Vesicular stomatitis.

Vesicular stomatitis is an infrequent yet important vesicular disease of cattle, horses, and swine. Periodic outbreaks of this disease in the United States have caused economic losses in cattle herds because of decreased production, movement restrictions, and trade embargoes. Vesicular stomatitis causes clinical signs indistinguishable from those of foot-and-mouth disease. It is of utmost importance that appropriate samples are collected from clinical cases of vesicular disease in cattle and swine so a rapid laboratory diagnosis can be made.     

Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.     

水疱性口炎研究进展

水疱性口炎(VS)是由水疱性口炎病毒(VSV)引起的人畜共患的重大动物疫病.VS病毒生态学复杂,易感动物较多,传播媒介种类广,病毒可在一定区域内长期存在.VSV主要呈嗜上皮性,大量流涎是家畜感染最重要的临床症状,其特征为口腔黏膜、乳房皮肤及蹄冠部皮肤出现水泡及糜烂,人感染后出现类似流感的症状.由于其临床症状与口蹄疫不易区别,发病时容易引起国际贸易恐慌,因此,对VS诊断防治的研究有着重要的社会经济和公共卫生意义.文章从分子生物学特征、流行病学、诊断和防控4个方面对水疱性口炎的研究进行了综述.     

TaqMan~T-PCR对水疱性口炎病毒的鉴定检测

水疱性口炎分为印第安那型 ( Indiana)和新泽西型 ( New Jersey)。这两种血清型病毒的快速和可靠的鉴别对该病的诊断、检疫、分子流行病学调查和监测至关重要。文章按照 VSV核蛋白基因序列 ,设计了一对两型通用引物和两型各自特异性探针。研究建立了 VSV实时荧光定量 PCR检测方法 ,对 VSV细胞培养物、人工感染实验动物组织、血清样品 ,以及系列稀释的不同 TCID50 样品、其它相关或相似病毒进行鉴定 ,同时与常规 PCR、病毒分离试验作了比较。Taq Man○R RT-PCR的特异性和敏感性相当于或优于对照方法。重复性和稳定性试验证实 ,该方法可靠。每个试验中设立阳性、阴性对照和标准稀释度对照 ,使试验结果可对病毒 RNA作准确定量 ,并可在 4h内获得结果。研究结果表明 ,Taq Man○R RT-PCR方法是一种特异性强、敏感性高、快速安全的定量检测方法。因此 ,可以作为 VSV的快速检测和定型。     

Molecular weapons against agricultural vulnerability and the war on terror

The multiple reports in this issue of the Journal from the Agenda for Action conference, coupled with the analysis by the National Academy of Sciences, the National Research Council, and the Auditor General (UK) on bioterror preparedness and homeland security, highlight the immediate need for rapid disease detection and advanced diagnostic capabilities to protect the public health, animal agriculture, and the numerous associated economies in the United States. In response to the potentially devastating consequences that could arise, there is an acute need for rapid detection of a variety of the lethal foreign animal diseases, such as foot-and-mouth disease virus (FMDV), highly pathogenic strains of avian influenza, classical swine fever, rinderpest, exotic Newcastle disease virus (END), and domestic, vesicular look-alike diseases that include bluetongue, epizootic hemorrhagic disease, vesicular stomatitis, bovine herpes IBR, contagious ecthyma, bovine herpes mammilitis virus, vesicular exanthema, malignant catarrhal fever, and papular stomatitis. Some striking advances are occurring in the creation of rapid technology, including microfluidics, robotics, miniaturization, and biostabilization that are quickly being applied to the development of rapid microbial detection assays. These are now providing important weapons to combat this agricultural vulnerability.     

A型塞尼卡病毒检测方法及疫苗研究进展

A型塞尼卡病毒（Senecavirus A,SVA）也称为塞尼卡谷病毒（Seneca Valley virus,SVV）,属于小RNA病毒科塞尼卡病毒属成员。SVA主要引起猪的水泡性疾病,与口蹄疫、水泡性口炎、猪水泡病等临床症状相似,可导致新生仔猪急性死亡,严重影响养猪业发展。自2015年广东省发生SVA感染以来,中国多省份陆续有该病发生的报道。当前,中国因猪群缺乏针对SVA的免疫屏障,加之该病传染性较强,存在大范围暴发的潜在风险。如何有效防控SVA感染是迫切需要解决的问题。目前已开发出多种SVA诊断方法用于进行实验室及现场条件下早期的鉴别诊断。SVA的分离鉴定、原位杂交和免疫组化可用于检测病原体的存在及其与组织内形态学变化关系;血清学诊断方法包括基于不同结构蛋白的间接ELISA方法、竞争ELISA方法、均相光激化学发光免疫技术和病毒中和试验,用免疫学方法检测抗体有助于了解SVA感染进程,是临床诊断的主要手段;病毒核酸检测方法主要有PCR技术、等温扩增技术、基因组测序等分子生物学技术,在病毒感染的早期快速检测及检测新发病毒中具有重要作用。目前仍无商品化疫苗预防SVA感染,但科研人员已研发出了灭活疫苗、弱毒疫苗、核酸疫苗和亚单位疫苗等多种具有潜力的候选疫苗。笔者系统总结了SVA检测方法及疫苗研发的最新进展,以期为SVA感染的防控提供参考依据。     

猪水疱性口炎病毒基因及其疫苗的研究进展

猪水疱性口炎是由水疱性口炎病毒引起的高度接触传染性的病毒性疾病。其临床特征为猪的唇部,鼻及口腔等处发生水疱,并从口中不断向外流涎,有时常常还发生在蹄冠和趾间皮肤上,其症状主要以水疱为主。该病在全球许多地区造成广泛流行。近年来,由于产品贸易量的增加,猪水疱性口炎病毒也陆续的传入我国。由于该病与猪水疱病、猪口蹄疫和猪水疱性疹等病毒性疾病容易混淆,因此对该病做出准确地诊断与防制显得尤为重要。在VSV疫苗的研究方面,主要是灭活疫苗和弱毒疫苗的研究,而在新型疫苗的研究方面很少。本文主要综述了猪水疱性口炎病毒的基因及其疫苗的研究进展,为进一步了解和预防该病提供参考依据。     

Trypsin treatment of bovine ova after in vitro exposure to vesicular stomatitis virus

Preimplantation bovine ova were exposed in vitro to vesicular stomatitis virus, Indiana serotype, to document adherence of the virus to the zona pellucida. To determine the efficacy of this treatment, some of the ova were treated with trypsin after exposure to the virus. Vesicular stomatitis virus was isolated from 5 of 10 groups of zona pellucida-intact ova after 12 sequential washes without trypsin treatment. Vesicular stomatitis virus was also isolated from 4 of 11 groups of zona pellucida-intact ova after trypsin treatment.     

应用RT-PCR方法快速检测水泡性口炎病毒

针对水泡性口炎病毒(VSV)的两种血清型设计了2对引物，建立RT-PCR方法，用于检测VSV。VSV接种细胞出现明显的细胞病变，经RT-PCR检测为阳性，而检测口蹄疫、猪水泡病均为阴性，说明引物具有较好的特异性。     

多重PCR同时检测口蹄疫病毒、猪水疱病病毒和水疱性口炎病毒

根据基因库中的口蹄疫病毒（FM—DV）,猪水疱病病毒（SVDV）和水疱性口炎病毒（VSV）各基因序列,设计了与FMDV,SVDV和VSV互补的3对特异性引物,对样品中的cDNA模板进行了多重PCR扩增及反应条件的优化,结果同时得到与设计相符合的3条特异性条带,分别为189bp,125bp和300bp。用这3对引物对病毒样品cDNA模板进行多次扩增,均能稳定得到与设计相符合的3条特异性条带。本试验能特异、敏感、快速地鉴定FMDV,SVDV和VSV。     

The molecular diagnosis of porcine viral diseases: a review

The worldwide occurrence and re-occurrence of transboundary diseases like foot-and-mouth disease or classical swine fever indicates that there is a high need for the development of powerful, robust and high-capacity new diagnostic methods, which are able to detect the causative agents before they could spread to large populations and cause tremendous losses. This article reports the experiences of a research group on the development of molecular methods for the improved diagnosis of a range of porcine viral diseases, including diseases on List A of the Office International des Epizooties (OIE). Nucleic acid hybridisation and various polymerase chain reaction (PCR) assays have been applied for routine diagnosis of a large range of viral diseases. During the last one-and-a-half decade more than 40 nested PCR assays have been developed to detect a variety of DNA and RNA viruses. False positive and negative results are avoided by the use of special tools, practices and internal controls of amplification (mimics). Recently, real-time PCR methods (TaqMan, molecular beacons, Primer-Probe Energy Transfer system) have been developed for the diagnosis of a wide range of diseases, such as foot-and-mouth disease, swine vesicular disease and vesicular stomatitis. Multiplex PCR packages have been developed for the simultaneous detection of eight important viruses of swine. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the diagnostic procedures have become rapid, robust and automated. In order to standardise the real-time PCR assays, the rules of OIE are considered. By following the five steps of OIE standardisation and validation, the new diagnostic procedures are nationally and internationally standardised and harmonised. The rapid, powerful and internationally standardised molecular diagnosis contributes to the reduction of losses caused by the transboundary viral diseases in swine populations.     

Viral infections and bovine mastitis: a review

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.     

口蹄疫病毒感染细胞的研究进展

口蹄疫是引起偶蹄动物的急性发热性水泡性疾病,具有高度接触传染性,对发病国家和地区经济有破坏性作用和不良的政治影响。口蹄疫病原体为小RNA病毒科的口蹄疫病毒,是一类单股正链RNA病毒,该病毒有多种血清型及其亚型,相互之间无交叉保护力或保护力极其有限。目前,口蹄疫病毒感染的分子机理还不是很清楚。口蹄疫病毒感染细胞的过程主要包括病毒与细胞的吸附、病毒穿透细胞壁进入细胞、病毒粒子的脱衣壳、病毒RNA的翻译转录、病毒基因组的复制以及病毒粒子的成熟过程,最后是成熟的病毒粒子衣壳包装成为完整病毒。文章就口蹄疫病毒感染细胞的过程做一概述。     

水疱性口炎病毒RT-LAMP快速检测方法的研究

根据逆转录环介导等温扩增(RT-LAMP)原理,针对水疱性口炎病毒(VSV)糖蛋白G基因序列中的6个区域设计内外各1对特异引物,建立扩增VSV糖蛋白(G)基因的RT-LAMP方法。扩增产物电泳呈特异的阶梯状条带分布,扩增产物加SYBR GREEN I染色呈特征性的黄绿色,肉眼可直接观察判定。同时,还建立了可以进行定量检测的实时RT-LAMP方法。特异性试验显示,本方法可快速检验鉴别VSV与口蹄疫病毒(FMDV)和猪水泡病病毒(SVDV)。敏感性试验显示,建立的实时RT-LAMP方法检测VSVRNA的最低检测量为0.01 PFU,比实时荧光RT-PCR显著提高。建立的LAMP方法可检测p-VSVNJ质粒DNA的最低量为6.36×10-3pg/μL(1.4×103copies/μL),比PCR也显著提高。综合表明,本研究建立RT-LAMP检测VSV的方法具有特异、敏感、快速、简便的特点,具有开发应用前景。     

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。     

Vesicular Diseases: Recent Advances and Concepts of Control

Most of this review is devoted to foot-and-mouth disease, considering only briefly vesicular stomatitis and vesicular exanthema of swine.     

Herpetiform genital lesions in a heifer with mucosal disease

A 14-month-old heifer with a 17-day history of unresponsive bloody diarrhea was necropsied. There were focal, pink-red erosions of the nares and hard palate; ulcers and fissures of the tongue; and multiple ulcerative lesions of the alimentary canal. Interdigital skin of both rear limbs was ulcerated and bleeding; and the margins of the vulva contained punctiform red ulcers. The gross lesions were consistent with mucosal disease. Histopathology and laboratory testing ruled out rinderpest, foot-and-mouth disease, and vesicular stomatitis, and identified bovine virus diarrhea virus to be the cause of this disease. Lesions of the vulva similar to those seen in some stages of infectious pustular vulvovaginitis were negative for bovine herpesvirus-1 and tested positive for bovine viral diarrhea virus antigen by immunohistochemistry.