Classical swine fever virus non-structural protein 4A recruits dihydroorotate dehydrogenase to facilitate viral replication.

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.

Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B.

Mx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione S-transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B.IMPORTANCE Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo, but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities.

Important roles of C-terminal residues in degradation of capsid protein of classical swine fever virus.

BACKGROUND: Capsid (C) protein plays an important role in the replication of classical swine fever virus (CSFV). The ubiquitin proteasome system (UPS) involves in replication of many viruses via modulation of viral proteins. The relationship of CSFV with UPS is poorly understood and the impact of 26S proteasome on C protein has never been reported before. METHODS: In this study, fused C protein with an EGFP tag is expressed in PK-15 and 3D4/2 cells. MG132 and 3-methyladenine (3-MA) are used to detect the roles of 26S proteasome and autophagolysosome in expression levels of C protein. Truncated and mutant C proteins are used to find the exact residues responsible for the degradation of C protein. Immunoprecipitaion is performed to find whether C protein is ubiquitinated or not. RESULTS: C-EGFP protein expresses in a cleaved form at a low level and is degraded by 26S proteasome which could be partly inhibited by MG132. C-terminal residues play more important roles in the degradation of C protein than N-terminal residues. Residues 260 to 267, especially M260 and L261, are crucial for the degradation. In addition, C-terminal residues 262 to 267 determine cleavage efficiency of C protein. CONCLUSIONS: CSFV C protein is degraded by 26S proteasome in a ubiquitin-independent manner. Last 8 residues at C-terminus of immature C protein play a major role in proteasomal degradation of CSFV C protein and determine the cleavage efficiency of C protein by signal peptide peptidase (SPP). Our findings provide valuable help for fully understanding degradation process of C protein and contribute to fully understanding the role of C protein in CSFV replication.

Molecular chaperone Jiv promotes the RNA replication of classical swine fever virus.

The nonstructural protein 2 (NS2) of classical swine fever virus (CSFV) is a self-splicing ribozyme wherein the precursor protein NS2-3 is cleaved, and the cleavage efficiency of NS2-3 is crucial to the replication of viral RNA. However, the proteolytic activity of NS2 autoprotease may be achieved through a cellular chaperone called J-domain protein interacting with viral protein (Jiv) or its fragment Jiv90, as evidence suggests that Jiv is required for the proper functioning of the NS2 protein of bovine viral diarrhea virus. Hence, the expression of Jiv may be correlated with the replication efficiency of CSFV RNA. We investigated the expression levels of Jiv and viral RNA in CSFV-infected cells and tissues using Real-time RT-PCR or Western blot analysis. The obtained results show that Jiv90 possibly plays an important role in the lifecycle of CSFV because the distribution of Jiv90 protein shows a positive correlation with the viral load of CSFV. Furthermore, the overexpression or knockdown of Jiv90 in swine cells can also significantly promote or decrease the viral load, respectively. The detection of Flow cytometry shows that the overexpression of Jiv90 prolongs the G1 phase of cell cycles but has no effect on apoptosis. These findings are likely to be of benefit in clarifying the pathogenesis of the CSFV.

FKBP8 interact with classical swine fever virus NS5A protein and promote virus RNA replication.

The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling and host cellular responses via to its ability to interact with various cellular proteins. FKBP8 is also reported to promote virus replication. Here, we show that NS5A specifically interacts with FKBP8 through coimmunoprecipitation and GST-pulldown studies. Additionally, confocal microscopy study showed that NS5A and FKBP8 colocalized in the cytoplasm. Overexpression of FKBP8 via the eukaryotic expression plasmid pDsRED N1 significantly promoted viral RNA synthesis. The cells knockdown of FKBP8 by lentivirus-mediated shRNA markedly decreased the virus replication when infected with CSFV. These data suggest that FKBP8 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of FKBP8 protein functions may be beneficial for developing new strategies to treat CSFV infection.

Exosomes secreted by CSFV-infected cells evade neutralizing antibody to activate innate immune responses and establish productive infection in recipient cells.

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.

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

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

Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation, assembly of infectious virus particles, and regulation of cellular signaling pathways. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism involved has not been elucidated. Here, we reported that NS5A directly interacted with NF-κB essential modulator (NEMO), a regulatory subunit of the IκB kinase (IKK) complex, to inhibit the NF-κB signaling pathway. Further investigations showed that the zinc finger domain of NEMO and the aa 126-250 segment of NS5A are essential for the interaction between NEMO and NS5A. Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation. In addition, NS5A blocked the K63-linked polyubiquitination of NEMO, thus inhibiting IKK phosphorylation, IκBα degradation, and NF-κB activation. These findings revealed a novel mechanism by which CSFV inhibits host innate immunity, which might guide the drug design against CSFV in the future.

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5' untranslated region (5'UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/µL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protect Pigs against a Virulent CSFV Challenge.

Classical swine fever (CSF) remains one of the most economically significant viral diseases affecting domestic pigs and wild boars worldwide. To develop a safe and effective vaccine against CSF, we have constructed a triple gene-deleted pseudorabies virus (PRVtmv)-vectored bivalent subunit vaccine against porcine circovirus type 2b (PCV2b) and CSFV (PRVtmv+). In this study, we determined the protective efficacy of the PRVtmv+ against virulent CSFV challenge in pigs. The results revealed that the sham-vaccinated control group pigs developed severe CSFV-specific clinical signs characterized by pyrexia and diarrhea, and became moribund on or before the seventh day post challenge (dpc). However, the PRVtmv+-vaccinated pigs survived until the day of euthanasia at 21 dpc. A few vaccinated pigs showed transient diarrhea but recovered within a day or two. One pig had a low-grade fever for a day but recovered. The sham-vaccinated control group pigs had a high level of viremia, severe lymphocytopenia, and thrombocytopenia. In contrast, the vaccinated pigs had a low-moderate degree of lymphocytopenia and thrombocytopenia on four dpc, but recovered by seven dpc. Based on the gross pathology, none of the vaccinated pigs had any CSFV-specific lesions. Therefore, our results demonstrated that the PRVtmv+ vaccinated pigs are protected against virulent CSFV challenge.

Cholesterol Biosynthesis Modulates CSFV Replication.

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) has resulted in severe losses to the pig industry worldwide. It has been proposed that lipid synthesis is essential for viral replication, and lipids are involved in viral protein maturation and envelope production. However, the specific crosstalk between CSFV and host cell lipid metabolism is still unknown. In this study, we found that CSFV infection increased intracellular cholesterol levels in PK-15 cells. Further analysis demonstrated that CSFV infection upregulated PCSK9 expression to block the uptake of exogenous cholesterol by LDLR and enhanced the cholesterol biosynthesis pathway, which disrupted the type I IFN response in PK-15 cells. Our findings provide new insight into the mechanisms underpinning the pathogenesis of CSFV and hint at methods for controlling the disease.

Complete genome sequences of classical swine fever virus: Phylogenetic and evolutionary analyses.

The classical swine fever virus (CSFV) outbreaks cause colossal losses of pigs and drastic economic impacts. The current phylogenetic CSFV groups were determined mainly based on the partial genome. Herein, 203 complete genomic sequences of CSFVs collected worldwide between 1998 and 2018 available on the GenBank database were retrieved for re-genotyping and recombination analysis. The maximum likelihood phylogenetic tree determined two main groups, GI and GII, with multiple sub-genotypes. The "strain 39" (GenBank ID: AF407339), previously identified as belonging to sub-genotypes 1.1 or 2.2 based on the partial sequences, is found to be genetically distinct and independent, forming a new lineage depicted as GI-2.2b. Ten potential natural recombination events were identified, seven of which were collected in China and found involved in the genetic diversity of CSFVs. Importantly, the vaccine strains and highly virulent strains were all involved in the recombination events, which would induce extra challenges to vaccine development. These findings alarm that attenuated vaccines should be applied with discretion and recommend using subunit vaccines in parallel with other preventive strategies for better management of CSFVs.

Seroprevalence of antibodies to classical swine fever virus and porcine reproductive and respiratory syndrome virus in healthy pigs in Hunan Province, China.

Classical swine fever (CSF) and porcine reproductive and respiratory syndrome (PRRS) are responsible for major economic losses and represent a threat to the swine industry worldwide. Routine surveillance serology for CSF and PRRS viruses is critical to maintaining the health status of sow farms in Hunan Province, which is one of the top pig production provinces in China. The aim of our study was to investigate the serological statistics of CSF virus (CSFV) and PRRS virus (PRRSV) in Hunan Province. The cohort serum samples were collected from vaccinated and unvaccinated pigs. Our findings showed that the average rates of CSFV and PRRSV antibody seropositivity were 82.2% (95% CI: 80.1-84.3) and 84.8% (95% CI: 82.5-87.1), respectively, in the immunized group and that these rates were higher than those in the unvaccinated group (58.6% for CSFV and 47.8% for PRRSV). Additionally, the level of CSFV antibody in piglet serum declined gradually with age, whereas PRRSV-specific antibody level increased initially (1 to 2 weeks old) and then declined with age (2 to 4 weeks old). In summary, we investigated the difference in CSFV/PRRSV antibody levels among piglets at various weeks old (1 to 4 weeks) to further establish the duration of maternal immunity in piglets. In addition, routine monitoring of CSFV/PRRSV antibodies in immunized pigs was carried out to evaluate the efficacy of vaccination.

Extracellular vesicles originating from autophagy mediate an antibody-resistant spread of classical swine fever virus in cell culture.

Free spread is a classical mode for mammalian virus transmission. However, the efficiency of this transmission approach is generally low as there are structural barriers or immunological surveillances in the extracellular environment under physiological conditions. In this study, we systematically analyzed the spreading of classical swine fever virus (CSFV) using multiple viral replication analysis in combination with antibody neutralization, transwell assay, and electron microscopy, and identified an extracellular vesicle (EV)-mediated spreading of CSFV in cell cultures. In this approach, intact CSFV virions are enclosed within EVs and transferred into uninfected cells with the movement of EVs, leading to an antibody-resistant infection of the virus. Using fractionation assays, immunostaining, and electron microscopy, we characterized the CSFV-containing EVs and demonstrated that the EVs originated from macroautophagy/autophagy. Taken together, our results showed a new spreading mechanism for CSFV and demonstrated that the EVs in CSFV spreading are closely related to autophagy. These findings shed light on the immune evasion mechanisms of CSFV transmission, as well as new functions of cellular vesicles in virus lifecycles.Abbreviations: 3-MA: 3-methyladenine; CCK-8: Cell Counting Kit-8; CSF: classical swine fever; CQ: chloroquine; CSFV: classical swine fever virus; DAPI, 4-,6-diamidino-2-phenylindole; EVs: extracellular vesicles; hpi: h post infection; IEM: immunoelectron microscopy; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MOI: multiplicity of infection; MVs: microvesicles; ND50: half neutralizing dose; PCR: polymerase chain reaction; PBS: phosphate-buffered saline; SEC: size-exclusion chromatography; siRNA: small interfering RNA; TEM: transmission electron microscopy.

Curcumin inhibits classical swine fever virus replication by interfering with lipid metabolism.

Although previous reports have shown that Curcumin inhibits many viruses, including some important members of different genera of Flaviviridae family (Japanese encephalitis virus, dengue virus and hepatitis C virus), the antiviral activity of curcumin against Classical swine fever virus (CSFV), which belongs to Pestivirus genus, is still unclear. In this study, we found that curcumin inhibited CSFV replication in a dose-dependent manner, but had no effect on virus adsorption and entry. Furthermore, the results showed that curcumin inhibited the expression of FASN, one of the key enzymes of fatty acid synthesis pathway, thereby, causing the reduction of the production of LDs upon infection. To this end, we detected transcription factor 6 (ATF6), the key factor of regulating lipid metabolism along with other related molecules (CHOP and GPR78) and found that curcumin significantly impaired the gene synthesis of ATF6, while CSFV infection promoted ATF6 expression. Therefore, it is confirmed that curcumin inhibited CSFV replication by interfere lipid metabolism. In addition, our subsequent studies found that curcumin played an antiviral role by promoting the innate immune independent of NF-κB signaling pathway. Taken together, our finding highlights that curcumin is a potential candidate drug against CSFV for controlling CSF.

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

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

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

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

Abrogation of the RNase activity of Erns in a low virulence classical swine fever virus enhances the humoral immune response and reduces virulence, transmissibility, and persistence in pigs.

The prevalence of low virulence classical swine fever virus (CSFV) strains makes viral eradication difficult in endemic countries. However, the determinants for natural CSFV attenuation and persistence in the field remain unidentified. The aim of the present study was to assess the role of the RNase activity of CSFV Erns in pathogenesis, immune response, persistent infection, and viral transmission in pigs. To this end, a functional cDNA clone pPdR-H30K-36U with an Erns lacking RNase activity was constructed based on the low virulence CSFV field isolate Pinar de Rio (PdR). Eighteen 5-day-old piglets were infected with vPdR-H30K-36U. Nine piglets were introduced as contacts. The vPdR-H30K-36U virus was attenuated in piglets compared to the parental vPdR-36U. Only RNA traces were detected in sera and body secretions and no virus was isolated from tonsils, showing that RNase inactivation may reduce CSFV persistence and transmissibility. The vPdR-H30K-36U mutant strongly activated the interferon-α (IFN-α) production in plasmacytoid dendritic cells, while in vivo, the IFN-α response was variable, from moderate to undetectable depending on the animal. This suggests a role of the CSFV Erns RNase activity in the regulation of innate immune responses. Infection with vPdR-H30K-36U resulted in higher antibody levels against the E2 and Erns glycoproteins and in enhanced neutralizing antibody responses when compared with vPdR-36U. These results pave the way toward a better understanding of viral attenuation mechanisms of CSFV in pigs. In addition, they provide novel insights relevant for the development of DIVA vaccines in combination with diagnostic assays for efficient CSF control.

Reduced Virus Load in Lungs of Pigs Challenged with Porcine Reproductive and Respiratory Syndrome Virus after Vaccination with Virus Replicon Particles Encoding Conserved PRRSV Cytotoxic T-Cell Epitopes.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe respiratory distress and reproductive failure in swine. Modified live virus (MLV) vaccines provide the highest degree of protection and are most often the preferred choice. While somewhat protective, the use of MLVs is accompanied by multiple safety issues, why safer alternatives are urgently needed. Here, we describe the generation of virus replicon particles (VRPs) based on a classical swine fever virus genome incapable of producing infectious progeny and designed to express conserved PRRSV-2 cytotoxic T-cell epitopes. Eighteen pigs matched with the epitopes by their swine leucocyte antigen-profiles were vaccinated (N = 11, test group) or sham-vaccinated (N = 7, control group) with the VRPs and subsequently challenged with PRRSV-2. The responses to vaccination and challenge were monitored using serological, immunological, and virological analyses. Challenge virus load in serum did not differ significantly between the groups, whereas the virus load in the caudal part of the lung was significantly lower in the test group compared to the control group. The number of peptide-induced interferon-γ secreting cells after challenge was higher and more frequent in the test group than in the control group. Together, our results provide indications of a shapeable PRRSV-specific cell-mediated immune response that may inspire future development of effective PRRSV vaccines.

The R614E mutation of mouse Mx1 protein contributes to the novel antiviral activity against classical swine fever virus.

Mx proteins are interferon-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses. We previously demonstrated that porcine Mx1 protein (poMx1) inhibited the replication of classical swine fever virus (CSFV), an economically important Pestivirus, and that mouse Mx1 did so as well. It is unknown why the nucleus-localizing mouse Mx1 inhibits CSFV replication which occurs in the cytoplasm. To the end, we assessed the anti-CSFV actions of wild type mouse Mx1 and seven previously reported mutants (K49A, G83R, A222V, A516V, G540E, R614E and ΔL4) and identified the molecular mechanism of R614E action against CSFV replication. A series of experiments revealed that mmMx1 (R614E) mutant reposted to the cytoplasm and interacted with the CSFV nucleocapsid protein (Core), thereby inhibiting viral replication. These findings broaden our understanding of the function of Mx protein family members against CSFV and suggest that the relative conservation of Mx1 among species is the basis of broad-spectrum antiviral properties.