Detection Of Classical Swine Fever Virus Research Articles

Rapid Differential Detection of Wild-Type Classical Swine Fever Virus and Hog Cholera Lapinized Virus Vaccines by TaqMan MGB-Based Dual One-Step Real-Time RT-PCR.

To establish a rapid real-time RT-PCR method for differentiating wild-type classical swine fever virus (CSFV) strains from vaccine strains (HCLV), we designed a universal primer targeting the NS3 gene to detect wild-type CSFV strains and vaccine strains simultaneously, and two TaqMan-MGB probes were designed to differentiate between wild-type and vaccine strains. After optimizing the RT-qPCR conditions, a rapid dual TaqMan-MGB RT-qPCR method for the detection and identification of CSFV and HCLV was developed. The results showed that method could specifically detect CSFV and HCLV with no cross-reactivity with other swine pathogens. The analytic sensitivity for the NS3 gene of CSFV and HCLV were 1.67 × 101 copies/μL, respectively. For precision testing, the repeatability and reproducibility of the test was less than 2%. This method was successfully used for the rapid detection of 193 biological samples collected from CSFV-vaccinated pigs. This fast and accurate detection technology can be used for the detection of CSFV and is suitable for differentiating between wild-type CSFV strains and vaccine strains.

Novel approach for detecting classical swine fever virus from swabs of wild boar cut tails using nested real-time PCR.

We devised a method to detect the classical swine fever virus (CSFV) in tail-wiped swabs from wild boars. The CSFV gene in swabs was detected with high sensitivity using nested real-time polymerase chain reaction (PCR), which is a combination of reverse transcription-PCR (RT-PCR) and real-time PCR. We compared CSFV gene detection from boar tissue using the conventional and our tail-wiped swab method. The tail-wiped swab method showed sensitivity and specificity of 100% (26/26) and 98.8% (172/174), respectively compared to the conventional method. Thus, the swab-based CSFV detection method was considered to have detection sensitivity comparable to that of conventional methods. Additionally, we conducted surveillance for CSFV in wild boars on Awaji Island. CSFV was detected in 10.7% (45/420) of samples.

Enhancing classical swine fever virus identification: the advantages of Field-LAMP testing.

Classical swine fever virus (CSFV) identification has witnessed significant advancements with the development of rapid reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays. However, conventional RT-LAMP assays for CSFV diagnosis are hindered by a laborious RNA extraction step. Moreover, the need for thermal incubators and expensive micropipettes has limited their application in field settings. Addressing these challenges, our study presents a groundbreaking solution-an electro-free and point-of-care (POC) tool known as the field-LAMP assay-for the rapid clinical detection of CSFV. By eliminating the RNA extraction requirement, advancing the colorimetric read-out and lyophilized reaction reagents, our field-LAMP assay streamlines the diagnostic process, saving valuable time and effort. This novel approach also overcomes the dependency on electric-dependent thermal incubators and expensive micropipettes, making it practical and accessible for use in the field. The successful development of the field-LAMP assay marks a significant milestone in CSFV detection. This electro-free and POC tool offers several advantages, including its ability to deliver rapid results without compromising accuracy, facilitating prompt response and containment measures.

Electrochemical deposition of Pyrazine-Templated 2D Ru@MOFs nanosheets for sensitive electrochemiluminescent immunoassay of classical swine fever virus

Electrochemical deposition technique for room-temperature fabrication of functionalized metal–organic frameworks (MOFs) have attracted increasing attention in many important applications. Herein, a sensitive electrochemiluminescent (ECL) immunosensor was fabricated for the determination of classical swine fever virus (CSFV) based on electrochemical synthesis and deposition of pyrazine-templated ruthenium (II)-MOFs (Pyz-Ru@MOFs) thin films on the electrode surface. In this protocol, the 2D Pyz-Ru@MOFs nanosheets were synthesized under green water phase condition from the carboxyl-rich tris(4,4′-dicarboxylic acid-2,2′-bipyridyl) ruthenium(II) (Ru(dcbpy)32+) as ECL luminophore and organic ligand to coordinate with zinc ions, and pyrazine as a structure-directing agent to control the formation of sheetlike structure. Due to packaging plenty of Ru(dcbpy)32+ molecules in the frameworks, the mesoporous thin films of Pyz-Ru@MOFs exhibited excellent ECL performance. Using this 2D Pyz-Ru@MOFs as a sensing platform, a “signal-off” ECL immunosensor was constructed for CSFV analysis, which displayed high sensitivity and good selectivity for CSFV detection in the range of 10 fg mL−1 –10 ng mL−1 with a detection limit of 5.4 fg mL−1. In addition, the prepared immunosensor was successfully utilized for the satisfactory detection of CSFV in serum samples, which has potential application in biological analysis and clinical diagnosis. This proposed woke also provided a simple and feasible electrochemical synthesis strategy to construct 2D luminescent MOFs immobilized ECL luminophores, offering significant inspiration to develop highly efficient ECL materials at room temperature with ECL sensing applications.

Development of a multiplex qRT-PCR assay for detection of classical swine fever virus, African swine fever virus, and Erysipelothrix rhusiopathiae.

Classical swine fever virus (CSFV), African swine fever virus (ASFV), and Erysipelothrix rhusiopathiae (E. rhusiopathiae) remain endemic in many parts of China. Co-infections make distinguishing their clinical symptoms and pathological changes difficult. This study developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) that can simultaneously detect CSFV, ASFV, and E. rhusiopathiae. Three sets of primers and probes were designed to target the CSFV 5΄ untranslated region, ASFV p72 gene, and E. rhusiopathiae 16sRNA gene. Multiplex qRT-PCR for simultaneous differential detection of these three pathogens was developed after optimizing reaction parameters such as annealing temperature, primer and probe concentrations, amplification cycles, etc. The multiplex qRT-PCR could detect CSFV, ASFV, and E. rhusiopathiae simultaneously but could not amplify other porcine pathogens. The assay's limit of detection (LOD) was 2.89 × 102 copies/μL for CSFV, ASFV, and E. rhusiopathiae. All correlation coefficients (R2) at higher than 0.99, and the amplification efficiency was 98, 90, and 84%, respectively. All correlation coefficients (R2) were higher than 0.99, and the efficacy of amplification was 84%. In a repeatability test utilizing standard recombinant plasmids, the intra- and inter-assay coefficients of variation (CVs) were less than 2.27 and 3.79 percent, respectively. Lastly, 150 clinical samples were used to evaluate the assay's applicability in the field. The positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33%, 0, and 3.33%, respectively. And no co-infection among the three pathogens was found. The concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits reached 100%. This study's multiplex qRT-PCR could provide a rapid, sensitive, and specific method for the simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae.

Differentiation of Classical Swine Fever Virus Virulent and Vaccine Strains by CRISPR/Cas13a.

ABSTRACTAs a notifiable terrestrial and aquatic animal disease listed by World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]), classical swine fever (CSF) has caused great economic losses to the swine industry worldwide during recent decades. Differentiation of infected and vaccinated animals (DIVA) is urgent for eradication of CSF. In this study, a diagnostic platform based on CRISPR/Cas13a was established with the ability to differentiate between classical swine fever virus (CSFV) virulent and vaccine strains. In combination with reverse transcription recombinase-aided amplification (RT-RAA), the detection limit for CSFV synthetic RNA templates reached 3.0 × 102 copies/μL. In addition, with boiling and chemical reduction, heating unextracted diagnostic samples to obliterate nucleases (HUDSON) treatment was introduced to inactivate nucleases and release viral genome, achieving robust pretreatment of tested sample before CRISPR/Cas13a detection without the need to extract viral nucleic acids. HUDSON-RT-RAA-CRISPR/Cas13a can directly detect cell cultures of virulent Shimen strain and vaccine hog cholera lapinized virus (HCLV) strain, with the detection limit of 3.5 × 102 copies/μL and 1.8 × 102 copies/μL, respectively, which was equally sensitive to nested PCR (nPCR) and 100 times more sensitive than antigen enzyme-linked immunosorbent assay (ELISA). Meanwhile, HUDSON-RT-RAA-CRISPR/Cas13a showed no cross-reactivity with bovine viral diarrhea virus (BVDV), atypical porcine pestivirus (APPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2), exhibiting good specificity. At last, a total of 50 pig spleen samples with suspected clinical signs were also assayed with HUDSON-RT-RAA-CRISPR/Cas13a, nPCR, and antigen ELISA in parallel. HUDSON-RT-RAA-CRISPR/Cas13a showed 100.0% with nPCR and 82.0% coincident rate with antigen ELISA, respectively.IMPORTANCE Classical swine fever (CSF) is a World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]) notifiable terrestrial and aquatic animal disease, causing great economic losses to the swine industry worldwide during the past decades. Due to the use of the most effective and safe attenuated live vaccine for CSF prevention, differentiation of infected and vaccinated pigs is vital work, as well as a bottleneck for eradication of CSF. Methods with the ability to precisely differentiate classical swine fever virus (CSFV) virulent strains from vaccine strain hog cholera lapinized virus (HCLV) are urgently needed. Combining the high sensitivity of isothermal recombinase-aided amplification (RAA) with the accurate molecular sensing ability of Cas13a, we presented a novel method for CSFV detection without the need to extract viral nucleic acids, which showed great advantage to traditional detection methods for precise differentiation of CSFV virulent strains and vaccine strain, providing a novel powerful tool for CSF eradication.

Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples.

African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nucleic acids together with an impurity-tolerant enzyme, we established a multiplex assay of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for simultaneous detection of ASF virus (ASFV), CSF virus (CSFV) and swine internal control derived genes in a sample without the need for prior purification of viral nucleic acids. We applied this method to test serum and tissue samples of infected pigs and wild boars and compared the statistical sensitivities and specificities with those of standard molecular diagnostic methods. When a serum was used as a test material, the newly established assay showed 94.4% sensitivity for both and 97.9 and 91.9% specificity for ASFV and CSFV detection, respectively. In contrast, the results were 100% identical with those obtained by the standard methods when a crude tissue homogenate was used as a test material. The present data indicate that this new assay offers a practical, quick, and reliable technique for differential diagnosis of ASF and CSF where geographical occurrences are increasingly overlapping.

Evaluation of the sensitivity of the classical swine fever surveillance system in two free zones in Colombia.

Infection with the classical swine fever virus (CSFV) causes a disease in pigs that ranges from a hyperacute form in which animals die in a few hours to subclinical disease. Due to this wide range of virulence, several complementary surveillance strategies should be implemented for the early detection of the disease. The objective of the present study was to determine the sensitivity of the surveillance system to detect CSFV outbreaks in a free zone (Zone 1) and in a zone undergoing an eradication process (Zone 2) in Colombia. Stochastic scenario tree models were used to describe the population and surveillance structures and to determine the probability of CSFV detection. The total sensitivity of the surveillance system in the case of a single infected farm in Zone 1 was 31.4% (CI 95%: 7.2-54.1) and in the case of 5 infected farms was 85.2% (CI 95%: 67.3-93.7), while in Zone 2 the sensitivities were 27.8% (CI 95%: 6.4-55.1) and 82.5% (CI 95%: 65-92.9), respectively. The on-farm passive surveillance shows the highest sensitivity for detection of a single CSFV infected farm in both zones (22.8% in Zone 1 and 22.5% in Zone 2). The probability of detection was higher in a family / backyard premise than on a commercial farm in both zones. The passive surveillance at slaughterhouse had a sensitivity of 5.3% and 4.5% for the detection of a single infected farm in Zone 1 and 2, respectively. Active surveillance presented a range of sensitivity between 2.2% and 4.5%. In conclusion, the sensitivity of the surveillance in the two studied zones was quite high, one of reasons for this good sensitivity being the sentinel network based on the voluntary participation of 5,500 collaborators that were trained for the identification and notification of diseases of national interest.

Evidence of transboundary transmission of viral pathogens through pigs transported from Myanmar to Mizoram, India

In Mizoram, pigs used to migrate through the porous international border from Myanmar. It is speculated that the migrated animals may act as potential carriers of various microbial pathogens. But so far no scientific evidence is available in this aspect. The present study was conducted on the detection of serum antibodies and molecular detection of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) and rotavirus in the pigs. A total of 204 blood and 209 rectal swabs were collected from apparently healthy pigs of Champhai district of Mizoram transported from Myanmar. All the serum samples were subjected to ELISA based detection of antibodies against CSFV, PRRSV and PCV2. The whole blood/serum/feces were processed for extraction of viral nucleic acids and subjected to PCR/RT-PCR for detection of CSFV, PRRSV, PCV2 and rotavirus. A total of 105 (51.47%), 12(5.88%) and 36 (17.65%) serum samples were found to be positive for antibodies against CSFV, PRRSV and PCV2, respectively. In addition, 17 (8.33%) sera were recorded positive for both CSFV and PCV-2 antibodies and 9 (4.41%) sera were positive for CSFV, PCV-2 and PRRSV antibodies. By specific PCR, group A rotavirus was found to be most prevalent (8.33%) followed by PCV-2 (7.84%), CSFV (4.41%) and PRRSV (0.9%). All the rotaviruses were detected as Group A only. Of the 204 animals, 2 (0.98%) pigs showed co-infection with all the four viruses under the study. A total of 5 (2.45%) pigs were positive for co-infection with CSFV and PCV-2 and 4 (1.96%) pigs were positive for co-infection with PCV-2 and rotavirus. The present study provided laboratory based evidence on transmission of porcine viral pathogens from Myanmar to Mizoram.

On-Site Determination of Classical Swine Fever Virus (CSFV) by a Fluorescent Microsphere-Based Lateral Flow Immunoassay Strip (FM-LFIAs) Based on Monoclonal Antibodies

Classical swine fever (CSF) is a highly infectious and lethal multisystem disease caused by the classical swine fever virus (CSFV). This virus has resulted in huge economic losses in the porcine industry. Rapid detection of CSFV is needed in order to prevent and control the early transmission of CSFV. In this study, nine hybridoma cell lines that secrete monoclonal antibodies against CSFV (anti-CSFV-mAb) were prepared using the cell fusion and cell surface fluorescent immunosorbent assay (CSFIA). A rapid, sensitive, and specific fluorescent microsphere-based lateral flow immunoassay strip (FM-LFIAs) based on the use of paired CSFV-mAbs was developed. The sensitivity of the FM-LFIAs for CSFV detection was 5.28 ng/mL with a linear range from 9.77 to 625 ng/mL. During clinical sample assays, the FM-LFIAs results were consistent with the measurements obtained using the reverse transcription – polymerase chain reaction (RT-PCR), highlighting the practical application of FM-LFIAs for the on-site detection of CSFV.

Development of a fluorescent probe-based real-time reverse transcription recombinase-aided amplification assay for the rapid detection of classical swine fever virus.

Classical swine fever (CSF), which is caused by the CSF virus (CSFV), remains one of the most economically important diseases of the global swine industry. Rapid and reliable detection of CSFV is critical for controlling CSF. In this study, a novel fluorescent probe-based real-time reverse transcription recombinase-aided amplification (rRT-RAA) assay, targeting a highly conserved position within the 5' non-translated region (5'NTR) among all CSFV genotypes, was developed for the detection of CSFV. The assay is highly specific to CSFV and does not cross react with other important viruses. Sensitivity analysis revealed that the assay could detect two 50% tissue culture infectious dose (TCID50 ) of CSFV RNA per reaction at 95% probability, which is comparable to that of a documentary reverse transcription quantitative PCR (RT-qPCR) assay for CSFV. The rRT-RAA assay exhibited good reproducibility, with intra- and inter-assay coefficient of variation values of<8.0%. Of the 135 samples (including 102 clinical tissue samples and 33 different cell culture isolates of CSFV), 50 and 52 samples were tested positive for CSFV by rRT-RAA and RT-qPCR, respectively. The coincidence rate between the two assays was 98.5% (133/135). Further linear regression analysis showed a significant correlation between the rRT-RAA and RT-qPCR assays with an R2 value of 0.8682. Interestingly, the amplification products of the rRT-RAA assay could be directly observed with naked eyes under a portable blue light imager, making it possible for an on-site testing. Our results indicate that the rRT-RAA assay is a robust diagnostic tool for the rapid detection of CSFV.

Pestivirus spillover effect: molecular detection of bovine viral diarrhea virus in domestic and feral pigs

ABSTRACT: Pestivirus infections are important in the livestock industries, with infection occurring in cattle, sheep and pigs. The Pestivirus genus of the family Flaviviridae, includes four recognized species: bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2), border disease virus (BDV), and classical swine fever virus (CSFV). All pestivirus species can infect pigs, therefore accurate and specific pestivirus detection and differentiation is of great importance to assure control measures in swine populations. The aim of the study was the molecular detection of different pestiviruses in domestic and feral pigs. A total of 527 samples (92 pigs and 435 wild boars) were tested for pestiviruses detection using molecular assays. Eleven positive samples (6 wild boars and 5 domestic pigs) were identified using panpestivirus primers targeting the 5’- UTR region of the pestivirus RNA genome. Further all the positive samples were sequentially tested for detection of CSFV, BVDV-1 and BVDV-2 using specific primers. All RNAs were identified as positives for BVDV-1 and no amplification signals were obtained from BVDV-2 and CSFV. The current detection of BVDV-1 in clinical swine specimens highlights the important risk factor of swine population as reservoir and consequently carrier for BVDV.

Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots.

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

Survey for pestiviruses in backyard pigs in southern Brazil.

The Pestivirus genus comprises species that affect animal health and productivity worldwide. Members of the Suidae family are hosts for classical swine fever virus (CSFV), an important pathogen tracked by the World Organization for Animal Health (OIE). However, swine are also susceptible to other pestivirus species that can result in disease or compromise CSFV detection. We searched for pestivirus infection in swine sera collected from 320 backyard pig herds in southern Brazil. We used reverse-transcription PCR primers for Bungowannah virus; atypical porcine pestivirus (APPV); and a panpestivirus pair that detects bovine viral diarrhea virus (BVDV)-1, -2, and HoBi-like pestivirus (HoBiPeV), border disease virus (BDV), and CSFV. Two samples were positive using the panpestivirus primer pair and were classified as BVDV-1d and -2a, respectively. Serum samples were tested for virus neutralization against BVDV-1a, -1b, and -2 strains, resulting in 28 (4.4%) positive samples. Of those, 16 samples had the highest titers against BVDV-1a (2), BVDV-1b (5), and BVDV-2 (9). Our results indicate that Bungowannah virus, APPV, CSFV, BDV, and HoBiPeV have not been circulating in these specific backyard swine populations. However, ruminant pestiviruses were detected and must be considered in future pestivirus control programs conducted in Brazil.

Effective surveillance for early classical swine fever virus detection will utilize both virus and antibody detection capabilities

Early recognition and rapid elimination of infected animals is key to controlling incursions of classical swine fever virus (CSFV). In this study, the diagnostic characteristics of 10 CSFV assays were evaluated using individual serum (n = 601) and/or oral fluid (n = 1417) samples collected from −14 to 28 days post inoculation (DPI). Serum samples were assayed by virus isolation (VI), 2 commercial antigen-capture enzyme-linked immunosorbent assays (ELISA), virus neutralization (VN), and 3 antibody ELISAs. Both serum and oral fluid samples were tested with 3 commercial real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays. One or more serum samples was positive by VI from DPIs 3 to 21 and by antigen-capture ELISAs from DPIs 6 to 17. VN-positive serum samples were observed at DPIs ≥ 7 and by antibody ELISAs at DPIs ≥ 10. CSFV RNA was detected in serum samples from DPIs 2 to 28 and in oral fluid samples from DPIs 4 to 28. Significant differences in assay performance were detected, but most importantly, no single combination of sample and assay was able to dependably identify CSFV-inoculated pigs throughout the 4-week course of the study. The results show that effective surveillance for CSFV, especially low virulence strains, will require the use of PCR-based assays for the detection of early infections (<14 days) and antibody-based assays, thereafter.

An immuno-chromatographic lateral flow assay (LFA) for rapid on-the-farm detection of classical swine fever virus (CSFV).

Classical swine fever (CSF) is a highly contagious and potentially fatal disease of domestic pigs. Classical swine fever is routinely diagnosed by clinical signs, serology, detection of CSF virus (CSFV) nucleic acid by PCR and virus isolation. Most of the current CSF diagnostic methods are expensive and have an extended turnaround time. In the majority of the CSF endemic countries, lack of easy access to diagnostic facilities is a major problem for swine producers trying to obtain early diagnosis and often results in the entire herd being infected. The acute form of CSF can show non-specific signs of illness, leaving CSF often undiagnosed. Hence there is an urgent need for a rapid and reliable pen side diagnostic assay for the better detection and control of this economically important disease of swine. We developed an immuno-chromatographic lateral flow assay (LFA) for on the farm detection of CSFV. A CSFV isolate [CSFV/AP/TRP2/2009 (TS2)] of genotype 1.1 was used for the production of monoclonal antibodies (mAbs) for the LFA's development. The virus detection level of the LFA device was 36.8 TCID50/ml of CSFV. The sensitivity and specificity of LFA in comparison with PCR were 80.36% and 87.10%, respectively. The positive and negative predictive values of the LFA device were 91.84% and 87.10%, respectively. In conclusion, the CSFV-LFA is a reliable and convenient resource for preliminary on the farm detection of classic swine fever.

Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates

Detection of Classical swine fever virus (CSFV) nucleic acid in archival formalin fixed tissue samples and their use for phylogenetic analysis was investigated. Ten samples were examined for the presence of CSFV nucleic acid by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of 5'UTR and E2 gene. RT-PCR was found positive for 5'UTR fragment in eight samples while only one tissue samples showed amplification for E2 gene target fragment. For molecular epidemiology of the disease, 5'UTR PCR product of sample from Darbhanga (Bihar), was cloned and sequenced. The sequence was compared with the sequences available in database. The phylogenetic analysis reveals that the isolate belongs to subgroup 2.2 sharing 98.7% nucleotide identities with Vietnamese isolate (CaTh05-1, AB252170), indicating towards the possible origin of genogroup 2.2 CSFV isolates involved in the outbreak from Vietnam. From the study, it can be concluded that the tissue samples collected and stored in buffer formalin for years can be used to detect CSFV nucleic acid. Results are also suggestive of that the 5'UTR region of genome is more suitable target for RT-PCR based detection of CSFV in archival formalin fixed specimens. The study also indicates the potential of archival formalin fixed tissues for molecular epidemiology and genotyping of the CSF virus.

Development and application of real-time TaqMan RT-PCR assay for improved detection of classical swine fever virus in slaughtered pigs

Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a well-characterized sensitive assay that can deliver a rapid and accurate diagnosis before the onset of clinical signs. With this objective, a real-time fluorogenic-probe hydrolysis (TaqMan) reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for rapid and specific detection of classical swine fever virus (CSFV) and applied on samples derived from infected slaughtered pigs. A pair of PCR primers targeting 5’ -non-coding region (CSFL1 and CSFR1) in conjunction with a CSFV-specific fluorogenic probe (CSFP1) was designed and assessed in real-time PCR. During PCR, when the target of interest was present, the CSFV specific FAM-labeled TaqMan probe annealed to the amplicon between the forward and reverse primers and was subsequently cleaved via the 5¢-3¢ exonuclease activity of the DNA polymerase resulting in the release of the fluorescent reporter dye. This assay was found to be rapid and strain-specific for CSFV detection.

Detection system based on magnetoelastic sensor for classical swine fever virus

In this paper, a magnetoelastic (ME) sensing system for the detection of classical swine fever virus (CSFV) is presented. The detection system comprises a test paper and a measurement circuit. The test paper consists mainly of an ME biosensor to detect the CSFV. Based on the impedance analysis technique, the measurement circuit is designed to measure the resonance frequency of the ME biosensor. The anti-CSFV IgG is immobilized onto the ME sensor surface to form the ME biosensor through a physical absorption approach. The experimental results show that the shift in the resonance frequency of the ME biosensor increases with the augmentation of the CSFV concentration. The effectiveness of the combination between the anti-CSFV IgG and CSFV is confirmed by the scanning electron microscopy (SEM) images, the sandwich-based enzyme-linked immunosorbent assay (ELISA) analysis, the interference study and the reference biosensor test method. The resonance frequency shift is linearly proportional to the concentration in the range from 0 to 2.5μg/ml, and becomes sub-linear at higher concentrations. The ME biosensor for CSFV detection has a sensitivity of about 95Hz/μg·ml−1, with a detection limit of 0.6μg/ml.

Development of single dilution immunoassay to detect E2 protein specific classical swine fever virus antibody

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in swine. The disease is endemic in different parts of the world and vaccination is the only way to protect pigs from CSFV infection. The virus surface protein E2 is the major immunogenic protein eliciting protective immunity against CSFV infection in swine. The whole virus antigen cannot differentiate CSFV from other pestiviruses as it cross reacts with border disease and bovine viral diarrhoea viruses. Commercial available ELISA is based on the whole CSFV particle and can lead to false positive results. Moreover, the available commercial ELISA is not cost effective. In the present study, a recombinant E2 protein based single serum dilution ELISA was developed which showed enhanced sensitivity, specificity and accuracy as compared to commercial CSFV detection ELISA. The recombinant E2 protein based ELISA could be an alternate to existing diagnostics against CSFV infection in pigs.