河南省牛病毒性腹泻病毒地方株的分离及鉴定

从河南省不同地区规模化肉牛场牛病毒性腹泻(BVD)疑似病例中采集病料,将处理好的6份病料接种MDBK细胞,并盲传4代,得到了两株可产生细胞病变的病毒,经测定该两株病毒的TCID50分别为10-6.59/0.1ml和10-6.51/0.1ml;琼脂扩散试验表明该两株病毒均能与牛病毒性腹泻病毒(BVDV)OregonC24标准阳性血清反应,出现沉淀线,且均能被BVDV标准阳性血清中和;电镜观察到圆形、有囊膜、直径为40～60nm、囊膜表面有突起的病毒粒子,与BVDV颗粒基本一致。动物回归试验表明,用两株分离毒攻毒的2头3月龄犊牛均出现了和BVD自然病例相似的临床症状,并检测其抗原和抗体,结果均为阳性,从而确定分离的两株病毒均为BVDV,分别将其命名为HN-1和HN-2株。     

绵羊BVDV的细胞培养与鉴定初报

<正> 牛病毒性腹泻—粘膜病(BVD—MD)病毒(简称BVDV)是国外牛群中常见的一种病原体,在我国亦有检出,但引致羔羊腹泻尚少报道。本文则是从腹泻羔羊所获的BVDV作细胞培养及其荧光抗体鉴定的报告。     

一株牛病毒性腹泻/黏膜病强毒株的分离与鉴定

从云南某牛场疑似BVDV感染的病料通过接种MDBK细胞分离到1株BVDV,将其命名为BVDV/W。该分离毒株连传15代均不产生CPE,为NCP型BVDV。通过电镜检测可观察到直径为 40～60nm病毒粒子。该分离病毒能被牛病毒性腹泻标准阳性血清中和,且能被BVDV IFA荧光抗体识别;针对常用作BVDV基因分型的5′-UTR设计特异性引物,经RT-PCR可扩增出288bp特异性片段。将所测的目的片段序列与中参考序列进行同源性比较,结果显示与293株和Y2株同源关系较近,分别为90.0%和89.9%,与2014年以来我国报道的分离株同源性在88%左右,具有一定的代表性。5′-UTR遗传进化分析证实该分离毒株为BVDV-1型。动物回归试验显示,该分离毒株可引起出现体温升高、腹泻、粘膜病等典型的BVD/MD症状,表明该毒株为1株BVD强毒株。     

牛睾丸原代细胞在牛病毒性腹泻/粘膜病中和试验上的应用

牛病毒性腹泻/粘膜病(BVD—MD),是我国进口检疫应检疫病之一。常用的实验室诊断方法有中和试验和血清病毒分离。由于犊牛睾丸原代细胞(BT细胞)对BVD—MD病毒最敏感,常用于病毒分离。而在检测中和抗体效价时,人们常用牛肾次代细     

牛病毒性腹泻的研究概况

牛病毒性腹泻 /粘膜病 ( Bovine viral diarrhea/mucosal disease,BVD/MD) ,简称牛病毒性腹泻( BVD)或牛粘膜病 ( BMD) ,是由牛病毒性腹泻 /粘膜病病毒 ( BVD/MDV)感染牛引起的以发热、粘膜糜烂溃疡、白细胞减少、腹泻、咳嗽及怀孕母牛流产或产出畸形胎儿为主要特征的一种传染病。 1 946年Olafson等首次报道病毒性腹泻病。1 95 3年 Ramsey和 Chiver发现粘膜病。 1 961年 Gillespie等研究证明 ,这两种病毒是有共同抗原性的同种病毒 [1] ,1 971年由美国兽医协会将其统一命名为“牛病毒性腹泻 /粘膜病”。BVD/MD呈世界性分布 ,在许…     

河南省肉牛病毒性腹泻的流行动态调查研究

从河南省15个县市的规模化肉牛场和散养户随机采取血清671份熏直肠粪拭子645份熏分别用微量细胞中和试验和双抗体夹心ELISA试剂盒检测牛病毒性腹泻/黏膜病(BVD/MD)的抗体和抗原。检测结果表明:十五个县市牛群的BVD/MD平均抗体阳性率为22.9%,其中肉牛抗体阳性率为21.0%鸦BVD/MD病毒抗原阳性检出率为7.22%～23.80%,从而证实中原地区肉牛群中已普遍存在有BVD/MD。     

宁夏地区牛病毒性腹泻流行情况调研报告

牛病毒性腹泻/黏膜病（BVD）是由牛病毒性腹泻病毒（BVDV）引起的一种传染病。为调查宁夏地区BVD的流行情况,对制订防控措施提供数据支持,采集宁夏地区疑似感染BVDV且未进行疫苗免疫的1 500 头牛的血清进行抗体检测,对临床症状明显的295 头牛用肛拭子进行抗原检测。结果显示,牛病毒性腹泻抗体阳性率87.60%,抗原阳性率2.71%,存在较为严重的BVDV感染。     

疑似牛病毒性腹泻──粘膜病病毒细胞培养及其抗体对猪瘟牛睾丸细胞苗生产影响的观察

1993年猪瘟牛睾丸细胞苗生产过程中出现细胞病变、毒价不高．猪瘟、牛病毒性腹泻─粘膜病病毒、猪细小病毒（HCV、BVD／MDV、PPV）三价荧光抗体及其他中间监测结果观察．初步认为，长沙市部分乳牛场已铁牛病毒性腹泻─粘膜病病毒感染．     

青海省牛病毒性腹泻/黏膜病血清学调查

<正>牛病毒性腹泻/黏膜病(BVD/MD)是由牛病毒性腹泻病毒(BVDV)引起的,主要发生于牛的一种急性、热性传染病。BVDV感染牛的典型症状是牛病毒性腹泻、急慢性黏膜病、发热以及随后的产奶量下降。病毒可通过胎盘感染,从而可导致怀孕母牛的流产和死产,产下存活的小牛可出现免疫耐受,并会终生排[1]     

96孔板BVD/MD血清荧光抗体查毒方法介绍

随着我国进出口贸易量的加大,牛病毒性腹泻—粘膜病(BVD/MD)病毒的荧光抗体检查工作量也越来越繁重。目前,我国口岸动植物检痊所系统多用青霉素瓶、玻片等常规查毒方法,操作相当繁琐。特别是BVD/MD中和试验微量法被广为采用以后,上     

牛病毒性腹泻病毒RT-PCR检测方法的建立及应用

根据GenBank中登录的牛病毒性腹泻病毒(BVDV)基因序列,设计合成了1对特异性引物,建立了检测BVDV的RT-PCR方法。通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法可从BVDV标准毒株Oregon C24V中扩增出471 bp的特异性片段,而对猪瘟病毒、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒、牛副流感病毒、MDBK正常细胞的扩增结果均为阴性。经对标准毒株的细胞毒进行检测,其敏感度达10^-1 TCID₅₀/mL。应用该方法对临床腹泻病牛各脏器样品进行检测,结果比病毒分离方法更为敏感,操作简便。表明建立的RT-PCR方法具有特异、灵敏、高效、快速的特点,可用于BVDV的临床检测及流行病学监测。     

猪瘟病毒单克隆抗体的研制和应用Ⅰ.抗猪瘟兔化弱毒单克隆抗体杂交瘤细胞株的建立与鉴定

以聚乙二醇(PEG,MW6000)沉淀法部分纯化的猪瘟兔化弱毒中国株(SFV-C)免疫BALB/C小鼠,取其脾脏制备脾细胞与SP2/0骨髓瘤细胞融合,经ELISA检测和有限稀释法克隆化筛选出9株对SFV特异的单克隆抗体(McAb)杂交瘤细胞。它们产生的McAb仅对SFV-C株发生特异性反应,而与SFV-S及BV DV Oregon株不发生反应.相加试验表明,9个McAb分别针对不同的抗原决定簇。所有的McAb均不具有沉淀反应特性。     

Retrospective genome analysis of a live vaccine strain of bovine viral diarrhea virus

A live bovine viral diarrhea (BVDV) vaccine, marketed as a derivate of the Oregon C24V strain, was used between the end of the 1960s and the beginning of the 1990s in Central Europe. Since laboratory investigations of mucosal disease cases in vaccinated animals suggested recombinations between the vaccine and wild type variants of BVDV, and recombinational nucleotide sequences seemed distinct from BVDV Oregon C24V, the aim of the present retrospective study was to analyze the genomes of pre-registration (termed here BVDV-Xpre) and of marketed (BVDV-X) batches of the vaccine. The results of the complete genome analysis of BVDV-Xpre confirmed that the original virus strain used at the start of the vaccine production was Oregon C24V. Surprisingly, the analysis of the complete nucleotide sequence of the BVDV-X marketed vaccine revealed that this strain belongs to the BVDV 1b subgroup, with a 93.7% nucleotide sequence homology to BVDV reference strain Osloss. The homology to BVDV Oregon C24V was significantly lower (77.4%), and a thorough sequence scanning showed that the genome of BVDV-X had not derived from Oregon C24V. These data indicate the very likely scenario that a strain different to Oregon C24V was picked up during the in vitro or in vivo passages for vaccine development. Despite of the virus-switch, the BVDV-X vaccine continuously maintained its innocuity and efficacy, as proven by the regular quality testing data, and the presence of the foreign virus remained unnoticed over many years. The results of this work emphasize that the contamination of commercially available live vaccines with exogenous BVDV strains is a real risk factor, and a unequivocal analysis, including molecular methods, is needed to verify their authenticity.     

Studies on Virus Diarrhea and Mucosal Disease of Cattle

Outbreaks of virus diarrhea and mucosal disease were studied and certain clinical and pathological criteria applied in making a diagnosis of one or the other disease. An attempt was made to verify the diagnosis in each case by serological means in field outbreaks and by transmission of the disease experimentally in calves using post-mortem material.

Serological studies in field outbreaks produced inconclusive results. Quite consistent results were obtained from transmission trials. Experimental calves developed diphasic temperature rises, leukopenia, and oral hyperemia with or without erosions. Specific antibody induced in experimental calves was capable of neutralizing the standard Oregon C24V strain of virus diarrhea. It was therefore apparent that the viral isolates obtained from nine outbreaks in this study were closely related.

It was concluded that there were not two diseases, but one, and that according to priority should be called virus diarrhea.

     

牛病毒性腹泻病毒侵染宿主细胞的电镜观察

用电镜观察了牛病毒性腹泻病毒Oregon C24株在侵染新生犊牛睾丸细胞中的形态发生。成熟的病毒颗粒呈直径约为50nm的球形颗粒，内含直径约为30nm的核芯。病毒侵染宿主细胞后，在胞质内复制，通过糙面内质网膜出芽成熟，病毒可通过侵染细胞外排或在细胞死亡后含有病毒颗粒的空泡破溃而释放到胞外。     

Pathogenicity of a bovine viral diarrhoea virus strain in pregnant sows: short communication

The biological properties of bovine viral diarrhoea virus (BVDV) strain Oregon C24V were studied after intranasal and subcutaneous infection of pregnant sows. This virus strain is widely used in Hungary for immunising cattle against bovine viral diarrhoea (BVD). Based upon the results of the clinical, gross pathological, histopathological and virological examinations it can be established that the given strain caused asymptomatic infection and serological conversion in sows that were in the second third of gestation. The virus caused clinically apparent disease in some of the piglets born at term, which indicates that it had crossed the placenta. More than half (57%) of the live-born piglets died within 60 days of birth. The sows and their progeny did not shed the virus. BVDV infection has great differential diagnostic importance in pigs, as classical swine fever (CSF) virus strains of reduced virulence cause similar clinical symptoms and gross and histopathological changes.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of bovine viral diarrhea virus antibody in cattle.

A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.     

牛病毒性腹泻/粘膜病病毒在不同细胞上生长增殖的比较

牛病毒性腹泻／粘膜病病毒（ＢＶＤ／ＭＤ）Ｏｒｅｇｏｎ　Ｃ２４Ｖ弱毒株抗原的制备一直以在犊牛仔细胞上生长为主，为了使该病毒有更广泛的宿主细胞，对该病毒株在犊牛仔细胞上和睾丸细胞上生长增殖情况作了对比，结果证明，该病毒在两种细胞上的增殖情况无差异。     

山西一例牛病毒性腹泻病例病原分离鉴定及测序分析

为查找引起山西某牛场疑似牛病毒性腹泻病例的病因,对送检的9份牛鼻腔棉拭子样品,经处理后进行了多病原PCR或RT-PCR检测、病原分离、特征性细胞病变观察、效价测定、RT-PCR鉴定及基因测序分析。结果显示:从9份样品中检出6份BVDV核酸阳性,IBRV、BRSV、BPIV3、支原体均阴性,病料上清接种MDBK细胞进行培养,传至F5代发现MDBK细胞出现特征性病变,表现为细胞变亮、圆缩、拉网并逐渐形成空泡,效价为10-5.42TCID50/0.1mL,BVDV RT-PCR鉴定阳性,将其命名为BVDV-SX2020株,测序发现其5’UTR与BVDV Oregon C24V等参考毒株同属BVDV基因1a亚型。结果证实该起病例由BVDV-1a毒株感染引起。     

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。