Attenuation of a virulent type 2 bovine viral diarrhea virus

The purpose of this study was to produce an attenuated bovine viral diarrhea virus (BVDV) type 2 strain as a tool for identifying potential virulence markers in the BVDV2 genome. The attenuation of the virulent strain, BVDV2-24515, was accomplished by in vivo and in vitro passage. The strain was initially used to infect an elk (Cervus elaphus) [J. Wildl. Dis. 35 (1999) 671], re-isolated at 7 days post-inoculation from serum, and then subsequently passaged 56 times in cell culture. Two groups of calves were inoculated intranasally with either BVDV2-24515 or the putative attenuated virus, designated BVDV2-LATT. Calves inoculated with BVDV2-24515 had cumulative clinical scores which ranged from 6 to 53. Clinical signs in these calves consisted of anorexia, depression, dehydration, diarrhea (±bloody), and pneumonia. Several calves developed leukocytopenia, primarily a neutrocytopenia, and presented lesions of enteritis or pneumonia at necropsy. In contrast, cattle inoculated with BVDV2-LATT had cumulative clinical scores which ranged from 0 to 2. This was not significantly different from that of controls which received no virus (range: 0–1). Calves inoculated with BVDV2-LATT produced high neutralizing antibody titers against BVDV2. Thus, in addition to its potential use as a tool for identifying virulence markers, the attenuated virus is also worthy of further study as a candidate virus for inclusion in a modified-live vaccine.     

3株牛病毒性腹泻病毒的分离鉴定及其基因分型

为确定牛呼吸道传染病的病原以及牛病毒性腹泻病毒(BVDV)在临床健康牛群中是否存在持续性感染,本研究采集了内蒙古自治区和黑龙江省两个牛场牛鼻拭子46份及肺脏样品8份,采用MDBK细胞进行病毒分离培养,经BVDV特异性引物RT-PCR检测有3份样品为阳性。利用间接免疫荧光检测3株阳性样品,可以观察到特异性荧光,表明分离得到3株BVDV,分别命名为480、A0583和Lung-6。进一步研究显示480、A0583和Lung-6均为非致细胞病变型。对3株分离病毒的5'端非编码区和Npro基因进化树分析显示3株病毒均属于BVDV1型,其中480株属于BVDV1c亚型,A0583株和Lung-6株同属于BVDV1m亚型。本研究首次在国内同一个牛场分离到BVDV1m(A0583)和BVDV1c(480)两个基因亚型。本研究为我国BVDV-1型不同基因亚型的抗原关系分析及BVDV疫苗的研制奠定了基础。     

Prevalence of serum antibodies to bovine herpesvirus-1 and bovine viral diarrhea virus in beef cattle in Uruguay

Our objective was to determine the prevalence of serum antibodies to bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea (BVD) virus in beef cattle in Uruguay. A random sample of 230 herds selected with probability proportional to population size based on the number of cattle was chosen from a list frame of all registered livestock farms as of June 1999. Sera from up to 10 heifers, cows and bulls (up to 30 sera total per herd) were collected on selected farms between March 2000 and March 2001 and evaluated by means of enzyme-linked immunosorbent assays (ELISAs). Overall, 6358 serum samples were evaluated. We also collected data on previous diagnosis of BHV-1 or BVD infections and on the use of vaccines against these agents.

The estimated prevalence of exposure to BHV-1 and BVD at the herd level for the Uruguayan beef population was 99% and 100%, respectively. Approximately 37% of beef cattle in Uruguay have been exposed to BHV-1 and 69% to BVD virus. Only 3% of beef herds in Uruguay regularly (typically, annually) use vaccines against either of these agents.     

Economic risk analysis model for bovine viral diarrhea virus biosecurity in cow-calf herds

A stochastic model was designed to calculate the cost-effectiveness of biosecurity strategies for bovine viral diarrhea virus (BVDV) in cow-calf herds. Possible sources of BVDV introduction considered were imported animals, including the calves of pregnant imports, and fenceline contact with infected herds, including stocker cattle raised in adjacent pastures. Spread of BVDV through the herd was modeled with a stochastic SIR model. Financial consequences of BVDV, including lost income, treatment costs, and the cost of biosecurity strategies, were calculated for 10 years, based on the risks of a herd with a user-defined import profile. Results indicate that importing pregnant animals and stockers increased the financial risk of BVDV. Strategic testing in combination with vaccination most decreased the risk of high-cost outbreaks in most herds. The choice of a biosecurity strategy was specific to the risks of a particular herd.     

Prevalence and risk factors associated with bovine viral diarrhea virus infection in dairy herds in Jordan

A cross-sectional study was carried out to determine the seroprevalence and to identify risk factors associated with bovine viral diarrhea virus (BVDV) infection in 62 non-vaccinated dairy herds (671 cows) in Jordan between January and June 2007. Information regarding herd management was recorded through a personal interview with farmers. Antibodies against BVDV were detected using an indirect ELISA test. Chi-square analysis and multivariable logistic regression model were used to identify risk factors for BVDV seropositivity. The true prevalence of antibodies against BVDV in individual cows and cattle herds was 31.6% and 80.7%, respectively. The seroprevalence of BVDV in medium and large size herds was significantly higher than that in smaller herds. There was no significant difference in BVD seroprevalence between different age groups. Random-effects logistic regression model revealed two major factors associated with seropositivity to BVDV; exchange of visits between adjacent farm workers and not isolating newly purchased animals before addition to the herd. The seroprevalence of BVDV in cows located in the northern Jordanian governorates was significantly higher than that in other studied governorates. Results of this study indicated that BVDV is highly prevalent in Jordan and BVDV infection could be controlled by livestock-trade control, and applying strict biosecurity measures in the dairy farms.     

二重RT-PCR同时检测VSV与BVDV核酸

水泡性口炎病毒(VSV)与牛病毒性腹泻病毒(BVDV)具有相近的传播途径与类似的检测方法，本文参照文献报道的基因序列，设计合成了两对能分别扩增VSV(202bp)、BVDV(341bp)基因片段的引物，并对PCR扩增条件进行优化，建立了二重RT-PCR方法，可同时检测VSV与BVDV病毒核酸。VSV产物经测序显示与报道的核酸序列同源性为88．6％。二重RT-PCR同时检测VSV与BVDV经济、快速、敏感、特异，可用于实验研究和流行病学调查。     

Long-term clinicopathological characteristics of alpacas naturally infected with bovine viral diarrhea virus type Ib

Background: Substantial bovine viral diarrhea virus (BVDV)‐related production losses in North American alpaca herds have been associated with BVDV type Ib infection. Objectives: To classify and differentiate the long‐term clinicopathological characteristics of BVDV type Ib infection of alpaca crias, after natural virus exposure. We hypothesized that persistently infected (PI) alpacas specifically demonstrate growth retardation, clinicopathological evidence of opportunistic infections, and early mortality. Animals: Thirty‐five crias naturally exposed to BVDV (18 acute, 3 chronic, 14 PIs), and 19 healthy cohort controls of 5 northeastern alpaca farms were prospectively evaluated over 2 years (September 2005–September 2008). Methods: Observational cohort‐control study. Results: Chronically (viremia >3 weeks) and PI crias demonstrated significantly lower birth weights, decreased growth rates, anemia, and monocytosis compared with control animals. Common clinical problems of PI alpacas included chronic wasting, diarrhea, and respiratory disease. Median survival of PI alpacas that died was 177 days (interquartile range, 555) with a case fatality rate of 50% within 6 months of life. Transplacental infection was confirmed in 82% (9/11) of pregnant females on 1 farm, resulting in the birth of 7 PI crias (7/10 deliveries; 1 animal was aborted). Mean gestation at the beginning and end of BVDV exposure was 64 and 114 days, respectively. Conclusions and Clinical Importance: Natural BVDV type 1b infection during early pregnancy resulted in a high incidence of PI offspring. Although PI alpacas may have distinct clinical characteristics, verification of persistent viremia in the absence of endogenous, neutralizing antibodies is essential to differentiate persistent from chronic infection.     

牛病毒性腹泻病毒抗原捕获ELISA检测方法的标准化研究

用纯化牛病毒性腹泻病毒免疫蛋鸡制备出的卵黄抗体作为包被抗体,采用自制的单抗为一抗,建立牛病毒性腹泻病毒抗原捕获ELISA方法。通过试验确定,抗牛病毒性腹泻病毒卵黄抗体最佳包被浓度为1:50;McAb最适稀释浓度为1:10,HRP-羊抗鼠IgG工作浓度为1:800。通过引入牛病毒性腹泻病毒质控血清进行质控检验,该方法所得检测结果均在质量控制范围内,达到预定标准化要求。标准化的抗原捕获ELISA方法具有特异、灵敏、可靠、方便、快捷等特点,可广泛应用推广,为我国牛病毒性腹泻病毒监测提供了行之有效的技术手段。     

牛病毒性腹泻病毒E2蛋白的多克隆抗体制备及鉴定

为制备牛病毒性腹泻病毒(BVDV)重组E2蛋白的兔源多克隆抗体,本研究利用表达BVDV E2蛋白的重组质粒pET30a-E2转化E.coli BL21(DE3),经诱导表达获得重组E2蛋白。Western blot检测显示纯化蛋白能够与BVDV参考阳性血清反应。以纯化的重组E2蛋白免疫新西兰白兔制备多克隆抗体,病毒中和试验测定其中和效价为1:2048,间接免疫荧光和western blot试验表明其具有良好的反应性和特异性。本研究制备的BVDV重组E2蛋白兔源多克隆抗体可应用于BVDV的检测,同时为进一步建立检测BVDV E2蛋白的ELISA方法奠定基础。     

Serological relationships among subgroups in bovine viral diarrhea virus genotype 1 (BVDV-1)

Bovine viral diarrhea virus (BVDV) has various economic impacts associated with diarrhea, poor performance, an increase in the frequency of other infections and lethal outcomes. Both genotypes, namely BVDV-1 and BVDV-2, as well as different subgroups within these genotypes have been reported worldwide. Understanding the serological differences among the BVDV subgroups is important for disease epidemiology and prevention as well as vaccination programs. The aim of this study was to determine the serological relatedness among the subgroups in BVDV-1. For that purpose, sheep hyperimmune sera were collected against representative strains from 6 of the subgroups of BVDV-1 (BVDV-1a, -1b, -1d, -1f, -1h and -1l). The serum samples that gave the peak antibody titer to the homologous strains were used to perform cross neutralization assays. The highest homologous antibody titer (1:5160) was obtained against BVDV-1h. Regarding the cross neutralizing (heterologous) antibodies, the lowest titer (1:20) was produced by the BVDV-1f antiserum against the BVDV-1a and BVDV1-b viruses. The highest cross neutralizing titer (1:2580) achieved by the BVDV-1h antiserum was against the BVDV-1b strain. The cross neutralization results indicated particular serological differences between the recently described subgroup (BVDV-1l) and BVDV-1a/-1b, which are widely used in commercial vaccines. Considering the cross neutralization titers, it is concluded that selected BVDV-1l and BVDV-1h strains can be used for the development of diagnostic and control tools.     

牛病毒性腹泻病毒基因组结构与蛋白功能研究进展

牛病毒性腹泻病毒为黄病毒科(Flaviviridae)瘟病毒属(Pestivirus)的代表种,这个属的成员中还包括羊边界病毒(BDV)和猪瘟病毒(CSFV)。病毒基因组为单股正链RNA。论文就牛病毒性腹泻病毒已定位的4种结构蛋白、8种非结构蛋白以及3′和5′非翻译区的基因结构特征及其编码蛋白的合成与功能研究进行了综述,为牛病毒性腹泻的防控和疫苗研制提供参考。     

牛病毒性腹泻病毒双抗体夹心ELISA检测方法的建立

将牛病毒性腹泻病毒超免疫血清以常规方法提取IgG，采用过碘酸钠法标记辣根过氧化物酶（HRP），建立了从粪样中检测牛病毒性腹泻病毒抗原的双抗体夹心ELISA。结果，抗体的最佳包被量为150μg／mL，酶标抗体最适工作浓度为1：200；封闭液为50mL／L的兔血清；待检粪样及酶标抗体的感作时间为37℃ 120min；底物显色时间为室温15min。应用建立的检测方法对河北省8个大中型奶牛场298份乳牛腹泻粪样进行了检测，结果，阳性检出率为42．6％。     

牛病毒性腹泻病毒JY株分离鉴定

为了对疑似含有牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的种公牛精液进行检测并分离病毒,本研究采用细胞培养、免疫荧光及纳米PCR技术,对采自吉林省某牛病毒性腹泻(BVD)发病牛场中使用的种公牛精液进行检测与病毒分离。共采公牛精液8份,接种牛肾细胞系(MDBK)进行分离培养。分离得到阳性毒株为非致细胞病变(NCP)型,测得第4代病毒效价为10^6.25TCID₅₀/mL。纳米PCR检测5′-UTR和E2基因,测序后与GenBank上已发表的BVDV流行毒株核酸序列比对和进化分析。结果表明,分离毒株属于BVDV-1型,与BVDVJL株亲缘关系最近,5′-UTR核苷酸同源性为100%,E2基因核苷酸同源性为99.3%,命名为BVDVJY株。研究显示,本次采集的种公牛精液携带BVDV-1型毒株。该牛场BVD的发生疑似与种公牛精液带毒有关,对牛场BVD的防治起到警示作用。     

牛病毒性腹泻病毒(BVDV)抗原捕获ELISA检测方法的建立

用牛病毒性腹泻病毒(BVDV)单克隆抗体包被酶标板,以兔多克隆抗体作为夹心抗体,建立BVDV抗原捕获ELISA(AC-ELISA)检测方法,优化反应条件并对该方法的稳定性等指标进行了测试和评价。结果表明,单抗最佳包被质量浓度为5μg/mL,兔多抗血清最佳质量浓度为10μg/mL;单抗在4℃包被12~24 h,多抗在37℃作用1 h为双抗体的最佳反应条件;酶标抗体最适稀释度1∶10000,最适作用时间为1 h;采用1%BSA和1%明胶分别在抗体包被后和加入待检抗原反应后进行两次封闭效果好。用AC-ELISA方法检测临床采集的11份牛腹泻病料和12份健康牛组织样品,同时以病毒分离和RT-PCR检测方法做对比,3种方法符合率很高。研究表明AC-ELISA方法稳定性好,可用于BVDV的临床快速检测。     

牛病毒性腹泻的鉴定以及治疗

为了鉴定甘肃武威地区某牛场中牛病毒性腹泻病的发病情况,分别采集了13只病牛的血液样品、粪便样品配合诊断,通过血清学诊断方法和PCR鉴定的方法对样本进行了检测,结果显示血清学方法中的13份血液样本有8份为阳性,粪便样本提取RNA,进行RT-PCR同样可以检测到8份样本中扩增得到片段大小为267bp的条带,检测的13份样本有8份样本的血清学检测和粪便PCR检测均为阳性,本次检测的阳性率为61.54%,结果表明本次检测的甘肃武威地区某养殖场的牛病毒性腹泻阳性率相对较高,需要加强牛病毒性腹泻的防控,采取科学的手段治理该类疾病降低其对养牛产业的损失。     

牛病毒性腹泻病毒BVDV-JL株的分离与鉴定

本研究从吉林某牛场表现严重腹泻症状濒死牛的胸腺病料样品中分离一株病毒,该病毒在MDBK细胞中盲传4代无细胞病变产生,而通过RT-PCR和间接免疫荧光试验、微量血清中和试验检测表明该分离病毒株为牛病毒性腹泻病毒(BVDV),并命名为BVDV-JL.将BVDV-JL株F4代细胞培养液(10<'7.13>TCID<,50>/...     

牛病毒性腹泻病毒生物型转化分子机制的研究进展

从病毒基因与宿主细胞基因的重组、病毒基因的复制与重排、病毒基因的重复复制和序列插入、病毒基因的缺失和点突变几个方面阐述了牛病毒性腹泻病毒（BVDV）由非致细胞病变型（NCP）向致细胞病变型（CP）转化的分子变异机制。总结了前人对NCP型向CP型BVDV转化研究的结果，归纳了5种生物型转化形式，揭示了BVDV具有高变异性。     

牛病毒性腹泻病毒CC13B株全基因组序列及分析

从长春地区某牛场发生疑似为牛病毒性腹泻-黏膜病的病牛粪样中分离到1株病毒,经序列测定为牛病毒性腹泻病毒命名为BVDV CC13B株。核苷酸序列的测定结果显示,CC13B毒株的完全基因组序列由12 265个核苷酸组成,其中5′端非编码区包含380个核苷酸,3′端非编码区包含188个核苷酸。病毒基因组含有1个大的读码框架,编码1个由3 898个氨基酸组成的前体多聚蛋白。序列对比结果显示,CC13B毒株的核苷酸和氨基酸序列与国外CP-5A毒株同源性最高,分别为为96.2%和97.3%;而与国内分离株JZ05-1的同源性最低,分别为69.8%和71.0%。系统进化树分析结果表明,CC13B毒株与国内分离的长春184、Xinjiang-3156和H等分离株归类为BVDV基因Ⅰ型的Ib基因亚型。结果表明,长春地区近年发生的牛病毒性腹泻-黏膜病依然主要由BVDV基因Ⅰ型毒株引起。     

Experimental infection of early pregnant cows with bovine viral diarrhea virus: Transmission of virus to the reproductive tract and conceptus

The objective of this study was to chronologically investigate the effect of bovine viral diarrhea virus (BVDV) infection on early pregnant cows before placenta formation. Six cows were intravenously inoculated with either BVDV (treated, n = 4) or growth medium (control, n = 2) (day 0) at day 26 of pregnancy. Two treated cows and one control cow were euthanized on day 3 post-infection and the remaining animals were euthanized on day 6. BVDV was isolated from maternal tissues such as lymphoid or reproductive tissues of treated animals on days 3 and 6 post-infection. Additionally, one treated cow autopsied on day 6 post-infection had evidence of infectious BVDV in the allantoic membranes, allantoic fluid and embryos. In three treated cows, a significant decline in progesterone concentration was also observed post-infection while in control cows they remained constant. Therefore, BVDV can infect bovine embryos before placenta formation and may affect progesterone profiles in cows during early pregnancy.     

Evaluation of bovine viral diarrhea virus control using a mathematical model of infection dynamics

A mathematical model for infection with bovine viral diarrhea virus (BVDV) was created comprising a series of coupled differential equations. The model architecture is a development of the traditional model framework using susceptible, infectious and removed animals (the SIR model). The model predicts 1.2% persistent infection (within the range of field estimates) and is fairly insensitive to alterations of structure or parameter values. This model allows us to draw important conclusions regarding the control of BVD, particularly with respect to the importance of persistently infected (PI) animals in maintaining BVD as an endemic entity in the herd. Herds without PI animals are likely to experience episodic reproductive losses at intervals of two to three years, unlike herds with PI animals which will not see such marked episodic manifestations of infection. Instead, these herds will experience an initial peak of disease which will settle to low-level chronic reproductive losses. The model indicates that vaccine coverage for herd immunity (to avoid episodic manifestations of disease) need be only 57% without PI animals, although 97% coverage is required when PI animals are present. Analysis of model behavior suggests, a program of detection and removal of PI animals may enhance the effectiveness of a vaccine program provided these animals are in the herd for 10 days or less. The best results would be seen with PI animals in the herd for 5 or fewer days.