Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

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.

Enhanced passive surveillance for early detection of African and classical swine fevers.

African swine fever (ASF) and classical swine fever (CSF) are transboundary animal diseases (TADs) of pigs. Much effort and resources are regularly put into preventing these diseases' introduction in free areas. Passive surveillance activities bring the highest chances for the early detection of TAD incursions because they are routinely and widely conducted at farms, and because these activities focus on the time between introduction and when the first sample is sent for diagnostic testing. The authors proposed the implementation of an enhanced passive surveillance (EPS) protocol based on collecting data through participatory surveillance actions using an objective and adaptable scoring system to aid the early detection of ASF or CSF at the farm level. The protocol was applied in two commercial pig farms for ten weeks in the Dominican Republic, which is a CSF- and ASF-infected country. This study was a proof of concept, based on the EPS protocol to aid detection of substantial variations in the risk score triggering testing. One of the followed farms had score variation, which triggered testing of the animals, although the test results were negative. The study enables assessment of some of the weaknesses associated with passive surveillance and provides lessons applicable to the problem. Results demonstrate the potential for overcoming some issues preventing the broad application of EPS protocols and suggest that standardised approaches may contribute to the early detection of CSF and ASF introductions.

La peste porcine africaine (PPA) et la peste porcine classique (PPC) sont des maladies animales transfrontalières touchant les porcs. De nombreux efforts et ressources sont régulièrement alloués pour prévenir l'introduction de ces maladies dans des zones indemnes. Les activités de surveillance passive offrent les meilleures perspectives de détection précoce des incursions de maladies animales transfrontalières parce qu'elles sont menées de manière systématique et exhaustive dans les élevages, et parce qu'elles se concentrent sur la période entre l'introduction de la maladie et le moment où le premier échantillon est envoyé au laboratoire pour analyse. Les auteurs proposent la mise en oeuvre d'un protocole de surveillance passive renforcée fondé sur la collecte de données via des actions de surveillance participative utilisant un système de notation objectif et adaptable, en vue d'une détection précoce de la PPA et de la PPC dans les élevages. Ce protocole a été appliqué en République dominicaine, pays infecté par la PPA et la PPC, dans deux élevages porcins commerciaux pendant dix semaines. Cette étude était destinée à valider le principe de la méthode et se fondait sur le protocole de surveillance passive renforcée pour mieux détecter les variations substantielles de la note de risque qui conduisent à tester les animaux. L'un des élevages suivis a présenté une variation de cette note, ce qui a conduit à tester les animaux mais les tests se sont révélés négatifs. L'étude permet d'évaluer certaines des faiblesses associées à la surveillance passive et apporte des enseignements applicables à ce problème. Les résultats illustrent le potentiel de l'approche à surmonter certaines des problématiques empêchant l'application extensive des protocoles de surveillance passive renforcée. Ils suggèrent également que des approches normalisées pourraient contribuer à la détection précoce des cas d'introduction de la PPC et de la PPA.

La peste porcina africana (PPA) y la peste porcina clásica (PPC) son enfermedades animales transfronterizas que afectan al cerdo. Periódicamente se dedican grandes esfuerzos y cuantiosos recursos a evitar que estas enfermedades penetren en zonas que están exentas de ellas. Las actividades de vigilancia pasiva son las más eficaces para detectar con prontitud toda incursión de enfermedades animales transfronterizas, no solo por la regularidad y amplitud con que se llevan a cabo en las explotaciones, sino también porque inciden específicamente en el intervalo entre la penetración de una enfermedad y el momento en que se envía la primera muestra para que sea sometida a pruebas de diagnóstico. Los autores propusieron que se aplicara un protocolo de vigilancia pasiva reforzada que reposaba en la obtención de datos mediante actividades de vigilancia participativa, empleando para ello un sistema objetivo y adaptable de puntuación que ayudaba a detectar con prontitud la presencia en las explotaciones de PPA o PPC. Dicho protocolo fue aplicado a lo largo de diez semanas en dos explotaciones porcinas industriales de la República Dominicana, país en el que ambas infecciones están presentes. El estudio, que sirvió para poner a prueba la idea, pasaba por la aplicación del protocolo de vigilancia pasiva reforzada para ayudar a detectar variaciones sustanciales de la puntuación del nivel de riesgo que activa la realización de pruebas. En una de las explotaciones estudiadas se produjo una variación de la puntuación, cosa que activó la realización de pruebas en los animales, aunque estas arrojaron resultado negativo. El estudio aquí descrito permite evaluar algunos de los puntos débiles de la vigilancia pasiva y extraer enseñanzas aplicables al problema. Los resultados demuestran que es posible salvar algunas de las dificultades que impiden la aplicación generalizada de protocolos de vigilancia pasiva reforzada y dejan pensar que quizá el uso de planteamientos normalizados pueda ayudar a detectar con prontitud los casos de penetración de PPC o PPA.

A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies.

Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodies derived from camelids with naturally missing light chains can overcome these deficiencies and are an excellent alternative to conventional antibodies. In this study, a nanobody (Nb)-AviTag fusion protein was constructed, and the feasibility of its use as a high-sensitivity probe in a blocking ELISA (bELISA) for classical swine fever virus (CSFV) was investigated. The CSFV E2 recombinant protein expressed by the CHO expression system exhibited good reactogenicity and immunogenicity and induced the production of high CSFV antibody levels in rabbits. Three different clones of Nbs were successfully isolated using a phage display system in alpaca, and an Nb1-AviTag fusion protein was successfully expressed using an Escherichia coli expression system. The purified Nb1-AviTag fusion protein was then biotinylated in vitro to obtain Nb1-biotin. A novel bELISA was developed for the detection of CSFV antibodies in clinical serum using Nb1-biotin as a probe. The cutoff value of bELISA was 32.18%, the sensitivity of bELISA was higher than that of the bELISA kit with IDEXX antibody, and the coincidence rate was 94.7%. A rapid, low-cost, highly sensitive and highly specific CSFV E2 antibody-based bELISA method was successfully established and can be used for the serological evaluation of CSFV E2 subunit vaccines and the ELISA-based diagnosis of CSFV infection. IMPORTANCE Currently, the epidemic situation of classical swine fever (CSF) is sporadic, and cases of atypical swine fever are on the rise in China. Therefore, it is necessary to accurately eliminate suspected cases by using highly sensitive and specific diagnostic techniques. In our study, a rapid, low-cost, highly sensitivity, highly reliable and reproducible, and highly specific classical swine fever virus (CSFV) E2 antibody-based blocking ELISA method was successfully established by using the phage display system and the Nb1-AviTag fusion expression platform. It provides a new technique for serological evaluation of CSFV vaccines and ELISA-based diagnosis of CSFV infection.

Classical swine fever (CSF): towards new challenges / La peste porcine classique (PPC) : vers de nouveaux défis

Classical swine fever (CSF) is a highly contagious swine-specific disease which may have a huge economic impact for porcine production. CSF is caused by a virus belonging to the Pestivirus genus, which has expanded for the past 5 years with the discovery of new species whose genetic proximity to the CSF virus could further complicate laboratory diagnosis. The various forms of the disease, and in particular the increased frequency of attenuated forms, linked to an evolution of CSF virus strains towards a reduction in their virulence, delay clinical diagnosis. Thus, a long period may elapse before an outbreak is detected, allowing the virus to circulate longer, with the risk of spreading to distant geographical areas. Efforts must be maintained in terms of surveillance and diagnostic tools development in order to detect CSF virus infection early and thus limit the spread of the disease and facilitate control measures.

La peste porcine classique (PPC) est une maladie très contagieuse, spécifique des suidés, qui continue à constituer une menace pour la production porcine malgré un statut indemne de la plupart des pays de l'Union européenne. La PPC est causée par un virus de la famille des Flaviviridae appartenant au genre Pestivirus, en extension depuis les cinq dernières années avec la découverte de nouvelles espèces, notamment chez le porc ou autres animaux de rente dont la proximité génétique avec le virus de la PPC pourrait davantage compliquer le diagnostic de laboratoire. La diversité des formes de la maladie, et notamment la fréquence accrue des formes atténuées et inapparentes liée à une évolution des souches du virus de la PPC vers une réduction de leur virulence, retarde le diagnostic clinique. Ainsi, une longue période peut s'écouler avant la détection d'un foyer, permettant au virus de la PPC de circuler plus longuement, avec le risque de diffuser vers des zones géographiques éloignées des premiers cas d'infection. Les efforts doivent être maintenus en termes de surveillance et de développement d'outils de diagnostic afin de détecter précocement une infection par le virus de la PPC et ainsi limiter la propagation de la maladie et faciliter les mesures de contrôle.

Establishment and application of a solid-phase blocking ELISA method for detection of antibodies against classical swine fever virus.

BACKGROUND: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. OBJECTIVES: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. METHODS: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. RESULTS: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. CONCLUSIONS: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

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

As 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.

A Novel Competitive ELISA for Specifically Measuring and Differentiating Immune Responses to Classical Swine Fever C-Strain Vaccine in Pigs.

Classical swine fever can be controlled effectively by vaccination with C-strain vaccine. In this study, we developed a novel competitive enzyme-linked immunosorbent assay (cELISA) based on a C-strain Erns specific monoclonal antibody (mAb 1504), aiming to serologically measure immune responses to C-strain vaccine in pigs, and finally to make the C-strain become a DIVA-compatible vaccine. The cELISA system was established based on the strategy that mAb 1504 will compete with the C-strain induced antibodies in the pig serum to bind the C-strain Erns protein. The cELISA was optimized and was further evaluated by testing different categories of pig sera. It can efficiently differentiate C-strain immunized from wild-type CSFV-infected pigs and lacks cross-reaction with other common swine viruses and viruses in genus Pestivirus such as Bovine viral diarrhea virus (BVDV). The C-strain antibody can be tested in pigs 7-14 days post vaccination with this cELISA. The sensitivity and specificity of the established cELISA were 100% (95% confidence interval: 95.60 to 100%) and 100% (95% confidence interval: 98.30 to 100%), respectively. This novel cELISA is a reliable tool for specifically measuring and differentiating immune responses to C-strain vaccine in pigs. By combining with the wild-type CSFV-specific infection tests, it can make the C-strain have DIVA capability.

Development and validation of an in vitro titrimetric method for determination of classical swine fever viruses in PK-15 cells.

Classical swine fever (CSF) is a highly contagious notifiable disease of pigs caused by CSF virus of Flaviviridae family. Previously, lapinized vaccines were used for the disease control, which has now been replaced with cell culture vaccines. Determination of virus titre is the key factor for development and quality control testing of classical swine fever (CSF) cell culture vaccines. Since CSFV is a non- cytopathic virus, an accurate method for the titration of this virus in cell culture has not yet been reported. Here we present a full proof method of titration of CSF cell culture viruses employing Fluorescent Antibody Technique (FAT) in 24 well plate cover slip culture of PK-15 cells. CSFV monoclonal antibodies (Mab) used in the test bind to the CSF virus particles in the cell cytoplasm of the infected cells and the immune-fluorescence signal is produced by subsequent binding of FITC conjugate with Mab. In this newly developed method, apple green fluorescence is observed in the cytoplasm of the infected cells as the virus multiplies only in the cytoplasm. The nucleus as well as the uninfected cells cytoplasm is stained red without any traces of green fluorescence. Thus, the test clearly differentiates a CSFV infected cell from the uninfected cells in the vicinity, if any, and also from the uninfected controls. The test can also quantify the accurate titres of CSF live viruses in the cell culture vaccines and hence it has wide application in routine virus titration applied for manufacturing of CSF cell culture vaccines, determination of accurate multiplicity of infection (m.o.i.) during infection and quality control of vaccines by the testing laboratories.

An Improved Immunochromatographic Strip Based on Plant-Derived E2 for Detection of Antibodies against Classical Swine Fever Virus.

Vaccination is an effective method to control the spread of classical swine fever virus (CSFV), which is a major cause of economic losses to the swine industry. Although serological detection assays are commonly used to assess immune status, current methods for monitoring of antibodies (Abs) are time-consuming, expensive, and require cell culture and virus manipulation. To address these problems, the E2 protein of CSFV was expressed in transgenic rice seeds as a labeled antigen for the development of an immunochromatographic test strip (ICTS) for rapid, precise, and cost-effective detection of Abs. The ICTS has a reasonable sensitivity of 1:128,000 for detection of serum Abs against CSFV and no cross-reactivity with Abs of other porcine viruses. The similarity of the results between the proposed ICTS and a commercial enzyme-linked immunosorbent assay was 94.1% (128/136) for detection of serum Abs from immunized animals and 92.3% (72/78) for detection of maternally derived Abs. The proposed assay was successfully used to monitor Abs against E2 of both pigs and rabbits immunized with a live attenuated vaccine or an E2 subunit vaccine. The results confirmed that the ICTS can be applied to detect Ab levels in animals with different immunological backgrounds. The ICTS based on plant-derived E2 is a relatively inexpensive, rapid, and accurate assay for detection of Abs against CSFV and avoids the risk of contamination by animal products. IMPORTANCE The E2 protein of classical swine fever virus (CSFV) was expressed in transgenic rice endosperms as a diagnostic antigen for use with a rapid colloidal gold assay for the detection of antibodies (Abs) against CSFV. This improved test was used to monitor Abs against the E2 protein in both pigs and rabbits immunized with a live attenuated vaccine or E2 subunit vaccine. The assay successfully detected Ab levels in serum samples from piglets with different immunological backgrounds. In contrast to current E2 protein-based diagnostic methods using Escherichia coli or insect cells as expression systems, plant-derived E2 avoids the limitations of low immunogenicity of eukaryotic expression systems and potential contamination of fetal bovine serum with bovine viral diarrhea virus in cell culture.

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus.

BACKGROUND: Classical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV. METHODS: The CSFV Erns and truncated E2 (tE2, residues 690-865) of BVDV were expressed in E. coli and purified by Ni-NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT). RESULTS: Indirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively. CONCLUSION: The newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

Development of a one-step multiplex qRT-PCR assay for the detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since coinfections of ASFV, CSFV and APPV occur in certain pig herds, it is necessary to accurately and differentially detect these pathogens in field-collected samples. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT-PCR) was developed for the simultaneous and differential detection of ASFV, CSFV and APPV. RESULTS: The one-step multiplex qRT-PCR presented here was able to simultaneously detect ASFV, CSFV and APPV but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, etc. The limit of detection (LOD) of the assay was 2.52 × 101 copies/µL for ASFV, CSFV and APPV. A repeatability test using standard recombinant plasmids showed that the intra- and interassay coefficients of variation (CVs) were less than 2%. An assay of 509 clinical samples collected in Guangxi Province, southern China, from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58, 12.57 and 3.54%, respectively, while the coinfection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91, 1.38, 0.98%, respectively. Phylogenetic analysis based on the nucleotide sequences of the partial ASFV p72 gene showed that all ASFV strains from Guangxi Province belonged to genotypes I and II. CONCLUSION: A one-step multiplex qRT-PCR with high specificity, sensitivity and repeatability was successfully developed for the simultaneous and differential detection of ASFV, CSFV and APPV.

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.

Characterization of a Cytopathogenic Reporter CSFV.

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5'-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus.

African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 102 copies/µL for ASFV and 6.34 × 101 copies/µL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018-2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV.

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.

Evaluation of sampling methods for effective detection of infected pig farms during a disease outbreak.

Emergency surveillance following an outbreak of transboundary animal diseases such as classical swine fever (CSF), is conducted to find another new infection as early as possible. Although larger sample sizes can help achieve higher disease surveillance sensitivity, the sample size is limited by the availability of resources in an emergency situation. Moreover, the recent CSF outbreak reported in Japan was associated with fewer clinical signs; this emphasizes the importance of detecting infected farms by surveillance. In this study, we aimed to identify effective and labor-efficient sampling methods showing high probabilities of detecting infection, by simulating infection and sampling in pigsties. We found that impartial sampling, which involves selection of pigs to be sampled from the four corners and the center of the pigsty, and random sampling showed comparable probabilities of detection. Impartial sampling involves sample collection without pig identification and random selection. Owing to its simplicity, impartial sampling is labor-efficient and thus a possible substitute for random sampling. In a group-housing pigsty, testing five pigs from five pens showed a higher detection probability than testing five pigs from one pen. These results suggest preferable surveillance methods for conducting emergency surveillance of infectious diseases.

Classical swine fever virus: the past, present and future.

Classical swine fever (CSF) is among the most relevant viral epizootic diseases of swine. Due to its severe economic impact, CSF is notifiable to the world organisation for animal health. Strict control policies, including systematic stamping out of infected herds with and without vaccination, have permitted regional virus eradication. Nevertheless, CSF virus (CSFV) persists in certain areas of the world and has re-emerged regularly. This review summarizes the basic established knowledge in the field and provides a comprehensive and updated overview of the recent advances in fundamental CSFV research, diagnostics and vaccine development. It covers the latest discoveries on the genetic diversity of pestiviruses, with implications for taxonomy, the progress in understanding disease pathogenesis, immunity against acute and persistent infections, and the recent findings in virus-host interactions and virulence determinants. We also review the progress and pitfalls in the improvement of diagnostic tools and the challenges in the development of modern and efficacious marker vaccines compatible with serological tests for disease surveillance. Finally, we highlight the gaps that require research efforts in the future.

Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR.

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/µL for CSFV and 36 copies/µL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

Serum Lipidomics Analysis of Classical Swine Fever Virus Infection in Piglets and Emerging Role of Free Fatty Acids in Virus Replication in vitro.

Lipids metabolism plays a significant role in cellular responses to virus pathogens. However, the impact of lipids metabolism in CSFV infection is not yet confirmed. In the present study, for the fist time, we performed serum lipidomics analysis of piglets infected with CSFV based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and identified 167 differentially expressed lipid metabolites. Interestingly, free fatty acids (FFAs) accumulated significantly in these metabolites, accompanied by an increase in sphingolipids and a decrease in glycerolipids and glycerophospholipids, suggesting that CSFV infection markedly changed the serum lipid metabolism of piglets. FFAs are the principal constituents of many complex lipids and are essential substrates for energy metabolism. Based on this, we focused on whether FFAs play a prominent role in CSFV infection. We found that CSFV infection induced FFAs accumulation in vivo and in vitro, which is due to increased fatty acid biosynthesis. Meanwhile, we discovered that alteration of cellular FFAs accumulation by a mixture of FFAs or inhibitors of fatty acid biosynthesis affects progeny virus production in vitro. Furthermore, in the absence of glucose or glutamine, CSFV still has replication capacity, which is significantly reduced with the addition of fatty acid beta oxidation inhibitors, suggesting that the process of FFAs enter the mitochondria for beta oxidation to produce ATP is necessary for virus replication. Finally, we demonstrated CSFV induced FFAs accumulation results in impaired type I IFN signaling-mediated antiviral responses by down-regulating RIG-I-like receptors (RLRs) signaling molecules, which may represent a mechanism of CSFV replication. Taken together, these findings provide the first data on lipid metabolites during CSFV infection and reveal a new view that CSFV infection requires FFAs to enhance viral replication.