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Pseudorabies virus (PRV) and classical swine fever virus (CSFV) are both economically important pathogens threatening the pig industry in many countries. The triple-gene-deleted variant of PRV, herein referred to as rPRVTJ-delgE/gI/TK, has exhibited pronounced efficacy and safety profiles. This underscores its viability as a prospective vaccine vector. However, the generation of specific anti-E2 antibodies necessitates elevated immunization doses and extended durations when the extracellular domain of the E2 protein of CSFV is secreted via the recombinant rPRVTJ-delgE/gI/TK vector. To enhance the presentation of exogenous antigens by antigen-presenting cells (APCs), we engineered the E2 protein expressed on the surface of PRV particles in this study. The recombinant virus expressing the E2 protein with a heterogonous transmembrane domain was generated in the backbone of rPRVTJ-delgE/gI/TK and designated as rPRVTJ-UL44-E2. The E2 gene was fused to the 3' terminus of the UL44 gene utilizing P2A, a self-cleaving peptide sequence. The electron microscopy showed that the E2 protein was anchored on the surface of the viral particles of rPRVTJ-delgE/gI/TK-E2. The insertion of the E2 gene did not alter the native biological characteristics of the viral vector. Rabbits immunized with 10 7 median tissue culture infective doses (TCID 50 ) of rPRVTJ-UL44-E2 exhibited a rapid seroconversion to anti-E2 specific antibodies within 7 days post-immunization (dpi). All the rabbits immunized with the rPRVTJ-UL44-E2 had generated antibodies specific to E2 prior to the administration of the booster immunization. However, the immunized rabbits were not protected from the CSFV C-strain challenge. Nevertheless, this strategy has notably achieved rapid induction of E2-specific non-neutralizing antibodies. These findings provide insights that the design of rPRVTJ-UL44-E2 requires optimization, thereby indicating a promising avenue for augmenting vaccine-induced immune responses., 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) is a severe disease caused by the highly contagious CSFV. Our previous study demonstrated that exosomes from CSFV-infected cells contained significant amounts of viral genome and Core (C) protein and were infectious. To further elucidate the mechanisms underlying the formation of these infectious exosomes, we investigated the intracellular transport of the C protein in this study. We first identified the synchronized transport of the C protein and viral genome to exosomes, distinguishing it from other structural proteins. This suggests that the C protein likely binds to the viral genome and is transported to exosomes as a nucleocapsid. Subsequently, Co-IP and co-localization experiments confirmed the interaction between the host Myosin 1B (MYO1B) protein and the C protein. Key interaction sites were identified by generating and analyzing various C protein point mutations and truncation variants. The results indicate that specific sites at the N-terminus of the C protein significantly impact its interaction with MYO1B. Ultimately, by modulating MYO1B expression, we found that MYO1B knockdown significantly reduced the C protein and viral genome content in exosomes, leading to a decrease in CSFV titers. These findings underscore the critical role of MYO1B in facilitating the transport of the C protein and viral genome into exosomes during CSFV infection. Overall, this study explores the mechanism of infectious exosome formation during CSFV infection, revealing the critical role of the host MYO1B in this process. This is the first study to identify the involvement of MYO1B in viral infection, not only offering important insights into host-virus interactions but also identifying a new target for antiviral drug development., 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.)

African swine fever (ASF), a highly infectious and devastating disease affecting both domestic pigs and wild boars, owes its etiology to African swine fever virus (ASFV). ASFV encodes more than 165 proteins. However, novel immunogenic proteins remain unknown. This study aimed to determine the antigenicity of the F317L protein (pF317L) of ASFV. The results revealed that pF317L was able to react with convalescent pig sera, indicating that pF317L could be a candidate antigen. The antigenic potential of pF317L expressed by rHCLV-F317L, a recombinant virus in the backbone of C-strain (a lapinized live attenuated classical swine fever virus) was further investigated in rabbits and pigs. The results revealed that antibodies and cell-mediated immune responses against pF317L were induced in either rabbits or pigs inoculated with rHCLV-F317L. Importantly, anti-pF317L antibodies from rabbits or pigs immunized with rHCLV-F317L significantly inhibited ASFV replication in vitro. In conclusion, pF317L demonstrates favorable immunogenic properties, positioning it as a promising candidate for the development of protective antigens in the ongoing endeavor to formulate efficacious ASF vaccine strategies., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest 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.)

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 virus (CSFV), an obligate intracellular pathogen, hijacks cellular metabolism to evade immune surveillance and facilitate its replication. The precise mechanisms by which CSFV modulates immune metabolism remain largely unknown. Our study reveals that CSFV infection disrupts serine metabolism, which plays a crucial role in antiviral immunity. Notably, we discovered that CSFV infection leads to the deacetylation of PHGDH, a key enzyme in serine metabolism, resulting in autophagic degradation. This deacetylation impairs PHGDH's enzymatic activity, reduces serine biosynthesis, weakens innate immunity, and promotes viral proliferation. Molecularly, CSFV infection induces the association of HDAC3 with PHGDH, leading to deacetylation at the K364 site. This modification attracts the E3 ubiquitin ligase RNF125, which facilitates the addition of K63-linked ubiquitin chains to PHGDH-K364R. Subsequently, PHGDH is targeted for lysosomal degradation by p62 and NDP52. Furthermore, the deacetylation of PHGDH disrupts its interaction with the NAD + substrate, destabilizing the PHGDH-NAD complex, impeding the active site, and thereby inhibiting de novo serine synthesis. Additionally, our research indicates that deacetylated PHGDH suppresses the mitochondria-MAVS-IRF3 pathway through its regulatory effect on serine metabolism, leading to decreased IFN-β production and enhanced viral replication. Overall, our findings elucidate the complex interplay between CSFV and serine metabolism, revealing a novel aspect of viral immune evasion through the lens of immune metabolism., Importance: Classical swine fever (CSF) seriously restricts the healthy development of China's aquaculture industry, and the unclear pathogenic mechanism and pathogenesis of classical swine fever virus (CSFV) are the main obstacle to CSF prevention, control, and purification. Therefore, it is of great significance to explore the molecular mechanism of CSFV and host interplay, to search for the key signaling pathways and target molecules in the host that regulate the replication of CSFV infection, and to elucidate the mechanism of action of host immune dysfunction and immune escape due to CSFV infection for the development of novel CSFV vaccines and drugs. This study reveals the mechanism of serine metabolizing enzyme post-translational modifications and antiviral signaling proteins in the replication of CSFV and enriches the knowledge of CSFV infection and immune metabolism., Competing Interests: The authors declare no conflict of interest.

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.)

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.)

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC 50 ) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF., Importance: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo , but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV., Competing Interests: The authors declare no conflict of interest.

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&#95;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.)

Classical swine fever (CSF) is caused by the classical swine fever virus (CSFV), which poses a threat to swine production. The activation of host innate immunity through linker proteins such as tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) is crucial for the induction of the NF-κB pathway. Recent research has revealed the involvement of mitochondrial antiviral-signaling protein (MAVS) in the interaction with TRAF2, 3, 5, and 6 to activate both the NF-κB and IRF3 pathways. This study revealed that CSFV infection led to the upregulation of TRAF1 mRNA and protein levels; moreover, TRAF1 overexpression inhibited CSFV replication, while TRAF1 knockdown promoted replication, highlighting its importance in the host response to CSFV infection. Additionally, the expression of RIG-I, MAVS, TRAF1, IRF1, and ISG15 were detected in PK-15 cells infected with CSFV, revealing that TRAF1 plays a role in regulating IRF1 and ISG15 within the RIG-I pathway. Furthermore, Co-IP, GST pull-down, and IFA analyses demonstrated that TRAF1 interacted with MAVS and co-localized in the cytoplasm during CSFV infection. Ultimately, TRAF1 acted as a novel member of the TRAF family, bound to MAVS as a linker molecule, and functioned as a mediator downstream of MAVS in the RIG-I/MAVS pathway against CSFV replication.

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.)

Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae . However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae . In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

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.

Simple Summary: African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine pseudorabies virus (PRV) are important pathogens circulating in pig herds in many countries. Pigs infected with these viruses show similar signs, such as increased body temperature and loss of appetite, and are prone to respiratory symptoms, digestive symptoms, neurological symptoms, and/or abortion in pregnant sows. To accurately detect these viruses and differentially diagnose these diseases, four pairs of specific primers and TaqMan probes were designed aiming at the B646L (p72) gene of ASFV, the 5′ untranslated region (5′UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV. After optimizing the reaction conditions, a multiplex real-time quantitative RT-PCR (RT-qPCR) assay was developed for the differential detection of ASFV, CSFV, PRRSV, and PRV. The assay was further validated to test 3116 clinical samples, and the positivity rates of ASFV, CSFV, PRRSV, and PRV were 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively. The assay provides a highly specific, sensitive, and reproducible method for the detection of ASFV, CSFV, PRRSV, and PRV. African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV) induce similar clinical signs in infected pigs, including hyperthermia, anorexia, hemorrhage, respiratory distress, neurological symptoms, and/or abortions in pregnant sows. The differential diagnosis of these diseases relies on laboratory examinations. In this study, a quadruplex RT-qPCR was established using four pairs of specific primers and probes aimed at the B646L (p72) gene of ASFV, the 5′ untranslated region (5′UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV for the detection and differentiation of ASFV, CSFV, PRRSV, and PRV. The assay exhibited great sensitivity with limits of detection (LODs) of 134.585, 139.831, 147.076, and 142.331 copies/reaction for ASFV, CSFV, PRRSV, and PRV, respectively. The assay exclusively identified ASFV, CSFV, PRRSV, and PRV, yielding negative results for the other control swine viruses used in this study. The intra-assay and inter-assay coefficients of variation (CVs) were not higher than 1.12%, indicating good reproducibility of the assay. The quadruplex RT-qPCR assay was used to analyze 3116 clinical tissue samples from pigs in Guangxi province, China, from April 2023 to September 2024. ASFV, CSFV, PRRSV, and PRV had positivity rates of 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively, demonstrating a coincidence rate of ≥99.45% with the previously described RT-qPCR assays, which were also used to test these same samples. The established assay was rapid, sensitive, and accurate in detecting and differentiating ASFV, CSFV, PRRSV, and PRV. [ABSTRACT FROM AUTHOR]

Abstract 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 oC and have a shelf life of at least 36 months at 2–8 oC. 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 oC for 12 months; (2) 30oC and 37 oC 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.

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.)

Classical swine fever virus (CSFV) and porcine productive and respiratory syndrome virus (PRRSV) both are significant infectious pathogens in pigs and pose great threats to the healthy development of the pig industry. PRRSV infection often reduces the antibody level of the CSFV attenuated vaccine and even leads to immune failure. In order to elucidate the potential mechanism of CSFV proliferation inhibition by PRRSV and screen out drugs that enhance the vaccine immune effect, we conducted experiments in the PAM39 cell line that can simultaneously support both PRRSV and CSFV infection. The results showed that PRRSV infection could induce gasdermin D (GSDMD) cleavage, promote cell pyroptosis, increase IL-1β secretion, and then inhibit CSFV replication. However, Astragalus polysaccharide treatment could reverse this phenomenon. The results elucidate the molecular mechanism of CSFV vaccine immune failure caused by PRRSV co-infection from the perspective of pyroptosis and provide a scientific basis for the prevention and control of clinical co-infection diseases., Competing Interests: Declaration of Competing Interest No conflict of interest exists in the submission of this manuscript, and the manuscript has been approved by all authors for publication. We also declare that the work described has not been submitted elsewhere for publication. Declaration of Competing Interest No conflict of interest exists in the submission of this manuscript, and the manuscript has been approved by all authors for publication., (Copyright © 2024. Published by Elsevier B.V.)

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 (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. African swine fever (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 classical swine fever virus 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. [Open Vet J 2024; 14(10.000): 2497-2508]

Classical swine fever (CSF) remains a challenge for pig farming industrial lover the world despite the measures taken. The last CS case in the Russian Federation was reported in 2020, however, the threat of the disease emerging still persists. A set of anti-epidemic measures including mainly preventive vaccination and annual diagnostic monitoring using molecular-genetic and serological methods is required for CSF virus introduction prevention and rapid eradication of potential disease out breaks. Therefore, areal-time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Therefore, areal time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Modified primers (locked nucleic acids containing conformationally blocked nucleosides) providing a higher affinity to the DNA matrix and physico-chemical stability and a FAM-labeled TaqMan probe were selected for 5’-untranslatedregion of the genome. The following validation parameters were defined: accuracy, repeatability, reproducibility, specificity and sensitivity. For comparative analysis of the developed as say sensitivity, swabs, samples of organs and tissues collected from pigs experimentally infected with an epizootic strain of the classical swine fever virus (spleen, kidney, liver, blood, lymph nodes, rectal and oral smears), animal-contaminated feed and virus-containing material with known virus titres were also tested in parallel with coded test systems No. x1 andx2. The developed assay was shown to have 100% diagnostic sensitivity and detection limit of 0,23 lgCCID50/cm3. Therewith, there sults of analysis of test systems No. x1, x2 based on above parameters were lower that could give rise to false positive real-time RT-PCR results and incorrect diagnosis. Thus, described assay can be used for extensive monitoring of classical swine fever in the Russian Federation

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).)

Abstract Classical swine fever virus (CSFV) p7 viroporin plays crucial roles in cellular ion balance and permeabilization. The antiviral drug amantadine effectively inhibits viral replication by blocking the activity of CSFV p7 viroporin. However, little information is available for the binding mode of amantadine with CSFV p7 viroporin, due to the lack of a known polymer structure for CSFV p7. In this study, we employed AlphaFold2 to predict CSFV p7 structures. Subsequently, we conducted a docking study to investigate the binding sites of amantadine to CSFV p7. Computational analysis showed that CSFV p7 forms a pore channel in a hexameric structure. Furthermore, molecular dynamics (MD) simulations and mutant analyses further suggest that CSFV p7 likely exists as a hexamer. Docking studies and MD simulations showed that amantadine interacts with the hydrophibic regions of tetramer and pentamer, as well as with the hydrophobic pore channel of the hexamer. Considering the potential hexameric assembly of CSFV p7, along with docking results, MD simulations, and the characteristics of the gated ion channels, we propose a model of CSFV p7 ion channel based on its hexameric configuration. In this model, residues E21, Y25, and R34 are suggested to selectively recruit and dehydrate ions, while residues L28 and L31 likely act as hydrophobic constrictors, thereby restricting the free movement of water. The binding of amantadine to residues I20, E21, V24 and Y25 effectively blocks ion transport. However, this proposed molecular model requires experimental validation. Our findings give a structural insight into the models of CSFV p7 as an ion channel and provide a molecular explanation for the inhibition effects of amantadine on CSFV p7-mediated ion channel conductance.

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.

Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) are two of the most devastating and economically significant pathogens affecting pig populations worldwide. Administration of a combination of vaccines against swine pathogens has been demonstrated to be as efficacious as the administration of single vaccines. In this study, we developed and tested a novel bivalent subunit vaccine against CSFV and PCV2. The safety and efficacy of this vaccine were demonstrated in mice and specific pathogen-free (SPF) piglets. In addition to investigating the serological responses after immunization, challenge studies with both viruses were also conducted. The results showed that this CSFV/PCV2 bivalent vaccine elicited a high level of neutralizing antibodies against both viruses and provided protection in challenge studies. In conclusion, the CSFV/PCV2 bivalent vaccine is safe and effective against CSFV or PCV2 challenge., (© 2024. The Author(s).)

Classical swine fever virus (CSFV) is a highly pathogenic RNA virus belonging to the Flaviviridae family that can cause deadly classical swine fever (CSF) in pigs. However, the molecular details of virus replication in the host are still unclear. Our previous studies have reported that several Rab proteins mediate CSFV entry into host cells, but it is unknown whether CSFV hijacks other Rab proteins for effective viral infection. Here, we systematically studied the role of Rab14 protein in regulating lipid metabolism for promoting viral assembly. First, Rab14 knockdown and overexpression significantly affected CSFV replication, indicating the essential role of Rab14 in CSFV infection. Interestingly, Rab14 could significantly affect virus replication in the late stage of infection. Mechanistically, CSFV NS5A recruited Rab14 to the ER, followed by ceramide transportation to the Golgi apparatus, where sphingomyelin was synthesized. The experimental data of small molecule inhibitors, RNA interference, and replenishment assay showed that the phosphatidylinositol-3-kinase (PI3K)/AKT/AS160 signaling pathway regulated the function of Rab14 to affect the transport of ceramide. More importantly, sphingomyelin on the Golgi apparatus contributed to the assembly of viral particles. Blockage of the Rab14 regulatory pathway induced the reduction of the content of sphingomyelin on the Golgi apparatus, impairing the assembly of virus particles. Our study clarifies that Rab14 regulates lipid metabolism and promotes CSFV replication, which provides insight into a novel function of Rab14 in regulating vesicles to transport lipids to the viral assembly factory. IMPORTANCE The Rab protein family members participate in the viral replication of multiple viruses and play important roles in the virus infection cycle. Our previous research focused on Rab5/7/11, which regulated the trafficking of vesicles in the early stage of CSFV infection, especially in viral endocytosis. However, the role of other Rab proteins in CSFV replication is unclear and needs further clarification. Strikingly, we screened some Rabs and found the important role of Rab14 in CSFV infection. Virus infection mobilized Rab14 to regulate the vesicle to transport ceramide from the ER to the Golgi apparatus, further promoting the synthesis of sphingomyelin and facilitating virus assembly. The treatment of inhibitors showed that the lipid transport mediated by Rab14 was regulated by the PI3K/AKT/AS160 signaling pathway. Knockdown of Rab14 or the treatment with PI3K/AKT/AS160 inhibitors reduced the ceramide content in infected cells and hindered virus assembly. Our study is the first to explain that vesicular lipid transport regulated by Rab promotes CSFV assembly, which is conducive to the development of antiviral drugs., Competing Interests: The authors declare no conflict of interest.

The Hybridization Chain Reaction (HCR) is an isothermal amplification technique widely used for sensing nucleic acids and small molecules. Despite its effectiveness, conventional linear HCR exhibits relatively slow kinetics and insufficient sensitivity. To address this challenge, we have innovatively combined HCR with DNAzyme technology to enhance nucleic acid detection. In this novel approach, the presence of a target molecule triggers the formation of DNAzyme, leading to the cleavage of substrate S, the initiation of HCR, and the production of DNA nanowires and labeled DNAzyme. The newly generated DNAzyme continuously cleaves substrate S, promoting sequential HCR amplification and significantly enhancing the fluorescence signal. This system offers a simple, sensitive, selective, and versatile method for nucleic acid detection, with a detection limit as low as 5 pM. When tested on classical swine fever virus (CSFV) samples, the system demonstrated detection accuracy comparable to RT-qPCR and exhibited superior repeatability.

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.)

Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65–229 of DHODH and the amino acids 25–40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV. [ABSTRACT FROM AUTHOR]

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).)

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.)

The classical swine fever virus (CSFV) particle consists of three glycoproteins, all of which have been shown to be important proteins involved in many virus functions, including interaction with several host proteins. One of these proteins, E2, has been shown to be directly involved with adsorption to the host cell and important for virus virulence. Using the yeast two-hybrid system, we have previously shown that CSFV E2 specifically interacts with the (DOCK7) dedicator of cytokinesis, a scaffolding protein. In this report, the interaction between E2 and DOCK7 was evaluated. To confirm the yeast two-hybrid results and to determine that DOCK7 interacts in swine cells with E2, we performed co-immunoprecipitation and proximity ligation assay (PLA). After demonstrating the protein interaction in swine cells, E2 amino acid residues Y65, V283, and T149 were determined to be critical for interaction with Dock7 by using a random mutated library of E2 and a reverse yeast two-hybrid approach. That disruption of these three residues with mutations Y65F, V283D, and T149A abrogated the Dock7-E2 protein interaction. These mutations were then introduced into a recombinant CSFV, E2DOCK7v, by a reverse genomics approach using the highly virulent CSFV Brescia isolate as a backbone. E2DOCKv was shown to have similar growth kinetics in swine primary macrophages and SK6 cell cultures to the parental Brescia strain. Similarly, E2DOCK7v demonstrated a similar level of virulence to the parental Brescia when inoculated in domestic pigs. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes indistinguishable from that produced by the parental strain. Therefore, interaction between CSFV E2 and host DOCK7 is not critically involved in the process of virus replication and disease production.

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 (RTPCR) 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. [ABSTRACT FROM AUTHOR]

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.

ABSTRACT Classical swine fever virus (CSFV), an obligate intracellular pathogen, hijacks cellular metabolism to evade immune surveillance and facilitate its replication. The precise mechanisms by which CSFV modulates immune metabolism remain largely unknown. Our study reveals that CSFV infection disrupts serine metabolism, which plays a crucial role in antiviral immunity. Notably, we discovered that CSFV infection leads to the deacetylation of PHGDH, a key enzyme in serine metabolism, resulting in autophagic degradation. This deacetylation impairs PHGDH’s enzymatic activity, reduces serine biosynthesis, weakens innate immunity, and promotes viral proliferation. Molecularly, CSFV infection induces the association of HDAC3 with PHGDH, leading to deacetylation at the K364 site. This modification attracts the E3 ubiquitin ligase RNF125, which facilitates the addition of K63-linked ubiquitin chains to PHGDH-K364R. Subsequently, PHGDH is targeted for lysosomal degradation by p62 and NDP52. Furthermore, the deacetylation of PHGDH disrupts its interaction with the NAD+ substrate, destabilizing the PHGDH-NAD complex, impeding the active site, and thereby inhibiting de novo serine synthesis. Additionally, our research indicates that deacetylated PHGDH suppresses the mitochondria-MAVS-IRF3 pathway through its regulatory effect on serine metabolism, leading to decreased IFN-β production and enhanced viral replication. Overall, our findings elucidate the complex interplay between CSFV and serine metabolism, revealing a novel aspect of viral immune evasion through the lens of immune metabolism.IMPORTANCEClassical swine fever (CSF) seriously restricts the healthy development of China’s aquaculture industry, and the unclear pathogenic mechanism and pathogenesis of classical swine fever virus (CSFV) are the main obstacle to CSF prevention, control, and purification. Therefore, it is of great significance to explore the molecular mechanism of CSFV and host interplay, to search for the key signaling pathways and target molecules in the host that regulate the replication of CSFV infection, and to elucidate the mechanism of action of host immune dysfunction and immune escape due to CSFV infection for the development of novel CSFV vaccines and drugs. This study reveals the mechanism of serine metabolizing enzyme post-translational modifications and antiviral signaling proteins in the replication of CSFV and enriches the knowledge of CSFV infection and immune metabolism.