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.     

A multiplex nested RT-PCR for the detection and differentiation of wild-type viruses from C-strain vaccine of classical swine fever virus

A multiplex nested RT-PCR (RT-nPCR) was developed for the detection and differentiation of classical swine fever virus (CSFV). A fragment of 447 or 343 bp was amplified from the genomic RNA of C-strain or virulent Shimen strain, respectively, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed samples of C-strain and Shimen. When detecting several wild-type isolates representative of different subgroups (1.1, 2.1, 2.2, and 2.3) circulating in Mainland China and samples from pigs experimentally infected with Shimen strain, the RT-nPCR resulted in an amplification pattern similar to Shimen. No amplification was achieved for uninfected cells, or cells infected with bovine viral diarrhea virus (BVDV), and other viruses of porcine origin. The RT-nPCR was able to detect as little as 0.04 pg of CSFV RNA. The restrictive fragment length polymorphism (RFLP) demonstrated unique patterns of wild-type viruses and C-strain. Among the 133 field samples, 42 were tested to contain wild-type viruses and 18 showing presence of C-strain. The RT-nPCR can be used to detect and differentiate pigs infected with wild-type CSFV from those vaccinated with C-strain vaccine, thus minimizing the risk of culling vaccinates during outbreaks.     

Preclinical diagnosis of African swine fever in contact-exposed swine by a real-time PCR assay

A fluorogenic probe hydrolysis (TaqMan) PCR assay for African swine fever virus (ASFV) was developed and evaluated in experimentally infected swine. This sensitive and specific one-step single-tube assay, which can be performed in 2 h or less, detected viral DNA in tonsil scraping samples 2 to 4 days prior to onset of clinical disease. Thus, the assay would have application for preclinical diagnosis of African swine fever and surveillance and/or emergency management of a disease outbreak.     

A multiplex RT-PCR for rapid and simultaneous detection of porcine teschovirus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex RT-PCR (mRT-PCR) assay was developed and evaluated for its ability to detect multiple viral infections of swine simultaneously. One pair of primers was selected carefully for each of the following three RNA viruses: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine teschovirus (PTV). Each target produced a specific amplicon with a size of 451bp (PRRSV), 343bp (CSFV), or 163bp (PTV). The sensitivity of the mRT-PCR using purified plasmid constructs containing the specific viral target fragments was 2.02 x 102, 2.90 x 103, and 6.16 x 103 copies for PRRSV, CSFV, and PTV, respectively. Among 69 clinical samples from Heilongjiang, Jilin, and Henan provinces, co-infection by PRRSV and CSFV was 4.4%, co-infection by PRRSV and PTV was 11.6%, co-infection by PTV and CSFV was 13.0%, and co-infection by the three viruses was 8.7%. In conclusion, the mRT-PCR should be useful for routine molecular diagnosis and epidemiology.     

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.     

Pathogenicity and kinetics of virus propagation in swine infected with the cytopathogenic classical swine fever virus containing defective interfering particles

Summary.  To analyze the pathogenicity and in vivo kinetics of the cytopathogenic (cp) classical swine fever virus (CSFV) WB82 strain, which is composed of cp defective interfering (DI) particles and noncytopathogenic (noncp) helper virus (WB82/E⁺ strain), WB82 and WB82/E⁺ strains were administered separately to domestic pigs. After inoculation with either strain, all pigs showed typical symptoms of classical swine fever (CSF), such as leucopenia and high fever. There were few differences in clinical signs and survival times between each group. However, the appearance of some symptoms of CSF had a tendency to be delayed following infection with the WB82 strain, when compared with the WB82/E⁺ strain. Virus isolation and detection of subgenomic (sg) and full-length viral (flv) RNA by RT-PCR was carried out using sera, 10% homogenized organs and oral, nasal and rectal swabs. Both noncytopathogenic helper virus and cp DI particles were detected in samples from pigs infected with the WB82 strain, but only noncp phenotype virus was isolated from pigs infected with the WB82/E⁺ strain. Interestingly, the cp DI particles appeared six to seven days later than helper virus in sera from pigs infected with the WB82 strain. Although active cp DI particles could not be isolated from swabs, sg RNA as well as flv RNA was detected in swabs from animals infected with the WB82 strain. These results suggest that progeny cp DI particles are replicated from parent DI particles after noncp virus replication, and subsequently discharged from infected animals. Furthermore, propagation of DI particles or replication of sg RNA, following propagation of helper virus, appears to inhibit the appearance of CSF symptoms induced by virulent helper CSFV. Received January 14, 2002; accepted August 19, 2002     

Molecular chaperone Jiv promotes the RNA replication of classical swine fever virus

The nonstructural protein 2 (NS2) of classical swine fever virus (CSFV) is a self-splicing ribozyme wherein the precursor protein NS2-3 is cleaved, and the cleavage efficiency of NS2-3 is crucial to the replication of viral RNA. However, the proteolytic activity of NS2 autoprotease may be achieved through a cellular chaperone called J-domain protein interacting with viral protein (Jiv) or its fragment Jiv90, as evidence suggests that Jiv is required for the proper functioning of the NS2 protein of bovine viral diarrhea virus. Hence, the expression of Jiv may be correlated with the replication efficiency of CSFV RNA. We investigated the expression levels of Jiv and viral RNA in CSFV-infected cells and tissues using Real-time RT-PCR or Western blot analysis. The obtained results show that Jiv90 possibly plays an important role in the lifecycle of CSFV because the distribution of Jiv90 protein shows a positive correlation with the viral load of CSFV. Furthermore, the overexpression or knockdown of Jiv90 in swine cells can also significantly promote or decrease the viral load, respectively. The detection of Flow cytometry shows that the overexpression of Jiv90 prolongs the G1 phase of cell cycles but has no effect on apoptosis. These findings are likely to be of benefit in clarifying the pathogenesis of the CSFV.     

Enzyme-linked immunosorbent assay based on a chimeric antigen bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus infection

The antigenic region (residues 109 to 160) of classical swine fever virus (CSFV) protein E(rns) and the N-terminal antigenic region (residues 1 to 136) of protein E2 were constructed in the form of a fused, chimeric protein, C21E(rns)E2, for use as an enzyme-linked immunosorbent assay (ELISA) antigen for the serodiagnosis of CSFV infection. Tested with 238 negative-field (CSFV-free) sera from Canadian sources, the specificity of the ELISA was determined to be 93.7%. All 20 sera from experimentally infected pigs representing a variety of animals, virus strains, and days postinfection (dpi; range, 7 to 210) were detected as positive (100%). In contrast, an ELISA based on an E(rns) fragment (E(rns)(aa 109-160)) or an E2 fragment (E2(aa 1-221)) identified only 18 (90%) of 20 sera from infected pigs as positive, missing two targets collected at 7 dpi. These data suggest that use of the chimeric antigen C21E(rns)E2 would improve serodiagnostic sensitivity and allow for the detection of CSFV infection as early as 7 dpi.     

Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection

The diagnostic properties of several assays on live animals were studied using data from different experiments. These experiments involved 128 classical swine fever virus (CSFV) infected pigs (weaner pigs, fatteners and sows). Since all pigs in the study were infected with CSFV, only the proportion of test positive results and the time until a test positive result is obtained were evaluated. The RT-nPCR detected the highest proportion of infected pigs (98.9%), whereas the Antigen ELISA gave the worst detection results (74.7%). Within the group of test positive animals, infection was detected earliest using the leukocyte count and latest using Antigen ELISA. Using the virus neutralisation test, antibodies against CSFV were detectable on average 7.6 days after the onset of viraemia in virus isolation in whole blood. Using survival analysis, the time until the first positive diagnosis and the proportion of detected animals were combined in one test. Results showed that RT-nPCR performed significantly better than either virus isolation in different blood fractions or antigen ELISA. It is concluded that the RT-nPCR technique is the best diagnostic tool available for early detection of a classical swine fever infection.     

Establishment and application of bicistronic classical swine fever virus genomes for foreign gene expression and complementation of E2 deletion mutants

Bicistronic genomes of the classical swine fever virus (CSFV) strain Alfort/187 (A187) were established by insertion of a second cistron consisting of an internal ribosome entry site of the encephalomyocarditis virus and a coding sequence in the 3' untranslated region of the genome. Introduction of the selectable marker gene for neomycin phosphotransferase into the second cistron of the CSFV replicon A187 Delta E2-CAT allowed the establishment of porcine SK-6 cell lines constitutively expressing the respective bicistronic replicon RNA. In cells transfected with RNA representing the full-length viral genome and containing the gene coding for bacterial enhanced green fluorescence protein (EGFP) in the second cistron infectious bicistronic virus was synthesized. Expression of EGFP in cells infected with this virus indicated the potential of CSFV as a viral vector. Finally, after insertion of the sequence encoding the signal peptide of the CSFV E2 protein followed either by the E2 or the E2-p7 sequence into the replicon A187 Delta E2 which carries an in frame deletion of 465 nucleotides in the E2 gene, infectious viruses vA187 Delta E2-IRES-sigE2 and vA187 Delta E2-IRES-sigE2p7, respectively, were obtained. This shows that E2 deletion mutants can be complemented by expression of E2 from a separate cistron.     

Simultaneous detection of Classical swine fever virus and North American genotype Porcine reproductive and respiratory syndrome virus using a duplex real-time RT-PCR

Classical swine fever and porcine reproductive and respiratory syndrome are both notifiable diseases of the World Organization for Animal Health (OIE). The two diseases exhibit indistinguishable clinical symptoms and sometimes co-exist in swine herds. In this study, a duplex real-time RT-PCR for simultaneous detection of Classical swine fever virus (CSFV) and North American (NA) genotype Porcine reproductive and respiratory syndrome virus (PRRSV) based on two differently labeled TaqMan probes was developed and evaluated. The detection limit of the assay was 3.2 TCID(50) or 13 RNA copies for CSFV and 1.8 TCID(50) or 10 RNA copies for PRRSV, about 50 times more sensitive than conventional RT-PCRs. The duplex real-time RT-PCR was capable of specifically detecting different subgroups of wild-type CSFV and different strains of NA-genotype PRRSV, whereas a number of non-CSFV/PRRSV porcine viruses and bovine pestivirus were tested negative. Out of 155 field samples, 16 were tested positive for CSFV, 73 were positive for PRRSV, and 13 were co-infected with the two viruses. These results were 99.4% in agreement with those using conventional RT-PCRs. Therefore, the assay provides sensitive and simultaneous detection and differentiation of CSFV and PRRSV.     

Detection of economically important viruses in boar semen by quantitative RealTime PCR technology

The objective of this study was to develop quantitative real-time polymerase chain reaction (ReTi-PCR) tests for the detection of five economically important viruses in swine semen namely, pseudorabies virus (PRV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each ReTi-PCR test was validated for specificity, analytical sensitivity (detection limits), and experimental infection studies were performed to compare the conventional virus isolation methods with the newly developed ReTi-PCR tests.

All five developed ReTi-PCR tests are very rapid compared to virus isolation, highly specific, and even more sensitive (lower detection limits) than conventional virus isolation methods for the detection of mentioned viruses in semen. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by ReTi-PCR tests than by virus isolations. The high throughput of these rapid ReTi-PCR tests makes it possible to screen large number of semen samples for the presence of viruses prior to insemination. This is a substantial advantage, in particular for boar semen the quality of which deteriorates quickly after storage.

In general, the newly developed ReTi-PCR tests are valuable tools for the early, reliable and rapid detection of five economically important viruses, namely PRV, CSFV, FMDV, SVDV, and PRRSV in boar semen. These ReTi-PCR tests will improve the control of viral diseases transmitted via semen.     

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.