫疫苗免疫效果评估及进一步研究其与疫苗保护力（PD₅₀）的相关性奠定基础。     

Propagation and quantitation of animal herpesviruses in eight cell culture systems

A comparative study was carried out to determine the relative sensitivities of eight different cell culture systems to six different herpesviruses of animals. The cells used were: OFL (ovine fetal lung), ML (mink lung), FK (ferret kidney), PTK-2 (potoroo kidney), TEK (turkey embryo kidney), ED (equine dermal), BT (bovine turbinate), and PK15 (porcine kidney). The viruses tested were: PRV (pseudorabies) of swine, CPHV (caprine herpesvirus), IBRV (infectious bovine rhinotracheitis virus), DN-599 strain of bovine herpesvirus type 4, EHV-1 (equine herpesvirus), and CHV (canine herpesvirus). On the basis of virus titers obtained and the time of appearance of CPE (cytopathic effects), ML cells were found to be the most useful because of their sensitivity to all six viruses tested. BT and OFL cells were also found to be highly sensitive to all viruses with the exception of CHV.     

Development of a Cell Culture Method for Quantal Assay of Strain I-2 of Newcastle Disease Virus

Repeated titrations of strains of Newcastle disease virus (NDV) are more conveniently undertaken in cell cultures rather than in embryonated eggs. This is relatively easy with mesogenic and velogenic strains that are cytopathic to various cell lines, but is difficult with avirulent Australian isolates that are poorly cytopathic. Strain V4 for example has been shown to be pathogenic iin vitro only to of chicken embryo liver cells. Strain I-2 was reported to produce cytopathic effect (CPE) on chicken embryo kidney (CEK) cells. The present studies confirmed this observation and developed a quantal assay. CEK cells infected with strain I-2 developed CPE characterized by degeneration, rounding, granularity and vacuolation, and the formation of synctia. End points were readily established by microscopic examination of fixed and stained cells. In virus infectivity studies on strain I-2, where multiple titrations are required and where large numbers of samples are used, titration using CEK cell grown in microtitre plates is recommended. Such studies may not be feasible in embryonated eggs.     

Advance on Foot-and-mouth Disease Virus

Foot-and-mouth disease (FMD) is an acute,febrile and highly contagious animal disease caused by foot-and-mouth disease virus (FMDV),and has been recognized as the most important constraint to international trade in animals and animal products.An outstanding feature for FMDV infection is that the FMDV infected animals may remain as a carrier state,some of the animals exposed to FMDV may have a long term asymptomatic infection.This article will review the advance of FMDV in the following aspects,epidemiology,etiology and pathogenesis.     

口蹄疫病毒研究进展

口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)引起的一种急性、热性、高度接触传染性动物疫病,是全球范围内家畜及其产品贸易最大的羁绊。FMDV通过逃避宿主的免疫监视建立持续性感染,使患畜持续向外界排毒,成为传染源。作者查阅了近几年FMDV的国内外研究进展,对其流行病学、病原学及致病机理进行了概述。