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Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 μg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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 × 10 2 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 × 10 2 copies/μL and 1.8 × 10 2 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.

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 E rns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV., Methods: The CSFV E rns and truncated E2 (tE2, residues 690-865) of BVDV were expressed in E. coli and purified by Ni-NTA affinity chromatography, respectively. Employing E rns 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 E rns 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 E rns and tE2 -based ELISAs. Compared to VNT, the CSFV E rns -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., (© 2022. The Author(s).)

Classical swine fever virus (CSFV) is a member of the genus Pestivirus, which causes serious economic losses. The re-emergence of the disease in Japan in 2018 has increased awareness of CSFV. In this study, Balb/c mice were immunized with plant-derived E2 protein, and four monoclonal antibodies (mAbs) 4B11, 7B3, 11A5 and 6F3 were generated. Two of these mAbs, 4B11 and 7B3, effectively blocked CSFV infection of PK-15 cells. Both mAbs recognized a novel linear epitope, 256 CLIGNTTVKVHASDER 271 . The neutralizing ability of anti-CSFV serum decreased 63%, when pre-incubated with the linear peptide at 200 μg/mL. Structural analysis showed that this linear epitope is present at the border of Domain C and Domain D on the surface of the E2 protein. Alignment of amino acid sequences showed that the epitope was conserved in different subgroups of CSFV but not in other members of the Pestivirus genus. Consistently with the analysis above, this epitope distinguished antibodies against CSFV from those against bovine viral diarrhea virus (BVDV). Our study provides an ideal candidate peptide for new vaccine design and differential diagnosis of CSFV. These findings will contribute to the control and eradication of classical swine fever., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) B646L gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10–15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized B646L gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32–64-fold for direct PCR, while only a 2–4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future. [ABSTRACT FROM AUTHOR]

• A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. • The detection limit of mRT-PCR was in agreement with the singleplex RT-PCR. • The mRT-PCR were successfully used to detect clinical specimens. • The mRT-PCR developed is a useful tool for clinical diagnosis of single virus and/or mixed infections in swine. A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed and evaluated for its specificity and sensitivity. The expected product from the single viral template was amplified by mRT-PCR and no spurious PCR amplification occurred from the genomic RNA or DNA of other pathogens. For single virus or different combinations of two viruses the detection limit of mRT-PCR was consistent with a single RT-PCR wtith 1 × 103 copies. For different combinations of the three viruses or four viruses, sensitivity of PEDV detection partially decreased. All of positive clinical specimens by the mRT-PCR were identically confirmed using Taqman RT-qPCR. Therefore, the mRT-PCR is a useful tool for epidemiological studies and laboratory diagnosis of single virus and/or mixed infections in swine. [ABSTRACT FROM AUTHOR]