Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus

A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.     

Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR

Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.     

Development of a one step real time RT-PCR assay to detect and quantify Dugbe virus

A one-step real time quantitative RT-PCR (qRT-PCR) assay was developed to detect all published Dugbe virus (DUGV) genomes of the Nairovirus genus. Primers and probes were designed to detect specific sequences on the most conserved regions of the S segment. The limit of detection of the assay was 10 copies per reaction which is an improvement of 3 log(10)FFU/mL over the sensitivity of conventional RT-PCR. The specificity of the primers and probe was confirmed with the closely related Nairoviruses CCHFV and Hazara virus, and on the non-related viruses Coronavirus and Influenza A virus. This qRT-PCR assay was used to screen nucleic acids extracted from 498 ticks collected in the Republic of Chad. One sample was found positive suggesting that DUGV is present in this part of the world. The molecular assay developed in this study is sensitive, specific and rapid and can be used for research and epidemiological studies.     

Real-time RT-PCR assay for rapid and specific detection of classical swine fever virus: comparison of SYBR Green and TaqMan MGB detection methods using novel MGB probes

Real-time PCR is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. The basic principle is the recurring measurement of a fluorescent signal, which is proportional to the amount of amplification product. In our trial two detection systems were tested for classical swine fever virus (CSFV) detection and for its discrimination from other pestiviruses; non-specific dsDNA-binding dye SYBR Green and specific fluorogenic TaqMan MGB probes. Real-time RT-PCR assays were evaluated for diagnostic sensitivity and specificity by different pestiviral reference and field strains. With both approaches, SYBR Green and TaqMan probes, respectively, all of the CSFV strains isolated on cell culture were detected and also clearly distinguished from other pestiviruses. However, the established one-step real-time TaqMan RT-PCR assay was shown to be more appropriate for pestivirus quantitation, it reduces the risk of contamination and is less time consuming.     

Validation of two commercial real-time RT-PCR kits for rapid and specific diagnosis of classical swine fever virus

Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, "pestivirus specificity", reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.     

Detection of classical swine fever virus in the ovaries of experimentally infected sows

Six sows were infected intranasally with a Korean isolate of classical swine fever virus (CSFV). The distribution of virus in ovarian tissues was then assessed for 21 days by in-situ hybridization and immunohistochemistry. CSFV was detected in the ovaries between 7 and 21 days post-inoculation (dpi) by both methods, but the labelling was particularly intense and widespread at 7 dpi. CSFV nucleic acid and antigen were located almost exclusively within the cytoplasm of cells shown by haematoxylin and eosin staining to be macrophages, which were numerous in atretic follicles. Small numbers of CSFV nucleic acid-positive cells with distinctly round morphology and oval nuclei, resembling monocytes, were also observed in the blood vessels of sows at 7 and 14 dpi. CSFV nucleic acid and antigen were not observed in primordial, primary or secondary follicles from infected sows at 7, 14 or 21 dpi. The results suggest that CSFV replicates in circulating peripheral monocytes and gains access to ovarian tissues from the bloodstream, and that this contributes to the distribution of CSFV in macrophages throughout the atretic follicles.     

Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus

Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF.     

Phylogenetic analysis of classical swine fever virus in Taiwan

Summary. Two envelope glycoprotein (E^rns and E2) regions of the classical swine fever virus (CSFV) were amplified by RT-PCR and sequenced directly from 158 specimens collected between 1989 and 2003 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and the E^rns region provided better discrimination than the E2 region. One hundred and fifteen isolates out of the 158 isolates were clustered within subgroup 2.1 (further classified as 2.1a and 2.1b) and 2.2, which were considered to be likely of the introduced strains, whereas the remaining 43 isolates were clustered within subgroup 3.4 and were considered to be of the endemic strains. The subgroup 2.1a viruses were first detected in 1994 and predominated from 1995 onwards. However, subgroup 3.4 viruses were prevalent in the early years, not being isolated after 1996. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (E^rns–E2) displayed better resolution than that based on the complete open reading frame.     

Evidence of natural recombination in classical swine fever virus

Classical swine fever (CSF) virus, one member of the family Flaviviridae is the pathogen of CSF, an economically important and highly contagious disease of pigs. Although homologous recombination has been demonstrated in many other members of the family, it is unknown whether there is recombination in natural populations of CSFV. To detect possible recombination events, we performed a phylogenetic analysis of 25 full-length CSFV strains isolated all over the world. Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning. A mosaic virus, CSFV 39 (AF407339) isolated in China was found. And its two putative parental-like strains CSFV Shimen (AF333000) and GXWZ02 (AY367767) were identified. Our work revealed that homologous recombination occurred in natural CSFV populations, generating genetic diversity. This would provide some insights for the role homologous recombinant plays in CSFV evolution.     

Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay

Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).     

Plaque assay for African swine fever virus on swine macrophages

Summary.  A plaque assay developed to detect the infection of African Swine Fever Virus on swine macrophages is described. Plaques were generated by all of the virus isolates tested. The method is suitable not only for virus titration but also for the selection of clones in protocols for isolation/purification of recombinant viruses. Received December 28, 2001; accepted February 20, 2002 Published online April 26, 2002     

A real time RT-PCR assay for the specific detection of Peste des petits ruminants virus

Peste des petits ruminants virus (PPRV) causes a devastating disease of small ruminants present across much of Africa and Asia. Recent surveillance activities and phylogenetic analyses have suggested that the virus is an emerging problem as it is now being detected in areas previously free of the disease. As such, the virus not only is threatening small ruminant production and agricultural stability in the developing world, but also poses an economic threat to livestock in the European Union (EU) through introduction from European Turkey and North Africa. This report describes the development of a high throughput, rapid, real time RT-PCR method for the sensitive and specific detection of PPRV using robotic RNA extraction. This assay targets the nucleocapsid (N) gene of PPRV and has been shown to detect all four genetic lineages of PPRV in tissues, ocular and nasal swabs and blood samples collected in the field. The lowest detection limit achieved was approximately 10 genome copies/reaction, making this assay an ideal tool for the sensitive and rapid detection of PPRV in diagnostic laboratories.