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.

Generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious swine disease leading to significant economic losses worldwide. Vaccines are widely used to control the disease, and no CSFV-specific antivirals are currently available. To facilitate anti-CSFV molecule discovery, we developed a reporter virus CSFV-N(pro)Fluc stably expressing the firefly luciferase (Fluc) gene in the N(pro) gene. The reporter virus enabled more sensitive and convenient detection of the N(pro) protein expression and the viral replication by luciferase reporter assay than by traditional methods. The CSFV N(pro) protein was detectable as early as 4.5h post-infection. As a proof-of-concept for its utility in rapid antiviral screening, this reporter virus was used to quantify anti-CSFV neutralizing antibodies of 50 swine sera and to assess 12 small interfering RNAs targeting different regions of the CSFV genome. The results were comparable to those obtained by traditional methods. Taken together, the reporter virus CSFV-N(pro)Fluc represents a useful tool for rapid and quantitative screening and evaluation of antivirals against CSFV.

The potential of antiviral agents to control classical swine fever: a modelling study.

Classical swine fever (CSF) represents a continuous threat to pig populations that are free of disease without vaccination. When CSF virus is introduced, the minimal control strategy imposed by the EU is often insufficient to mitigate the epidemic. Additional measures such as preemptive culling encounter ethical objections, whereas emergency vaccination leads to prolonged export restrictions. Antiviral agents, however, provide instantaneous protection without inducing an antibody response. The use of antiviral agents to contain CSF epidemics is studied with a model describing within- and between-herd virus transmission. Epidemics are simulated in a densely populated livestock area in The Netherlands, with farms of varying sizes and pig types (finishers, piglets and sows). Our results show that vaccination and/or antiviral treatment in a 2 km radius around an infected herd is more effective than preemptive culling in a 1 km radius. However, the instantaneous but temporary protection provided by antiviral treatment is slightly less effective than the delayed but long-lasting protection offered by vaccination. Therefore, the most effective control strategy is to vaccinate animals when allowed (finishers and piglets) and to treat with antiviral agents when vaccination is prohibited (sows). As independent control measure, antiviral treatment in a 1 km radius presents an elevated risk of epidemics running out of control. A 2 km control radius largely eliminates this risk.

Intra-host variation structure of classical swine fever virus NS5B in relation to antiviral therapy.

Classical swine fever (CSF) is one of most important diseases of the Suidea with severe social economic consequences in case of outbreaks. Antivirals have been demonstrated, in recent publications, to be an interesting alternative method of fighting the disease. However, classical swine fever virus is an RNA virus which presents a challenge as intra-host variation and the error prone RNA dependent RNA polymerase (RdRp) could lead to the emergence/selection of resistant variants hampering further treatment. Therefore, it was the purpose of this study to investigate the intra-host variation of the RdRp gene, targeted by antivirals, in respect to antiviral treatment. Using the non-unique nucleotide changes, a limited intra-host variation was found in the wild type virus with 2 silent and 2 non-synonymous sites. This number shifted significantly when an antiviral resistant variant was analyzed. In total 22nt changes were found resulting in 14 amino acid changes whereby each genome copy contained at least 2 amino-acid changes in the RdRp. Interestingly, the frequency of the mutations situated in close proximity to a region involved in antiviral resistance in CSFV and bovine viral diarrhea virus (BVDV) was elevated compared to the other mutations. None of the identified mutations in the resistant variant and which could potentially result in antiviral resistance was present in the wild type virus as a non-unique mutation. In view of the spectrum of mutations identified in the resistance associated region and that none of the resistance associated mutations reported for another strain of classical swine fever for the same antiviral were observed in the study, it can be suggested that multiple mutations confer resistance to some degree. Although the followed classical approach allowed the analysis the RdRp as a whole, the contribution of unique mutations to the intra-host variation could not be completely resolved. There was a significant difference in de number of unique mutations found between: 1/wild type virus and the antiviral resistant variant and 2/between both and the number to be expected from the error rate of the RT-PCR process. This indicates that the some of the unique mutations contributed to the intra-host variation and that the antiviral pressure also shifted this pattern. This is important as one of the non-synonymous mutations found in the resistant variant and which was located in the antiviral resistance associated region, was present in the wild type virus as a unique mutation. The findings presented in this study not only show the importance of intra-host variation analysis but also warrants further research certainly in view of the potential inclusion of antivirals in a control/eradication strategy.

Approved and experimental countermeasures against pestiviral diseases: Bovine viral diarrhea, classical swine fever and border disease.

The pestiviruses, bovine viral diarrhea virus (BVDV), classical swine fever (CSFV) and border disease virus, are important livestock pathogens in many countries, but current vaccines do not completely prevent the spread of infection. Control of pestiviral diseases is especially difficult due to the constant viremia and viral shedding of persistently infected (PI) animals, which must be identified and eliminated to prevent disease transmission. Existing vaccines are limited by the delay between vaccination and the onset of protection, the difficulty of differentiating serologically between vaccinated and naturally infected animals and the need for broad vaccine cross-protection against diverse virus strains. Antiviral therapy could potentially supplement vaccination by providing immediate protection in the case of an outbreak. Numerous compounds with in vitro antiviral activity against BVDV have been identified through its role as a surrogate for hepatitis C virus. Fewer drugs active against CSFV have been identified, but many compounds that are effective against BVDV will likely inhibit CSFV, given their similar genomic sequences. While in vitro research has been promising, the paucity of efficacy studies in animals has hindered the commercial development of effective antiviral drugs against the pestiviruses. In this article, we summarize the clinical syndromes and routes of transmission of BVD, CSF and border disease, discuss currently approved vaccines, review efforts to develop antiviral therapies for use in outbreak control and suggest promising directions for future research.

Proof of concept for the reduction of classical swine fever infection in pigs by a novel viral polymerase inhibitor.

5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative of a class of imidazopyridines with potent in vitro antiviral activity against pestiviruses including classical swine fever virus (CSFV). This study analysed whether the lead compound, BPIP, was able to reduce virus replication in infected piglets. The compound, administered in feed, was readily bioavailable and was well tolerated. Eight specific-pathogen-free pigs received a daily dose of 75 mg kg(-1) (mixed in feed) for a period of 15 consecutive days, starting 1 day before infection with the CSFV field isolate Wingene. BPIP-treated pigs developed a short, transient viraemia (one animal remained negative) and leukopenia (three animals did not develop leukopenia). Virus titres at peak viraemia (7 days post-infection) were markedly lower (approximately 1000-fold) than in untreated animals (P=0.00005) and the viral genome load in blood was also significantly lower (P

Prostaglandin A1 inhibits replication of classical swine fever virus.

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 micrograms/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.