Development of classical swine fever virus E2-protein based indirect ELISA for detection of antibodies against the virus in pigs.

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.

Classical swine fever virus inhibits serine metabolism-mediated antiviral immunity by deacetylating modified PHGDH.

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-ß 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.

Development of an indirect ELISA for the immunoprotection evaluation of E2 antibodies against classical swine fever virus.

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.

Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection.

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.

Safety and DIVA Capability of Novel Live Attenuated Classical Swine Fever Marker Vaccine Candidates in Pregnant Sows.

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 Erns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in Erns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in Erns combined with a 5-168 amino acids deletion of Npro (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.

Assessment of the Safety Profile of Chimeric Marker Vaccine against Classical Swine Fever: Reversion to Virulence Study.

Chimeric marker vaccine candidates, vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, 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 Erns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE-/PhoPeV Erns 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 Erns was significantly higher than that of vGPE-/PhoPeV Erns 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.

Recent decline in hepatitis E virus prevalence among wild boars in Japan: Probably due to countermeasures implemented in response to outbreaks of classical swine fever virus infection.

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.

Pathogenicity of genotype 2.1 classical swine fever virus isolated from Japan in 2019 in pigs.

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.

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.

IDO1 promotes CSFV replication by mediating tryptophan metabolism to inhibit NF-κB signaling.

Tryptophan metabolism plays a crucial role in facilitating various cellular processes essential for maintaining normal cellular function. Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn), thereby initiating the degradation of Trp. The resulting Kyn metabolites have been implicated in the modulation of immune responses. Currently, the role of IDO1-mediated tryptophan metabolism in the process of viral infection remains relatively unknown. In this study, we discovered that classical swine fever virus (CSFV) infection of PK-15 cells can induce the expression of IDO1, thereby promoting tryptophan metabolism. IDO1 can negatively regulate the NF-κB signaling by mediating tryptophan metabolism, thereby facilitating CSFV replication. We found that silencing the IDO1 gene enhances the expression of IFN-α, IFN-ß, and IL-6 by activating the NF-κB signaling pathway. Furthermore, our observations indicate that both silencing the IDO1 gene and administering exogenous tryptophan can inhibit CSFV replication by counteracting the cellular autophagy induced by Rapamycin. This study reveals a novel mechanism of IDO1-mediated tryptophan metabolism in CSFV infection, providing new insights and a theoretical basis for the treatment and control of CSFV.IMPORTANCEIt is well known that due to the widespread use of vaccines, the prevalence of classical swine fever (CSF) is shifting towards atypical and invisible infections. CSF can disrupt host metabolism, leading to persistent immune suppression in the host and causing significant harm when co-infected with other diseases. Changes in the host's metabolic profiles, such as increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, can also influence virus replication. Mammals utilize various pathways to modulate immune responses through amino acid utilization, including increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, thereby limiting viral replication. Therefore, this study proposes that targeting the modulation of tryptophan metabolism may represent an effective approach to control the progression of CSF.

Novel approach for detecting classical swine fever virus from swabs of wild boar cut tails using nested real-time PCR.

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.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

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.

PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation.

Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.

Classical swine fever virus NS5A protein activates autophagy via the PP2A-DAPK3-Beclin 1 axis.

IMPORTANCE: Autophagy is a conserved degradation process that maintains cellular homeostasis and regulates native and adaptive immunity. Viruses have evolved diverse strategies to inhibit or activate autophagy for their benefit. The paper reveals that CSFV NS5A mediates the dissociation of PP2A from Beclin 1 and the association of PP2A with DAPK3 by interaction with PPP2R1A and DAPK3, PP2A dephosphorylates DAPK3 to activate its protein kinase activity, and activated DAPK3 phosphorylates Beclin 1 to trigger autophagy, indicating that NS5A activates autophagy via the PP2A-DAPK3-Beclin 1 axis. These data highlight a novel mechanism by which CSFV activates autophagy to favor its replication, thereby contributing to the development of antiviral strategies.

N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

The gC1qR Binding Site Mutant PCV2 Is a Potential Vaccine Strain That Does Not Impair Memory CD4+ T-Cell Generation by Vaccines.

PCV2 has been reported to reduce the protective effects of various vaccines on immunized pigs. Our previous studies showed that the interaction of Cap and host protein gC1qR mediated the PCV2 infection-induced suppression of immune response. Thus, we wondered whether the gC1qR binding site mutant PCV2RmA could be a vaccine strain and whether this mutant PCV2RmA impairs other vaccines. Herein, we showed that PCV2 infection reduced the classic swine fever virus (CSFV) vaccine-induced generation of memory CD4+ T cells through the interaction of Cap with gC1qR. PCV2RmA can effectively induce the production of PCV2-specific antibodies, neutralizing antibodies, and peripheral blood lymphocyte proliferation in piglets at the same levels as the commercial inactivated PCV2 vaccine. The PCV2RmA-induced anti-PCV2 immune responses could eliminate the serum virus and would not lead to pathological lesions like wild-type PCV2. Moreover, compared to the commercial inactivated PCV2 vaccine, PCV2RmA is capable of inducing more durable protective immunity against PCV2 that induced production of PCV2-specific antibodies and neutralizing antibodies for a longer time via stronger induction of memory CD4+ T cells. Importantly, PCV2RmA infection did not impair the CSFV vaccine-induced generation of memory CD4+ T cells. Collectively, our findings showed that PCV2 infection impairs memory CD4+ T-cell generation to affect vaccination and provide evidence for the use of PCV2RmA as an efficient vaccine to prevent PCV2 infection. IMPORTANCE PCV2 is one of the costliest pathogens in pigs worldwide. Usage of PCV2 vaccines can prevent the PCV2 infection-induced clinical syndromes but not the viral spread. Our previous work found that PCV2 infection suppresses the host type I interferon innate immune response and CD4+ T-cell-mediated Th1 immune response through the interaction of Cap with host gC1qR. Here, we showed that the gC1qR binding site mutant PCV2RmA could effectively induce anti-PCV2 immunity and provide more durable protective immunity against wild-type PCV2 infection in pigs. PCV2RmA would not impair the generation of memory CD4+ T cells induced by classic swine fever virus (CSFV) vaccines as wild-type PCV2 did. Therefore, PCV2RmA can serve as a potential vaccine strain to better protect pigs against PCV2 infection.

Removal of the Erns RNase Activity and of the 3' Untranslated Region Polyuridine Insertion in a Low-Virulence Classical Swine Fever Virus Triggers a Cytokine Storm and Lethal Disease.

In this study, we assessed the potential synergistic effect of the Erns RNase activity and the poly-U insertion in the 3' untranslated region (UTR) of the low-virulence classical swine fever virus (CSFV) isolate Pinar de Rio (PdR) in innate and adaptive immunity regulation and its relationship with classical swine fever (CSF) pathogenesis in pigs. We knocked out the Erns RNase activity of PdR and replaced the long polyuridine sequence of the 3' UTR with 5 uridines found typically at this position, resulting in a double mutant, vPdR-H30K-5U. This mutant induced severe CSF in 5-day-old piglets and 3-week-old pigs, with higher lethality in the newborn (89.5%) than in the older (33.3%) pigs. However, the viremia and viral excretion were surprisingly low, while the virus load was high in the tonsils. Only alpha interferon (IFN-α) and interleukin 12 (IL-12) were highly and consistently elevated in the two groups. Additionally, high IL-8 levels were found in the newborn but not in the older pigs. This points toward a role of these cytokines in the CSF outcome, with age-related differences. The disproportional activation of innate immunity might limit systemic viral spread from the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms. Infection with vPdR-H30K-5U resulted in poor neutralizing antibody responses compared with results obtained previously with the parent and RNase knockout PdR. This study shows for the first time the synergistic effect of the 3' UTR and the Erns RNase function in regulating innate immunity against CSFV, favoring virus replication in target tissue and thus contributing to disease severity. IMPORTANCE CSF is one of the most relevant viral epizootic diseases of swine, with high economic and sanitary impact. Systematic stamping out of infected herds with and without vaccination has permitted regional virus eradication. However, the causative agent, CSFV, persists in certain areas of the world, leading to disease reemergence. Nowadays, low- and moderate-virulence strains that could induce unapparent CSF forms are prevalent, posing a challenge for disease eradication. Here, we show for the first time the synergistic role of lacking the Erns RNase activity and the 3' UTR polyuridine insertion from a low-virulence CSFV isolate in innate immunity disproportional activation. This might limit systemic viral spread to the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms, thus contributing to disease severity. These results highlight the role played by the Erns RNase activity and the 3' UTR in CSFV pathogenesis, providing new perspectives for novel diagnostic tools and vaccine strategies.

Cellular ESCRT components are recruited to regulate the endocytic trafficking and RNA replication compartment assembly during classical swine fever virus infection.

As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.

ARF1 with Sec7 Domain-Dependent GBF1 Activates Coatomer Protein I To Support Classical Swine Fever Virus Entry.

Classical swine fever virus (CSFV), a positive-sense, enveloped RNA virus that belongs to the Flaviviridae family, hijacks cell host proteins for its own replication. We previously demonstrated that Golgi-specific brefeldin A (BFA) resistance factor 1 (GBF1), a regulator of intracellular transport, mediates CSFV infection. However, the molecular mechanism by which this protein regulates CSFV proliferation remains unelucidated. In this study, we constructed a series of plasmids expressing GBF1 truncation mutants to investigate their behavior during CSFV infection and found that GBF1 truncation mutants containing the Sec7 domain could rescue CSFV replication in BFA- and GCA (golgicide A)-treated swine umbilical vein endothelial cells (SUVECs), demonstrating that the effect of GBF1 on CSFV infection depended on the activity of guanine nucleotide exchange factor (GEF). Additionally, it was found that ADP ribosylation factors (ARFs), which are known to be activated by the Sec7 domain of GBF1, also regulated CSFV proliferation. Furthermore, we demonstrated that ARF1 is more important for CSFV infection than other ARF members with Sec7 domain dependence. Subsequent experiments established the function of coatomer protein I (COP I), a downstream effector of ARF1 which is also required for CSFV infection by mediating CSFV invasion. Mechanistically, inhibition of COP I function impaired CSFV invasion by inhibiting cholesterol transport to the plasma membrane and regulating virion transport from early to late endosomes. Collectively, our results suggest that ARF1, with domain-dependent GBF1 Sec7, activates COP I to facilitate CSFV entry into SUVECs. IMPORTANCE Classical swine fever (CSF), a highly contact-infectious disease caused by classical swine fever virus (CSFV) infecting domestic pigs or wild boars, has caused huge economic losses to the pig industry. Our previous studies have revealed that GBF1 and class I and II ARFs are required for CSFV proliferation. However, a direct functional link between GBF1, ARF1, and COP I and the mechanism of the GBF1-ARF1-COP I complex in CSFV infection are still poorly understood. Here, our data support a model in which COP I supports CSFV entry into SUVECs in two different ways, depending on the GBF1-ARF1 function. On the one hand, the GBF1-ARF1-COP I complex mediates cholesterol trafficking to the plasma membrane to support CSFV entry. On the other hand, the GBF1-ARF1-COP I complex mediates CSFV transport from early to late endosomes during the entry steps.

The Unique Glycosylation at Position 986 on the E2 Glycoprotein of Classical Swine Fever Virus Is Responsible for Viral Attenuation and Protection against Lethal Challenge.

Classical swine fever (CSF) is an economically important disease of pigs caused by classical swine fever virus (CSFV). The live attenuated vaccine C-strain (also called HCLV strain) against CSF was produced by multiple passages of a highly virulent strain in rabbits. However, the molecular determinants for its attenuation and protection remain unclear. In this study, we identified a unique glycosylation at position 986 (986NYT988) on the E2 glycoprotein Domain IV of C-strain but not (986NYA988) the highly virulent CSFV Shimen strain. We evaluated the infectivity, virulence, and protective efficacy of the C-strain-based mutant rHCLV-T988A lacking the glycosylation and Shimen strain mutant rShimen-A988T acquiring an additional glycosylation at position 986. rShimen-A988T showed a significantly decreased viral replication ability in SK6 cells, while rHCLV-T988A exhibited a growth kinetics indistinguishable from that of C-strain. Removal of the C-strain glycosylation site does not affect viral replication in rabbits and the attenuated phenotype in pigs. However, rShimen-A988T was attenuated and protected the pigs from a lethal challenge at 14 days postinoculation. In contrast, the rHCLV-T988A-inoculated pigs showed transient fever, a few clinical signs, and pathological changes in the spleens upon challenge with the Shimen strain. Mechanistic investigations revealed that the unique glycosylation at position 986 influences viral spreading, alters the formation of E2 homodimers, and leads to increased production of neutralizing antibodies. Collectively, our data for the first time demonstrate that the unique glycosylation at position 986 on the E2 glycoprotein is responsible for viral attenuation and protection. IMPORTANCE Viral glycoproteins involve in infectivity, virulence, and host immune responses. Deglycosylation on the Erns, E1, or E2 glycoprotein of highly virulent classical swine fever virus (CSFV) attenuated viral virulence in pigs, indicating that the glycosylation contributes to the pathogenicity of the highly virulent strain. However, the effects of the glycosylation on the C-strain E2 glycoprotein on viral infectivity in cells, viral attenuation, and protection in pigs have not been elucidated. This study demonstrates the unique glycosylation at position 986 on the C-strain E2 glycoprotein. C-strain mutant removing the glycosylation at the site provides only partial protection against CSFV challenge. Remarkably, the addition of the glycan to E2 of the highly virulent Shimen strain attenuates the viral virulence and confers complete protection against the lethal challenge in pigs. Our findings provide a new insight into the contribution of the glycosylation to the virus attenuation and protection.