牛病毒性腹泻-粘膜病病毒TaqMan荧光定量RT-PCR检测方法的建立及初步应用

为建立-种牛病毒性腹泻-粘膜病病毒（BVDV）的快速检测方法,根据GenBank中登录的BVDV基因序列,针对5’UTR的保守序列,设计合成了一对特异性引物和一条TaqMan荧光探针。通过对反应条件和反应体系进行优化,建立了一种能快速定量检测BVDV的荧光定量RT—PCR检测方法。通过对20份胎牛血清样品和猪瘟细胞苗半成品进行检测,对该方法的特异性、敏感性和重复性进行试验。结果显示,建立的方法能检测到BVDV,而对CSFV、PRRSV和MDBK细胞的扩增结果均为阴性,具有高度的特异性。在所检测的20份样品中,BVDV阳性6份（30％）。对所构建的标准品进行检测,在10^2-10^8拷贝／μL的范围内可以得到良好的动力学曲线,能检测低至10^3-10^4拷贝／mL的病毒量。表明所建立的检测方法具有快速、特异、灵敏、重复性好等特点,可用于临床及科研中对BVDV的快速定量检测和对牛血清及猪瘟兔化弱毒疫苗等生物制品中是否污染BVDV进行监测。     

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。     

First finding of genetic and antigenic diversity in 1b-BVDV isolates from Argentina

Infection with Bovine Viral Diarrhea Viruses (BVDV) in cattle results in a wide range of clinical manifestations, ranging from mild respiratory disease to fetal death and mucosal disease, depending on the virulence of the virus and the immune and reproductive status of the host. In this study 30 Argentinean BVDV isolates were characterized by phylogenetic analysis. The isolates were genotyped based on comparison of the 5′ untranslated region (5′ UTR) and the E2 gene. In both phylogenetic trees, 76% of the viruses were assigned to BVDV 1b, whereas BVDV 1a, 2a and 2b were also found. Eight of the BVDV 1b isolates were further characterized by cross-neutralization tests using guinea pig antisera and sera from bovines vaccinated with two different commercial vaccines. The results demonstrated the presence of a marked antigenic diversity among Argentinean BVDV isolates and suggest the need to incorporate BVDV 1b isolates in diagnostic strategies.     

The diagnosis of enzootic bovine leukosis

This paper reviews the clinical and virological diagnostic procedures for enzootic bovine leukosis (EBL). The clinical diagnosis must be always confirmed by a specific laboratory test for Bovine Leukaemia Virus (BLV). Many virological tests were proposed. The sensitivity of all the diagnostic methods is sufficient to do an early detection of a BLV infection on an individual base. Advantages of the highly sensitive methods like RIA and ELISA appear when the samples to be tested have naturally very low antibody titers (individual milk, bulk milk, pooled sera).     

History of the control of foot and mouth disease

From the many existing documents on the history of foot and mouth disease, it is possible to describe the practical measures adopted for disease surveillance and control from ancient times until the 20th century.

Surveillance was based on diagnosis or post-mortem examination, and also on knowledge of the conditions under which infection occurred: aetiology, pathogenesis, mode of infection, susceptible species, virulent material, etc. The historical facts are assembled and compared, with comments on each of these points.

Control was based upon the application of isolation, then slaughter or aphtisation, then vaccination. A study of these various procedures makes it possible to compare their efficacy.

Résumé

D'après les nombreux documents existant sur l'histoire de la fièvre aphteuse il est possible de décrire les mesures adoptées pour suivre l'évolution de la maladie et la maîtriser, depuis l'Antiquité jusqu'au XXème siècle.

La surveillance de la maladie était fondée sur le diagnostic clinique ou l'examen des lésions, mais aussi sur la connaissance des condition de l'infection: étiologie, pathogénie, mode de transmission du contage, matières virulentes, etc. Les faits historiques concernant ces différents points sont rassemblés, comparés et commentés.

La maîtrise de la fièvre aphteuse a été assurée soit par des mesures d'isolement, puis d'abattage sanitaire, soit par des opérations d'aphtisation puis de vaccination. L'étude de ces différentes stratégies permet de comparer leur efficacité respective.     

Comparison of three serotests for the detection of pseudorabies antibodies in pigs

The serum-neutralization test (SN), enzyme-linked immunosorbent assay (ELISA) and the radial immunodiffusion enzyme assay (RIDEA) were compared for the detection of pseudorabies (PRV) antibodies in swine sera. A total of 1285 serum samples were tested. All three tests were considered useful in determining the PRV antibody status of swine on a herd basis, but available evidence supports the continued use of SN as the definitive test because of possible false positive reactions associated with ELISA and RIDEA.     

Effects of pseudorabies virus infection upon cytotoxicity and antiviral activities of porcine alveolar macrophages

Alveolar macrophages (AM) infected with Pseudorabies virus (PRV) were compared to noninfected AM for cytotoxicity against foreign or transformed cells and production of interferon (IFN). Five PRV strains were used to infect AM including strains that are known to be highly virulent for pigs, i.e. strain 4892 and strain S-62 as well as strains that are regarded as mild or nonvirulent, i.e. BUK and Bartha. The multiplicity of infection ranged from 0.005 to 0.05 TCID₅₀/cell. The target cells in the cytotoxicity assays were either chicken red blood cells, PRV-infected vero cells, or human myeloblastoma cells (K562 cell line). For the producton of IFN, AM cultures were treated with polyinosinic: polycytidylic acid (Poly I:C) diluted in tissue culture media at a concentration of 5 μg/10⁶ cells. Culture supernatants were collected at various times poststimulation and tested for antiviral activity using the Vesicular Stomatitis Virus replication inhibition test. Swine AM were able to lyse chicken red blood cells in an antibody-independent way but not in an antibody-dependent way, whereas lysis of PRV-infected vero cells was accomplished both ways. The cytotoxicity against chicken red blood cells was reduced in the PRV-infected AM as compared to noninfected cells, particularly in AM infected with virulent PRV strains. Specific ⁵¹Cr release values for AM infected with S-62 and 4892 strains were 14 and 19, while the noninfected AM had values of 36. Similarly, in the antibody-dependent cytotoxicity assay against PRV-infected vero cells there was no activity of AM against K562 cells. The production of IFN was readily stimulated with Poly I:C. The optimal time for supernatant collection was between 12 and 16h poststimulation. The antiviral activity was abrogated by treatment of the supernatant with antiserum against human leukocyte IFN; it was therefore considered to be due to interferon-alpha (IFN) released from the macrophages. The antiviral activity present in supernatants of PRV-infected AM was reduced compared to noninfected AM. The difference between AM cultures infected with virulent strains of PRV and noninfected AM cultures was statistically significant at P 0.025. The results provide support to the premise that the role of AM in lung defense can be compromised by PRV infection.     

The pathogenesis and immunology of bluetongue virus infection of ruminants

Bluetongue (BLU) virus is transmitted from infected to susceptible ruminants by hematophagous vector midges (Culicoides species). Cattle are important reservoir hosts of the virus because infection typically is asymptomatic and characterized by prolonged cell associated viremia, and because at least some species of insect vector preferentially feed on cattle. Interaction of BLU virus with the cell membrane of erythrocytes in infected cattle likely facilitates both prolonged viremia as well as infection of the insect vector. BLU disease is most common in sheep and some wildlife species. A variety of host, agent and environmental factors clearly can influence expression of disease in these species. The pathogenesis of BLU virus infection of cattle and sheep is remarkably similar, thus the basis for expression of disease in sheep but not cattle remains to be firmly established. Some difference in susceptibility of endothelial cells to infection in the two species is one potential explanation.

Ruminants develop a variety of antiviral responses after BLU virus infection. Antibodies to outer capsid protein VP2 are responsible for virus neutralization, and confer resistance to reinfection with the homologous serotype of BLU virus. Antibodies to epitopes on proteins which are common to all viruses of the BLU serogroup form the basis of current diagnostic serologic tests. Cell mediated responses have been incompletely characterized, in part because BLU virus replicates within dividing lymphocytes and virus-mediated cytolysis inhibits in vitro blastogenesis. Immunological competence of ruminants to BLU virus arises prior to midgestation, and suggestions that persistent immune tolerant BLU virus infection occurs after in utero exposure of cattle have not been substantiated and are not consistent with recent findings.     

Effects in calves of mixed infections with bovine viral diarrhea virus and several other bovine viruses

The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form of mucosal disease being produced.

Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection of calves with BVDV and PI-3 virus or BVDV and IBR virus.

From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis of the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus.     

Foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD.

Résumé

Le virus de la fièvre aphteuse est un aphtovirus de la famille des Picornaviridae et l'agent de la maladie animale la plus importante sur le plan économique. En tant que picornavirus typique, le virus de la fièvre aphteuse est nu, sous forme d'icosaèdre et son génome comprend un acide ribonucléique monobrin avec environ 8500 nucléotides et une polarité positive. L'acide ribonucléique de ce virus est infectieux et il se réplique par l'intermédiaire d'un brin d'ARN moins, complémentaire. La réplication de l'acide nucléique de ce virus conduit à des erreurs, de telle sorte que les populations virales comprennent un ensemble de mutants (quasi espèce) plutôt qu'une séquence génomique bien définie. Par suite, le virus de la fièvre aphteuse est génétiquement et antigéniquement varié. Ceci entraîne des difficultés importantes pour le diagnostic, la prévention et la maîtrise de la fièvre aphteuse. Une connaissance plus approfondie de la complexité et de l'évolution de la population de ce virus a conduit à des besoins pour une nouvelle génération de vaccines aphteux. Ceci est lié au débat actuel sur le choix d'une politique de vaccination ou de non-vaccination dans la lutte contre la fièvre aphteuse.     

Effect of administration of anti-bursal extract or perfusate serum on the antibody production in chickens

Antisera to bursal extracts or perfusates were prepared and the influence of such sera on antibody production in chickens was investigated by the injection of antisera during the embryonic stage. Antisera to cyclophosphamide treated bursal extracts or bursal perfusates were injected on the 15th day of embryogenesis. The level of antibodies produced by chickens treated by these antisera was equal to the controls but IgG antibodies were totally absent. These results suggested that the administration of these antisera inhibited the differentiation of IgM antibody producing cells.