牛病毒性腹泻病毒对新西兰白兔致病性分析及E2蛋白免疫效果的评价

【目的】 研究牛病毒性腹泻病毒（BVDV）感染对新西兰白兔的致病性以及BVDV E2重组蛋白的免疫效果。【方法】 将实验室培养保存的BVDV病毒纯化并按照Reed-Muench法测定其病毒滴度。在致病性试验中,将10只新西兰白兔随机分为感染组和对照组,每组5只。感染组用1 mL纯化的BVDV病毒攻毒（滴鼻500 μL、耳缘静脉注射500 μL）,对照组用等体积的生理盐水处理,连续3 d,每天1次,每天观察各组兔的临床症状并测量体温;分别于接种病毒后第6、9、12、15、17天通过耳缘静脉采集血液检测血常规;感染病毒第17天采集鼻拭子进行RT-PCR鉴定,采集后剖杀并采集气管、肺脏、脾脏和小肠组织,制备病理切片观察病理变化。在免疫效果评价试验中,将10只新西兰白兔随机分为免疫组和对照组,每组5只,免疫组用E2重组蛋白（1 mg/只）与佐剂混合后经肌内多点注射免疫新西兰白兔,对照组接种等体积生理盐水;共免疫2次,2次免疫间隔为14 d。在一免后0、7、14、21、28 d采集血清,通过间接ELISA方法检测血清中抗重组蛋白特异性抗体水平;在一免后第28天按致病性试验中方法攻毒,在攻毒第17天采集鼻拭子进行RT-PCR鉴定,采集气管、肺脏、脾脏和小肠组织制备病理切片观察病理变化及免疫组织化学检测。【结果】 纯化后BVDV的病毒滴度为4.16×10⁶ TCID₅₀/mL。与对照组相比,感染组部分新西兰白兔6 d内活动减少,采食略微减少,6 d后逐渐恢复正常,在感染第13天出现腹泻症状,从第5天开始体温略微升高,但均在正常范围内波动。与对照组相比,在攻毒第6和9天,感染组白细胞和血小板分别显著和极显著降低（P<0.05;P<0.01）;在攻毒第12、15和17天,感染组白细胞、血小板和淋巴细胞均极显著降低（P<0.01）。鼻拭子RT-PCR检测为阳性,气管、肺脏、脾脏及小肠组织表现出轻度至重度的组织病理学变化。间接ELISA检测结果表明,在一免后7 d时,血清抗体滴度为1：16~1：32;在一免后28 d时,血清抗体滴度为1：256~1：512;免疫攻毒组新西兰白兔鼻拭子经RT-PCR检测为阴性;组织病理学观察显示,免疫攻毒组气管及肺脏表现出轻微的组织病理学变化。免疫组化检测结果显示,免疫组结果均呈阴性,对照组结果均为阳性。【结论】 通过滴鼻及耳缘静脉注射BVDV的方式可以构建新西兰白兔致病模型,BVDV E2亚单位疫苗能够刺激机体产生特异性抗体,起到免疫防御的作用。     

Research Progress on Molecular Biology of Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) is one of the most important pathogenic viruses which mainly causes bovine viral diarrhea disease (BVD). BVDV can not only cause serious clinical symptoms, but also lead to decrease of immunity of livestock and infect other pathogens, resulting in significant increase of morbidity and mortality of sick animals, and causes significant losses to the cattle industry. With the development of molecular biology theory and technology in recent years, the research on BVDV has been deepening, and some new understandings have been made to the molecular biology of the virus. In this paper, the progress of molecular biology of BVDV in recent years is described from three aspects of the composition and function of virus, the epidemic situation of BVDV gene and the genetic and mutation of BVDV gene.     

牛病毒性腹泻病毒一步 RT-LAMP快速检测方法的建立

[目的]建立一种快速、敏感、特异的检测牛病毒性腹泻病毒（BVDV）反转录环介导等温扩增（RT-LAMP）方法,为诊断与监测BVDV 提供准确可靠工具。[方法]根据GenBank公布的BVDV序列,在其保守序列区域设计了一套 LAMP引物,优化了反应时间与反应温度,建立了检测 BVDV的 RT-LAMP方法。[结果]该方法能在56℃等温条件下40分钟完成扩增,扩增结果可通过凝胶电泳梯形带判定。该方法具有良好的特异性和敏感性,最低能检测到3.74×100 copies/μl的病毒 RNA,而且与其他病毒无交叉反应。[结论]该方法能用于牛病毒性腹泻病毒的诊断与监测。     

牛病毒性腹泻病毒、牛呼吸道合胞体病毒和牛副流感病毒3型三重RT-PCR检测方法的建立及应用

根椐GenBank中牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)和牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV-3)3种病毒基因序列,设计合成引物,建立3种病毒的三重RT-PCR方法。用这3对引物对同一样品中的BVDV、BRSV和BPIV-3核酸模板进行三重RT-PCR扩增,结果显示:可同时扩增BVDV的466 bp,BRSV的735 bp和BPIV-3的258 bp的特异性片段,而对其他4种病原的PCR扩增结果均为阴性;敏感性测定结果表明,该三重RT-PCR技术能检出10 pg的BVDV、1 pg的BPIV-3和10 pg的BRSV模板。用37份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示:两者的总符合率为100%。结果表明:建立的多重RT-PCR检测方法,具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。     

牛病毒性腹泻病毒弱毒活疫苗免疫持续期的研究

为检测牛病毒性腹泻病毒(BVDV)弱毒活疫苗在免疫牛体内抗体产生及其消长规律,评价弱毒疫苗的保护效力,并确定免疫持续期,本试验对免疫试验牛每头颈部肌肉接种BVDV SM株弱毒疫苗104.5TCID50,监测血清抗体效价,进行免疫持续期的确定。在疫苗免疫后的6个月、9个月和12个月,分别抽取5头免疫组和5头对照组牛,采用BVDV-JL强毒株进行攻毒试验,每头牛攻毒剂量为6×107.0TCID50/mL。结果显示疫苗免疫后12个月时血清中和抗体效价仍维持在1∶1048以上。攻毒结果显示,在3个不同时间点进行强毒攻击后,免疫组所有动物白细胞数量都没有下降也没有分离到病毒,而对照组动物白细胞数下降均超过30%,6个月和9个月时动物血清中均能分离到病毒,而12个月对照组动物由于年龄大,没有分离到病毒,因此暂定此疫苗的免疫持续期为9个月。     

河南牛病毒性腹泻-黏膜病流行病学调查

为探讨河南省牛病毒性腹泻病毒的流行情况,用牛病毒性腹泻病毒抗体检测试剂盒(IDEXX)对本省部分地区6个奶牛场、5个肉牛养殖场的181份牛奶样品和395份血清样品进行BVD抗体的检测.结果检出阳性牛奶106份,阳性率最高为93.3％,最低的为5.0％,平均阳性率为58.6％.其中有5个规模化奶牛场牛奶样品阳性率均在75％以上.肉牛血清样品共检出阳性124份,最高阳性率为74.12％,最低5.80％,平均阳性率31.39％.结果表明,该病在所调查地区中奶牛和肉牛均有感染,不同牛场间感染率差异较大,其中规模化奶牛的感染率较高.表明应采取相应的净化措施对该病进行控制.     

Genotypic characterization of Chilean llama (Lama glama) and alpaca (Vicugna pacos) pestivirus isolates

Llamas and alpacas are domesticated South American camelids (SACs) important to ancestral population in the Altiplano region, and to different communities worldwide where they have been introduced. These ungulates have shown to be susceptible to several livestock viral pathogens such as members of the Pestivirus genus, in particular Bovine Viral Diarrhea (BVDV), but there is little data available on Pestivirus infections in SACs. In this study we aimed to detect and identify Pestivirus genotypes and subgroups infecting SACs in both wild and confined environments. Samples were collected from 136 llamas and 30 alpacas from different areas in the Chilean Altiplano (wild animals), and from 22 llamas and 26 alpacas diagnosed as Pestivirus positive from the Metropolitana region in Chile (confined animals). Seroneutralization tests showed titers lower than 2 in all 166 samples from Chilean Altiplano. These samples were also negative to BVDV isolation, indicating that these animals have not been exposed to Pestivirus. After reactivation of positive samples from the Metropolitana region, the 5′ non-codifying region (5′NCR) and E2 glycoprotein were amplified by RT-PCR from the Pestivirus genome. Viral sequences were pairwise compared and phylogenetic trees were constructed. The 5′NCR analysis showed that all 12 sequenced isolates belonged to BVDV-1. Of particular interest, isolates from eight llama and two alpaca were BVDV-1j and two alpacas were BVDV-1b. In agreement with these results, E2 phylogenetic analysis rendered a similar grouping indicating that all 16 isolates belong to BVDV-1. However, the lower availability of E2 sequences determines the creation of a smaller number of sub-groups than the 5′NCR sequences. Based on the E2 sequences, the 5′NCR BVDV 1j group consisting of all the llamas and 3 alpacas are completely included in the E2 BVDV 1e group. Due to the universal availability of the 5′NCR segment, we propose the classification of these Chilean llamas and alpacas Pestivirus isolates as BVDV 1j and BVDV 1b respectively. Thus, this is the first time BVDV-1j is obtained in SACs. In addition, these results indicate Pestivirus infection in llamas and alpacas is associated with bovine population as genotypes and sub-groups are the same as those affecting Chilean livestock.     

The use of a mass-action model to validate the output from a stochastic simulation model of bovine viral diarrhoea virus spread in a closed dairy herd

The spread of bovine virus diarrhoea virus (BVDV) in a closed dairy herd maintained under typical management conditions is studied using two approaches. In the first instance a stochastic computer model is used to simulate the month-to-month changes in the infection status of each animal. These results are contrasted with the results of a mass-action model which uses three differential equations. A comparison of the two approaches indicates that the results are in broad agreement. The stochastic approach has the benefit of providing an estimate of the probability of the infection becoming extinct and the herd becoming BVDV-free for different herd sizes.     

从幼鹿顽固性腹泻病料中检出牛病毒性腹泻——粘膜病病毒

应用双抗体夹心ＥＬＩＳＡ对长春、前郭、伊通、双阳等地区送检的２８份幼鹿顽固性腹泻病料进行了牛病毒性腹泻病毒（ＢＶＤＶ）检测。应用电镜对部分ＥＬＩＳＡ阳性和阴性样品进行负染观察,结果６份阳性样品中均见有ＢＶＤＶ粒子,４份阳性样品未观察到ＢＶＤＶ粒子。结果表明,ＢＶＤＶ可能是引起幼鹿顽固性腹泻的重要病因。     

牛病毒性腹泻病毒研究进展

牛病毒性腹泻病毒是黄病毒科瘟病毒属的代表种,主要感染牛并引发疾病,造成严重的经济损失.自该病毒发现至今,已有60余年研究历史,随着生物学理论及技术不断发展,特别是反向遗传技术以及蛋白组学在病毒研究领域的应用,人们对该病毒产生了一些新的认识.论文从牛病毒性腹泻病毒的病毒粒子结构组成及功能、病毒复制周期及蛋白裂解过程、细胞损伤及免疫逃逸机制三个方面阐述了近几年牛病毒性腹泻病毒分子生物学研究的进展,重点讨论了病毒复制周期、多聚蛋白裂解、致细胞病变效应和免疫逃逸机制.