Isolation of cytopathic and noncytopathic bovine viral diarrhea virus from the spleen of cattle acutely and chronically affected with bovine viral diarrhea

Both cytopathic and noncytopathic bovine viral diarrhea virus (BVDV) were isolated from 16 of 17 bovine spleens representing 11 herds that had experienced acute BVD and from 12 of 21 bovine spleens from 1 herd affected with chronic BVD. It was concluded that isolation of cytopathic and noncytopathic BVDV from the same spleen probably indicates that an animal with a persistent, noncytopathic BVDV infection was superinfected with a cytopathic BVDV. The prevalence (greater than 70%) of 2 viruses in the spleen of cattle with acute or chronic BVD suggested that persistent infection with noncytopathic BVDV may be an important factor in the pathogenesis of BVD.     

Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus

Eight healthy cattle that were persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) were inoculated with cell culture fluids that contained noncytopathic or cytopathic BVDV. A severe disease occurred after inoculation with cytopathic BVDV. The clinical signs, lesions, and immune response were consistent with those of clinical BVDV infections.     

Genetic comparison of ovine and bovine pestiviruses

Viral RNA oligonucleotide fingerprinting was used to compare genetic relationship among pestiviruses originating from ovine or bovine host species. Ovine pestiviruses, including reference border disease virus and 2 border disease isolates originating from natural pestivirus infections of sheep, appeared to have a more distant genetic relationship among themselves than with certain bovine pestiviruses. A closer genetic relatedness was evident between border disease virus and 3 noncytopathic bovine pestiviruses, including Draper bovine viral diarrhea virus (BVDV), a BVDV isolate that originated from aborted bovine fetuses, and a virus that was isolated from the serum of a calf that had a chronic BVDV infection. Four noncytopathic bovine viruses, including Draper BVDV and 3 field isolates, were closely related. Reference Oregon C24V BVDV, a cytopathic virus, was closely related to only 1 of the 7 noncytopathic viruses in this study.     

牛病毒性腹泻病毒致病机制研究进展

牛病毒性腹泻（bovine viral diarrhea,BVD）和黏膜病（mucosal disease,MD）均是由牛病毒性腹泻病毒（bovine viral diarrhea virus,BVDV）感染引发的传染病,严重威胁世界养牛业的发展。文章概述了BVDV分型及其分子生物学特征,并从急性感染、经胎盘或子宫感染、持续性感染和黏膜病4个方面总结了近期国内外BVDV致病机制的研究进展。根据序列保守性及是否致细胞病变可将BVDV分为两种基因型和两种生物型,其中,新发现的"HoBi"株归类为瘟病毒属。BVDV基因进化很快,基因组编码4种结构蛋白和8种非结构蛋白,编码蛋白在病毒的复制、翻译及在宿主致病过程中发挥重要作用。BVDV致病机制复杂,急性感染会造成病毒血症、繁殖障碍、免疫抑制等,急性感染牛发生腹泻的原因与BVDV感染胃肠道的肌层、黏膜下层并干扰肠道神经的正常功能相关,非致细胞病变型（NCP）BVDV是造成急性感染的病因。胚胎感染BVDV取决于病毒首次侵袭时胎儿在子宫内的生长阶段。NCP型BVDV具有抑制胎儿体内产生Ⅰ型干扰素的能力,致使该病毒在宿主中得以生存并形成持续性感染牛,当持续性感染牛再次感染与NCP型BVDV高度同源的致细胞病变型（CP）毒株时直接诱发黏膜病。两种生物型的产生是发生持续性感染和黏膜病的重要因素,NCP型可向CP型BVDV进行转化。本综述有助于发现控制BVD-MD传播的新途径,为消灭该病和新型疫苗的研制提供参考。     

Molecular specificity of the antibody responses of cattle naturally and experimentally infected with cytopathic and noncytopathic bovine viral diarrhea virus biotypes

The specificity of the humoral IgG response of cattle naturally or experimentally infected with bovine viral diarrhea virus (BVDV) was studied by radioimmunoprecipitation. Serum samples were tested against radiolabeled lysates of cells infected with cytopathic and noncytopathic biotypes of BVDV. A biotype-specific serologic marker was not detected. The specificity of the IgG induced in cows naturally or experimentally infected with either BVDV biotype was essentially the same. A strong IgG response to the 2 glycoproteins (56 to 58 kilodaltons, [kD] and 48 kD) of both biotypes and to the major polypeptides was induced in infected cells: 118 kD and 80 kD by cytopathic BVDV and only 118 kD by noncytopathic BVDV. The most consistent difference among cattle was the presence of IgG specific for the 37-kD polypeptide. Sequential serum sample collection after spontaneous and induced infections with either BVDV biotype did not indicate specific IgG markers for determining infection history. Sera from cattle with a confirmed diagnosis of mucosal disease and lacking neutralizing antibodies to BVDV usually lacked (greater than 80%) nonneutralizing BVDV-specific IgG. One animal had substantial amounts of IgG to the 80-kD polypeptide. Other cattle had less readily detectable amounts of IgG specific for 80-kD or 37-kD viral polypeptides.     

Experimental primary postnatal bovine viral diarrhea viral infections in six-month-old calves

Eight clinically healthy calves were inoculated intranasally, four with either noncytopathic or four with cytopathic bovine viral diarrhea virus, and were necropsied 5 or 12 days post-inoculation. The most frequent gross lesion associated with noncytopathic or cytopathic viral infection was proximal colonic mural edema. Consistent microscopic findings were acute to subacute tracheitis, mild enterocolitis with edema, petechial hemorrhages of mesenteric lymph nodes with mild follicular lymphocytic depletion, and paracortical lymphocytic hyperplasia. At necropsy, cytopathic virus was recovered from 4/4 calves and noncytopathic virus was isolated from 2/4 calves. Neutralizing antibodies to noncytopathic bovine viral diarrhea virus were detected in the two calves from which noncytopathic virus was not recovered. Immunohistochemical analysis of lymphoid tissues demonstrated a small, randomly distributed population of mononuclear cells that contained bovine viral diarrhea viral antigen in 7/8 calves.     

Comparison of cytopathic and noncytopathic isolates of bovine viral diarrhea virus by oligonucleotide fingerprinting.

The molecular technique of RNA fingerprinting was used to characterize the genomes of 5 isolates of bovine viral diarrhea virus (BVDV): 2 viral pairs from the same animal, BVD-ILN/BVD-ILC and BVD-TGAN/BVD-TGAC, and the cytopathic viral prototype, BVD-NADL. Oligonucleotide patterns from the viruses were compared, and unique and overlapping oligonucleotides were identified. A comparison of the fingerprints indicated that the genome of each virus was distinguishable by the T1 RNase oligonucleotide fingerprinting technique. The greatest similarity observed was between oligonucleotides from BVD-ILC and BVD-ILN. Eighteen large oligonucleotides were conserved in all 5 BVDV isolates studied. We found that within a pair of BVDV, the cytopathic fingerprint was different from the noncytopathic fingerprint, indicating that cytopathic and noncytopathic BVDV may be distinct viruses.     

Frequency of persistent bovine viral diarrhea virus infection in selected cattle herds

Sera and blood buffy coat samples were obtained from 3,157 cattle in 66 selected herds. Antibodies to bovine viral diarrhea (BVD) virus were detected in 89% of the serum samples by immunoprecipitation or virus-neutralization tests. Cytopathic or noncytopathic BVD viruses were isolated from blood buffy coat samples from 60 cattle in 6 herds. A second blood buffy coat sample was obtained from 54 of the 60 cattle 2 months after the initial sampling, and BVD virus was isolated again from each cow. The 54 cattle were considered persistently infected with BVD virus. The frequency of persistent infection was 1.7%.     

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.     

Lesions and localization of viral antigen in tissues of cattle with experimentally induced or naturally acquired mucosal disease, or with naturally acquired chronic bovine viral diarrhea.

Tissues from cattle that died of experimentally induced mucosal disease (n = 3), naturally acquired mucosal disease (n = 6), or naturally acquired chronic bovine viral diarrhea (n = 4) were examined. Consistent findings were lymphocytic depletion of lymphoid tissues, degeneration of myenteric ganglion cells, and mild adrenalitis. Intracytoplasmic viral antigen was detected in myenteric ganglia and in endocrine glandular cells. Noncytopathic virus was isolated from all cattle, and cytopathic virus was isolated from 12 of 13 cattle.     

牛病毒性腹泻病毒侵染细胞机制的研究进展

牛病毒性腹泻病毒(BVDV)是反刍动物和猪体内广泛存在的危害动物健康的重要病原体.BVDV感染牛后主要引起牛的持续性感染、免疫耐受、免疫抑制、繁殖障碍及急慢性黏膜病等临床症状,给养牛业造成重大的损失.其致病机理非常复杂,给该病的治疗和根除带来极大的困难.随着分子病毒学研究的发展以及对猪瘟病毒和黄病毒科其他成员的研究,人们在BVDV分子水平和细胞水平的研究方面也取得了一些进展.就此,作者从BVDV入侵细胞、在细胞内的复制以及与宿主蛋白分子相互作用等方面进行综述,有助于阐明BVDV致病和在体内持续存活的机制,为该病的防治和疫苗研发提供新的思路和对策.     

Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus

Nine steers persistently infected with noncytopathic bovine viral diarrhea (BVD) virus were allotted into 3 groups (3 cattle/group). Cattle in group A were vaccinated with a modified-live BVD virus vaccine of porcine cell origin, cattle in group B with a modified-live BVD virus vaccine of bovine cell origin, and cattle in group C with a killed BVD virus vaccine of bovine cell origin. Detrimental effects due to vaccination were not seen. Six weeks after vaccination, the steers were challenge exposed with a cytopathic BVD virus. All steers developed mucosal disease after challenge exposure, produced antibodies that neutralized various isolates of BVD virus, and remained persistently infected until death. Steers given killed virus vaccine had a minimal neutralizing-antibody response and developed mucosal disease as quickly as reported for challenge-exposed, nonvaccinated, persistently infected cattle. Steers given modified-live virus vaccines had higher neutralizing-antibody response and longer intervals from challenge exposure to development of mucosal disease. The specificity of the neutralizing-antibody response differed between groups of vaccinated cattle.     

BVDV对后备牛生长发育状况及繁殖性能的影响

牛病毒性腹泻/黏膜病(BVD/MD)是一种严重危害奶牛健康的病毒性传染病,其病原为牛病毒性腹泻病毒(BVDV)。BVDV感染牛后主要表现两种状态,即一过性感染(TI)和持续性感染(PI)。BVD在牛场的流行,可严重影响奶牛的生产性能、繁殖性能及牛群健康状况,对奶牛的影响可表现为流产、胎儿畸形、腹泻和免疫抑制等。怀孕母牛在特定妊娠阶段感染BVDV后,可娩出PI犊牛,部分PI犊牛能像正常犊牛一样生长发育至成年,但其生长发育状况和繁殖性能较同龄健康牛差异十分明显。为评价BVDV对后备牛生长发育及繁殖状况的影响,笔者采用ELISA方法检出北京地区28个规模化奶牛场141头BVDV-PI牛,并与同龄健康牛生长发育及繁殖数据相比较,结果表明,BVDV-PI后备牛各月龄段的体高、体重均低于健康后备牛,其首次输精日龄、配准日龄、耗精量明显高于健康后备牛,而一次情期受胎率显著低于健康后备牛。数据显示,BVDV严重影响后备牛的生长发育及繁殖状况。     

New concepts in the pathogenesis, diagnosis and control of diseases caused by the bovine viral diarrhea virus

The new information on the pathogenesis and epidemiology of mucosal disease of cattle is reviewed. It is now known that clinical mucosal disease occurs only in cattle which were infected with a pestivirus in early gestation and were born with persistent viral infection and specific immunotolerance. These animals may be clinically normal at birth but may develop fatal mucosal disease, perhaps following superinfection with another pestivirus, usually between 6 and 24 months of age. They may also remain clinically normal indefinitely and breed successfully. The progeny from persistently infected females will similarly be persistently viremic, and maternal families of such animals may be established.

Congenital defects may occur when infection of the fetus occurs in mid-gestation. Although fetuses may be infected in utero in late gestation, the infections do not persist, the fetuses develop antibodies, and they appear to suffer no ill-effects. Postnatal infection can result in subclinical disease (bovine viral diarrhea) with a normal immune response; the virus may also be responsible for enhanced susceptibility to other infections, diarrhea in newborn calves, and reproductive failure.

Prevention of the economically important diseases caused by the virus is dependent upon the identification and elimination of persistently viremic animals, which are reservoirs of infection, and the vaccination of immunocompetent females at least three weeks before breeding. However, because of serotypic differences between strains, there is some doubt whether vaccination will reliably provide protection against the transplacental fetal infections that are important in the pathogenesis of this disease. There is no substantial evidence to warrant the vaccination of feedlot cattle.

     

Apoptosis inhibitors delay the cytopathic effect of bovine viral diarrhoea virus (BVDV)

Based on their action in cell culture, two biotypes of bovine viral diarrhoea virus (BVDV) can be distinguished. The noncytopathic (ncp) BVDV isolated from persistently infected animals cause no visible damage to cultured bovine cells. In contrast, cytopathic (cp) BVDV induces severe damage and apoptosis in cell cultures. Cp BVDV can be isolated from cattle suffering from mucosal disease (MD) and is associated with the severe lesions that primarily affect the gastrointestinal tract. To get an insight into the molecular events during BVDV induced cytopathic effect (CPE), the effect of three chemical reagents (3-aminobenzamide, ascorbic acid and N-acetyl-leucyl-leucyl-methional) with completely different mode of actions in infected cells was analysed. All three substances were able to delay the cytopathic effect induced in permissive bovine cells.     

Monitoring bovine viral diarrhea virus vaccines for adventitious virus, using T1 ribonuclease viral RNA oligonucleotide fingerprinting.

Viral RNA oligonucleotide fingerprinting was used to discriminate 3 cytopathic vaccine bovine viral diarrhea viruses (BVDV) grown in medium supplemented with serum contaminated with noncytopathic BVDV from the same 3 viruses grown in cell culture free of BVDV. Oligonucleotide fingerprinting also effectively discriminated between reference Singer BVDV, NADL BVDV, and New York-1 BVDV grown in BVDV-free noncontaminated or BVDV-contaminated cell cultures. Oligonucleotide fingerprint mapping of viral RNA maybe used to determine the purity of virus stocks, as well as that of BVDV vaccines.     

Detection of bovine viral diarrhea virus genome in leukocytes from persistently infected cattle by RNA-cDNA hybridization.

A bovine viral diarrhea virus (BVDV) cDNA library was constructed. One cloned complementary DNA sequence was used as a probe to detect BVDV RNA by hybridization in infected cell cultures and in mononuclear leukocytes from persistently infected cattle by dot blot and in situ hybridization. The cDNA probe hybridized with all cytopathic and noncytopathic BVDV isolates tested. The hybridization results were consistent with results obtained using conventional subculturing and immunofluorescent staining methods and by inoculation of seronegative test cattle.     

Immunologic and virologic findings in a bull chronically infected with noncytopathic bovine viral diarrhea virus

Depressed lymphocyte blastogenesis in response to mitogen stimulation, depressed iodination of protein by neutrophils, and enhanced ingestion of Staphylococcus aureus by neutrophils were detected in a bull with chronic bovine viral diarrhea (BVD). Before developing chronic BVD, the bull was vaccinated with a killed cytopathic BVD virus. Neutralizing antibodies specific for the vaccine virus were detected in serum specimens obtained from the bull immediately before death. A noncytopathic BVD virus was isolated from the spleen after death. The immunologic and virologic findings in this bull supported reported research findings on the pathogenetic mechanisms involved in chronic BVD and mucosal disease.     

Effect of bovine viral diarrhea virus in the feedlot.

It could be argued that bovine viral diarrhea virus (BVDV) is one of the most economically significant infectious pathogens of feedlot cattle. Although the direct economic losses caused by this virus have not been well quantified, the role it plays as an immunosuppressive agent and as a potentiator for other diseases, most notably bovine respiratory disease, have been well documented. It is also a difficult disease for the feedlot veterinarian to control effectively.Individual cattle persistently infected with BVDV often serve as the source of infectious virus within a group of feedlot cattle, and the ultimate responsibility for preventing persistent infections in cattle rests with the cow-calf producer and not with the feedlot owner. The enormous impact of the virus on the livestock industry has led the Academy of Veterinary Consultants to draft a position statement that resolves that the beef and dairy industries adopt measures to control and target eventual eradication of BVDV from North America.     

Development of a neutralising antibody response to an inoculated cytopathic strain of bovine virus diarrhoea virus

Fourteen clinically healthy cattle that were persistently infected with non-cytopathic bovine virus diarrhoea virus (BVDV) and three BVDV-free cattle were inoculated with one of three cytopathic BVDV strains. Mucosal disease developed in 12 of the viraemic cattle, resulting in a moribund condition 17 to 99 days after inoculation. Two of the viraemic cattle remained clinically healthy until the end of the experiment, 14 months after inoculation. The BVDV-free cattle did not develop clinical signs after inoculation. From each cow with mucosal disease a noncytopathic and a cytopathic BVDV strain were isolated from tissue specimens collected post mortem. All the cattle developed moderate to high levels of neutralising antibodies against the cytopathic BVDV strain with which they were inoculated. The antibodies from 10 of the 12 cattle with mucosal disease did not react with the cytopathic BVDV strains isolated post mortem, and antibodies from none of them reacted with the non-cytopathic BVDV isolates. Antibody responses to the inoculated BVDV strains developed earlier in the viraemic cattle than in the BVDV-free cattle.