Improved isolation of rinderpest virus in transformed bovine T lymphoblast cell lines.

Bovine T lymphoblast cell lines transformed by the protozoan Theileria parva were compared with bovine kidney (BK) and Vero cells for their ability to isolate various strains of rinderpest virus from tissues and infected secretions. All of the strains of rinderpest virus that were tested, including attenuated cell-culture, caprinised and lapinised vaccines, and both mild and virulent pathogenic strains, readily induced syncytial cytopathic effect (cpe) in T lymphoblasts. The cpe could often be detected within one day of inoculation of lymphoblasts, whereas it took three to 14 days to appear in Vero and BK cells. Using lymphoblasts it was possible to reisolate rinderpest virus from nine of 42 swabs collected from three cattle experimentally infected with an isolate from a recent outbreak of mild disease whereas the same swabs yielded only one reisolate on BK cells. It was also possible using the lymphoblasts to detect infectious virus in the ocular, nasal and oral secretions of goats and rabbits infected with caprinised and lapinised virus, respectively. Peste des petits ruminants virus appeared to grow as rapidly as rinderpest virus in the lymphoblasts whereas canine distemper virus readily induced cpe on first passage but less readily on subsequent passage. Measles virus induced relatively little cpe when inoculated into lymphoblasts and did not appear to passage in these cells. The lymphoblasts are easy to maintain in culture and since they rapidly recovered 11 isolates from 37 diagnostic samples could prove useful in laboratories carrying out rinderpest diagnosis.     

Sensitivity and specificity of different methods for the isolation of Salmonella from pigs.

Three different selective enrichment media, Rappaport-Vassiliadis broth (RV), selenite broth (SB) and Müller-Kauffmann tetrathionate broth (MKTB), in combination with plating on modified brilliant green agar (BGA), were compared for the isolation of Salmonella from samples of pig feces. These conventional methods were also compared with a new ELISA kit in conjunction with RV and SB enrichment. Of the conventional methods, enrichment in RV had a higher sensitivity and selectivity than SB and MKTB. Recovery of S. typhimurium from MKTB was significantly poorer than recovery of other serotypes. The combination of RV enrichment and ELISA was as good as the conventional method involving RV enrichment, with a similar high sensitivity and specificity.     

Replication of classical swine fever virus strains and isolates in different porcine cell lines

Classical swine fever virus (CSFV) is an economically important pathogen of domestic pigs and wild boar. Due to the highly variable clinical picture of CSF, laboratory methods are essential for an unambiguous diagnosis. Virus isolation using cell culture is still considered the gold standard. It is based on the incubation of permissive cells with organ or leukocyte preparations followed by antigen detection. In the "EU Diagnostic Manual for CSF Diagnosis", the permanent cell line PK(15) (porcine kidney) is recommended. In the European Reference Laboratory (EURL) a clone of this cell line, PK(15)A, and the STE (swine testicular epitheloid) cell line are in use for propagation of CSFV. The aim of this work was to assess the relative ability of eleven permanent cell lines derived from various organs of wild boar and domestic pig, respectively, to support the replication of different strains and isolates in comparison to these cell lines. An avirulent and a highly virulent laboratory CSFV strain, and several recent field isolates from domestic pigs and wild boars were used. Titers were determined after one, two and three virus passages, and after 48 and 120 h of incubation. Of the eleven cell lines analyzed, two were found that replicated all the tested CSFV strains and field isolates. Those may be useful for improving diagnosis of CSFV and for preparing low-passaged virus stocks of new isolates.     

Sensitivity of seven different types of cell cultures to three serotypes of foot-and-mouth disease virus.

The ability of bovine tongue origin foot-and-mouth disease virus serotypes A, O and C to replicate in seven different types of cell cultures was studied. Primary and secondary calf thyroid cells were equivalent in susceptibility to bovine kidney cell cultures passaged up to five times. Calf thyroid cells lost their susceptibility after two passages. Cryopreserved bovine kidney cell cultures passaged three and four times were equivalent in susceptibility to sensitive calf thyroid and bovine kidney cells. Susceptibility to foot-and-mouth disease virus serotype C was most variable among the cells tested. Lamb testicle and porcine kidney cells were susceptible to foot-and-mouth disease virus while goat and calf testicle and calf lung cells were refractory.     

Alternative cell lines for the propagation of lumpy skin disease virus

In our Institute lumpy skin disease virus is grown on primary lamb testis cells for isolation, identification and vaccine production. However, the availability of lambs in Kenya has been seriously reduced over the past few years. This has led to an increase in the cost of using primary lamb testis cells. This study was undertaken to investigate other primary cell lines, which are easily available and provide an equivalent or better yield of lumpy skin disease virus. Foetal bovine muscle (FBM) cells were found to be an adequate alternative for lamb testis cells.     

鸡传染性喉气管炎病毒适应性细胞的筛选

为了克服鸡传染性喉气管炎弱毒活疫苗产品制备中存在的缺陷,通过对BHK-21细胞、Vero细胞、DF1细胞、原代鸡胚成纤维细胞(CEF)、原代鸡胚肝细胞、原代鸡胚肾细胞等六种细胞进行病毒适应性培养,采用PCR和免疫荧光(IFA)跟踪检测的方法成功筛选出具有稳定病毒滴度的病毒适应性细胞——鸡胚肝细胞。然后采用新的克隆方法缩短了克隆时长并对筛选的鸡胚肝细胞进行克隆纯化,通过与原代鸡胚肝细胞进行比较,结果显示本试验所采用的克隆方法具有高几率的特点,并且能够克隆出高活性的鸡胚肝细胞,并能够繁殖较高效价的鸡传染性喉气管炎病毒,为解决鸡传染性喉气管炎弱毒疫苗在生产上存在的瓶颈问题提供了重要的基础条件。     

动物狂犬病病毒巢式RT-PCR检测方法的建立

以GenBank收录的多基因型狂犬病病毒(RV)N基因序列为参考,设计了简并的PCR引物,建立了能够有效扩增7种基因型RV基因组片段的巢式RT-PCR(nested RT-PCR)方法,命名为RVN371.该方法能够检测到4.6个TCID50的SRV9固定毒,对犬、猪、蝙蝠及牛的狂犬病阳性脑组织样品均表现出良好的特感性:扩增产物目的带位置正确,未见非特异性扩增条带.对比试验表明,RVN371对街毒脑组织样品检测的灵敏度较乳鼠脑内接种试验(MIT)高出100倍;在对3种鼠脑固定毒、12种犬脑街毒样品的检测中,与RV检测的金标方法--免疫荧光试验(FAT)完全符合.RVN371为动物狂犬病的实验室诊断和流行病学研究提供了有利的工具.     

狂犬病快速诊断方法的建立和应用

根据狂犬病病毒核蛋白基因保守区序列设计1对引物，建立了用于狂犬病病毒特异性核酸检测的RT-PCR技术。该技术可从狂犬病病毒CVS株、8202株和SRV9株的含毒细胞培养物及鼠脑组织中。扩增出443bp的核酸片段，检出核酸的敏感性约为3Pg。对30份不同种动物脑组织的检测结果与小鼠脑内接种试验（MIT）的测定结果完全吻合，但前者可在3h内直接对组织匀浆进行诊断，具有快速、简便和敏感的优点。     

狂犬病病毒入胞途径的初步探索

狂犬病病毒(rabies virus,RABV)具有高度嗜神经性,通过识别细胞膜上特异性受体并借助内吞途径侵入细胞。为了探究其入胞途径,本研究在人神经瘤细胞(SH-SY5Y)和非洲绿猴肾上皮细胞(Vero)上,应用氯丙嗪(chlorpromazine)、制霉菌素(nystatin)、巴弗洛霉素a1(bafilomycin a1)、dynasore等抑制剂处理细胞后,进行RABV感染,通过检测病毒N基因拷贝数和RABV效价,对RABV入胞途径进行初步探究。结果显示,RABV通过网格蛋白介导、低pH值和发动蛋白(dynamin)依赖途径侵入SH-SY5Y和Vero细胞,但不通过小窝蛋白依赖途径入胞。这些结果为进一步研究狂犬病病毒感染机制和药物靶点研究提供了新的数据。     

狂犬病诊断基因芯片的建立和检测应用的初步研究

根据已发表的狂犬病病毒（RV）的序列,设计合成能扩增高度保守核（N）蛋白片断的一对引物。通过生物素标记PCR技术,将核（N）蛋白基因片断作为探针点在硝酸素纤维膜上,制作成疾病诊断基因芯片。取160份可疑动物的血液,提取核酸作模板进行PCR扩增,将其产物与诊断基因芯片进行特异性逆向点杂交检测;并用蔗糖密度梯度离心和凝胶层析纯化狂犬病病毒作抗原,建立检测RV抗体的间接ELISA。把以上两种方法应用于临床,结果基因芯片的检测率比ELISA方法和（RT）PCR要高15％左右,表明建立的狂犬病诊断基因芯片具有更高的灵敏度和特异性。     

Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degrees C. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degrees C in 5% CO(2) atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable tool in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied.     

狂犬病弱毒SRV9对街毒暴露小鼠的治疗效果

为评估狂犬病弱毒SRV9对狂犬病街毒暴露小鼠的治疗效果,首先将SRV9通过脑内和肌肉接种3周龄ICR小鼠检测其安全性,然后应用狂犬病街毒接种小鼠,随后在暴露后不同时间用SRV9治疗,研究其保护率.结果显示,SRV9对3周龄ICR小鼠安全,在街毒暴露后1、2、3d,SRV9的保护率分别为70％、60％、30％,而相同暴露时间下单剂量灭活疫苗联合免疫球蛋白的保护率分别是30％、20％、10％.对应用SRV9治疗而成活小鼠的中和抗体检测,发现所有小鼠的抗体水平均达到0.5 IU以上,这些数据初步显示活弱毒SRV9具有用于暴露后治疗的潜力.     

Replication of infectious bursal disease virus in continuous cell lines

Three mammalian continuous cell lines--MA-104, Vero, and BGM-70--were tested for their ability to support replication of infectious bursal disease virus (IBDV). Selected tissue-culture-adapted vaccine strains and tissue-culture-adapted field isolates of IBDV replicated in the MA-104, Vero, and BGM-70 cells; cytopathic effects were most pronounced in the BGM-70 cells. The cytopathic effects of the viruses in BGM-70 cells and chicken embryo fibroblast (CEF) cultures were similar. Virus-neutralization titers of selected serum samples determined in BGM-70 cultures compared well with those obtained from CEF cultures.     

狂犬病病毒LAMP检测方法的建立及初步应用

针对狂犬病病毒基因Ⅰ型核蛋白保守区设计4条识别靶序列上6个位点的特异性引物,在链置换聚合酶(Bst酶)作用下,60℃恒温1 h内完成扩增。反应特异性强、灵敏度高。扩增产物可通过琼脂糖凝胶电泳、显色反应或浊度比较进行判定。通过对反应条件的优化,病毒基因的的最低检出量可达到101拷贝数。该方法可用于狂犬病病毒的实验室检测和临床初步诊断。     

Evaluation of five different methods for routine diagnosis of rabies

Brain tissue from 187 animals of different species was investigated by means of fluorescent antibody test, peroxidase anti-peroxidase technique, mouse inoculation test and cell culture technique for a diagnosis of rabies. With peroxidase anti-peroxidase technique the rabies specific reaction comprised inclusion bodies and a granular staining of the cytoplasm of affected cells. A specific positive reaction was found only in neurons, in which perikaryon as well as cell processes were affected. Fluorescent antibody test and peroxidase anti-peroxidase technique detected 98% each, mouse inoculation test 95% and cell culture technique 81% of the rabies positive animals. In conclusion, peroxidase anti-peroxidase technique allows a highly reliable diagnosis of rabies when only formalin-fixed and paraffin-embedded material is available. Histopathological alterations comprising Negri bodies, inflammatory and degenerative lesions were encountered in 53% of the rabies positive brains.