Development of a triplex TaqMan real-time RT-PCR assay for differential detection of wild-type and HCLV vaccine strains of classical swine fever virus and bovine viral diarrhea virus 1.

In this study, genomic sequences of pestiviruses available in GenBank were aligned to design three primer pairs and TaqMan probes: two targeting the NS5A region of the viral genome of classical swine fever virus (CSFV) for the differentiation of wild-type CSFV and hog cholera lapinized vaccine (HCLV) vaccine, and one targeting the 5'-untranslated region of bovine viral diarrhea virus 1 (BVDV-1). With these primers and probes, a triplex TaqMan real-time RT-PCR assay was developed for differentiating wild-type CSFV, the HCLV strain, and BVDV-1. The detection limit of the assay was 4.5 TCID(50) for wild-type CSFV, 10 TCID(50) for HCLV-strain CSFV, and 3.2 TCID(50) for BVDV-1. The triplex real-time RT-PCR had at least 98% (248 samples) agreement with other RT-PCR methods. The assay provides a sensitive tool for simultaneous detection and differentiation of wild-type CSFV and HCLV from BVDV-1.

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.

Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China.

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65°C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis.

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

This study describes evaluation of a real-time PCR assay based on primer-probe energy transfer (PriProET) technology for detection of classical swine fever virus (CSFV). The PriProET technology allows melting curve analysis following PCR amplification and thus provides a higher specificity. The assay was compared with a TaqMan assay by testing a total of 203 samples including 175 clinical specimens and 28 batches of Hog Cholera Lapinized Virus (HCLV) vaccine. The two assays gave the same results for 184 (91%) samples. Compared with the TaqMan assay, 19 additional samples were found to be positive for CSFV using the PriProET assay. In an RNA mixture of both wild type CSFV and C-strain vaccine, the melting curves displayed only one curve: either a wild type-like or a vaccine-like depending on the dominating RNA. The PriProET assay can be a routine molecular tool or a confirmative tool for diagnosis of classical swine fever (CSF), especially in the case of samples that yield an inconclusive result by the TaqMan assay.

Validation of a loop-mediated isothermal amplification assay for visualised detection of wild-type classical swine fever virus.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method applied and adapted to the detection of a number of pathogens. A LAMP assay was developed for the specific detection of wild-type classical swine fever virus (CSFV). Based on an alignment of genomic sequences of pestiviruses available in GenBank, four primers were selected targeting six positions in the NS5B gene region of the viral genome. The assay was performed in a simple water bath at a constant temperature of 62 degrees C, and after adding SYBR Green I, the results were visualised by the naked eye. This assay allowed easy applicability in a laboratory that is equipped simply, or even on site, close to the outbreaks and under field conditions. For confirmation, the results could also be visualised under UV light or by separation of the amplified products on 2% agarose gels. The detection limit of the assay was 2.5 medium tissue culture infectious dose (TCID(50)). The assay was validated with 116 clinical samples from vaccinated swineherds and 53 blood samples from experimental infections, and the results were comparable to a real-time RT-PCR assay. In summary, the LAMP assay provides a simple, rapid, and sensitive tool for the detection of wild-type CSFV under field conditions.

In vitro inhibition of the replication of classical swine fever virus by capsid-targeted virus inactivation.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious fatal disease of swine. Few effective antiviral drugs are currently available against CSFV infections. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against CSFV infections, we expressed the CSFV capsid protein (Cap) fused with the nuclease of Staphylococcus aureus (SN) in Escherichia coli and investigated its effects on the replication of CSFV in PK-15 cells. The results indicated that the fusion protein Cap-SN showed a strong Ca(2+)-dependent nuclease activity and inhibited the replication of CSFV in a dose-dependent manner, with complete inhibition at a concentration of 15 microg/ml, whereas the Cap fused with an enzymatically inactive SN (Cap-SN*) showed no nuclease activity or antiviral effects. Thus, the CTVI approach might be applicable to CSFV inhibition as a novel antiviral strategy.