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.

Self-Assembling E2-Based Nanoparticles Improve Vaccine Thermostability and Protective Immunity against CSFV.

Classical swine fever virus (CSFV) is a highly contagious pathogen causing significant economic losses in the swine industry. Conventional inactivated or attenuated live vaccines for classical swine fever (CSF) are effective but face biosafety concerns and cannot distinguish vaccinated animals from those infected with the field virus, complicating CSF eradication efforts. It is noteworthy that nanoparticle (NP)-based vaccines resemble natural viruses in size and antigen structure, and offer an alternative tool to circumvent these limitations. In this study, we developed an innovative vaccine delivery scaffold utilizing self-assembled mi3 NPs, which form stable structures carrying the CSFV E2 glycoprotein. The expressed yeast E2-fused protein (E2-mi3 NPs) exhibited robust thermostability (25 to 70 °C) and long-term storage stability at room temperature (25 °C). Interestingly, E2-mi3 NPs made with this technology elicited enhanced antigen uptake by RAW264.7 cells. In a rabbit model, the E2-mi3 NP vaccine against CSFV markedly increased CSFV-specific neutralizing antibody titers. Importantly, it conferred complete protection in rabbits challenged with the C-strain of CSFV. Furthermore, we also found that the E2-mi3 NP vaccines triggered stronger cellular (T-lymphocyte proliferation, CD8+ T-lymphocytes, IFN-γ, IL-2, and IL-12p70) and humoral (CSFV-specific neutralizing antibodies, CD4+ T-lymphocytes, and IL-4) immune responses in pigs than the E2 vaccines. To sum up, these structure-based, self-assembled mi3 NPs provide valuable insights for novel antiviral strategies against the constantly infectious agents.

Development and application of classical swine fever virus monoclonal antibodies derived from single B cells.

Vaccination with E2 subunit vaccines is currently the main measure to control classical swine fever virus (CSFV), which is an endemic disease, and detection of antibodies against CSFV E2 is the most effective way to evaluate herd immunity. In the present study, the E2 protein was expressed by a baculovirus expression system, and two monoclonal antibodies (mAbs), namely, 3A9 and 4F7, were successfully produced using techniques for the isolation of single B cells from splenocytes from mice immunized with the E2 protein. Moreover, two linear B-cell epitopes, 25GLTTTWKEYSHDLQL39 and 259GNTTVKVHASDERGP273, reactive to 3A9 and 4F7, respectively, were identified using epitope mapping of the E2 protein. In addition, the diagnostic performance of the two mAbs was evaluated using blocking enzyme-linked immunosorbent assay (bELISA), and the results showed that the two mAbs had high diagnostic specificity (96.08%, 94.38%) and diagnostic sensitivity (97.49%, 95.97%). Together, these findings identify two ideal candidate peptides and matching mAbs for a new method of CSFV diagnosis, which will contribute to the control and eradication of classical swine fever.

Host cell factors involved in classical swine fever virus entry.

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

A prospective CSFV-PCV2 bivalent vaccine effectively protects against classical swine fever virus and porcine circovirus type 2 dual challenge and prevents horizontal transmission.

Classical swine fever virus (CSFV) infection leading to CSF outbreaks is among the most devastating swine diseases in the pig industry. Porcine circovirus type 2 (PCV2) infection, resulting in porcine circovirus-associated disease (PCVAD), is also a highly contagious disease affecting pig health worldwide. To prevent and control disease occurrence, multiple-vaccine immunization is necessary in contaminated areas or countries. In this study, a novel CSFV-PCV2 bivalent vaccine was constructed and demonstrated to be capable of eliciting humoral and cellular immune responses against CSFV and PCV2, respectively. Moreover, a CSFV-PCV2 dual-challenge trial was conducted on specific-pathogen-free (SPF) pigs to evaluate vaccine efficacy. All of the vaccinated pigs survived and showed no clinical signs of infection throughout the experimental period. In contrast, placebo-vaccinated pigs exhibited severe clinical signs of infection and steeply increased viremia levels of CSFV and PCV2 after virus challenge. Additionally, neither clinical signs nor viral detections were noted in the sentinel pigs when cohabitated with vaccinated-challenged pigs at three days post-inoculation of CSFV, indicating that the CSFV-PCV2 bivalent vaccine completely prevents horizontal transmission of CSFV. Furthermore, conventional pigs were utilized to evaluate the application of the CSFV-PCV2 bivalent vaccine in field farms. An adequate CSFV antibody response and a significant decrease in PCV2 viral load in the peripheral lymph nodes were observed in immunized conventional pigs, suggesting its potential for clinical application. Overall, this study demonstrated that the CSFV-PCV2 bivalent vaccine effectively elicited protective immune responses and the ability to prevent horizontal transmission, which could be a prospective strategy for controlling both CSF and PCVAD in commercial herds.

Structural Glycoprotein E2 of Classical Swine Fever Virus Critically Interacts with Host Protein Torsin-1A during the Virus Infectious Cycle.

The classical swine fever virus (CSFV) glycoprotein E2 is the major structural component of the virus particle. E2 is involved in several functions, such as virus adsorption to the cell, the elicitation of protective immune responses, and virus virulence in swine. Using a yeast two-hybrid system, we previously identified the swine host protein Torsin-1A, an ATPase protein residing in the endoplasmic reticulum and inner nucleus membrane of the cell, as a specific binding partner for E2. The interaction between Torsin-1A and E2 proteins was confirmed to occur in CSFV-infected swine cells using three independent methods: coimmunoprecipitation, confocal microscopy, and proximity ligation assay (PLA). Furthermore, the E2 residue critical to mediate the protein-protein interaction with Torsin-1A was identified by a reverse yeast two-hybrid assay using a randomly mutated E2 library. A recombinant CSFV E2 mutant protein with a Q316L substitution failed to bind swine Torsin-1A in the yeast two-hybrid model. In addition, a CSFV infectious clone harboring the E2 Q316L substitution, although expressing substantial levels of E2 protein, repetitively failed to produce virus progeny when the corresponding RNA was transfected into susceptible SK6 cells. Importantly, PLA analysis of the transfected cells demonstrated an abolishment of the interaction between E2 Q316L and Torsin-1A, indicating a critical role for that interaction during CSFV replication.IMPORTANCE Structural glycoprotein E2 is an important structural component of the CSFV particle. E2 is involved in several virus functions, particularly virus-host interactions. Here, we characterized the interaction between CSFV E2 and swine protein Torsin-1A during virus infection. The critical amino acid residue in E2 mediating the interaction with Torsin-1A was identified and the effect of disrupting the E2-Torsin-1A protein-protein interaction was studied using reverse genetics. It is shown that the amino acid substitution abrogating E2-Torsin-1A interaction constitutes a lethal mutation, demonstrating that this virus-host protein-protein interaction is a critical factor during CSFV replication. This highlights the potential importance of the E2-Torsin-1A protein-protein interaction during CSFV replication and provides a potential pathway toward blocking virus replication, an important step toward the potential development of novel virus countermeasures.

Designing a novel E2-IFN-γ fusion protein against CSFV by immunoinformatics and structural vaccinology approaches.

Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: • An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. • A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. • The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.

Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes.

BACKGROUND: RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences-a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5' end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3' end. RESULTS: Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues. CONCLUSIONS: The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening.

A Polyuridine Insertion in the 3' Untranslated Region of Classical Swine Fever Virus Activates Immunity and Reduces Viral Virulence in Piglets.

Low-virulence classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Few data are available on the molecular determinants of CSFV virulence. The aim of the present study was to assess a possible role for CSFV virulence of a unique, uninterrupted 36-uridine (poly-U) sequence found in the 3' untranslated region (3' UTR) of the low-virulence CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3' UTR (vPdR-36U) and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of 20 5-day-old piglets were infected with vPdR-36U and vPdR-5U. Ten contact piglets were added to each group. Disease progression, virus replication, and immune responses were monitored for 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality, and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. Ninety percent of the piglets infected with vPdR-36U seroconverted, while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient alpha interferon (IFN-α) responses in serum after 1 week of infection, while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first 2 weeks. Taken together, these data show that the 3' UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission.IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3' untranslated region (3' UTR) plays an important role in flavivirus replication, the present study showed for the first time that a long polyuridine sequence acquired in the 3' UTR by an endemic CSFV isolate can activate immunity, control viral replication, and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants is also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.

Development and neutralization analysis of recombinant BVDVs expressing a CSF single chain antibody in vitro.

Although the immunization against swine fever (SF) is compulsory in China, it has still emerged in several areas at times. Herein, this study was conducted to develop an antibody vaccine which can clear the classical swine fever virus (CSFV) immediately after the pathogen invasion. Bovine viral diarrhoea virus (BVDV) infectious cDNA clone pASH28 was used to express a single-chain fragment variable (scFv) antibody against CSFV (CSFV/scFv) by reverse genetic technique. CSFV/scFv was inserted at the N-terminus of the C or Erns gene, generating two rBVDVs (rBVDV/C-CSFV/scFv and rBVDV/Erns-CSFV/scFv). Although both the rBVDVs could stably propagate on MDBK cells, different cellular characteristics existed. Obvious green fluorescence against the CSFV/scFv antibody could be visual on the cytomembrane or outside of the cells infected with rBVDV/Erns-CSFV/scFv, while much weaker fluorescence was observed in rBVDV/C-CSFV/scFv - infected cells. The CSFV/scFv antibodies induced by the two rBVDVs could recognize CSFV, but the rBVDV/Erns-CSFV/scFv induced stronger viral neutralization reaction. It was speculated that the neutralization activity might be associated with the expression location of CSFV/scFv antibody. The datas in this study provide evidence that rBVDV/Erns-CSFV/scFv may be engineered as a new antibody vaccine candidate against CSFV in the future.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

Activation of Dendritic Cells in Tonsils Is Associated with CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus.

Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163+CD14+ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-γ+ CD8 T cell response in C-strain vaccinated pigs, but not in pigs that received Alfort-187. Moreover, the frequency of CSFV-specific IFN-γ+ CD8 T cells was inversely correlated to the viral load in the tonsils of individual animals. Accordingly, we hypothesise that the activation of cDC1 is key in initiating local CSFV-specific CD8 T cell responses which curtail early virus replication and dissemination.

Sero-epidemiology of porcine parvovirus, circovirus, and classical swine fever virus infections in India.

Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industry in developing countries like India. For evaluating the true extent of porcine infections, a total of 1308 representative sera samples were collected from 92 different pig farms covering 8 North-Eastern states and Punjab state of Northern India during a period of 2 years (2011-2013). Sera samples were tested for the presence of antibodies against porcine parvovirus (PPV), porcine circovirus-2 (PCV-2), and classical swine fever virus (CSFV) using commercial enzyme-linked immunosorbent assay (ELISA) kits. In the North-Eastern states, the seroprevalence of CSFV in non-vaccinated animals was 6.30% and that of PCV2 and PPV was 6.28% and 1.24%, respectively. In Punjab, the seroprevalence of CSFV in non-vaccinated animals was 44.44% and seroprevalence of PCV-2 and PPV was 34.07% and 39.10%, respectively. Detection of antibodies against more than one virus revealed that 4.66% animals had co-infection with PCV-2 and PPV, 1.75% with CSF and PPV, 1.98% with CSF and PCV-2, and 1.75% with all the three viruses. The receiver operator characteristics (ROC) curve analysis depicted that piglet mortality, parvovirus, and CSFV were the most important parameters with an AUC value of 0.997, 0.897, and 0.973, respectively. Incidence of single or co-infection with different viruses showed that the occurrence of single infection was significantly more prevalent than co-infection. This study provides useful information to set up future epidemiologic, flock management, and public animal health policies for the prevention and control of PCV-2, PPV, and CSF in India.

Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle.

The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence.IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation.

For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.

Viperin inhibits classical swine fever virus replication by interacting with viral nonstructural 5A protein.

Classical swine fever virus (CSFV) is a single-stranded RNA flavivirus that can cause serious diseases in porcine species, including symptoms of infarction, systemic hemorrhage, high fever, or depression. Viperin is an important interferon-inducible antiviral gene that has been shown to inhibit CSFV, but the exact mechanisms by which it is able to do so remain poorly characterized. In the present study, we determined that CSFV infection led to viperin upregulation in PK-15 cells (porcine kidney cell). When viperin was overexpressed in these cells, this markedly attenuated CSFV replication, with clear reductions in viral copy number after 12 to 48 hours postinfection. Immunofluorescence microscopy revealed that the viral NS5A protein colocalized with viperin in infected cells, and this was confirmed via confocal laser scanning microscopy using labeled versions of these proteins, and by co-immunoprecipitation which confirmed that NS5A directly interacts with viperin. When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N-terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction. Together, our in vitro results highlight a potential mechanism whereby viperin is able to inhibit CSFV replication. These results have the potential to assist future efforts to prevent or treat systemic CSFV-induced disease, and may also offer more general insights into the antiviral role of viperin in innate immunity.

Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain.

Classical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by a de novo mechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCE Pigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genus Pestivirus, family Flaviviridae Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

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.

Coordinated expression of vascular endothelial growth factor A and urokinase-type plasminogen activator contributes to classical swine fever virus Shimen infection in macrophages.

BACKGROUND: The Shimen strain of classical swine fever (CSF) virus (CSFV) causes CSF, which is mainly characterised by disseminated intravascular haemorrhage. Macrophages are an essential component of innate immunity against pathogenic microorganisms; however, the role of macrophages in CSF pathogenesis remains unclear. To illuminate the infective mechanism of CSFV, we used gene co-expression networks derived from macrophages infected with CSFV Shimen and CSFV C as well as uninfected macrophages to screen key regulatory genes, and their contributions to the pathogenesis of CSF were discussed. RESULTS: Vascular endothelial growth factor A (VEGFA) and plasminogen activator, urokinase (PLAU, which encodes urokinase-type plasminogen activator [uPA]) were identified as coordinated genes expressed in macrophages by gene co-expression networks. Quantitative polymerase chain reaction and western blot analysis confirmed that VEGFA and PLAU were significantly up-regulated at both the transcription and translation levels after infection. Further, confocal microscopy analysis proposed that the VEGFA and uPA proteins were temporally co-localised with the CSFV protein E2. CONCLUSIONS: Our findings suggest that co-expression of VEGFA and PLAU in macrophages contributes to CSFV Shimen infection and serves as a significant avenue for the strain to form an inflammatory microenvironment, providing new insight into the mechanisms of CSF caused by a virulent strain.