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Mittelholzer C Johansson I Olsson AK Ronéus M Klingeborn B Belák S 《Journal of virological methods》2006,133(1):48-52
Recovery of infectious Equine arteritis virus (EAV) from the semen of persistently infected Swedish stallions was attempted by classical cell culture isolation and by transfection of extracted total RNA. Whereas virus from semen samples stored for several months at -20 degrees C or from extended semen could only be recovered by transfection of extracted RNA, isolation in cell culture was achieved readily with fresh, unextended semen stored at -70 degrees C or directly used after sampling. In parallel, the viruses were examined in the variable region of the large glycoprotein GP5 by nested RT-PCR and direct nucleotide sequencing. The resulting sequences were placed into a large phylogenetic tree from this region, demonstrating that Swedish strains belonged to very diverse phylogenetic groups. This represents the first report of recovery of infectious EAV from archived semen samples by RNA transfection. 相似文献
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Detection of equine arteritis virus in the semen of carrier stallions by using a sensitive nested PCR assay. 总被引:1,自引:0,他引:1 下载免费PDF全文
S A Gilbert P J Timoney W H McCollum D Deregt 《Journal of clinical microbiology》1997,35(8):2181-2183
A nested PCR, developed for the detection of equine arteritis virus (EAV) in semen, detected less than 2.5 PFU of EAV per ml of naturally infected seminal plasma. Based on results from testing 88 semen samples from 70 stallions, the sensitivity and specificity of the test were 100 and 97%, respectively. 相似文献
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K. Sekiguchi S. Sugita Y. Fukunaga T. Kondo R. Wada M. Kamada S. Yamaguchi 《Archives of virology》1995,140(8):1483-1491
Summary A polymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF 6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1000 times by performing nested PCR enabling the detection of RNA at a level of 0.5–5 PFU. Differentiation among the virus strains and the live vaccine was achieved by cutting the PCR-amplified products from three sets of primers with six restriction endonucleases. Using this procedure it was possible to distinguish among the seven EAV strains used. 相似文献
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C. Malavé Lara M. T. Gorriño C. Campelo P. Lardelli E. Suñen R. Cisterna 《European journal of clinical microbiology & infectious diseases》1992,11(8):740-744
The polymerase chain reaction (PCR) was used to detect hepatitis B virus DNA (HBV-DNA) in serum samples of 104 chronic HBV carriers. Of 22 patients positive for both HBV surface (HBsAg) and HBVe (HBeAg) antigens, seven were positive for HBV-DNA on dot blot hybridisation, and all 22 positive in the PCR. Of 41 HBsAg positive patients who had antibodies against HBeAg (anti-HBe), only three were positive for DNA-HBV on dot blot hybridisation, however DNA was detected in 30 of them with the PCR. Similarly, of 41 individuals with antibodies against HBsAg (anti-HBs), 23 yielded positive results in the PCR technique, although dot blot hybridisation detected HBV-DNA in only one patient. These results indicate that while serological and conventional DNA hybridisation assays are not sensitive enough to determine the infectivity of HBV chronic carriers, PCR is an accurate method for establishing the status and progression of disease in these patients. 相似文献
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U B Balasuriya E J Snijder L C van Dinten H W Heidner W D Wilson J F Hedges P J Hullinger N J MacLachlan 《Virology》1999,260(1):201-208
Virus derived from an infectious cDNA clone of equine arteritis virus (EAV030H) was intranasally inoculated into two stallions, neither of which subsequently developed clinical manifestations of equine viral arteritis (EVA). Virus was isolated from nasal swabs and mononuclear cells collected from both stallions =14 days p.i. and from the semen of one stallion only at 7 days p.i. Similarly, viral RNA was detected by RT nested-PCR in nasal swabs and mononuclear cells for =14 days p.i. and at 7 days p.i. in the semen of the one stallion. Both stallions seroconverted to EAV by 10 days p.i. and maintained high neutralizing antibody titers thereafter. Sequence and restriction digestion analysis demonstrated that the recombinant virus present in nasal swabs, mononuclear cells, and semen from the two stallions was identical to the infectious clone-derived virus that was used to inoculate them. Furthermore analysis of multiple clones derived by RT nested-PCR amplification from several samples indicated that the recombinant EAV030H virus was stable during replication in horses. These studies document for the first time that a recombinant virus derived from an infectious cDNA clone of a member of the order Nidovirales is replication competent in animals, and the genetic stability of the recombinant virus during in vivo replication indicates that it will be useful for the characterization of genetic determinants of virulence and persistence of EAV. The genetic conservation of the cloned recombinant virus during in vivo infection is similar to that which occurs during natural horizontal and vertical transmission of EAV in horses and contrasts with the heterogeneous virus population (quasispecies) that occurs in the semen of carrier stallions. 相似文献
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Mankoc S Hostnik P Grom J Toplak I Klobucar I Kosec M Barlic-Maganja D 《Journal of virological methods》2007,146(1-2):341-354
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Sexual and in-contact transmission of asinine strain of equine arteritis virus among donkeys. 总被引:1,自引:0,他引:1 下载免费PDF全文
J T Paweska D H Volkmann B J Barnard E D Chirnside 《Journal of clinical microbiology》1995,33(12):3296-3299
Two in a group of five naturally seropositive donkey stallions were found to shed equine arteritis virus (EAV) in their semen as demonstrated by virus isolation. Direct intramuscular inoculation of sonicated semen from one virus-shedding stallion (S3) caused clinical disease in two donkeys from which virus was recovered and in which seroconversion was detected. Sexual transmission was confirmed in two mares mated to S3 when after a febrile response during which EAV was isolated from huffy coats and nasal and ocular exudates, both mares were found to have seroconverted. In-contact transmission in a susceptible stallion was demonstrated after its exposure to a sexually infected mare. The 3' end of the asinine virus was amplified directly from donkey semen with EAV-specific primers, and its nucleotide sequence was found to be homologous to that of the prototype Bucyrus virus isolated from horses. These results indicate that EAV and its disease transmission are analogous in donkeys and horses. 相似文献
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Widespread presence of cytomegalovirus DNA in tissues of healthy trauma victims. 总被引:1,自引:0,他引:1 下载免费PDF全文
AIMS: To determine the localisation of human cytomegalovirus (CMV) DNA in abdominal aorta, spleen, and transplantable organs, such as kidney, pancreas, and liver, obtained from healthy individuals; to characterise the cell type(s) in these tissues that serve as a reservoir for latent CMV. METHODS: CMV DNA was detected by dot blot DNA hybridisation and in situ DNA hybridisation with a probe for CMV major immediate early sequences (UL123) and nested PCR with primers derived from the CMV major immediate early (IE) gene exon 4 (UL123ex4). Samples of liver, abdominal aorta, spleen, kidney, and pancreas were obtained at necropsy or from donor kidneys from healthy subjects. RESULTS: CMV DNA was detected in most tissue samples using dot blot hybridisation and nested PCR. In situ hybridisation demonstrated that, in addition to smooth muscle cells in the arterial wall, hepatocytes, tubular and glomerular kidney cells, splenic red pulp cells, and pancreatic acinar cells also harboured CMV DNA. CMV DNA was detected in seropositive and in some seronegative subjects. CONCLUSION: CMV DNA is widely distributed in organs of healthy subjects. CMV DNA was found in various cell types in several organs, suggesting that during latency, CMV DNA is present thoughout the body. 相似文献
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Smits CB Van Maanen C Glas RD De Gee AL Dijkstrab T Van Oirschot JT Rijsewijk FA 《Journal of virological methods》2000,85(1-2):65-73
Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection. 相似文献