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.

Biological Characteristics of Feline Calicivirus Epidemic Strains in China and Screening of Broad-Spectrum Protective Vaccine Strains.

Feline calicivirus (FCV) is one of the most important pathogens causing upper respiratory tract diseases in cats, posing a serious health threat to these animals. At present, FCV is mainly prevented through vaccination, but the protective efficacy of vaccines in China is limited. In this study, based on the differences in capsid proteins of isolates from different regions in China, as reported in our previous studies, seven representative FCV epidemic strains were selected and tested for their viral titers, virulence, immunogenicity, and extensive cross-protection. Subsequently, vaccine strains were selected to prepare inactivated vaccines. The whole-genome sequencing and analysis results showed that these seven representative FCV strains and 144 reference strains fell into five groups (A, B, C, D, and E). The strains isolated in China mainly fall into groups C and D, exhibiting regional characteristics. These Chinese isolates had a distant evolutionary relationship and low homology with the current FCV-255 vaccine strain. The screened FCV-HB7 and FCV-HB10 strains displayed desirable in vitro culture characteristics, with the highest virus proliferation titers (109.5 TCID50/mL) at 36 h post inoculation at a dose of 0.01 MOI. All five cats infected intranasally with FCV-HB7 or FCV-HB10 strains showed obvious clinical symptoms of FCV. The symptoms of cats infected with the FCV-HB7 strain were more severe than those infected with the FCV-HB10 strain. Both the single-strain inactivated immunization and combined bivalent inactivated vaccine immunization of FCV-HB7 and FCV-HB10 induced high neutralizing antibody titers in five cats immunized. Moreover, bivalent inactivated vaccine immunization protected cats from FCV-HB7 and FCV-HB10 strains. The cross-neutralizing antibody titer against seven representative FCV epidemic strains achieved by combined bivalent inactivated vaccine immunization was higher than that achieved by single-strain immunization, which was much higher than that achieved by commercial vaccine FCV-255 strain immunization. The above results suggest that the FCV-HB7 and FCV-HB10 strains screened in this study have great potential to become vaccine strains with broad-spectrum protective efficacy. However, their immune protective efficacy needs to be further verified by multiple methods before clinical application.

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.

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.

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.

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.

Genomic Characteristics and E Protein Bioinformatics Analysis of JEV Isolates from South China from 2011 to 2018.

Japanese encephalitis is a mosquito-borne zoonotic epidemic caused by the Japanese encephalitis virus (JEV). JEV is not only the leading cause of Asian viral encephalitis, but also one of the leading causes of viral encephalitis worldwide. To understand the genetic evolution and E protein characteristics of JEV, 263 suspected porcine JE samples collected from South China from 2011 to 2018 were inspected. It was found that 78 aborted porcine fetuses were JEV-nucleic-acid-positive, with a positive rate of 29.7%. Furthermore, four JEV variants were isolated from JEV-nucleic-acid-positive materials, namely, CH/GD2011/2011, CH/GD2014/2014, CH/GD2015/2015, and CH/GD2018/2018. The cell culture and virus titer determination of four JEV isolates showed that four JEV isolates could proliferate stably in Vero cells, and the virus titer was as high as 108.5 TCID 50/mL. The whole-genome sequences of four JEV isolates were sequenced. Based on the phylogenetic analysis of the JEV E gene and whole genome, it was found that CH/GD2011/2011 and CH/GD2015/2015 belonged to the GIII type, while CH/GD2014/2014 and CH/GD2018/2018 belonged to the GI type, which was significantly different from that of the JEV classical strain CH/BJ-1/1995. Bioinformatics tools were used to analyze the E protein phosphorylation site, glycosylation site, B cell antigen epitope, and modeled 3D structures of E protein in four JEV isolates. The analysis of the prevalence of JEV and the biological function of E protein can provide a theoretical basis for the prevention and control of JEV and the design of antiviral drugs.

Using Self-Assembling ADDomer Platform to Display B and T Epitopes of Type O Foot-and-Mouth Disease Virus.

Foot-and-mouth disease virus (FMDV) is a highly contagious and devastating virus that infects cloven-hoofed livestock and various wildlife species. Vaccination is the best measure to prevent FMD. ADDomer, as a kind of non-infectious adenovirus-inspired nanoparticle, has the advantage of high thermal stability. In this study, two dominant B-cell antigen epitopes (residues 129~160 and 200~213) and a dominant T-cell antigen epitope (residues 16~44) of type O FMDV were inserted into the ADDomer variable loop (VL) and arginine-glycine-aspartic acid (RGD) loop. The 3D structure of the recombinant protein (ADDomer-RBT) was simulated by homology modeling. First, the recombinant proteins were expressed by the baculovirus expression system and detected by western blot and Q Exactive mass spectrometry. Then the formation of VLPs was observed under a transmission electron micrograph (TEM). Finally, we evaluated the immunogenicity of chimeric VLPs with a murine model. Bioinformatic software analysis preliminarily corroborated that the chosen epitopes were successfully exposed on the surface of ADDomer VLPs. The TEM assay demonstrated the structural integrity of the VLPs. After immunizing, it was found that FMDV-specific antibodies can be produced in mice to induce humoral and cellular immune responses. To sum up, the ADDomer platform can be used as an effective antigen carrier to deliver antigen epitopes. This study presents one of the candidate vaccines to prevent and control FMDV.

Swine Enteric Coronavirus: Diverse Pathogen-Host Interactions.

Swine enteric coronavirus (SeCoV) causes acute gastroenteritis and high mortality in newborn piglets. Since the last century, porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) have swept farms all over the world and caused substantial economic losses. In recent years, porcine delta coronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV) have been emerging SeCoVs. Some of them even spread across species, which made the epidemic situation of SeCoV more complex and changeable. Recent studies have begun to reveal the complex SeCoV-host interaction mechanism in detail. This review summarizes the current advances in autophagy, apoptosis, and innate immunity induced by SeCoV infection. These complex interactions may be directly involved in viral replication or the alteration of some signal pathways.

Development of Diagnostic Tests Provides Technical Support for the Control of African Swine Fever.

African swine fever is a highly contagious global disease caused by the African swine fever virus. Since African swine fever (ASF) was introduced to Georgia in 2007, it has spread to many Eurasian countries at an extremely fast speed. It has recently spread to China and other major pig-producing countries in southeast Asia, threatening global pork production and food security. As there is no available vaccine at present, prevention and control must be carried out based on early detection and strict biosecurity measures. Early detection should be based on the rapid identification of the disease on the spot, followed by laboratory diagnosis, which is essential for disease control. In this review, we introduced the prevalence, transmission routes, eradication control strategies, and diagnostic methods of ASF. We reviewed the various methods of diagnosing ASF, focusing on their technical characteristics and clinical test results. Finally, we give some prospects for improving the diagnosis strategy in the future.