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Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus 总被引:1,自引:0,他引:1
McMenamy MJ McKillen J Reid SM Hjertner B King DP Adair B Allan G 《Journal of virological methods》2011,171(1):219-224
The design and development of a 5′ conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2 × 102 copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2 × 102 to 2 × 108 copies/μl. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease. 相似文献
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Sánchez-Cordón PJ Núñez A Salguero FJ Carrasco L Gómez-Villamandos JC 《Journal of comparative pathology》2005,132(4):249-260
This study characterized the cell-mediated immune response in pigs inoculated with the Alfort 187 isolate of classical swine fever (CSF) virus. Quantitative changes in the T-lymphocyte population (CD3(+), CD4(+) and CD8(+)) and qualitative changes in cytokine expression (IL-2, IL-4 and IFNgamma) by these cells in serum, thymus and spleen were demonstrated. These changes coincided spatially and temporally with previously described quantitative and qualitative changes in monocyte-macrophage populations, thus demonstrating the contribution of the two cell populations to lymphoid depletion. Moreover, examination of cytokine expression in thymus and spleen samples revealed a type 1 cell-mediated immune response in the early and middle stages of the experiment, giving way to a type 2 immune response towards the end of the experiment; these findings, which accorded with the serological results and lymphopenia, may influence the delayed humoral response characteristic of CSF. 相似文献
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Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus 总被引:7,自引:0,他引:7
Reid SM Ferris NP Hutchings GH King DP Alexandersen S 《Journal of virological methods》2004,116(2):169-176
Differential detection of swine vesicular disease virus (SVDV) from the other vesicular disease viruses of foot-and-mouth disease (FMD), vesicular stomatitis (VS) and vesivirus is important as the vesicular lesions produced by these viruses are indistinguishable in pigs. Two independent sets of primers and probe, designed from nucleotide sequences within the 5' untranslated region (UTR) of the SVDV genome, were evaluated in a real-time (5' nuclease probe-based or fluorogenic) PCR format. Although both primers/probe sets failed to detect one isolate, the assays successfully amplified RNA extracted from epithelial suspensions (ES) and cell culture grown virus preparations from clinical samples representing all currently designated phylogenetic groups of SVDV. Furthermore, no cross-reactivity was demonstrated when these primer/probe sets were tested with RNA prepared from all seven serotypes of FMD virus (FMDV) and from selected isolates of VS virus (VSV), vesivirus and teschoviruses. These assays provide sensitive and rapid alternatives to supplement the routine procedures of ELISA and virus isolation for SVDV diagnosis. The two independent sets of primers/probe can be used routinely while only one of the primers/probe sets would typically be used in SVDV diagnosis during an outbreak. 相似文献
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Oem JK Yeh MT McKenna TS Hayes JR Rieder E Giuffre AC Robida JM Lee KN Cho IS Fang X Joo YS Park JH 《Journal of comparative pathology》2008,138(4):204-214
Experimental infection of susceptible cattle and pigs showed that the O/SKR/AS/2002 pig strain of foot-and-mouth disease virus (FMDV) causes an infection that is highly virulent and contagious in pigs but very limited in cattle. Pigs directly inoculated with, or exposed to swine infected with, strain O/SKR/AS/2002 showed typical clinical signs, including gross vesicular lesions in mouth and pedal sites. In addition, FMDV was isolated from, and FMDV genomic RNA was detected in, blood, serum, nasal swabs and oesophageal-pharyngeal (OP) fluid early in the course of infection. Antibodies against the non-structural protein (NSP) 3ABC were detected in both directly inoculated and contact pigs, indicating active virus replication. In contrast, the disease in cattle was atypical. After inoculation, lesions were confined to the infection site. A transient viraemia occurred 1 and 2 days after inoculation, and this was followed by the production of antibodies to NSP 3ABC, indicating subclinical infection. No clinical disease was seen, and no antibodies to NSP 3ABC were present in contact cattle. Additionally, no virus or viral nucleic acid was detected in blood, nasal swab and OP fluid samples from contact cattle. Thus, the virus appeared not to be transmitted from infected cattle to contact cattle. In its behaviour in pigs and cattle, strain O/SKR/AS/2002 resembled the porcinophilic FMDV strain of Cathay origin, O/TAW/97. However, the latter, unlike O/SKR/AS/2002, has reduced ability to grow in bovine-derived cells. The porcinophilic character of O/TAW/97 has been attributed to a deletion in the 3A coding region of the viral genome. However, O/SKR/AS/2002 has an intact 3A coding region. 相似文献
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Reid SM King DP Shaw AE Knowles NJ Hutchings GH Cooper EJ Smith AW Ferris NP 《Journal of virological methods》2007,140(1-2):166-173
Marine caliciviruses form a distinct lineage within the genus Vesivirus (family Caliciviridae). This group includes vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) and other related viruses which have been proposed to be marine in origin isolated from a variety of terrestrial and marine animals. Rapid and reliable detection of marine caliciviruses is important as these viruses appear to be widespread and can cause vesicular disease in a wide variety of susceptible hosts including pigs and experimentally infected cattle where clinical signs cannot be easily distinguished from foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). A real-time RT-PCR assay targeting conserved nucleotide sequences in the RNA-dependent RNA polymerase (3D) region of the genome successfully detected cell culture-grown virus preparations of more than thirty marine calicivirus serotypes. Only the atypical SMSV serotypes 8 and 12 failed to be detected, which provided further indication of genetic divergence between these and the other calicivirus serotypes said to be marine in origin. The real-time RT-PCR assay also specifically amplified RNA from samples collected following experimental inoculation of pigs with VESV. No cross-reactivity was demonstrated when the assay was tested with RNA prepared from representative viruses of FMD, SVD and VS. The real-time RT-PCR assay described is a sensitive and specific tool for detection and differential diagnosis of these viruses from other vesicular-disease causing viruses. 相似文献
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Mengelle C Sandres-Sauné K Miédougé M Mansuy JM Bouquies C Izopet J 《Journal of medical virology》2004,74(3):459-462
Herpes simplex virus infections may be diagnosed by several techniques, including conventional cell culture and the polymerase chain reaction (PCR). This prospective study compares the analytical performances and usefulness of an in-house real-time PCR method and the Light Cycler HSV (1/2) detection kit (Roche Diagnostics, Mannheim, Germany). The results of both PCRs were then compared to those obtained by conventional cell culture. A total of 313 samples were tested (70 dermal samples, 81 cerebrospinal fluids (CSF), 47 ocular, 42 anogenital, 34 throat swabs, and 33 oral samples, 3 whole blood, 2 biopsies, and 1 bronchoalveolar lavage). Samples for molecular assays were extracted twice with the MagNa Pure instrument (Roche Molecular Biochemicals, Mannheim, Germany) and tested blind in parallel by the two PCR methods. Most (226) samples were also examined by cell culture. Forty three samples were found positive by both PCRs, whereas 267 were negative. The HSV-1 and -2 typing of positive samples was identical. Three of the samples were positive in the in-house PCR and negative in the Light Cycler HSV (1/2) detection kit. There was no statistically significant difference between the two tests. Only one sample gave an invalid result due to negative PCR and negative internal control result. Seven samples were positive by both real-time PCRs and negative in conventional culture. The PCRs were significantly (P < 0.05) more sensitive. The results show good agreement between the two real-time PCR methods, with the molecular tests being more sensitive than cell culture. 相似文献
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Christine Laroche Emmanuel B. Drouet Pierre Brousset Christiane Pain Andre Boibieux Francois Biron Josette Icart Gerard A. Denoyel Alain Niveleau 《Journal of medical virology》1995,46(1):66-74
A polymerase chain reaction (PCR) assay for the detection of Epstein-Barr virus (EBV) sequences in various clinical samples, especially peripheral blood leukocytes (PBL) and serum, was carried out and the results obtained were compared with specific EBV serology. One hundred seventy patients were enrolled in the study: 89 healthy blood donors, 22 asymptomatic patients, 36 individuals with primary EBV infection (including 19 patients with infectious mononucleosis [IM]), 22 HIV-infected subjects (including 4 with hairy oral leukoplakia, 3 with central nervous disorders, and 15 with non-Hodgkin's lymphoma). All the serum samples from the healthy blood donors were negative, In patients with IM and in AIDS-non Hodgkin's lymphoma (ARNHL), PCR was strongly positive in leukocytes (>2, 000 genome equivalents/l 04 cells), which was correlated with detectable amounts of EBV DNA in serum. The overall positivity rate of PCR in serum was 58.8%, 68%, and 73% of cases for non-IM primary EBV infections, IM, and ARNHL, respectively. In two cases of EBV primary infection, the viral DNA was detected in serum, respectively 1 month and 2 months before IgM positivity and IgG rise. In one case of ARNHL followed up for several months, PCR (viral load of 2, 000 genome equivalents/104 cells) became positive concurrently with appearance of lymphoma. In immunocompromised individuals, PCR EBV, if carried out in larger prospective studies, could be considered as a tumor marker, useful for predicting EBV-driven lymphoma and follow-up therapy. © 1995 Wiley-Liss, Inc. 相似文献
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Fabbiani M Sali M Di Cristo V Pignataro G Prete V Farina S D'Avino A Manzara S Dal Verme LZ Silveri NG Cauda R Delogu G Fadda G Di Giambenedetto S 《Journal of medical virology》2011,83(12):2057-2065
Since several characteristics of pandemic influenza A (H1N1) virus infection remain to be determined, this study aimed to describe clinical features and complications of patients infected with H1N1. Subjects affected by influenza-like illnesses and a control group of asymptomatic patients were enrolled prospectively at an Emergency Department from October 2009 to April 2010. At enrollment, clinical data and nasopharyngeal swabs for virological analyses were obtained. Ill subjects were followed until recovery and swabs were collected weekly in patients infected with H1N1. Of 318 patients enrolled, 92 (28.9%) were positive to H1N1. Patients infected with H1N1 were mainly young adults and complained classic influenza-like symptoms. Fever was observed for a median time of 5 (IQR 3-7) days. Hospitalization occurred in 27.7% with 2% requiring intensive care unit admission: median length of hospitalization was 6 days (IQR 5-9). Pneumonia was diagnosed in 19.6% of patients. A similar proportion of lower airways involvement and of clinical complications was observed in subjects testing positive or negative for H1N1. However, patients infected with H1N1 were younger and hospitalized for a shorter period as compared to the control group (P = 0.002 and P = 0.045, respectively). Older age, asthma/chronic obstructive pulmonary disease and hypertension were associated with an increased risk of pneumonia. Viral shedding was observed for at least 1 week in 21.3% of patients. Asymptomatic infection was uncommon (1.1%). Respiratory syndromes caused by H1N1 and factors associated with disease severity were investigated and compared to influenza-like illnesses of other origin. Such findings might contribute to improve clinical and epidemiological management of the disease. 相似文献
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To identify the genetic determinants of virulence for swine vesicular disease virus, a panel of recombinant and site-directed mutant viruses were constructed from cDNA clones of a virulent J1′73 strain and an avirulent H/3′76 strain. Initial studies mapped the genetic determinants of virulence to either or both of the two sites at nucleotide (nt) 2842, encoding VP1-132, and nt 3355, encoding 2A-20. To determine their relative importance with regard to virulence, viruses mutated at either of these two sites from the avirulent to the virulent genotype and vice versa were tested in pigs. Viruses, mutated at nt 2842 to the virulent genotype (vSVLS104MJ1) or mutated at nt 3355 to the virulent genotype (vSVLS201MJ1), slightly recovered virulence but were very weak compared with viruses with site-directed mutations at both sites (vSVLS104/201MJ1). On the other hand, viruses, mutated at nt 2842 to the avirulent genotype (vSVLS104M00) or mutated at nt 3355 to the avirulent genotype (vSVLS201M00), did not have attenuated virulence. Sequence analysis of viruses recovered from inoculated pigs revealed that reversion at nt 3355 to the virulent genotype occurred in pigs which had been inoculated with vSVLS201M00. These results suggested that both amino acids determined the virulent phenotype, but that the 2A-20 site might be the major determinant for virulence. 相似文献
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Vesikari T Beran J Durviaux S Stainier I El Idrissi M Walravens K Devaster JM 《Journal of clinical virology》2012,53(1):22-28