牛传染性鼻气管炎可视化LAMP检测方法的建立和应用

根据牛传染性鼻气管炎病毒(IBRV)gB基因保守序列(GenBank Accession No. DQ006857.1),利用Primer ExplorerV4软件设计3对LAMP引物,通过反应体系的优化、敏感性试验和特异性试验,建立IBRV的LAMP检测方法,并对393份临床样本进行了检测应用。结果显示,建立的IBRV LAMP方法在65℃、50 min条件下可扩增出LAMP特征性梯状条带,并可通过颜色变化判定结果。该方法可以检测到10copies/μL的质粒DNA,与nested-PCR方法的敏感性相当,比PCR方法敏感1000倍,且与牛病毒性腹泻病毒、猪伪狂犬病毒、水泡口炎病毒等均无交叉反应。应用该方法检测301份鼻腔拭子和92份血清样本,阳性率分别为87.6%和58.8%,表明鼻腔棉拭子更适用于IBRV的临床检测。文中建立的IBRV LAMP方法具有可视化、快速、特异、灵敏性强的优势,适合基层和现场对临床样本进行快速检测,为IBR的防控提供了技术支撑。     

Further evidence for entry of infectious bovine rhinotracheitis virus into bovine kidney cells

The penetration of bovine kidney cells by infectious bovine rhinotracheitis virus, a member of the herpesvirus group, was investigated using the direct immunoferritin labeling technique. Electron microscopic examination of infected cells after 10 min at 37°C revealed fusion between viral envelope and cell membrane; the former reacted with the ferritin particles conjugated with antiviral antibody. However, shortly after penetration of the nucleocapsid, viral-specific antigenic sites on the plasma membrane were not detected by the immunoferritin technique. Antigenically reactive structures in a disorganized array were frequently detected extracellularly, situated above the penetration sites as indicated by the internalized nucleocapsids.     

Purification and buoyant density of infectious bovine rhinotracheitis virus.

A purification scheme for infectious bovine rhinotracheitis virus utilizing rate-zonal centrifugation in a 10-40% potassium tartrate gradient was described. The density of IBRV in the potassium tartrate gradient was found to be 1.22 g/cm3. Electron microscopic examination of purified virus preparations revealed homogeneous populations of enveloped virions with minute projections on the envelope surface.     

Effects of hormone-induced superovulation on latent infectious bovine rhinotracheitis virus in cattle

Twelve nulliparous, sexually mature heifers free of antibodies to infectious bovine rhinotracheitis (IBR) virus were exposed intranasally to Colorado strain IBR virus. After 3 mos, when the postexposure antibody titers had stabilized, the heifers were divided into three groups. Individuals in each group were treated with either saline, dexamethasone or follicle stimulating hormone (FSH) for five consecutive days. Blood samples were taken at predetermined intervals for isolation of virus, and for determination of serum cortisol levels. No changes occurred in the saline-treated group, except that one heifer had a slightly elevated serum neutralizing antibody titer. Recrudescense of typical clinical lesions was observed in the dexamethasone-treated group, and the IBR virus was isolated from nasal swab samples taken from all heifers. In the FSH-treated group, no changes occurred, with the exception of slightly reduced serum cortisol levels. Results indicate that FSH-induced superovulation does not cause reactivation of IBR virus in heifers previously infected by the intranasal route, and has no effect on serum neutralizing anti-IBR virus antibody titers.     

Trypsin treatment of bovine embryos after in vitro exposure to infectious bovine rhinotracheitis virus or bovine herpesvirus-4

The objectives of this study were to evaluate the efficacy of trypsin treatment for the removal/inactivation of infectious bovine rhinotracheitis virus (IBRV) adhering to zona pellucida-intact (ZP-I) bovine embryos and to determine if bovine herpesvirus-4 (BHV-4) adheres to ZP-I bovine embryos. When adherence of BHV-4 was demonstrated, an additional objective was to determine whether trypsin treatment removes or inactivates this virus. A total of 139 ZP-I embryos was collected from superovulated donor cows at 7 d after estrus. Embryos were exposed to 10(6) to 10(7) plaque-forming units (pfu) of either IBRV or BHV-4 for 1 to 2 h. Subsequently, approximately equal numbers of embryos exposed to each virus were either washed 12 times and the washes and embryos examined for the presence of infectious virus, or they were treated with trypsin and the embryos examined for the presence of infectious virus. Although the fourth wash was the last positive wash, an average of 18 pfu of virus was detected from each of six groups (a total of 24 embryos) after exposure to IBRV and washing. Infectious bovine rhinotracheitis virus was not isolated from any of nine trypsin-treated groups (a total of 43 embryos). The seventh wash was the last positive wash for any group after exposure to BHV-4, yet an average of 2 pfu of virus was detected from each of six groups (a total of 29 embryos) after washing. No BHV-4 was isolated from any of eight trypsin-treated groups (a total of 43 embryos). The study confirmed previous reports that IBRV adheres to the bovine ZP after in vitro exposure and that trypsin treatment is effective in keeping ZP-I embryos free of this virus. Adherence of BHV-4 to ZP-I bovine embryos was demonstrated for the first time. Trypsin treatment was also effective in removing this herpesvirus.     

Differentiation of strains of bovine infectious rhinotracheitis virus using restriction analysis]

The virus of infectious rhinotracheitis of cattle (BHV-1) causes the respiratory diseases, encephalitis, vulvovaginitis, abortions, etc. in sensitive animals. The attempts to differentiate the viral strains by virology and serology methods were unsuccessful. The restriction analysis makes possible to divide the strains into the three main groups designated BHV-1.1, BHV-1.2 and BHV-1.3. Majority of authors note the lack of correlation between the restriction pattern of DNA and the syndromes caused by the strains of the first and second groups while all the representatives of the third group were isolated in case of meningoencephalitis. The restriction analysis of BHV-1 strains isolated in the USSR by restriction endonucleases EcoRI, BamHI, HpaI and ClaI is presented. The obtained results show the absence of correlation between the restriction pattern of the strain and the syndrome caused by the viruses of all three groups. The representatives of the group BHV-1.3 that are not associated with the neurological syndrome are isolated for the first time.     

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells

Summary Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. Within 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed. This work was supported in part by Public Health Service Research Contract FDA 233-74-1035 and by Research Grant AI-08648 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.     

Experimental infection of wildebeest with the herpesvirus of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis.

Intravaginal inoculation of a wildebeest (Connochaetes taurinus) with a wildebeest strain of the infectious bovine rhinotracheitis/infectious pustular vulvovaginitis herpesvirus induced only mild vulvovaginitis. The same virus did not produce any disease in another wildebeest exposed intranasally. A wildebeest bull which was inoculated by preputial instillation developed mild posthitis. The virus was reisolated only from the sites of inoculation. A carrier state was initiated in a wildebeest inoculated only once, intravaginally. The presence of this virus in the various secretions is a potential source for venereal transmission.     

Malignant transformation of hamster cells following infection with bovine herpesvirus (infectious bovine rhinotracheitis virus.

Hamster embryo cells, following infection with IBR virus, showed malignant transformation. Hamsters of all ages, inbred or random bred, inoculated with two of the transformed cell lines developed solid tumors. Preliminary characterization of the tumors induced by one of the cell lines has indicated undifferentiated sarcomas. Viral specific antigen was detected in about 5% of the transformed cells and 10% of primary tumor cells in culture. Viral specific antibody was detected in the serum of tumor-bearing hamsters by the indirect immunofluorescent method, but no neutralizing antibodies were found. Infectious virus has not been recovered from either the transformed or tumor cells by cocultivation with bovine embryonic kidney cells.