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.

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.

Intracellular Vimentin Regulates the Formation of Classical Swine Fever Virus Replication Complex through Interaction with NS5A Protein.

Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Three-dimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. IMPORTANCE Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV.

Kinesin-1 Regulates Endocytic Trafficking of Classical Swine Fever Virus along Acetylated Microtubules.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an important and highly infectious pig disease worldwide. Kinesin-1, a molecular motor responsible for transporting cargo along the microtubule, has been demonstrated to be involved in the infections of diverse viruses. However, the role of kinesin-1 in the CSFV life cycle remains unknown. Here, we first found that Kif5B played a positive role in CSFV entry by knockdown or overexpression of Kif5B. Subsequently, we showed that Kif5B was associated with the endosomal and lysosomal trafficking of CSFV in the early stage of CSFV infection, which was reflected by the colocalization of Kif5B and Rab7, Rab11, or Lamp1. Interestingly, trichostatin A (TSA) treatment promoted CSFV proliferation, suggesting that microtubule acetylation facilitated CSFV endocytosis. The results of chemical inhibitors and RNA interference showed that Rac1 and Cdc42 induced microtubule acetylation after CSFV infection. Furthermore, confocal microscopy revealed that cooperation between Kif5B and dynein help CSFV particles move in both directions along microtubules. Collectively, our study shed light on the role of kinesin motor Kif5B in CSFV endocytic trafficking, indicating the dynein/kinesin-mediated bidirectional CSFV movement. The elucidation of this study provides the foundation for developing CSFV antiviral drugs. IMPORTANCE The minus end-directed cytoplasmic dynein and the plus end-directed kinesin-1 are the molecular motors that transport cargo on microtubules in intracellular trafficking, which plays a notable role in the life cycles of diverse viruses. Our previous studies have reported that the CSFV entry host cell is dependent on the microtubule-based motor dynein. However, little is known about the involvement of kinesin-1 in CSFV infection. Here, we revealed the critical role of kinesin-1 that regulated the viral endocytosis along acetylated microtubules induced by Cdc42 and Rac1 after CSFV entry. Mechanistically, once CSFV transported by dynein met an obstacle, it recruited kinesin-1 to move in reverse to the anchor position. This study extends the theoretical basis of intracellular transport of CSFV and provides a potential target for the control and treatment of CSFV infection.

The Small GTPase Rab14 Regulates the Trafficking of Ceramide from Endoplasmic Reticulum to Golgi Apparatus and Facilitates Classical Swine Fever Virus Assembly.

Classical swine fever virus (CSFV) is a highly pathogenic RNA virus belonging to the Flaviviridae family that can cause deadly classical swine fever (CSF) in pigs. However, the molecular details of virus replication in the host are still unclear. Our previous studies have reported that several Rab proteins mediate CSFV entry into host cells, but it is unknown whether CSFV hijacks other Rab proteins for effective viral infection. Here, we systematically studied the role of Rab14 protein in regulating lipid metabolism for promoting viral assembly. First, Rab14 knockdown and overexpression significantly affected CSFV replication, indicating the essential role of Rab14 in CSFV infection. Interestingly, Rab14 could significantly affect virus replication in the late stage of infection. Mechanistically, CSFV NS5A recruited Rab14 to the ER, followed by ceramide transportation to the Golgi apparatus, where sphingomyelin was synthesized. The experimental data of small molecule inhibitors, RNA interference, and replenishment assay showed that the phosphatidylinositol-3-kinase (PI3K)/AKT/AS160 signaling pathway regulated the function of Rab14 to affect the transport of ceramide. More importantly, sphingomyelin on the Golgi apparatus contributed to the assembly of viral particles. Blockage of the Rab14 regulatory pathway induced the reduction of the content of sphingomyelin on the Golgi apparatus, impairing the assembly of virus particles. Our study clarifies that Rab14 regulates lipid metabolism and promotes CSFV replication, which provides insight into a novel function of Rab14 in regulating vesicles to transport lipids to the viral assembly factory. IMPORTANCE The Rab protein family members participate in the viral replication of multiple viruses and play important roles in the virus infection cycle. Our previous research focused on Rab5/7/11, which regulated the trafficking of vesicles in the early stage of CSFV infection, especially in viral endocytosis. However, the role of other Rab proteins in CSFV replication is unclear and needs further clarification. Strikingly, we screened some Rabs and found the important role of Rab14 in CSFV infection. Virus infection mobilized Rab14 to regulate the vesicle to transport ceramide from the ER to the Golgi apparatus, further promoting the synthesis of sphingomyelin and facilitating virus assembly. The treatment of inhibitors showed that the lipid transport mediated by Rab14 was regulated by the PI3K/AKT/AS160 signaling pathway. Knockdown of Rab14 or the treatment with PI3K/AKT/AS160 inhibitors reduced the ceramide content in infected cells and hindered virus assembly. Our study is the first to explain that vesicular lipid transport regulated by Rab promotes CSFV assembly, which is conducive to the development of antiviral drugs.

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.

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.

Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus.

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as a cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture-adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig-expressing cells and areas of nonexpressing cells, and one single cell line revealed not to express any CD46pig The CD46pig-deficient porcine lymphoma cell line, known to facilitate CSFV replication, was the only porcine cell line nonpermissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig-independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized.IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and is highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in the absence of CD46pig Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

Fatty Acid Synthase Is Involved in Classical Swine Fever Virus Replication by Interaction with NS4B.

Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, relies on host machinery to complete its life cycle. Previous studies have shown a close connection between virus infection and fatty acid biosynthesis, mainly regulated by fatty acid synthase (FASN). However, the molecular action of how FASN participates in CSFV replication remains to be elucidated. In this study, two chemical inhibitors of the fatty acid synthesis pathway [5-(tetradecyloxy)-2-furoic acid (TOFA) and tetrahydro-4-methylene-2R-octyl-5-oxo-3S-furancarboxylic acid (C75)] significantly impaired the late stage of viral propagation, suggesting CSFV replication required fatty acid synthesis. We next found that CSFV infection stimulated the expression of FASN, whereas knockdown of FASN inhibited CSFV replication. Furthermore, confocal microscopy showed that FASN participated in the formation of replication complex (RC), which was associated with the endoplasmic reticulum (ER). Interestingly, CSFV NS4B interacted with FASN and promoted overexpression of FASN, which is regulated by functional Rab18. Moreover, we found that FASN regulated the formation of lipid droplets (LDs) upon CSFV infection, promoting virus proliferation. Taken together, our work provides mechanistic insight into the role of FASN in the viral life of CSFV, and it highlights the potential antiviral target for the development of therapeutics against pestiviruses. IMPORTANCE Classical swine fever, caused by classical swine fever virus (CSFV), is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. CSFV, like other (+)-strand RNA viruses, requires lipid and sterol biosynthesis for efficient replication. However, the role of lipid metabolism in CSFV replication remains unknown. Here, we found that fatty acid synthase (FASN) was involved in viral propagation. Moreover, FASN is recruited to CSFV replication sites in the endoplasmic reticulum (ER) and interacts with NS4B to regulate CSFV replication that requires Rab18. Furthermore, we speculated that lipid droplet (LD) biosynthesis, indirectly regulated by FASN, ultimately promotes CSFV replication. Our results highlight a critical role for de novo fatty acid synthesis in CSFV infection, which might help control this devastating virus.

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.

The ESCRT-I Subunit Tsg101 Plays Novel Dual Roles in Entry and Replication of Classical Swine Fever Virus.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine with high morbidity and mortality that negatively affects the pig industry worldwide, in particular in China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. The endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological metabolic processes, including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT uses to regulate the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in clathrin-mediated endocytosis of CSFV and is also involved in CSFV trafficking. Tsg101 assists the virus in entering the host cell through the late endosome (Rab7 and Rab9) and finally reaching the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and double-stranded RNA (dsRNA) or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not the Golgi, in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is a World Organization for Animal Health (OIE) notifiable disease and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genera Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for pestivirus inhibitors.

Isolation and Characterization of Porcine Astrovirus 5 from a Classical Swine Fever Virus-Infected Specimen.

Many new astroviruses have been identified in humans and other animals in recent years, but only a few have been successfully isolated for extensive biological study. Here, we report an unusual isolation of a porcine astrovirus 5 (PAstV5) strain from a clinical classical swine fever virus (CSFV)-infected tissue sample. Incubation of porcine PK-15 cells with an extract of the CSFV-positive tissue resulted in unexpected cytopathic effects (CPEs), and high-throughput viromic sequencing identified PAstV5 and porcine circovirus type 2 (PCV2) as well as CSFV in the culture. After clearance of CSFV and PCV2, a pure PAstV5 strain, named PAstV5-AH29-2014, was obtained. Analysis revealed virus of typical astroviral morphology with a genome of 6,448 nucleotides, sharing 84.3 to 88.9% nucleotide identity with previously published PAstV5 strains. A mechanistic study showed that CSFV coinfection was likely an important factor for successful isolation by significantly enhancing PAstV5 replication in PK-15 cells via suppression of a type I interferon response. Altogether, PAstV5-AH29-2014, as the first isolated PAstV5 strain, will provide critical material for the investigation of the biological and pathogenic properties of this virus as well as for future development of relevant biological and diagnostic reagents.IMPORTANCE Porcine astroviruses are mainly associated with gastroenteritis and neurological diseases in pigs, and five genotypes have been identified (PAstV1-5). However, the clinical manifestations of genotypes other than PAstV1 have not yet been determined because of the failure of in vitro virus isolation. Here, we report a surprising isolation of a PAstV5 strain from a clinical classical swine fever virus (CSFV)-infected tissue sample, which can stably passage in PK-15 cells, and coinfection with CSFV significantly enhanced the replication of PAstV5, possibly through suppression of beta interferon production. Thus, the first isolated PAstV5 strain will be useful for investigating the biological and pathogenic properties of this virus, and the findings obtained in this study provide new insights into defining the interaction mechanism between CSFV and PAstV5.

P108 and T109 on E2 Glycoprotein Domain I Are Critical for the Adaptation of Classical Swine Fever Virus to Rabbits but Not for Virulence in Pigs.

The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs, in contrast with the highly virulent CSFV Shimen strain. Previously, we demonstrated that P108 and T109 on the E2 glycoprotein (E2P108-T109) in domain I (E2DomainI) rather than R132, S133, and D191 in domain II (E2DomainII) determine C-strain's adaptation to rabbits (ATR) (Y. Li, L. Xie, L. Zhang, X. Wang, C. Li, et al., Virology 519:197-206, 2018). However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that E2P108-T109 or E2DomainI but not E2DomainII of C-strain renders vSM-HCLVErns adaptive to rabbits, suggesting that E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2P108-T109-Erns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2P108-T109-Erns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.

Epidemiological investigation of porcine circovirus type 2 and its coinfection rate in Shandong province in China from 2015 to 2018.

BACKGROUND: Porcine circovirus type 2 (PCV2) is one of the crucial swine viral pathogens, caused porcine circovirus associated diseases (PCVAD). Shandong province is one of the most important pork producing areas and bears a considerable economic loss due to PCVAD. However, there is limited information on epidemiology and coinfection rate of PCV2 with other critical swine diseases in this area, such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV). RESULTS: Overall, 89.59% serum samples and 36.98% tissue samples were positive for PCV2 specified ELISA and PCR positive for PCV2, respectively. The coinfection rates of PCV2 with PRRSV, PRV, CSFV, and PEDV were 26.73%, 18.37%, 13.06%, and 3.47%, respectively. Moreover, genetic characteristic of PCV2 were analyzed based on the cap genes showing that PCV2d is the dominant sub-genotype circulating in the province. CONCLUSIONS: Our findings reveal that PCV2d, as the dominant strain, is prevailing in pig farms in Shandong province at high levels. There was a high frequency of coinfection of PCV2 and PRRSV.

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.

Porcine RING Finger Protein 114 Inhibits Classical Swine Fever Virus Replication via K27-Linked Polyubiquitination of Viral NS4B.

In the host, many RING domain E3 ligases have been reported to inhibit viral replication through various mechanisms. In a previous screen, we found that porcine RING finger protein 114 (pRNF114), a RING domain E3 ubiquitin ligase, inhibits classical swine fever virus (CSFV) replication. This study aimed to clarify the underlying antiviral mechanism of pRNF114 against CSFV. Upon CSFV infection, pRNF114 mRNA was upregulated both in vitro and in vivo CSFV replication was significantly suppressed in PK-pRNF114 cells stably expressing pRNF114 by the lentivirus-delivered system, whereas CSFV growth was enhanced in PK-15 cells with RNF114 knockout by the CRISPR/Cas9 system. The RING domain of pRNF114, which has E3 ubiquitin ligase activity, is crucial for its antiviral activity. Mechanistically, pRNF114 interacted with the CSFV NS4B protein through their C-terminal domains, which led to the K27-linked polyubiquitination and degradation of NS4B through a proteasome-dependent pathway. Collectively, these findings indicate that pRNF114 as a critical regulator of CSFV replication and uncover a mechanism by which pRNF114 employs its E3 ubiquitin ligase activity to inhibit CSFV replication.IMPORTANCE Porcine RING finger protein 114 (pRNF114) is a member of the RING domain E3 ligases. In this study, it was shown that pRNF114 is a potential anti-CSFV factor and the anti-CSFV effect of pRNF114 depends on its E3 ligase activity. Notably, pRNF114 targets and catalyzes the K27-linked polyubiquitination of the NS4B protein and then promotes proteasome-dependent degradation of NS4B, inhibiting the replication of CSFV. To our knowledge, pRNF114 is the first E3 ligase to be identified as being involved in anti-CSFV activity, and targeting NS4B could be a crucial route for antiviral development.

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.

Antiviral activity of ISG15 against classical swine fever virus replication in porcine alveolar macrophages via inhibition of autophagy by ISGylating BECN1.

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-α treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-α restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.