Characterization of bovine herpesviruses isolated from six sheep and four goats by restriction endonuclease analysis and radioimmunoprecipitation

Viral DNA from 10 herpesviruses isolated from 6 sheep and 4 goats were examined by restriction endonuclease analysis with respect to their relatedness to one another; to bovine herpesvirus type 6 (BHV-6), also known as caprine herpesvirus; and to 2 strains of bovine herpesvirus type 1 (BHV-1), known as infectious bovine rhinotracheitis virus (IBRV) and infectious pustular vulvovaginitis virus (IPVV). Viral proteins from the isolates were examined by radioimmunoprecipitation with anti-BHV-1/IBRV gnotobiotic calf (bovine) serum, anti-BHV-1/IBRV bovine hyperimmune serum, and anti-BHV-6 rabbit serum to evaluate their antigenic relatedness to each other. The goat isolates were obtained from animals with various disease conditions including respiratory tract disorders, vulvovaginitis, and wart-like lesions on the eyelid. The other isolates were from domestic sheep and came from aborted fetuses or from sheep with fatal pneumonia or proliferative lesions around lips and nose. All of the goats and 4 of the sheep from which the viral isolates were obtained had comingled with cattle. Purified DNA from each of the 10 field isolates and from BHV-1/IBRV, BHV-1/IPVV, and BHV-6 caprine herpesvirus was cleaved with restriction endonuclease Pst I. Five of 6 sheep isolates and 3 of 4 goat isolates yielded unique restriction patterns, ie, patterns that differed from each other by one or more bands. Sheep isolate DNA patterns were different from goat isolate patterns, and all restriction endonuclease analysis patterns were similar to the pattern for BHV-1/IBRV, but different from that for BHV-1/IPVV or for BHV-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection

A strain of bovine herpesvirus-4 (BHV-4) isolated from bovine cases of mammary pustular dermatitis was used for experimental infection of rabbits. The strain is serologically indistinguishable from the group prototype Movar 33/63 and from the American isolate DN599. Groups of rabbits were inoculated by various routes. Intravaginal and conjunctival inoculations resulted in vulvovaginitis and conjunctivitis, respectively, and in shedding of virus. The rabbits seroconverted for the virus, with high titers of antibodies (indirect fluorescent antibody test) that persisted throughout the experiment. Treatment with dexamethasone, beyond the acute infection, did not produce recrudescence of disease or shedding of the virus. Rabbits were killed at various times, from 3 to 6 months post-infection, and the virus was recovered from explant cultures of spleen and by cocultivation of spleen cells with bovine lung cells. These results demonstrate the usefulness of the rabbit as a model for studying the pathogenesis of BHV-4 infection in cattle.     

河南孟津地区6种引起奶牛流产病原的PCR检测及分析

为调查孟津地区引起奶牛流产的主要病原,本实验采集该地区8个规模化奶牛养殖场共52头新鲜流产胎牛,及其母体的血液、阴道分泌物和鼻腔分泌物。对所采集样本进行新孢子虫（N.caninum）、弓形虫(T.gondii)、胎儿三毛滴虫(T. foetus)、布氏杆菌(B.abortus)、牛传染性鼻气管炎病毒（IBRV）和牛病毒性腹泻病毒（BVDV）6种常见流产病原的PCR和RT-PCR检测。结果显示,被检样本总感染率为44.2%（23/52）,其中新孢子虫感染率最高,为32.7%（17/52）;IBRV次之,为13.5%（7/52）;胎儿三毛滴虫、布氏杆菌、弓形虫和BVDV感染率分别为1.9%、1.9%、1.9%和0。11.5%（6/52）的被检样本存在混合感染,其中以新孢子虫+IBRV混合感染为主（50%,3/6）。结合被检流产奶牛的临床特征及饲养管理等因素,推断新孢子虫是导致该地区奶牛流产的主要病原。     

Demonstration of infectious bovine rhinotracheitis virus antigen in paraffin sections

Nine pregnant heifers were inoculated intravenously with infectious bovine rhinotracheitis virus (IBRV) in the sixth month of pregnancy. Tissues were collected from the fetus of a heifer killed 13 days postinoculation (PI), from fetuses of 6 heifers that aborted 16-27 days PI, and from mummified fetuses of 2 heifers that aborted 53 and 85 days PI, respectively. Control tissues were obtained from the fetus of a non-inoculated heifer that was killed in the seventh month of gestation. Tissues were fixed in 10% formalin, embedded in paraffin, and examined for viral antigen by immunohistochemistry, using biotinylated second antibody and alkaline phosphatase-labeled avidin-biotin complex. Antigen was detected in at least 1 tissue from the fetus of each inoculated heifer. Positive tissues included lung, liver, spleen, kidney, adrenal, and placenta. In several fetuses, antigen was identified in tissues from which virus was not isolated in cell culture. This appeared to occur when tissues had only a few small foci of infection or when tissues were severely autolyzed. The observation of viral antigen in tissues from mummified fetuses indicates that this technique may be useful in diagnostic laboratories to detect IBRV infection in tissues that are not suitable for virus isolation or for examination by the cryostat tissue section-fluorescent antibody technique.     

传染性牛鼻气管炎病毒分离鉴定及特性分析

传染性牛鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)是牛的重要传染病病原,除能引起呼吸道疾病外,还可引起结膜炎、流产、脑炎和全身性感染。本试验从广东某牛场疑似IBRV感染牛中采取鼻拭子样品,用PCR和荧光PCR方法,初步诊断病牛感染IBRV。用MDBK细胞对初诊阳性病料进行病毒分离和鉴定,分离出1株传染性牛鼻气管炎病毒,命名为GD0109。细胞感染试验表明,该株病毒可以使MDBK细胞产生典型的圆缩、呈葡萄样聚集以及空洞等细胞病变。将GD0109与强毒的ATCCVR-2112TM株感染细胞裂解液分别接种MDBK细胞并盲传5代,对5代、10代、15代的病毒分别进行TCID50测定及中和试验,结果表明分离株和参考株VR-2112TM相似,提示该分离株为强毒株,并且具有良好的传代稳定性,为进一步疫苗生产打下良好的基础。     

Efficacy of viral components of a nonabortigenic combination vaccine for prevention of respiratory and reproductive system diseases in cattle

Efficacy and safety of components of an IM-administered vaccine for prevention of infectious bovine rhinotracheitis virus (IBRV), parainfluenza type-3 (PI-3) virus, bovine viral diarrhea virus (BVDV), and respiratory syncytial virus (RSV) infections and campylobacteriosis and leptospirosis were evaluated in cattle, including calves and pregnant cows. Challenge of immunity tests were conducted in calves for IBRV, PI-3 virus, or BVDV vaccinal components. All inoculated calves developed serum-neutralizing antibodies and had substantially greater protection (as measured by clinical rating systems) than did controls after challenge exposure to virulent strains of IBRV, PI-3 virus, BVDV, or RSV. In in utero tests, IBRV or bovine RSV vaccinal strains were inoculated into fetuses of pregnant cows. Histologic changes or abortions did not occur after fetal inoculation of the RSV vaccinal strain, and 10 of 14 fetuses responded serologically. Of 9 fetuses, one responded serologically to the IBRV vaccinal strain after in utero inoculation and was aborted 3 weeks later. In an immunologic interference test, 10 calves vaccinated with 2 doses of the multivalent vaccine, containing the 4 viral components and a Campylobacter-Leptospira bacterin, developed serum-neutralizing antibodies to IBRV, PI-3 virus, BVDV, and RSV without evidence of serologic interference. Under field conditions, 10,771 cattle, including 4,543 pregnant cows, were vaccinated. Vaccine-related abortions did not occur.     

Induction of persistent infection in mice and oncogenic transformation of mouse macrophages with infectious bovine rhinotracheitis virus

Infectious bovine rhinotracheitis virus (IBRV) established long-term persistent infection in intracerebrally inoculated athymic nude mice. After intraperitoneal injection into outbred mice, virus was isolated for only 3 days from spleens and livers. In vitro inoculation of outbred mouse spleen fragments with IBRV resulted in persistent infection and subsequent transformation of spleen macrophages. The IBRV-specific membrane and intracellular antigens were detected by indirect immunofluorescence techniques in transformed cells in early in vitro passages. The presence of IBRV genetic formation was confirmed by in situ hybridization. The IBRV-transformed mouse macrophages induced fibrosarcomas and cystic tumors in athymic nude mice. Infective virus could not be rescued from transformed cells by cocultivation with rabbit kidney cells, treatment with iododeoxyuridine, or ultraviolet irradiation.     

Evaluation of pregnant rabbits as a laboratory model for bovid herpesvirus-4 infection

A field strain (87-8363) of bovid herpesvirus-4 (BHV-4) isolated from an aborted bovine fetus was used to inoculate pregnant rabbits. Eleven rabbits in midgestation were alloted to 4 groups consisting of 3 infected groups and 1 control group. Rabbits were inoculated with BHV-4 or mock-infected cell culture preparations via IV, intravaginal, and intrauterine routes. Mild vulvovaginitis and endometritis were observed after intravaginal and IV inoculation of BHV-4, whereas intrauterine inoculation of BHV-4 resulted in abortion of hemorrhagic fetuses and nonsuppurative endometritis. Virus was successfully isolated from organ explants of fetal tissues. Rabbits seroconverted 1 week after infection as detected by results of an indirect immunofluorescence assay.     

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.     

Vaccinia viruses isolated from skin infection in horses produced cutaneous and systemic disease in experimentally infected rabbits

The susceptibility of rabbits to two isolates of Vaccinia virus (VACV) recovered from cutaneous disease in horses in Southern Brazil was investigated. Rabbits were inoculated in the ear skin with both VACV isolates, either in single or mixed infection. All inoculated animals presented local skin lesions characterized by hyperaemia, papules, vesicles, pustules and ulcers. Infectious virus was detected in the lungs and intestine of rabbits that died during acute disease. Histological examination of the skin revealed changes characteristic of those associated with members of the genus Orthopoxvirus. These results demonstrate that rabbits develop skin disease accompanied by systemic signs upon intradermal inoculation of these two equine VACV isolates, either alone or in combination, opening the way for using rabbits to study selected aspects of the biology and pathogenesis of VACV infection.     

牛传染性鼻气管炎病毒诱导MDBK细胞线粒体损伤模型的建立

为探讨牛传染性鼻气管炎病毒（IBRV）诱导MDBK细胞线粒体损伤的最适时间和浓度,本试验用0.5、1.0、1.5、2.0感染复数（multiplicity of infection,MOI） IBRV感染MDBK细胞6、12、24、36、48 h,采用透射电镜、流式细胞术等技术检测MMP和MPTP异常开放情况。结果显示,用不同MOI IBRV感染MDBK细胞12 h,感染组与对照组相比,MPTP均显著降低,且除2.0 MOI外均随接毒剂量的增加而降低;MMP在1.5 MOI后开始极显著降低;用1.5 MOI IBRV分别感染MDBK细胞6、12、24、36 h,感染组与对照组相比MPTP及MMP从6 h起均显著降低,且随着病毒感染时间的不断延长而降低。透射电镜结果显示,IBRV感染MDBK细胞6 h就可引起细胞线粒体损伤,出现部分空泡化,24 h线粒体完全空泡化,并且在线粒体周围可以看见病毒粒子。结果表明,IBRV感染可以诱导MDBK细胞线粒体损伤,且用1.5 MOI感染MDBK细胞6～24 h是诱导细胞线粒体损伤的最佳时间和浓度。该时间和浓度可用于IBRV相关药物研究,为进一步研究线粒体损伤提供理论依据。     

IBRV对牛单核巨噬细胞吞噬鸡红细胞功能的影响

为研究牛传染性鼻气管炎病毒（IBRV）感染对牛单核巨噬细胞吞噬功能的影响,应用淋巴细胞分离液获得牛外周血单个核细胞,通过贴壁培养成为单核巨噬细胞。 IBRV 感染单核巨噬细胞24 h后加入鸡红细胞,测定被感染细胞对鸡红细胞的吞噬率。结果显示,单核巨噬细胞被IBRV 感染后,对鸡红细胞的吞噬率显著降低（P<0.05）。     

牛传染性鼻气管炎病毒致病机制研究进展

牛传染性鼻气管炎病毒(IBRV)主要引起牛的呼吸道、生殖道等炎症反应,也可以引起呼吸困难及流产。IBRV的致病机制尚不清楚,目前发现IBRV的非结构蛋白和结构糖蛋白与病毒毒力相关,不但影响病毒的复制及对宿主细胞的感染,同时也与病毒的免疫逃逸密切相关。除此之外,IBRV通过诱导宿主细胞凋亡造成持续性感染及激活炎症复合体诱导严重的炎症反应,造成宿主广泛病理反应的发生。因此,探索IBRV毒力蛋白结构功能、IBRV感染诱导的细胞凋亡及宿主炎性复合体激活的分子机制,将成为未来IBRV研究的热点。     

抗牛传染性鼻气管炎病毒囊膜gD糖蛋白的单链抗体在昆虫细胞中表达及活性检测

为了获得具有生物学活性的牛传染性鼻气管炎病毒（infectious bovine rhinotracheitis virus,IBRV） gD蛋白特异性单链抗体,试验采用PCR方法扩增杂交瘤细胞的cDNA单链抗体重链可变区（VH）和轻链可变区（VL）基因,通过重叠延伸PCR及Linker序列获得完整的单链抗体基因,将单链抗体基因克隆至杆状病毒载体pFB-LIC-Bse中,构建重组转移载体pFB-VH-VL,将其转化至大肠杆菌DH10Bac感受态细胞中制备重组杆粒rBacmid-VH-VL,将重组杆粒rBacmid-VH-VL转染至Sf9细胞中获得携带单链抗体基因的重组杆状病毒。该重组杆状病毒在Sf9细胞中表达了分子质量约为30 ku的单链抗体蛋白。Western blotting结果显示,重组单链抗体蛋白能被His标签抗体特异性结合,也能特异性结合gD蛋白。间接免疫荧光试验结果显示,该单链抗体蛋白能够识别感染牛肾细胞MDBK中的IBRV。本研究在昆虫细胞中成功表达了具有识别IBRV的gD蛋白特异性单链抗体,为牛传染性鼻气管炎的诊断与治疗奠定了基础。     

Fatal herpesvirus infection in commercial rabbits

Acute mortality occurred in two unrelated rabbitries. In the rabbits examined, an unidentified herpesvirus caused lesions that have not been reported previously in this species. The primary lesions were multifocal hemorrhagic dermatitis on the face and back, localized pneumonia, and severe splenic necrosis. Large eosinophilic, intranuclear inclusion bodies that were observed in tissue sections of skin, spleen, and lung were identified as herpes-like viral particles by electron microscopy, and herpesvirus was cultured on rabbit kidney cells. Intramuscular injection of tissue culture fluid containing virus resulted in mortality and severe illness in two seven-week-old domestic rabbits four and six days postinfection, respectively. The gross and microscopic lesions were reproduced and herpeslike viral inclusions were observed in skin lesions. Herpesvirus was recovered from lung, trachea, spleen, liver, and from the thigh muscle at the site of inoculation. The experimental infection also activated severe pasteurella septicemia. The herpesvirus isolate needs further characterization.     

4 种呼吸道疫病血清学调查分析——以凯里市A奶牛场为例

[目的]为了解凯里市A奶牛场牛呼吸道疫病的流行情况。[方法]本次采用酶联免疫吸附试验（ELISA）对142 份奶牛血清样品进行牛呼吸道合胞体病毒（BRSV）、牛病毒性腹泻（BVDV）、牛传染性鼻气管炎（IBRV）、牛支原体感染抗体检测。[结果]凯里市A奶牛场4 种呼吸道疾病病原普遍存在,BRSV阳性率为41.5%（59/142）;BVDV阳性率为40.1%（57/142）;IBRV阳性率为45.0%（64/142）;支原体阳性率为33.8%（48/142）。犊牛4 种呼吸疾病单一感染率为15.1%~45.5%;BVDV感染最严重,为45.5%;成年牛单一感染率为33.8%~45.1%。此外,奶牛场存在IBRV、BRSV、BVDV、支原体多种病原体混合感染,二重感染率为18.3％~30.2％;BVDV+IBRV感染率最高,为30.2%;三重感染以BVDV+支原体+IBRV感染率最高,为19.7%;四重感染率为9.9%。[结论]凯里市A奶牛场存在4 种疫病,有不同程度的病原体感染,应加强对上述病原体的流行控制,提高奶牛场管理水平,减少奶牛场疫病发生和经济损失。     

Placental lesions caused by pseudorabies virus in pregnant sows

Pathologic and viral investigations were done on 13 fetal placentas and 23 aborted fetuses associated with naturally occurring pseudorabies in swine. Of the 13 fetal placentas examined, 7 (53.8%) had various degrees of necrotizing placentitis. The lesions were characterized by coagulative necrosis of the chorionic fossae and by intranuclear inclusions in degenerating trophoblasts and occasionally in mesenchymal cells. In addition, a mild inflammatory cell reaction was observed in the mesenchyma. Numerous viral particles, ultrastructurally indistinguishable from herpesvirus, were observed by electron microscopy in the affected chorionic membrane. Large aggregates of herpesvirus virions were demonstrated in the nucleus and cytoplasm of degenerated trophoblasts and mesenchymal cells. Of the 23 aborted fetuses examined, 22 (95.6%) had typical coagulative necrosis in the liver, spleen, adrenal glands, and visceral lymph nodes. Inclusions similar to those in the chorionic placenta were observed in the parenchymal cells of those organs, on the margins of necrotic areas. Pseudorabies virus was isolated from various organs of the aborted fetuses, but virus isolation from the placentas was not attempted. In a survey of 52 affected sows, sera from 49 (94.2%) neutralized the isolated virus. The findings indicated that the placental lesions caused by the virus were primary. The study also indicated the merit of routine examination of aborted fetal placentas and fetuses for diagnosis of pseudorabies.     

Expression and Biological Activity of Single Chain Antibody Against gD Glycoprotein of Infectious Bovine Rhinotracheitis Virus in Insect Cells

For preparation of bioactive single chain fragment variable (ScFv) which was targeted against envelope glycoprotein D (gD) of infectious bovine rhinotracheitis virus (IBRV),VH and VL genes were amplified from the cDNA of lymphocyte hybridoma, then VH and VL genes were integrated with a Linker by SOE-PCR,and the ScFv gene was cloned into the baculovirus vector pFB-LIC-Bse, which was transformed into Escherichia coli DH10Bac competent cells to construct a recombinant bacmid rBacmid-VH-VL. Finally,ScFv was expressed by transfected rBacmid-VH-VL into insect Sf9 cells, its molecular weight was about 30 ku. The result of Western blotting suggested that ScFv generated in Sf9 cells was specifically binds to His antibody,the protein also showed high affinity to gD of IBRV. The result of indirect immunofluorescence assay showed that ScFv could recognize IBRV which infected bovine kidney cells (MDBK). The study indicated that the recombinant ScFv was expressed in Sf9 cells, and could bind to gD of IBRV specifically. The result could provide the basis for the diagnosis and treatment of infection of IBRV.     

Clinical signs and diagnosis of a severe primary infection with BVDV subtype 1b in a dairy herd

As a result of a BVDV infection in a herd consisting of 95 adult cattle 17 cows aborted their calves within a period of 3.5 months, one third got severe diarrhoea, 3 cows died and an increased percentage of the cattle got lochiometra after calving or abortion. The disease was diagnosed by paired serological testing of cattle with diarrhoea or abortion and post mortem examination of several aborted calves. From one foetus BVDV virus was isolated and subsequently subtyped by sequencing. Of aborting cattle, the testing results were influenced by the interval between infection and abortion. These results indicate that a primary infection with BVDV subtype 1b can cause severe clinical symptoms in a dairy herd.     

Histological lesions in vascular tissues of bovine herpes virus type 4-infected rabbits

The gamma-herpes virus bovine herpes virus type 4 (BoHV-4) is distributed worldwide in cattle populations with unknown pathogenicity. Bovine endothelial cells were recently shown to be susceptible to BoHV-4 infection in vitro and this virus accelerated the cholesterol-induced atherosclerotic process in rabbits. In this study, the in vivo effect of BoHV-4 on cardiovascular tissue was investigated by intravenous infection of rabbits fed a cholesterol free diet. Inflammatory lesions of vascular tissue in aortic and valvular endothelial cells, and smooth muscle cells were detected by H&E staining, PCR, IF, EM immunohistochemistry, while virus isolation was used to detect virus particles. Acute and chronic vasculitis, signs of chronic endocarditis, with mononuclear cell accumulation and a fresh thrombus was found. Herpes viruses have already been thought to initiate cardio-vascular disorders, now this paper shows that a bovine gamma-herpes virus could also be a causative agent of vascular lesions in mammals fed a normal diet. BoHV-4-infection of rabbits could serve as a useful animal model for research into virus-induced human cardio-vascular diseases.