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Brazil is the fourth largest producer and exporter of pork in the world, standing out for its potential to increase pig production nationwide. Among the diseases that can severely compromise trade relations and cause significant losses in pig production in Brazil, Classical Swine Fever (CSF) is notable for being a highly contagious viral disease with high potential for spreading among domestic and wild pigs, wild boars, and peccaries. The present study aimed to identify high-risk regions for CSF in Brazil through spatial, temporal, and spatiotemporal analyses from 2000 to 2023. During this period, 3189 cases of CSF were detected, all reported exclusively in states within the CSF non-free zone. The Northeast region accounted for 90.31 % (2880/3189) of the total CSF cases, with Ceará reporting the highest number of cases at 1475 (46.25 %), of which 810 were reported in 2018 alone. The North region accounted for 9.69 % (309/3189) of the total cases, with the states of Pará and Amapá being the only ones to report cases of the disease. Temporal trend analysis identified an increase in Piauí (Annual Percentage Change [APC]: 25.58 %), Alagoas (APC: 28.01 %), and Ceará (APC: 17.99 %), a decreasing trend for Pernambuco (APC: 24.49 %), Paraíba (APC: 19.35 %), and Rio Grande do Norte (APC: 11.85 %), and stability in Maranhão (APC: 0.17 %), Pará (APC: 1.61 %), and Amapá (APC: 15.52 %). The spatiotemporal analysis identified the formation of two high-risk clusters: the primary cluster occurred in the Northeast region, specifically in the states of Ceará, Piauí, Rio Grande do Norte, Paraíba, Pernambuco, and Alagoas, between 2018 and 2019, with a total of 1556 CSF cases and Relative Risk (RRs) of 120.02. The secondary cluster was formed by the states of Rondônia, Acre, Amazônia, Roraima, Pará, Amapá, and Mato Grosso, located in the North and part of the Midwest region, for the year 2009, with a total of 309 cases and an RRs of 29.89. In conclusion, the Northeast region is considered high-risk for the emergence of new CSF cases, highlighting the states of Ceará and Piauí, which can be considered important sources of CSF virus spread to other Brazilian states, alerting authorities to the need for disease control actions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Classical Swine Fever (CSF) is still one of the most economically important viral diseases of pigs. In endemic countries, the disease is controlled mostly through vaccination; hence, the availability of safe and effective vaccines is of utmost importance. Vaccines intended for application in developing countries must also be thermally stable, since the infrastructure needed to maintain a cold chain in those countries is usually lacking. Porvac ® is a second-generation subunit marker vaccine against CSF that has demonstrates to be safe and protective. Previous studies have also shown that the vaccine is stable for 1 week at 37 o C and have a shelf life of at least 36 months at 2-8 o C. The aim of this work was to further explore the accelerated stability of Porvac ® by assessing the physicochemical properties of the emulsion, and the safety and immunogenicity of the vaccine subjected to more drastic conditions of thermal stress: (1) 25 o C for 12 months; (2) 30 o C and 37 o C for one month and (3) 15 days at 37 °C after the cap of the vials had been needle-punctured., Results: The vaccine subjected to all these conditions did not show significant changes in the physicochemical properties of the emulsion; did not produce local or systemic adverse reactions in pigs, and the chromatographic profile of the recovered antigen was preserved. All vaccinated swine developed neutralizing antibody titers ≥ 1:1000 at 28 days post vaccination., Conclusions: Porvac ® is stable in all the experimental conditions tested, even after cap puncture, and retains the capacity to induce high titers of neutralizing antibodies, well above the threshold of protection. These results reinforce the robustness of the vaccine, and support its use as a very attractive alternative to modified live vaccines in developing countries endemic for CSF., Competing Interests: Declarations Ethics approval and consent to participate The experiments were approved and conducted following the guidelines of the Ethics Committee on Animal Experimentation (CICUAL). Approved protocols numbers VE2CD-0120/2, VE2CD-0121/2 and VE2CD-0220/2. All procedures and samplings involving animals were carried out following the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Classical swine fever (CSF) is endemic in Cuba and is one of the major health problems of the Cuban swine industry. The current efforts to control the disease in Cuba include vaccination with Porvac ® , a subunit marker vaccine. Although the efficacy of Porvac against CSF virus (CSFV) subgenotype 1.4 has been extensively documented, little is known about the ability of the antibodies induced by this vaccine to neutralize other CSFV genotypes. In this study, sera collected from three pigs vaccinated with Porvac were able to efficiently neutralize CSFV strains belonging to genotypes 1, 2, and 3. The findings from this study indicate that additional in vivo studies are warranted to confirm the ability of this vaccine to protect pigs against CSFV genotypes 2 and 3., Competing Interests: Declarations. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. Crown.)

Classical swine fever (CSF) re-emerged in Japan in 2018, with the epidemic virus identified as genotype 2.1, which is moderately virulent and more difficult to detect and control than the highly virulent strain. Domestic pigs were administered with GPE - strain live attenuated vaccine (GPE - vaccine) for outbreak management. CP7_E2alf strain recombinant live vaccine (marker vaccine), approved for differentiation of infected from vaccinated animals (DIVA), was considered optional for obtaining CSF-free country certification issued by the World Organization for Animal Health. This study aimed to assess the efficacy of both vaccines in pigs through experimental challenge tests and evaluate the DIVA ability of the marker vaccine using two enzyme-linked immunosorbent assay (ELISA) antibody detection kits. Results showed that both GPE - and marker vaccines were effective against the Japanese epidemic strain; however, the ability of the ELISA antibody detection kits to discriminate the marker vaccine was limited. Therefore, to achieve CSF-free certification using vaccines with DIVA functionality, alternative detection methods and enhancement of the sensitivity and specificity of ELISA kits are needed., Competing Interests: Declaration of competing interest The authors declare the following financial interests which may be considered as potential competing interests. Maiko YAMASHITA reports drugs were provided by Matsuken Pharmaceutical Ind. Co., Ltd. (Japan) and Zoetis (USA). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Matsuken Pharmaceutical Ind. Co., Ltd. (Tokyo, Japan) and Zoetis, Inc. (Parsippany, NJ, USA) provided the vaccines used in this study free of charge., (Copyright © 2024. Published by Elsevier Ltd.)

Previous studies have emphasized the necessity of surveillance and control measures for hepatitis E virus (HEV) infection in wild boars, an important reservoir of HEV. To assess the current situation of HEV infection in wild boars in Japan, this study investigated the prevalence and genetic diversity of HEV among wild boars captured in 16 prefectures of Japan during 2018-2023. Serum samples from 968 wild boars were examined for anti-HEV IgG antibodies and HEV RNA. The prevalence of anti-HEV IgG varied geographically from 0 % to 35.0 %. HEV RNA was detected in 3.6 % of boars, with prevalence varying by prefecture from 0 % to 22.2 %. Genotype 3 was the most prevalent genotype (91.9 %), followed by genotype 4 (5.4 %), with one strain closely related to genotype 6. The prevalence of HEV infection among wild boars decreased from 2018/2019 to 2022/2023 with significant declines in levels of anti-HEV IgG antibodies (14.5 % vs. 6.2 %, P < 0.0001) and HEV RNA (7.6 % vs. 1.5 %, P < 0.0001). Regional analysis showed varying trends, with no HEV RNA-positive boars found in several regions in recent years. A plausible factor contributing to the decline in HEV infection is the application of countermeasures, including installing fences to prevent intrusion into pig farms, implemented in response to the emergence of classical swine fever virus (CSFV) infection in wild boars and domestic pigs, with incidents reported annually since 2018. Further investigation is warranted to explore the association between countermeasures to CSFV infection and the decrease in HEV infection among wild boars., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

To identify the best method of distributing vaccine baits for animals, the oral vaccine Flc-LOM-BE rns was buried or placed directly on the ground within a mountainous area measuring 163 ha. Wild boars were observed more often around sites where the oral vaccine was buried than around sites where the oral vaccine was placed directly on the ground. Only wild boars consumed both the bait and the vaccine formulation. To confirm whether the Flc-LOM-BE rns vaccine strain has DIVA function, it was spread or buried within a mountainous area of 2.3 ha that was frequented by grazing hybrid-wild boars. Among the 15 hybrid-wild boars captured before consuming the oral vaccine, two were positive for anti-CSFV E2, but negative for anti-BVDV E rns antibodies. The CSFV E2 and BVDV E rns antibody positivity rates in 46 hybrid-wild boars captured 6 weeks after distributing the oral vaccine were 56.5 % (26/46, 95 % CI: 42.2-69.7) and 52.1 % (24/46, 95 % CI: 38.1-65.8), respectively. However, the CSFV E2 and BVDV E rns antibody positivity rates at 28 weeks post-distribution of the oral vaccine decreased by 10.3 % and 13.7 %, respectively, when compared with those in hybrid-wild boars captured after 6 weeks. Since the antibody positivity rates induced by the oral vaccine decreased after 7 months, it is necessary to distribute the oral vaccine at least twice a year (early spring and late fall)., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dong-Jun An reports financial support was provided by Animal and Plant Quarantine Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Classical swine fever (CSF) is an economically important and highly contagious disease of pigs caused by CSF virus, genus Pestivirus. Serological diagnosis of the disease is highly valuable for surveillance and thereby containment of spread of the disease. In this study, we have demonstrated the development of CSFV envelope glycoprotein E2-based indirect ELISA (E2-iELISA) for the detection of CSFV specific antibodies. The full-length E2 protein was expressed in E. coli and the purified protein was used as a coating antigen in indirect ELISA for detecting CSFV specific antibodies in pigs. A panel of 506 pig sera samples was used to validate the ELISA and the results were highly comparable to the results obtained with the commercial antibody detection kit (PrioCHECK CSFV Ab kit). The in-house E2-iELISA demonstrated high diagnostic sensitivity (95.4%) and specificity (95.5%), highlighting its potential application for sero-surveillance or monitoring of the disease in the swine population., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Classical swine fever (CSF) re-emerged in Japan in 2018 for the first time in 26 years. The disease has been known to be caused by a moderately pathogenic virus, rather than the highly pathogenic virus that had occurred in the past. However, the underlying pathophysiology remains unknown. This study conducted an experimental challenge on specific pathogen-free (SPF) pigs in a naïve state for 2, 4, and 6 weeks and confirmed the disease state during each period by clinical observation, virus detection, and pathological necropsy. We revealed the pathological changes and distribution of pathogens and virus-specific antibodies at each period after virus challenge. These results were comprehensively analyzed and approximately 70% of the pigs recovered, especially at 4- and 6-week post-virus challenge. This study provides useful information for future countermeasures against CSF by clarifying the pathogenicity outcomes in unvaccinated pigs with moderately pathogenic genotype 2.1 virus., (© 2024 The Author(s). Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

The Chinese government's reclassification of Classical Swine Fever (CSF) from a class Ⅰ to a class Ⅱ animal infectious disease, now also including CSF under the disease eradication program, reflects the significant progress made through extensive immunization with CSF vaccines. In light of this advancement, there is an imperative need for an expedient and accurate method to assess the levels of immunoprotection against classical swine fever virus (CSFV) in vaccinated pigs, a critical component in the campaign to eradicate the disease. This study develops an indirect enzyme-linked immunosorbent assay (iELISA) based on a highly glycosylated E2 protein stable expressed in CHO-K1 mammalian cells. Statistical analysis revealed strong positive correlations between the iELISA and VNT results (r = 0.9063, p < 0.0001) that were much greater than those between the IDEXX ELISA and VNT results (r = 0.8126, p < 0.0001). Taking the VNT data as the standard, the consistency of the iELISA (κ =0.880) was greater than that of the IDEXX ELISA (κ =0.699). In summary, the iELISA provides a more efficient and precise method for assessing CSFV immunity in pigs. Its reliable detection of immunoprotection levels against CSFV makes it an essential tool for optimizing CSF vaccination strategies. Consequently, its application can significantly support the ongoing efforts to eradicate CSF., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Signal transducer and activator of transcription 1 (STAT1) plays a critical role in immune response, human STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. Classical swine fever virus (CSFV)-infected induce autophagy, leading to immune evasion. However, there are limited reports on the function of porcine STAT1 in autophagy during CSFV infection. There is also lack of suitable in vitro models for studying porcine STAT1. The objective of this study was to establish porcine PK-15 STAT1-/- and 3D4/21 STAT1-/- cell lines using the CRISPR/Cas9 system to investigate the function of the STAT1 in autophagy. The PK-15 STAT1-/- and 3D4/21 STAT1-/- cell lines, featuring homozygous knockout of STAT1 gene were successfully constructed using the CRISPR/Cas9 editing system. The knockout efficiency determined to be 82.4% and 81.1%, respectively. Infection with CSFV in porcine PK-15 STAT1-/- and 3D4/21 STAT1-/- cells led to an observable increase in autophagosomes as evidenced by transmission electron microscope. Additionally, STAT1 knockout (STAT1 -/- ) by the CRISPR/Cas9 system upregulated the expression of ULK1, Beclin1, and LC3 genes, thereby enhancing autophagy during CSFV infection. Conversely, overexpression of STAT1 downregulated the expression of ULK1, Beclin1, and LC3 genes, leading to inhibition of autophagy during CSFV infection.The application of an autophagy dual-fluorescent-tracking plasmid demonstrated that STAT1 knockout enhanced autophagy accumulation during CSFV infection, while STAT1 overexpression inhibited it. Moreover, the 3D4/21 STAT1-/- cell line proved to be a more suitable in vitro model compared to the PK-15 STAT1-/- cell line for elucidating the involvement of STAT1 in autophagy during CSFV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Liang, Min, Liang, Tian, Liu, Luo and Li.)

Classical swine fever (CSF), sometimes referred to as hog cholera, is a highly contagious, virally based, systemic illness that affects both domestic and wild pigs. The virus known as classical swine fever virus (CSFV) is a member of the Flaviviridae family, specifically the genus Pestivirus. This disease is thought to be endemic in many Asian countries that produce pork as well as in several countries in Central and South America, the Caribbean, and elsewhere. As previously indicated, depending on the virulence of the virus strain involved and several host circumstances, clinical indications of CSFV infection can vary greatly, ranging from abrupt fatality to an occult course. CSF diagnosis can be made by serological detection, antigen, RNA, and isolation. CSF's highly varied symptoms and post-mortem pathology resemble those of African swine fever (ASF). ASF, the kind of CSFV, the pig's age, and its susceptibility all affect the clinical symptoms. Pigs that contract CSFV, a highly infectious and economically significant virus. The great economic significance of the swine business makes the CSFV a potential bioterrorism threat. Live attenuated CSF vaccinations have been around for many years and are quite safe and effective. Controlling epidemics in CSFV-free zones requires quick action. Pigs that are impacted must be slaughtered, and the carcasses must be buried or burned., Competing Interests: The authors declare that there is no conflict of interest.

Chimeric marker vaccine candidates, vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns , have been generated and their efficacy and capability to differentiate infected from vaccinated animals were confirmed in previous studies. The safety profile of the two chimeric marker vaccine candidates, particularly in the potential reversion to virulence, was evaluated. Each virus was administered to pigs with a dose equivalent to the vaccination dose, and pooled tonsil homogenates were subsequently inoculated into further pigs. Chimeric virus vGPE - /PAPeV E rns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE - /PhoPeV E rns was detectable until the third passage and disappeared entirely by the fourth passage. The vGPE - strain, assessed alongside, consistently exhibited stable virus recovery across each passage without any signs of increased virulence in pigs. In vitro assays revealed that the type I interferon-inducing capacity of vGPE - /PAPeV E rns was significantly higher than that of vGPE - /PhoPeV E rns and vGPE - . In conclusion, the safety profile of the two chimeric marker vaccine candidates was affirmed. Further research is essential to ensure the stability of their attenuation and safety in diverse pig populations.

Classical Swine Fever (CSF), a highly contagious viral disease affecting pigs and wild boar, results in significant economic losses in the swine industry. In endemic regions, prophylactic vaccination and stamping-out strategies are used to control CSF outbreaks. However, sporadic outbreaks and persistent infections continue to be reported. Although the conventional attenuated CSF vaccines protect pigs against the disease, they do not allow for the differentiation of infected from vaccinated animals (DIVA), limiting their use as an eradication tool. In this study, three targeted attenuation strategies were employed to generate vaccine candidates based on the current prevalent CSFV group 2 strains GD18 and QZ07: a single deletion of H79 in E rns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in E rns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in E rns combined with a 5-168 amino acids deletion of N pro (GD18-ddNpro-ErnsH-KARD). Additionally, a negative serological marker with four substitutions in a highly conserved epitope in E2 recognized by the monoclonal antibody 6B8 was introduced in each candidate for DIVA purposes. The safety of these four resulting vaccine candidates was evaluated in pregnant sows. Two candidates, GD18-ddErnsHC-KARD and QZ07-sdErnsH-KARD were found to be safe for pregnant sows and unlikely to cause vertical transmission. Both candidates also demonstrated potential to be used as DIVA vaccines, as was shown using a proprietary blocking ELISA based on the 6B8 monoclonal antibody. These results, together with our previous work, constitute a proof-of-concept for the rational design of CSF antigenically marked modified live virus vaccine candidates.

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.

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., (© 2023 Australian Veterinary Association.)

The host genetic makeup plays a significant role in causing the within-breed variation among individuals after vaccination. The present study was undertaken to elucidate the genetic basis of differential immune response between high and low responder Landlly (Landrace X Ghurrah) piglets vis-à-vis CSF vaccination. For the purpose, E2 antibody response against CSF vaccination was estimated in sampled animals on the day of vaccination and 21-day post-vaccination as a measure of humoral immune response. Double-digestion restriction associated DNA (ddRAD) sequencing was undertaken on 96 randomly chosen Landlly piglets using Illumina HiSeq platform. SNP markers were called using standard methodology. Genome-wide association study (GWAS) was undertaken in PLINK program to identify the informative SNP markers significantly associated with differential immune response. The results revealed significant SNPs associated with E2 antibody response against CSF vaccination. The genome-wide informative SNPs for the humoral immune response against CSF vaccination were located on SSC10, SSC17, SSC9, SSC2, SSC3 and SSC6. The overlapping and flanking genes (500Kb upstream and downstream) of significant SNPs were CYB5R1, PCMTD2, WT1, IL9R, CD101, TMEM64, TLR6, PIGG, ADIPOR1, PRSS37, EIF3M, and DNAJC24. Functional enrichment and annotation analysis were undertaken for these genes in order to gain maximum insights into the association of these genes with immune system functionality in pigs. The genetic makeup was associated with differential immune response against CSF vaccination in Landlly piglets while the identified informative SNPs may be used as suitable markers for determining variation in host immune response against CSF vaccination in pigs., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Classical swine fever has been spreading across the country since its re-emergence in Japan in 2018. Gifu Prefecture has been working diligently to control the disease through the oral vaccine dissemination targeting wild boars. Although vaccines were sprayed at 14,000 locations between 2019 and 2020, vaccine ingestion by wild boars was only confirmed at 30% of the locations. Here, we predicted the vaccine ingestion rate at each point by Random Forest modeling based on vaccine dissemination data and created prediction surfaces for the probability of vaccine ingestion by wild boar using spatial interpolation techniques. Consequently, the distance from the vaccination point to the water source was the most important variable, followed by elevation, season, road density, and slope. The area under the curve, model accuracy, sensitivity, and specificity for model evaluation were 0.760, 0.678, 0.661, and 0.685, respectively. Areas with high probability of wild boar vaccination were predicted in northern, eastern, and western part of Gifu. Leave-One-Out Cross Validation results showed that Kriging approach was more accurate than the Inverse distance weighting method. We emphasize that effective vaccination strategies based on epidemiological data are essential for disease control and that our proposed tool is also applicable for other wildlife diseases., (© 2024. The Author(s).)

The early detection of classical swine fever (CSF) remains a key challenge, especially when outbreaks are caused by moderate and low-virulent CSF virus (CSFV) strains. Oral fluid is a reliable and cost-effective sample type that is regularly surveilled for endemic diseases in commercial pig herds in North America. Here, we explored the possibility of utilizing oral fluids for the early detection of CSFV incursions in commercial-size pig pens using two independent experiments. In the first experiment, a seeder pig infected with the moderately-virulent CSFV Pinillos strain was used, and in the second experiment, a seeder pig infected with the highly-virulent CSFV Koslov strain was used. Pen-based oral fluid samples were collected daily and individual samples (whole blood, swabs) every other day. All samples were tested by a CSFV-specific real-time RT-PCR assay. CSFV genomic material was detected in oral fluids on the seventh and fourth day post-introduction of the seeder pig into the pen, in the first and second experiments, respectively. In both experiments, oral fluids tested positive before the contact pigs developed viremia, and with no apparent sick pigs in the pen. These results indicate that pen-based oral fluids are a reliable and convenient sample type for the early detection of CSF, and therefore, can be used to supplement the ongoing CSF surveillance activities in North America.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bohórquez, Muñoz-Aguilera, Lanka, Coronado, Rosell, Alberch, Maddox and Ganges.)

Classical Swine Fever (CSF), caused by the Classical Swine Fever Virus (CSFV), inflicts significant economic losses on the global pig industry. A key factor in the challenge of eradicating this virus is its ability to evade the host's innate immune response, leading to persistent infections. In our study, we elucidate the molecular mechanism through which CSFV exploits m6A modifications to circumvent host immune surveillance, thus facilitating its proliferation. We initially discovered that m6A modifications were elevated both in vivo and in vitro upon CSFV infection, particularly noting an increase in the expression of the methyltransferase METTL14. CSFV non-structural protein 5B was found to hijack HRD1, the E3 ubiquitin ligase for METTL14, preventing METTL14 degradation. MeRIP-seq analysis further revealed that METTL14 specifically targeted and methylated TLRs, notably TLR4. METTL14-mediated regulation of TLR4 degradation, facilitated by YTHDF2, led to the accelerated mRNA decay of TLR4. Consequently, TLR4-mediated NF-κB signaling, a crucial component of the innate immune response, is suppressed by CSFV. Collectively, these data effectively highlight the viral evasion tactics, shedding light on potential antiviral strategies targeting METTL14 to curb CSFV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral N pro . High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or N pro -expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV N pro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent N pro -mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote N pro -mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its N pro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) re-emerged in Gifu Prefecture, central Japan, in September 2018 and is currently widespread in wild boar populations. Due to its widespread in wild boars, an oral mass vaccination strategy was initiated in March 2019, employing a commercial bait vaccine that is a live attenuated vaccine. To enhance the effectiveness of oral vaccination, it is crucial to determine the vaccine's effective spatial range. This understanding is essential for devising a comprehensive vaccination strategy, which should also include a preliminary investigation of wild boar habitats before vaccination. This study aimed to estimate the effective range of oral vaccination for wild boars against CSF by analyzing the geographical relationship between immune wild boars and vaccination points within the vaccination areas in Gifu Prefecture. This study utilized oral vaccination data from April 2021 to March 2022. The prevalence of CSF infections in wild boars remained below 5% in this period, suggesting limited disease transmission and immune wild boars were considered to be induced by the effect of vaccination. Two vaccination campaigns were conducted during this period, with almost 2000 vaccination points each. To investigate the factors associated with the intensity (i.e., density) of immune wild boar, the nearest distances to a vaccination point and to a susceptible wild boar were evaluated as explanatory variables. The Rhohat procedure and point process model were utilized to analyze the relationship between the intensity of immune wild boars and the explanatory variables. The point process model revealed a significant decrease in the intensity of immune wild boars when the distance from the nearest vaccination point exceeded 500 m, indicating that the effective spatial range of bait vaccination is within 500 m of the vaccination point. Although the distance to the nearest susceptible animal did not show significance in the model, Rhohat plots indicated that the intensity of immune wild boars decreased at distances greater than 1200 m from the nearest susceptible wild boar. This finding highlights the importance of investigating susceptible wild boar populations within a range of at least 1200 m from a vaccination point before implementation. The present study revealed the effective range of oral vaccination for wild boars against CSF and indicated the importance of investigating susceptible wild boar habitats around vaccination points before the implementation of vaccination. These findings may help improve the effectiveness of oral vaccinations., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

Classical swine fever (CSF) has been eradicated from Western and Central Europe but remains endemic in parts of Central and South America, Asia, and the Caribbean. CSF virus (CSFV) has been endemic in Cuba since 1993, most likely following an escape of the highly virulent Margarita/1958 strain. In recent years, chronic and persistent infections with low-virulent CSFV have been observed. Amino acid substitutions located in immunodominant epitopes of the envelope glycoprotein E2 of the attenuated isolates were attributed to positive selection due to suboptimal vaccination and control. To obtain a complete picture of the mutations involved in attenuation, we applied forward and reverse genetics using the evolutionary-related low-virulent CSFV/Pinar del Rio (CSF1058)/2010 (PdR) and highly virulent Margarita/1958 isolates. Sequence comparison of the two viruses recovered from experimental infections in pigs revealed 40 amino acid differences. Interestingly, the amino acid substitutions clustered in E2 and the NS5A and NS5B proteins. A long poly-uridine sequence was identified previously in the 3' untranslated region (UTR) of PdR. We constructed functional cDNA clones of the PdR and Margarita strains and generated eight recombinant viruses by introducing single or multiple gene fragments from Margarita into the PdR backbone. All chimeric viruses had comparable replication characteristics in porcine monocyte-derived macrophages. Recombinant PdR viruses carrying either E2 or NS5A/NS5B of Margarita, with 36 or 5 uridines in the 3'UTR, remained low virulent in 3-month-old pigs. The combination of these elements recovered the high-virulent Margarita phenotype. These results show that CSFV evolution towards attenuated variants in the field involved mutations in both structural and non-structural proteins and the UTRs, which act synergistically to determine virulence.

Importance: Autophagy is a conserved degradation process that maintains cellular homeostasis and regulates native and adaptive immunity. Viruses have evolved diverse strategies to inhibit or activate autophagy for their benefit. The paper reveals that CSFV NS5A mediates the dissociation of PP2A from Beclin 1 and the association of PP2A with DAPK3 by interaction with PPP2R1A and DAPK3, PP2A dephosphorylates DAPK3 to activate its protein kinase activity, and activated DAPK3 phosphorylates Beclin 1 to trigger autophagy, indicating that NS5A activates autophagy via the PP2A-DAPK3-Beclin 1 axis. These data highlight a novel mechanism by which CSFV activates autophagy to favor its replication, thereby contributing to the development of antiviral strategies., Competing Interests: The authors declare no conflict of interest.

Classical swine fever virus (CSFV) is an ancient pathogen that continues to pose a threat to animal agriculture worldwide. The virus belongs to the genus Pestivirus and the family Flaviviridae. It causes a multisystemic disease that affects only pigs and is responsible for significant economic losses. CSFV infection is probably a multistep process that involves the proteins in the virus envelope and more than one receptor in the membrane of permissive cells. To date, the cellular receptors essential for CSFV entry and their detailed functions during this process remains unknown. All the viral envelope proteins Erns, E1 and E2 are involved in the entry process to some extent and the experimental approaches conducted until now have helped to unveil their contributions. This review aims to provide an overview of current knowledge on cellular molecules described to be involved in CSFV entry, including complement regulatory protein 46 (CD46), heparan sulphate (HS), Laminin receptor, Integrin ß3, Annexin II, MERKT and ADAM17. This knowledge would not only help to understand the molecular mechanisms involved in pestivirus infection, but also provide a rational basis for the development of nonvaccinal alternatives for CSFV control., (© 2023. The Author(s).)

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.)

CSFV (classical swine fever virus) is currently endemic in developing countries in Asia and has recently re-emerged in Japan. Under the pressure of natural selection pressure, CSFV keeps evolving to maintain its ecological niche in nature. CSFV has evolved mechanisms that induce immune depression, but its pathogenic mechanism is still unclear. In this study, using transcriptomics and metabolomics methods, we found that CSFV infection alters innate host immunity by activating the interferon pathway, inhibiting host inflammation, apoptosis, and remodelling host metabolism in porcine alveolar macrophages. Moreover, we revealed that autophagy could alter innate immunity and metabolism induced by CSFV infection. Enhanced autophagy further inhibited CSFV-induced RIG-I-IRF3 signal transduction axis and JAK-STAT signalling pathway and blocked type I interferon production while reducing autophagy inhibition of the NF-κB signalling pathway and apoptosis in CSFV infection cells. Furthermore, the level of CSFV infection-induced glycolysis and the content of lactate and pyruvate, as well as 3-phosphoglyceraldehyde, a derivative of glycolysis converted to serine, was altered by autophagy. We also found that silencing HK2 (hexokinase 2), the rate-limiting enzyme of glycolytic metabolism, could induce autophagy but reduce the interferon signalling pathway, NF-κB signalling pathway, and inhibition of apoptosis induced by CSFV infection. In addition, inhibited cellular autophagy by silencing ATG5 or using 3-Methyladenine, could backfill the inhibitory effect of silencing HK2 on the cellular interferon signalling pathway, NF-κB signalling pathway, and apoptosis.

In several countries, classical swine fever (CSF) has not been detected in domestic pigs, but has been detected in wild boars, making the disease difficult to control. To overcome this problem, we inoculated pigs with a CSF live marker vaccine (Flc-LOM-BE rns strain), which has "distinguish infection from vaccinated animals (DIVA)" function, to determine whether it is suitable as an oral vaccine specifically for wild boars. Pigs inoculated intramuscularly or orally with the Flc-LOM-BE rns vaccine were challenged 2 or 4 weeks later, respectively, with virulent CSFV. Pigs administered the oral Flc-LOM-BE rns strain (10 5.0 and 6.0 TCID 50 /dose), and those vaccinated intramuscularly (10 3.0 TCID 50 /dose), had normal numbers of leukocytes and normal body temperature. Also, they generated protective neutralizing antibodies and anti-BVDV E rns antibodies. In addition, all pigs in these groups survived, with no CSFV RNA detected in feces, spleen, or other organs. Thus, the Flc-LOM-BE rns vaccine shows excellent safety and efficacy, while having DIVA function and suitability for oral inoculation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Importance: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express N pro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its N pro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving N pro -mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms., Competing Interests: The authors declare no conflict of interest.

Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the Pestivirus genus in the Flaviviridae family. CSFV causes a severe and highly contagious disease in pigs and is prevalent worldwide, threatening the pig farming industry. The detailed mechanisms of the CSFV life cycle have been reported, but are still limited. Some receptors and attachment factors of CSFV, including heparan sulfate (HS), laminin receptor (LamR), complement regulatory protein (CD46), MER tyrosine kinase (MERTK), disintegrin, and metalloproteinase domain-containing protein 17 (ADAM17), were identified. After attachment, CSFV internalizes via clathrin-mediated endocytosis (CME) and/or caveolae/raft-dependent endocytosis (CavME). After internalization, CSFV moves to early and late endosomes before uncoating. During this period, intracellular trafficking of CSFV relies on components of the endosomal sorting complex required for transport (ESCRT) and Rab proteins in the endosome dynamics, with a dependence on the cytoskeleton network. This review summarizes the data on the mechanisms of CSFV attachment, internalization pathways, and intracellular trafficking, and provides a general view of the early events in the CSFV life cycle.

C-strain, also known as the HCLV strain, is a well-known live attenuated vaccine against classical swine fever (CSF), a devastating disease caused by classical swine fever virus (CSFV). Vaccination with C-strain induces a rapid onset of protection, which is associated with virus-specific gamma interferon (IFN-γ)-secreting CD8 + T cell responses. The E2 protein of CSFV is a major protective antigen. However, the T cell epitopes on the E2 protein remain largely unknown. In this study, eight overlapping nonapeptides of the E2 protein were predicted and synthesized to screen for potential T cell epitopes on the CSFV C-strain E2 protein. Molecular docking was performed on the candidate epitopes with the swine leukocyte antigen-1*0401. The analysis obtained two highly conserved T cell epitopes, 90 STEEMGDDF 98 and 331 ATDRHSDYF 339 , which were further identified by enzyme-linked immunospot assay. Interestingly, the mutants deleting or substituting the epitopes are nonviable. Further analysis demonstrated that 90 STEEMGDDF 98 is crucial for the E2 homodimerization, while CSFV infection is significantly inhibited by the 331 ATDRHSDYF 339 peptide treatment. The two novel T cell epitopes can be used to design new vaccines that are able to provide rapid-onset protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Vaccination with E2 subunit vaccines is currently the main measure to control classical swine fever virus (CSFV), which is an endemic disease, and detection of antibodies against CSFV E2 is the most effective way to evaluate herd immunity. In the present study, the E2 protein was expressed by a baculovirus expression system, and two monoclonal antibodies (mAbs), namely, 3A9 and 4F7, were successfully produced using techniques for the isolation of single B cells from splenocytes from mice immunized with the E2 protein. Moreover, two linear B-cell epitopes, 25 GLTTTWKEYSHDLQL 39 and 259 GNTTVKVHASDERGP 273 , reactive to 3A9 and 4F7, respectively, were identified using epitope mapping of the E2 protein. In addition, the diagnostic performance of the two mAbs was evaluated using blocking enzyme-linked immunosorbent assay (bELISA), and the results showed that the two mAbs had high diagnostic specificity (96.08%, 94.38%) and diagnostic sensitivity (97.49%, 95.97%). Together, these findings identify two ideal candidate peptides and matching mAbs for a new method of CSFV diagnosis, which will contribute to the control and eradication of classical swine fever., (© 2023. L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE).)

Classical swine fever (CSF) is a severe infectious disease caused by the classical swine fever virus (CSFV) that poses significant challenges to the swine industry. α-ketoglutarate dehydrogenase (OGDH), the first rate-limiting enzyme of the tricarboxylic acid (TCA) cycle, catalyzes α-ketoglutarate (α-KG) to succinyl-CoA, playing a crucial role in glycometabolism. Our previous studies showed that CSFV disrupts the TCA cycle, resulting in α-KG accumulation. However, the interplay between CSFV and OGDH remains unclear. In this study, we found that CSFV significantly reduces OGDH protein levels and promotes α-KG secretion through OGDH in PK-15 cells. Furthermore, we observed CSFV C protein interacts with OGDH and revealed that CSFV utilizes NDP52/NBR1 to target OGDH protein degradation in the autophagy-lysosome pathway. We also unveiled that OGDH overexpression inhibits CSFV proliferation, whereas OGDH knockdown increases CSFV proliferation. Further investigation into the mechanisms of OGDH on CSFV replication revealed that OGDH regulates the AMPK-mTOR-autophagy pathway. Additionally, using the autophagy agonist/inhibitor, rapamycin/3-MA, we observed that OGDH modulates autophagy to regulate the IRF3-IFN-β network and CSFV replication. These findings shed light on the role of OGDH in CSFV infection and host metabolism, promoting the development of innovative strategies for combating CSFV and other viral infections via targeting metabolic pathways., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Infectious diseases at the wildlife-livestock-human interface have become a crucial issue, and evidence-based measures are growing increasingly important. One countermeasure against animal diseases in wildlife is using fencing to contain and reduce disease spread and transmission rates between wild populations; however, quantitative assessments on fencing are rare. Moreover, existing research on fencing has highlighted knowledge gaps on the social and ecological aspects relevant to the use and design of fences. To control the spread of classical swine fever, fences were installed from the east to west in Gifu Prefecture, Japan, by March 2019, with the aim of restricting wild boar movement. To clarify the process of installation and maintenance of the fences, we conducted semi-structured interviews with prefectural government officers in Gifu Prefecture. Additionally, we installed infrared-triggered cameras at fence locations with and without gaps to evaluate the fence permeability of mammals. We used a generalized linear mixed model to evaluate the relationship between the presence of gaps and the relative abundance and permeability of each mammal. Our findings showed that the occurrence of gaps was inevitable during the installation and management of wide-area perimeter fence in Japan, partly because of social factors. For example, fences could not be installed on roads that were frequently used by residents and were not adequately maintained owing to budgetary reasons in some cases. Analysis of footage from the infrared-triggered cameras revealed that several mammal species crossed the fence at gaps and even had the ability to cross the gapless parts of the fences. Wild boars crossed through the gaps regularly. It is possible that Sika deer, Japanese serows, raccoons, Japanese badgers, raccoon dogs, Japanese macaques, and feral cats crossed through fence gaps because their relative abundance was high at gap locations. In contrast, Japanese hares slipped through the fence mesh rather than crossing through the gaps. In conclusion, we suggest that coordination and collaboration among related parties, a sufficient supply of fence materials, and securing a budget for fence maintenance are important for fence installation and maintenance. Furthermore, as fence gaps are inevitable, technical development of countermeasures for these gaps could be effective., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masatsugu Suzuki reports financial support was provided by Environmental Restoration and Conservation Agency of Japan., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Classical swine fever and porcine reproductive and respiratory syndrome have seriously affected the development of the swine breeding industry in China. Vaccine immunization remains the main way to prevent these infections. The aim of this study was to establish an optimized protocol for vaccine immunization against classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV)., Methods: Blood samples were collected from the anterior vena cava of pigs after immunization, and blood indices, secreted levels of specific antibodies and neutralizing antibodies associated with humoral immunity, the proliferation capacity of T lymphocytes as a measure of cellular immunity, and secreted levels of IFN-γ and TNF-α were determined., Results: The results showed that simultaneous immunization against CSFV and PRRSV infections induced strong and specific humoral and T-cellular immune responses, high levels of cytokine IFN-γ secretion and delayed secretion of cytokine TNF-α. Moreover, significantly higher lymphocyte percentages and red blood cell and leukocyte counts were found in the group simultaneously immunized against CSFV and PRRSV. However, no statistically significant differences were observed in hemoglobin values, neutrophil counts, and median cell percentages among the S + PRRS, PRRS-S, and S-PRRS groups., Conclusion: This study demonstrated that simultaneous immunization against CSFV and PRRSV had the advantages of inducing a rapid, enhanced, and long-lasting immune response. These findings provide a theoretical basis for the establishment of a reasonable and optimized vaccine immunization protocol against CSFV and PRRSV in combination with a variety of other vaccine inoculations., (© 2023. The Author(s).)

A previous study proved that vGPE - mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE - vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE - vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein E rns of the GPE - vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns , were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE - vaccine strain. Vaccination at a dose of 10 4.0 TCID 50 with either vGPE - /PAPeV E rns or vGPE - /PhoPeV E rns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE - /PAPeV E rns and vGPE - /PhoPeV E rns affirmed their properties, as the vGPE - vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Three-dimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. IMPORTANCE Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV., Competing Interests: The authors declare no conflict of interest.

Exosomes, which are small membrane-enclosed vesicles, are actively released into the extracellular space by a variety of cells. Growing evidence indicates that exosomes derived from virus-infected cells can selectively encapsulate viral proteins, genetic materials, or even entire virions. This enables them to mediate cell-to-cell communication and facilitate virus transmission. Classical swine fever (CSF) is a disease listed by the World Organisation for Animal Health (WOAH) Terrestrial Animal Health Code and must be reported to the organisation. It is caused by classical swine fever virus (CSFV) belonging to the Flaviviridae family. Recent studies have demonstrated that extracellular vesicles originating from autophagy can facilitate the antibody-resistant spread of classical swine fever virus. However, due to the extreme difficulty in achieving a complete separation from virions, the role of exosomes during CSFV infection and proliferation remains elusive. In this study, we ingeniously chose to perform immunoprecipitation (IP) targeting the CSFV E2 protein, thereby achieving the complete removal of infectious virions. Subsequently, we discovered that the purified exosomes are shown to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes secreted by CSFV-infected cells can evade CSFV-specific neutralizing antibodies, establish subsequent infection, and stimulate innate immune system after uptake by recipient cells. In summary, exosomes play a critical role in CSFV transmission. This is of great significance for in-depth exploration of the characteristics of CSFV and its complex interactions with the host., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of N pro and substituted 3'UTR to generate the recombinant chimeric virus vC/SM3'UTR N -S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3'UTR N -S1NCOE to obtain vC/SM3'UTR N -tPAsS1NCOE and vC/SM3'UTR N -CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The N pro D136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-β induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-β induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3'UTR N -CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3'UTR N -CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

Classical Swine Fever (CSF) is still one of the most economically important viral diseases of pigs. The disease is controlled by vaccination in the endemic countries. Hence, availability or supply of efficacious and potent vaccine in the field settings is of utmost importance. Currently, as per requirement of any Pharmacopoea, a CSF vaccine must contain 100 PD 50 /dose which is determined by vaccinating pigs at 1/40 th and 1/160 th dilution of each dose followed by virulent challenge at 28 days post vaccination (dpv). Here, the control and the unprotected groups succumb to disease and need to be euthanized. Moreover, such challenge experiments are not feasible for each batch of the vaccine. In this communication, an alternate method of PD 50 dose calculation of live-attenuated CSF vaccines by measuring Serum Neutralizing Titre i.e Fluorescent Antibody Virus Neutralization (FAVN) titre of the vaccinated pigs at 28 dpv was established. This alternative method do not require the vaccinated pigs to be challenged. Serum samples, generated out of QC testing of eight batches of CSF vaccines in the laboratory, were tested and found that pigs having FAVN titre ≥10 were protected against challenge. Initially this test was optimized in serum samples of 12 animals and then validated with another 56 serum samples. It was found that the alternate method is 100% correlating with the challenge experiment. Thus, based on FAVN titre of the vaccinated animal serum, it can be predicted whether the pigs would or would not come through the challenge infection. Using the predicted status (protected/succumbed), PD50 can be calculated by applying Reed and Muench formula, hence alternate method can be used as routine QC test for potency of CSF vaccines. The newly developed assay was specific since no signal was observed in controls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC 50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1 st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV E rns -positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV E rns - and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Classical swine fever virus (CSFV) is an enveloped positive-sense RNA virus belonging to the Flaviviridae family. The virus utilizes cellular lipids and manipulates host lipid metabolism to ensure its replication, especially during virus invasion and replication steps. Therefore, identification of the molecular lipid metabolism pathways that are suitable targets is critical for the development of anti-CSFV therapeutics. In this study, we screened the anti-CSFV activity of 12 compounds targeting synthesis of cholesterol and fatty acids, cholesterol esters, and cholesterol transport. We found that 25-hydroxycholesterol (25HC), a regulator of cholesterol metabolism and transport, has potent anti-CSFV activity. Mechanistically, we showed that 25HC inhibited CSFV proliferation by blocking the entry of virions into porcine alveolar macrophages (3D4/21) by decreasing cholesterol abundance in the plasma membrane through activation of acyl-CoA:cholesterol acyltransferase (ACAT). Finally, we revealed that cholesterol 25-hydroxylase (CH25H), a redox enzyme that mediates 25HC production, also restricted CSFV infection via both enzyme activity-dependent and -independent mechanisms. Collectively, our results shed light on the mechanisms by which 25HC inhibits CSFV entry into cells and suggests a potential new therapeutic method against CSFV infection., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) live vaccine used in Japan, GPE - strain, is produced using guinea pig kidney (GPK)-derived primary culture cells. This means that a large number of guinea pigs are used to generate the primary GPK cells needed to produce the CSF live vaccine, and alternative solution is desired. Hence, we established two GPK cell lines capable of culturing the GPE - strain: spontaneously immortalized GPK (GPK-SI) cells were generated by repeated passaging of primary GPK cells, and the other cell line, artificially immortalized GPK (GPK-AI) cells, were obtained by introducing the SV40 large T antigen gene into primary GPK cells. Both cell lines were susceptible to the GPE - virus, and the virus grew more efficiently in GPK-SI cells at 37°C. When the culture temperature was set to 30°C, the virus titer reached 10 4.8 50% Tissue Culture Infectious Dose (TCID 50 )/mL in GPK-SI cells 7 days after virus inoculation at a multiplicity of infection (MOI) of 1, which was equivalent to that in cells cultured at 37°C. When the virus was inoculated at MOI <1, the virus titer 7 days after inoculation was higher when cultured at 30°C than when cultured at 37°C in both cell lines, reaching 10 5.63 TCID 50 /mL in GPK-SI cells. These results indicate that GPK-SI and GPK-AI cells can potentially replace primary GPK cells for the production of CSF live vaccines. This could also contribute to stable CSF vaccine production and animal welfare.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

The classical swine fever virus (CSFV) is a species member of the family Flaviviridae. CSFV is widely distributed in the world causing a severe impact on pig industry. This pathogen is considered restricted to domestic and wild suids. However, some reports from 2014 to 2018 showed the presence of the CFSV antigen in the bovine species. The virus was found in commercialized batches of fetal bovine serum (FBS) of Chinese origin and in bovine herds in in the provinces of Henan and Jiangsu, China, and in Tamil Nadu and Meghalaya, southern and north‑eastern states of India, respectively. Detection was done using antigen capture ELISA and RT‑PCR tests. In certain cases, animals with natural infection showed clinical signs and reproduction was also affected. Genetic characterization was performed considering the 5'‑UTR sequences of the bovine strains. In addition, the entire CSFV E2 genomic region could be amplified from two positive animals. The bovine strains were genetically related to the Chinese CSFV live attenuated hog cholera lapinized vaccine (HCLV) strain used in pigs, sharing sequence characteristics. The vaccine strain HCLV was widely used in China to protect bovines and yaks from bovine viral diarrhea, and, as a possible consequence, inducing an adaptation in cattle and a further natural diffusion. Furthermore, a contaminant strain from China was genetically distant from all other previously described genotypes of the CSFV. This suggests also the occurrence of micro evolutive step in the species related to geographical segregation. These observations deserve attention and further investigations, especially relevant in countries where CSFV control and eradication strategies are applied.