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1.
Wild rabies virus detection by plaque assay from naturally infected brains in different species 总被引：1，自引：0，他引：1
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. 相似文献
2.
Transmissible gastroenteritis (TGE) virus was detected in house flies (Musca domestica Linneaus) by staining with specific fluorescent antibody. The flies were collected within a swine confinement facility in which TGE was enzootic. Laboratory-reared flies were infected experimentally with TGE virus and the virus was recovered from the insects for 72 hours after infection. The TGE virus was identified both by the fluorescent antibody technique and by isolation in cell culture. The nature of plaque formation in cell monolayers inoculated with the virus passaged through flies changed from a large plaque (4 mm or greater in diameter) to a small plaque (1 mm in diameter) over the period. Large plaques were observed early after infection and were attributed to TGE virus mechanically carried by the flies. Small plaques occurred 8 to 12 hours after infection and were considered to be produced by virus replicated in the dipterous cell. 相似文献
3.
Specific pathogen free cats were infected by two feline calicivirus isolates of different plaque type, an extra-large plaque (ep) former and a minute plaque (mp) former. A comparison was made of the disease produced when these isolates were administered by either aerosol or direct intranasal instillation. With both isolates, aerosol infection produced lesions and gave rise to virus replication throughout the respiratory tract. The effects of intranasal infection were more confined to the upper respiratory tract and oropharynx. By both routes of infection the disease produced by the mp virus was clinically and pathologically less severe than that produced by the ep virus. 相似文献
4.
采用PCR方法对猪场猪瘟病毒、猪繁殖障碍与呼吸道综合征病毒、猪圆环病毒2型混合感染的诊断研究 总被引：6，自引：4，他引：6
采用猪瘟-猪繁殖障碍与呼吸道综合征病毒(CSFV-PRRSV)多重PCR诊断试剂盒和猪圆环病毒2 型(PCV 2)PCR诊断试剂盒对来自河南省不同地区的6个规模猪场送检的60份临床疑似病料进行了病原学PCR检测。结果表明：60份样品中CSFV、PRRSV、PCV-2感染的阳性率分别为38.3%、30%和43.3%；3种病毒共感染样品数目占所有样品的13.3%，CSFV PCV-2双感染的比例最高，达到31.7%，PRRSV-PCV-2双感染比例为18.3%，而CSFV-PRRSV双感染比例只有15%。自感染猪内脏器官和血清中均能成功检测出病毒。本研究结果表明PCR诊断可作为临床上这3种病的病原学快速、灵敏的诊断方法，并为猪场猪瘟、猪繁殖障碍与呼吸道综合征、猪圆环病毒2型3种病的流行病学和检测方法的研究奠定了一定的基础。 相似文献
5.
It is poorly understood why vaccines could not be developed for the control and prevention of African swine fever (ASF) virus infection. The aim of our study was to identify genes non-essential for ASF virus replication because there were indications that certain viral gene products, which apparently are non-essential for viral replication, conferred protection from death due to ASF. A cosmid library representing the genome of ASF virus strain France 64 was established and characterized. Then, in order to inactivate viral genes by insertion, the beta-galactosidase (beta-gal) gene was introduced either randomly or at specific locations of selected cloned DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted viral strain in order to allow the generation of recombinant progeny virus. Viable recombinant progeny was identified by at least one of the following means: (1) expression of beta-gal; (2) detection of beta-gal specific DNA by plaque hybridization, and (3) absence of a functional product of the inactivated gene. Presently, we are characterizing a recombinant virus with an insertionally inactivated thymidine kinase gene. 相似文献
6.
A plaque assay method was developed for Teschen disease virus using primary pig kidney cultures in which neutral red stain was incorporated in the agar overlay medium. Certain factors associated with the plaque-forming ability of the virus were standardized. Cultures allowed to adsorb the virus for 45-60 minutes at 28 and 37 C, gave maximal plaque count. The virus infected the cellular layer through the agar medium. Plating efficiency of the virus was enhanced by using a small volume of inoculum. Addition of serum to the medium was not required. Neutral red concentration in the agar overlay medium affected the number of plaques. Statistical analysis demonstrated the uniformity of the plaque counts and their linear relationship to virus concentration. Isolation of the virus by the plaque technique from infected spinal cord supernatant fluid was achieved. 相似文献
7.
A rapid, sensitive and specific semi-nested RT-PCR was developed to detect and differentiate virulent and avirulent strains of Newcastle disease virus (NDV). For a total of 67 NDV strains, the results obtained from the semi-nested RT-PCR were consistent with those from nucleotide sequence analysis, plaque forming assays, mean death time (MDT) measurements and intracerebral pathogenicity index (ICPI). Furthermore, 13 class I NDV strains can be characterized by the semi-nested RT-PCR approach, which was feasible by the conventional methods. The detection limit for the semi-nested RT-PCR was two plaque forming units (PFU) both for NDV strain F48E9 in allantoic fluid and for isolate APMV1/ch/ChinaND4031 in oral or cloacal swabs. In conclusion, this semi-nested RT-PCR method offers a new assay for the rapid detection and differentiation of NDVs. 相似文献
8.
A continuous bovine kidney cell line for routine assays of foot-and-mouth disease virus 总被引：4，自引：0，他引：4
L M Swaney 《Veterinary microbiology》1988,18(1):1-14
A continuous bovine kidney cell line, LF-BK, arose from primary bovine calf kidney cells that survived infection with a temperature-sensitive mutant of foot-and-mouth disease virus. No virus was recovered after the first passage. Cells of Passage 48 were inoculated into two steers which remained healthy and did not develop neutralizing antibodies to the virus. The karyotype of cells of the 53rd and 87th passages was similar and revealed that the cells were markedly transformed. The modal number of diploid chromosomes was 52 at both passage levels. LF-BK cells and primary bovine kidney cells were equally susceptible in plaque assays to each of the 7 types of foot-and-mouth disease virus. The cell line and primary bovine kidney cells were less susceptible than primary bovine thyroid cells to several subtypes of the virus in suspensions of tongue epithelium. The LF-BK continuous cell line is recommended for routine plaque assays or plaque neutralization tests as a substitute for primary bovine kidney cells. 相似文献
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10.
基于E184L基因的非洲猪瘟病毒实时荧光定量PCR检测方法的建立 总被引：1，自引：0，他引：1
非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)感染引起的一种高度接触性传染病。由于ASFV的感染机制极为复杂,基因型多,至今尚无有效疫苗用于防控,防止该病暴发主要依赖于早期快速诊断和控制。为建立一种高效快速、特异的ASFV检测方法,根据ASFV的E184L基因序列,设计了TaqMan荧光定量PCR引物及探针,建立了检测ASFV的TaqMan荧光定量PCR方法。结果表明,该方法设计的引物具有高度特异性,以构建的重组质粒为标准品建立的TaqMan荧光定量PCR方法的标准曲线具有良好的线性关系,线性相关系数为0.992,对ASFV核酸最低检测限为1.51拷贝,且与伪狂犬病病毒、猪细小病毒、猪圆环病毒2型等不存在交叉反应。建立的基于ASFV E184L基因实时荧光定量PCR检测方法能够快速、准确、特异地对ASFV核酸进行定量分析,丰富了ASFV的检测方法。 相似文献
11.
为分析使用棉绳采集口腔液监测猪场伪狂犬病（PR）方法的科学性,本研究拟通过实验室水平、动物试验及临床应用3个方面对使用棉绳采集口腔液监测猪场PR这一方法开展应用分析。首先对采样条件进行优化,并通过模拟试验测定棉绳对伪狂犬病病毒（PRV）的释放能力,通过动物试验测定病毒感染后检出时间,最后对临床样品进行检测分析。结果表明：使用直径为1.0 cm的棉绳,在早上喂料前采集口腔液样品,采样时间为20~30 min,可采集到更能满足试验要求的口腔液。病毒释放能力测定显示,棉绳对PRV的释放能力为50%左右,使用棉绳采集口腔液可检测到1个TCID50/0.1 mL的病毒含量。动物试验检测发现,猪群在感染后28 d中除第5天外,口腔液中病原含量均高于鼻拭子中病原含量,口腔液检测效果优于鼻拭子,且口腔液中病毒的检出时间（感染后第1天）早于血液中抗体转阳时间（感染后第7天）。临床样品检测分析结果表明,PRV疫苗免疫后也可通过口腔液检测到疫苗毒,并且存在无法通过口腔液检测感染PRV野毒后稳定猪中带毒的情况,因此口腔液检测方法应结合gE抗体检测,才可综合判断猪群是否为感染群体。综上,本研究优化了口腔液采集方法,并测定了棉绳对口腔液的释放能力和感染后检出时间,表明口腔液可作为较好的监测猪群PR的手段;口腔液监测方法需结合gE抗体检测来综合判断猪群是否为感染群体。 相似文献
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In vitro detection of equine arteritis virus from seminal plasma for identification of carrier stallions 总被引：1，自引：0，他引：1
Fukunaga Y Wada R Sugita S Fujita Y Nambo Y Imagawa H Kanemaru T Kamada M Komatsu N Akashi H 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2000,62(6):643-646
Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10(2.7) PFU/ml. In vitro detection of EAV by virus isolation supplemented with RT-PCR using seminal plasma was proved to be an effective alternative to the standard test mating as a diagnostic method for carrier stallions. 相似文献
14.
B W Parry S A Holloway M J Studdert 《Veterinary Clinics of North America: Small Animal Practice》1989,19(4):719-727
Feline leukemia virus is an oncogenic retrovirus that can result in a wide variety of neoplastic and non-neoplastic diseases, including immunosuppression. Diagnosis of FeLV infection can be achieved by several methods, including virus isolation; IFA assay of a peripheral blood smear; and detection of a viral protein (called p27) by ELISA testing of whole blood, plasma, serum, saliva, or tears. Commercially available ELISA kits have revolutionized FeLV testing and have become very popular as "in-house" procedures. This article discusses the interpretation of ELISA results and compares them with IFA assay findings. Feline immunodeficiency virus is a lentivirus that causes immunosuppression, but not neoplasia, in cats. It originally was called feline T-lymphotropic lentivirus. Differentiating FIV infection from the immunosuppressive type of FeLV infection requires virus isolation or serology. The most rapid method for diagnosis of FIV infection is ELISA testing for antiviral antibody. 相似文献
15.
Sheep were inoculated with 2 ml of 10(7) plaque forming units per ml of purified prototypes of the four United States serotypes (10, 11, 13 and 17) of bluetongue virus. Nine weeks following the initial inoculation, a challenge inoculation with homologous virus was done. Animals were followed for virus isolation and evidence of cell-mediated immunity by weekly lymphocyte stimulation tests (LST). Two dilutions (10 micrograms/ml and 1 microgram/ml) of pure virus from each of the purified serotypes were used as antigen as were the phytomitogens phytohemagglutinin, Concanavalin A, and pokeweed mitogen. LST data were analyzed by the analysis of variance method and reported as counts per minute and stimulation index (SI). Significant SI were observed following primary and secondary challenge with both homologous and heterologous virus. There was evidence of lymphocyte perturbations characterized by a sharp decrease in response to mitogens following primary and secondary challenge lasting for one week followed by a significant increase in blastogenesis three to four weeks after inoculation of virus. These results provide evidence that cell-mediated immunity is evident in bluetongue infection, that there is cross reactivity between viral serotypes and that BTV infection leads to perturbations in lymphocyte function including suppression of responses. An increase in the blastogenic response to phytomitogens correlated with viral clearance. 相似文献
16.
Fetuses and placental tissues were taken from pregnant ewes at intervals varying between eight and 21 days after inoculation with tissue suspensions from cases of Border disease. Virus isolation procedures involving the detection of a cytopathic effect in tissue cultures with or without interference tests produced universally negative results but interference tests, using a plaque technique with the NADL strain of bovine virus diarrhoea virus as a challenge virus, detected the presence of an agent in tissues from six out of 10 fetuses. Inoculated ewes allowed to proceed to term showed a serological response characteristic of Border disease infection, as measured by four different tests. Although hairy shaker lambs were not seen, the occurence of abortion and stillbirth due to causes other than bacterial agents, was an indication that the Border disease agent was present. Electron microscopy of fetal fluids failed to detect viral particles. 相似文献
17.
Immunohistochemical detection of swine influenza A virus in formalin-fixed and paraffin-embedded tissues. 
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下载免费PDF全文 An avidin-biotin complex (ABC) immunohistochemical method utilizing a commercially-available polyclonal antiserum to human influenza A virus was used to detect antigens of influenza A virus in formalin-fixed, paraffin-embedded tissues of swine. Influenza A antigens were immunohistochemically detected in 28/30 cases in which influenza A virus was demonstrated by virus isolation and in 5/22 cases suspected to be influenza A-infected by clinical and histological criteria, but from which the virus was not isolated. Viral antigen was not demonstrated in 30/30 cases not suspected clinically or histologically to be associated with influenza infection. This method is a convenient, sensitive, and specific means of influenza A virus detection and is applicable to both routine diagnosis of influenza A virus infection and to retrospective and prospective studies of the occurrence and the pathogenesis of this virus in pigs. 相似文献
18.
为分析使用棉绳采集口腔液监测猪场伪狂犬病(PR)方法的科学性,本研究拟通过实验室水平、动物试验及临床应用3个方面对使用棉绳采集口腔液监测猪场PR这一方法开展应用分析。首先对采样条件进行优化,并通过模拟试验测定棉绳对伪狂犬病病毒(PRV)的释放能力,通过动物试验测定病毒感染后检出时间,最后对临床样品进行检测分析。结果表明:使用直径为1.0 cm的棉绳,在早上喂料前采集口腔液样品,采样时间为20~30 min,可采集到更能满足试验要求的口腔液。病毒释放能力测定显示,棉绳对PRV的释放能力为50%左右,使用棉绳采集口腔液可检测到1个TCID50/0.1 mL的病毒含量。动物试验检测发现,猪群在感染后28 d中除第5天外,口腔液中病原含量均高于鼻拭子中病原含量,口腔液检测效果优于鼻拭子,且口腔液中病毒的检出时间(感染后第1天)早于血液中抗体转阳时间(感染后第7天)。临床样品检测分析结果表明,PRV疫苗免疫后也可通过口腔液检测到疫苗毒,并且存在无法通过口腔液检测感染PRV野毒后稳定猪中带毒的情况,因此口腔液检测方法应结合gE抗体检测,才可综合判断猪群是否为感染群体。综上,本研究优化了口腔液采集方法,并测定了棉绳对口腔液的释放能力和感染后检出时间,表明口腔液可作为较好的监测猪群PR的手段;口腔液监测方法需结合gE抗体检测来综合判断猪群是否为感染群体。 相似文献
19.
因马铃薯在栽培中容易受各种病毒混合侵染,对马铃薯产量或品质等个方面产生巨大的影响.为了确保种薯的质量,准确、快速的进行马铃薯病毒检测,成为马铃薯生产的关键。马铃薯病毒检测的方法有很多,基本上可以划分为生物学实验方法(指示植物法)、电子显微镜技术、免疫学方法和分子生物学检测方法几大类。本文分析了马铃薯有害生物风险分析及病毒检测技术问题。 相似文献
20.
John S. Munday Kristen A. Willis Matti Kiupel Fraser I. Hill Magdalena Dunowska 《Veterinary dermatology》2008,19(6):400-404
A 3‐year‐old cat from New Zealand developed three small raised non‐ulcerated plaques on the face. Serology detected antibodies against feline immunodeficiency virus (FIV). Histology of the plaque revealed epidermal hyperplasia with keratinocytes either distended with large blue‐grey cytoplasmic bodies or with shrunken nuclei surrounded by a clear halo. Papillomavirus (PV) antigen was detected immunohistochemically and feline viral plaque was diagnosed. Swabs were taken of both lesional and non‐lesional skin, and polymerase chain reactions were used to detect PV DNA. Three different PV DNA sequences were amplified, one from a Felis domesticus PV type 1 (FdPV‐1) previously amplified from a feline viral plaque, a second (FdPV‐JM) previously amplified from feline cutaneous squamous cell carcinomas, and a third FdPV‐MY that was not reported previously. All three sequences were amplified from swabs of both lesional and non‐lesional skin. These results extend the geographical range of FdPV‐1 outside North America and also demonstrate the ability of FdPV‐1 to asymptomatically infect feline skin. However, the detection of multiple PV sequences within both lesional and non‐lesional samples makes it difficult to determine whether or not any of the PVs caused feline viral plaque development in this cat. This is the first time PV DNA has been detected in a feline skin swab sample. Additionally, it is the first report of multiple PVs being detected in a single sample from a cat. This may suggest that FIV infection predisposes cats to cutaneous PV infection. 相似文献