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.

Correction: Li et al. African Swine Fever Virus: A Review. Life 2022, 12, 1255.

In the original publication [...].

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.

CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3.

IMPORTANCE: CSFV infection in pigs causes persistent high fever, hemorrhagic necrotizing multi-organ inflammation, and high mortality, which seriously threatens the global swine industry. Cell death is an essential immune response of the host against pathogen invasion, and lymphopenia is the most typical clinical feature in the acute phase of CSFV infection, which affects the initial host antiviral immunity. As an "old" virus, CSFV has evolved mechanisms to evade host immune response after a long genetic evolution. Here, we show that necroptosis is a limiting host factor for CSFV infection and that CSFV-induced autophagy can subvert this host defense mechanism to promote its sustained replication. Our findings reveal a complex link between necroptosis and autophagy in the process of cell death, provide evidence supporting the important role for CSFV in counteracting host cell necrosis, and enrich our knowledge of pathogens that may subvert and evade this host defense.

Plasma metabonomics of classical swine fever virus-infected pigs.

Classical swine fever (CSF) is an infectious disease caused by Classical swine fever virus (CSFV), which is characterized by depression, high fever, extensive skin bleeding, leukopenia, anorexia, alternating constipation, and diarrhea. Hemorrhagic infarction of the spleen is the main characteristic pathological change following CSFV infection. Large-scale outbreaks of CSF are rare in China and are mainly distributed regionally. The clinical symptoms of CSF are not obvious, and show variation from typical to atypical symptoms, which makes diagnosis based on clinical symptoms and pathology challenging. In recent years, the incidence of CSF-immunized pig farms in China has increased and new CSFV gene subtypes have appeared, posing new challenges to the prevention and control of CSF in China. Changes in metabolites caused by viral infection reflect the pathogenic process. Metabonomics can reveal the trace metabolites of organisms; however, plasma metabonomics of CSFV-infected pigs have rarely been investigated. Therefore, we used an established pig CSFV infection model to study changes in plasma metabolites. The results showed significant differences in forty-five plasma metabolites at different time periods after CSFV infection in pigs, with an increase in twenty-five metabolites and a decrease in twenty metabolites. These changed metabolites were mainly attributed to the tricarboxylic acid cycle, amino acid cycle, sugar metabolism, and fat metabolism. Thirteen metabolic pathways changed significantly in CSFV-infected pigs, including tricarboxylic acid cycle, inositol phosphate metabolism, glycine, serine and threonine metabolism,lysine degradation, alanine, aspartate and glutamic acid metabolism, pantothenate and CoA biosynthesis, ß-alanine metabolism, lysine degradation, arginine and proline metabolism, glycerolipid metabolism, phenylalanine metabolism, arachidonic acid metabolism, linoleic acid metabolism. Among these, changes in fatty acid biosynthesis and metabolism occurred at all time periods post-infection. These results indicate that CSFV infection in pigs could seriously alter metabolic pathways.

Virus-like particle vaccines with epitopes from porcine epidemic virus and transmissible gastroenteritis virus incorporated into self-assembling ADDomer platform provide clinical immune responses in piglets.

Introduction: Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are major intestinal coronaviruses that cause vomiting, diarrhea, dehydration, and mortality in piglets. These viruses coexist and lead to significant economic losses in the swine industry. Virus-like particles (VLPs) have emerged as promising alternatives to conventional inactivated vaccines due to their exceptional safety, efficacy, and ability to provide multi-disease protection with a single dose. Methods: Our study focused on specific antigenic epitopes from the PEDV S protein (SS2 and 2C10 regions) and the TGEV S protein (A and D sites) as target candidates. These epitopes were integrated into the ADDomer framework, and we successfully generated recombinant proteins AD, AD-P, AD-T, and AD-PT using the baculovirus expression vector system (BEVS). By meticulously optimizing conditions in High Five cells, we successfully expressed and purified the recombinant proteins. Subsequently, we developed the recombinant ADDomer-VLP vaccine and conducted a comprehensive evaluation of its efficacy in piglets. Results: Following ultrafiltration concentration and sucrose gradient centrifugation purification, the recombinant proteins self-assembled into VLPs as observed by transmission electron microscopy (TEM). Administration of the vaccine did not result in any adverse reactions in the immunized piglets. Additionally, no significant instances of fever were detected in any of the experimental groups, and there were no notable changes in average daily weight gain compared to the control group that received PBS. The recombinant ADDomer-VLP vaccines demonstrated strong immunogenicity, effectively stimulating the production of neutralizing antibodies against both PEDV and TGEV. Moreover, the recombinant ADDomer-VLP vaccine induced elevated levels of IFN-γ, IL-2, and IL-4, and enhanced cytotoxic T lymphocyte (CTL) activity in the peripheral blood of piglets. Discussion: These recombinant VLPs have demonstrated the ability to induce strong cellular and humoral immune responses in piglets, making them an incredibly promising platform for the rapid and simplified development of epitope vaccines.

Recombinant Porcine Interferon-α Decreases Pseudorabies Virus Infection.

Interferon (IFN) is a cell-secreted cytokine possessing biological activities including antiviral functioning, immune regulation, and others. Interferon-alpha (IFN-α) mainly derives from plasmacytoid dendritic cells, which activate natural killer cells and regulate immune responses. IFN-α responds to the primary antiviral mechanism in the innate immune system, which can effectively cure acute infectious diseases. Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV). The clinical symptoms of PRV are as follows: reproductive dysfunction among pregnant sows and high mortality rates among piglets. These pose a severe threat to the swine industry. Related studies show that IFN-α has broad applications in preventing and treating viral diseases. Therefore, a PRV mouse model using artificial infection was established in this study to explore the pathogenic effect of IFN-α on PRV. We designed a sequence with IFN-α4 (M28623, Genbank) and cloned it on the lentiviral vector. CHO-K1 cells were infected and identified using WB and RT-PCR; a CHO-K1 cell line with a stable expression of the recombinant protein PoIFN-α was successfully constructed. H&E staining and virus titer detection were used to investigate the recombinant protein PoIFN-α's effect on PR in BALB/c mice. The results show that the PoIFN-α has a preventive and therapeutic impact on PR. In conclusion, the recombinant protein can alleviate symptoms and reduce the replication of PRV in vivo.

Role of OGDH in Atophagy-IRF3-IFN-ß pathway during classical swine fever virus infection.

Classical swine fever (CSF) is a severe infectious disease caused by the classical swine fever virus (CSFV) that poses significant challenges to the swine industry. α-ketoglutarate dehydrogenase (OGDH), the first rate-limiting enzyme of the tricarboxylic acid (TCA) cycle, catalyzes α-ketoglutarate (α-KG) to succinyl-CoA, playing a crucial role in glycometabolism. Our previous studies showed that CSFV disrupts the TCA cycle, resulting in α-KG accumulation. However, the interplay between CSFV and OGDH remains unclear. In this study, we found that CSFV significantly reduces OGDH protein levels and promotes α-KG secretion through OGDH in PK-15 cells. Furthermore, we observed CSFV C protein interacts with OGDH and revealed that CSFV utilizes NDP52/NBR1 to target OGDH protein degradation in the autophagy-lysosome pathway. We also unveiled that OGDH overexpression inhibits CSFV proliferation, whereas OGDH knockdown increases CSFV proliferation. Further investigation into the mechanisms of OGDH on CSFV replication revealed that OGDH regulates the AMPK-mTOR-autophagy pathway. Additionally, using the autophagy agonist/inhibitor, rapamycin/3-MA, we observed that OGDH modulates autophagy to regulate the IRF3-IFN-ß network and CSFV replication. These findings shed light on the role of OGDH in CSFV infection and host metabolism, promoting the development of innovative strategies for combating CSFV and other viral infections via targeting metabolic pathways.

Evolutionary dynamics and adaptive analysis of Seneca Valley virus.

Over the past 20 years, the Seneca Valley virus (SVV) has emerged in various countries and regions around the world. Infected pigs display symptoms similar to foot-and-mouth disease and other vesicular diseases, causing severe economic losses to affected countries. In recent years, the number of SVV infections has been increasing in Brazil, China, and the United States. In this study, we comprehensively analyzed SVV genomic sequence data from the perspectives of evolutionary dynamics, phylogeography, and codon usage bias. We aimed to gain further insights into SVV's genetic diversity, spatiotemporal distribution patterns, and evolutionary adaptations. Phylogenetic analysis revealed that SVV has evolved into eight distinct lineages. Based on the results of phylogeographic analysis, it is speculated that the United States might have been the source of SVV, from where it subsequently spread to different countries and regions. Moreover, our analysis of positive selection sites in SVV capsid proteins suggests their potential importance in the process of receptor recognition. Finally, codon preference analysis indicates that natural selection has been a primary evolutionary driver influencing SVV codon usage bias. In conclusion, our in-depth investigation into SVV's origin, dissemination, evolution, and adaptation emphasizes the significance of SVV surveillance and control measures.

Relationship of environmental factors in pond water and dynamic changes of gut microbes of sea bass Lateolabrax japonicus.

The effect of structure of gut microbes on the health of host has attracted increasing attention. Sea bass Lateolabrax japonicus is an important farmed fish in China. The relationship of the dynamic changes of intestinal bacterial communities in L. japonicus and the cultural water environment is very important for healthy culture. Here, the diversity and abundance of the gut microbial communities of L. japonicus were evaluated during the culture using 16S rRNA Illumina sequencing. Both the opportunistic pathogens Aeromonas (1.68%), Vibrio (1.59%), and Acinetobacter (1.22%); and the potential probiotics Lactobacillus (2.27%), Bacillus (1.16%), and Lactococcus (0.37%) were distributed in the gut of L. japonicus. The increasing concentration of nitrogen of water environments with the increase of culture time significantly correlated with shifts in the microbial community structure: 40.04% of gut microbial changes due to nitrogen concentration. Higher concentrations of nitrogen showed a significantly negative correlation with intestinal probiotics in L. japonicus. The results indicate that the abundance of intestinal bacteria of L. japonicus is mainly driven by the changes of environmental factors (e.g., nitrogen), and it's very important that the linking environmental parameters with bacterial data of guts could be used as an early warning indicator in L. japonicus heath culture.

The regulation of cell homeostasis and antiviral innate immunity by autophagy during classical swine fever virus infection.

CSFV (classical swine fever virus) is currently endemic in developing countries in Asia and has recently re-emerged in Japan. Under the pressure of natural selection pressure, CSFV keeps evolving to maintain its ecological niche in nature. CSFV has evolved mechanisms that induce immune depression, but its pathogenic mechanism is still unclear. In this study, using transcriptomics and metabolomics methods, we found that CSFV infection alters innate host immunity by activating the interferon pathway, inhibiting host inflammation, apoptosis, and remodelling host metabolism in porcine alveolar macrophages. Moreover, we revealed that autophagy could alter innate immunity and metabolism induced by CSFV infection. Enhanced autophagy further inhibited CSFV-induced RIG-I-IRF3 signal transduction axis and JAK-STAT signalling pathway and blocked type I interferon production while reducing autophagy inhibition of the NF-κB signalling pathway and apoptosis in CSFV infection cells. Furthermore, the level of CSFV infection-induced glycolysis and the content of lactate and pyruvate, as well as 3-phosphoglyceraldehyde, a derivative of glycolysis converted to serine, was altered by autophagy. We also found that silencing HK2 (hexokinase 2), the rate-limiting enzyme of glycolytic metabolism, could induce autophagy but reduce the interferon signalling pathway, NF-κB signalling pathway, and inhibition of apoptosis induced by CSFV infection. In addition, inhibited cellular autophagy by silencing ATG5 or using 3-Methyladenine, could backfill the inhibitory effect of silencing HK2 on the cellular interferon signalling pathway, NF-κB signalling pathway, and apoptosis.

Transcriptome Profiling in Swine Macrophages Infected with African Swine Fever Virus (ASFV) Uncovers the Complex and Close Relationship with Host.

African swine fever virus (ASFV) is a pathogen to cause devastating and economically significant diseases in domestic and feral swine. ASFV mainly infects macrophages and monocytes and regulates its replication process by affecting the content of cytokines in the infected cells. There is a limited understanding of host gene expression and differential profiles before and after ASFV infection in susceptible cells. In this study, RNA-seq technology was used to analyze the transcriptomic change in PAMs infected with ASFV at different time points (0 h, 12 h, 24 h). As a result, a total of 2748, 1570, and 560 genes were enriched in group V12 h vs. MOCK, V24 h vs. MOCK, and V24 h vs. V12 h, respectively. These DEGs (differentially expressed genes) in each group were mainly concentrated in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways related to innate immunization and inflammation, including the NF-κB signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, IL-17 signaling pathway, cytokine-cytokine receptor interaction, and chemokine signaling pathway. Furthermore, the increased levels of IL-1ß, TNF-α, IKKß, CXCL2, and TRAF2 and decreased level of IκBα were validated through the qPCR method. These results suggested that ASFV infection can activate the NF-κB signaling pathway in the early stage. In general, this study provides a theoretical basis for further understanding the pathogenesis and immune escape mechanism of ASFV.

Immunogenicity and Immunoprotection of PCV2 Virus-like Particles Incorporating Dominant T and B Cell Antigenic Epitopes Paired with CD154 Molecules in Piglets and Mice.

Porcine circovirus type 2 (PCV2) is capable of causing porcine circovirus-associated disease (PCVAD) and is one of the major threats to the global pig industry. The nucleocapsid protein Cap encoded by the PCV2 ORF2 gene is an ideal antigen for the development of PCV2 subunit vaccines, and its N-terminal nuclear localization sequence (NLS) structural domain is essential for the formation of self-assembling VLPs. In the present study, we systematically expressed and characterized full-length PCV2 Cap proteins fused to dominant T and B cell antigenic epitopes and porcine-derived CD154 molecules using baculovirus and found that the Cap proteins fusing epitopes were still capable of forming virus-like particles (VLPs). Both piglet and mice experiments showed that the Cap proteins fusing epitopes or paired with the molecular adjuvant CD154 were able to induce higher levels of humoral and cellular responses, particularly the secretion of PCV2-specific IFN-γ and IL-4. In addition, vaccination significantly reduced clinical signs and the viral load of PCV2 in the blood and tissues of challenged piglets. The results of the study provide new ideas for the development of a more efficient, safe and broad-spectrum next-generation PCV2 subunit vaccine.

Advances in the differential molecular diagnosis of vesicular disease pathogens in swine.

Foot-and-mouth disease virus (FMDV), Senecavirus A (SVA) and swine vesicular disease virus (SVDV) are members of the family Picornaviridae, which can cause similar symptoms - vesicular lesions in the tissues of the mouth, nose, feet, skin and mucous membrane of animals. Rapid and accurate diagnosis of these viruses allows for control measures to prevent the spread of these diseases. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR are traditional and reliable methods for pathogen detection, while their amplification reaction requires a thermocycler. Isothermal amplification methods including loop-mediated isothermal amplification and recombinase polymerase amplification developed in recent years are simple, rapid and do not require specialized equipment, allowing for point of care diagnostics. Luminex technology allows for simultaneous detection of multiple pathogens. CRISPR-Cas diagnostic systems also emerging nucleic acid detection technologies which are very sensitivity and specificity. In this paper, various nucleic acid detection methods aimed at vesicular disease pathogens in swine (including FMDV, SVA and SVDV) are summarized.

HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway.

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is a highly contagious and fatal viral disease, posing a significant threat to the swine industry. Heat shock protein 90 kDa alpha class A member 1 (HSP90AA1) is a very conservative chaperone protein that plays an important role in signal transduction and viral proliferation. However, the role of HSP90AA1 in CSFV infection is unknown. In this study, we found that expression of HSP90AA1 could be promoted in PK-15 and 3D4/2 cells infected by CSFV. Over-expression of HSP90AA1 could inhibit CSFV replication and functional silencing of HSP90AA1 gene promotes CSFV replication. Further exploration revealed that HSP90AA1 interacted with CSFV NS5A protein and reduced the protein levels of NS5A. Since NS5A has an important role in CSFV replication and is closely related to type I IFN and NF-κB response, we further analyzed whether HSP90AA1 affects CSFV replication by regulating type I IFN and NF-κB pathway responses. Our research found HSP90AA1 positively regulated type I IFN response by promoting STAT1 phosphorylation and nuclear translocation processes and promoted the nuclear translocation processes of p-P65. However, CSFV infection antagonizes the activation of HSP90AA1 on JAK/STAT and NF-κB pathway. In conclusion, our study found that HSP90AA1 overexpression significantly inhibited CSFV replication and may inhibit CSFV replication by interacting with NS5A and activating JAK/STAT and NF-κB signaling pathways. These results provide new insights into the mechanism of action of HSP90AA1 in CSFV infection, which abundant the candidate library of anti-CSFV.

Interaction of SERINC5 and IFITM1/2/3 regulates the autophagy-apoptosis-immune network under CSFV infection.

The host restriction factor serine incorporator 5 (SERINC5) plays a key role in inhibiting viral activity and has been shown to inhibit classical swine fever virus (CSFV) infection. However, the action of SERINC5 in the interaction between host cells and CSFV remains poorly understood. This study found that SERINC5 represses CSFV-induced autophagy through MAPK1/3-mTOR and AKT-mTOR signalling pathways. Further research showed that SERINC5 promotes apoptosis by repressing autophagy. Likewise, it was demonstrated that SERINC5 interacting proteins IFITM1/2/3 inhibit CSFV replication and regulate autophagy in a lysosomal-associated membrane protein LAMP1-dependent manner. In addition, IFITM1/2/3 interference promotes the NF-κB signalling pathway for potential immunoregulation by inhibiting autophagy. Finally, the functional silencing of IFITM1/2/3 genes was demonstrated to enhance the inhibitory effect of SERINC5 on autophagy. Taken together, These data uncover a novel mechanism through SERINC5 and its interacting proteins IFITM1/2/3, which mediates CSFV replication, and provides new avenues for controlling CSFV.

Historical Evolutionary Dynamics and Phylogeography Analysis of Transmissible Gastroenteritis Virus and Porcine Deltacoronavirus: Findings from 59 Suspected Swine Viral Samples from China.

Since the beginning of the 21st century, humans have experienced three coronavirus pandemics, all of which were transmitted to humans via animals. Recent studies have found that porcine deltacoronavirus (PDCoV) can infect humans, so swine enteric coronavirus (SeCoV) may cause harm through cross-species transmission. Transmissible gastroenteritis virus (TGEV) and PDCoV have caused tremendous damage and loss to the pig industry around the world. Therefore, we analyzed the genome sequence data of these two SeCoVs by evolutionary dynamics and phylogeography, revealing the genetic diversity and spatiotemporal distribution characteristics. Maximum likelihood and Bayesian inference analysis showed that TGEV could be divided into two different genotypes, and PDCoV could be divided into four main lineages. Based on the analysis results inferred by phylogeography, we inferred that TGEV might originate from America, PDCoV might originate from Asia, and different migration events had different migration rates. In addition, we also identified positive selection sites of spike protein in TGEV and PDCoV, indicating that the above sites play an essential role in promoting membrane fusion to achieve adaptive evolution. In a word, TGEV and PDCoV are the past and future of SeCoV, and the relatively smooth transmission rate of TGEV and the increasing transmission events of PDCoV are their respective transmission characteristics. Our results provide new insights into the evolutionary characteristics and transmission diversity of these SeCoVs, highlighting the potential for cross-species transmission of SeCoV and the importance of enhanced surveillance and biosecurity measures for SeCoV in the context of the COVID-19 epidemic.

African Swine Fever Virus: A Review.

African swine fever (ASF) is a viral disease with a high fatality rate in both domestic pigs and wild boars. ASF has greatly challenged pig-raising countries and also negatively impacted regional and national trade of pork products. To date, ASF has spread throughout Africa, Europe, and Asia. The development of safe and effective ASF vaccines is urgently required for the control of ASF outbreaks. The ASF virus (ASFV), the causative agent of ASF, has a large genome and a complex structure. The functions of nearly half of its viral genes still remain to be explored. Knowledge on the structure and function of ASFV proteins, the mechanism underlying ASFV infection and immunity, and the identification of major immunogenicity genes will contribute to the development of an ASF vaccine. In this context, this paper reviews the available knowledge on the structure, replication, protein function, virulence genes, immune evasion, inactivation, vaccines, control, and diagnosis of ASFV.