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

In the original publication [...].

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.

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.

Viruses Hijack ERAD to Regulate Their Replication and Propagation.

Endoplasmic reticulum-associated degradation (ERAD) is highly conserved in yeast. Recent studies have shown that ERAD is also ubiquitous and highly conserved in eukaryotic cells, where it plays an essential role in maintaining endoplasmic reticulum (ER) homeostasis. Misfolded or unfolded proteins undergo ERAD. They are recognized in the ER, retrotranslocated into the cytoplasm, and degraded by proteasomes after polyubiquitin. This may consist of several main steps: recognition of ERAD substrates, retrotranslocation, and proteasome degradation. Replication and transmission of the virus in the host is a process of a "game" with the host. It can be assumed that the virus has evolved various mechanisms to use the host's functions for its replication and transmission, including ERAD. However, until now, it is still unclear how the host uses ERAD to deal with virus infection and how the viruses hijack the function of ERAD to obtain a favorable niche or evade the immune clearance of the host. Recent studies have shown that viruses have also evolved mechanisms to use various processes of ERAD to promote their transmission. This review describes the occurrence of ERAD and how the viruses hijack the function of ERAD to spread by affecting the homeostasis and immune response of the host, and we will focus on the role of E3 ubiquitin ligase.

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.

The Advances of Broad-Spectrum and Hot Anti-Coronavirus Drugs.

Coronaviruses, mainly including severe acute respiratory syndrome virus, severe acute respiratory syndrome coronavirus 2, Middle East respiratory syndrome virus, human coronavirus OC43, chicken infectious bronchitis virus, porcine infectious gastroenteritis virus, porcine epidemic diarrhea virus, and murine hepatitis virus, can cause severe diseases in humans and livestock. The severe acute respiratory syndrome coronavirus 2 is infecting millions of human beings with high morbidity and mortality worldwide, and the multiplicity of swine epidemic diarrhea coronavirus in swine suggests that coronaviruses seriously jeopardize the safety of public health and that therapeutic intervention is urgently needed. Currently, the most effective methods of prevention and control for coronaviruses are vaccine immunization and pharmacotherapy. However, the emergence of mutated viruses reduces the effectiveness of vaccines. In addition, vaccine developments often lag behind, making it difficult to put them into use early in the outbreak. Therefore, it is meaningful to screen safe, cheap, and broad-spectrum antiviral agents for coronaviruses. This review systematically summarizes the mechanisms and state of anti-human and porcine coronavirus drugs, in order to provide theoretical support for the development of anti-coronavirus drugs and other antivirals.

Viral Vector Vaccine Development and Application during the COVID-19 Pandemic.

With the accumulation of mutations in SARS-CoV-2 and the continuous emergence of new variants, the importance of developing safer and effective vaccines has become more prominent in combating the COVID-19 pandemic. Both traditional and genetically engineered vaccines have contributed to the prevention and control of the pandemic. However, in recent years, the trend of vaccination research has gradually transitioned from traditional to genetically engineered vaccines, with the development of viral vector vaccines attracting increasing attention. Viral vector vaccines have several unique advantages compared to other vaccine platforms. The spread of Omicron has also made the development of intranasal viral vector vaccines more urgent, as the infection site of Omicron is more prominent in the upper respiratory tract. Therefore, the present review focuses on the development of viral vector vaccines and their application during the COVID-19 pandemic.

The Role of Mitophagy in Viral Infection.

Mitophagy, which is able to selectively clear excess or damaged mitochondria, plays a vital role in the quality control of mitochondria and the maintenance of normal mitochondrial functions in eukaryotic cells. Mitophagy is involved in many physiological and pathological processes, including apoptosis, innate immunity, inflammation, cell differentiation, signal transduction, and metabolism. Viral infections cause physical dysfunction and thus pose a significant threat to public health. An accumulating body of evidence reveals that some viruses hijack mitophagy to enable immune escape and self-replication. In this review, we systematically summarize the pathway of mitophagy initiation and discuss the functions and mechanisms of mitophagy in infection with classical swine fever virus and other specific viruses, with the aim of providing a theoretical basis for the prevention and control of related diseases.

Development of Recombinase Aided Amplification Combined With Disposable Nucleic Acid Test Strip for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is the dominant causative agent of PCV2 systemic disease (PCV2-SD) in pigs. It can also associate with other diseases such as respiratory and enteric diseases, reproductive failure, porcine dermatitis and nephropathy syndrome in pigs. Currently, PCV2 infection is a considerable threat in the swine industry. Therefore, it is of great significance to prevent, control, and accurately detect PCV2 in pig farms. Recombinase aided amplification (RAA) technology is an isothermal nucleic acid amplification technology that could rapidly amplify the target gene fragment at a constant temperature. The amplification products labeled with specific molecules could be visually detected using the test strip with the corresponding antibody. In the present study, the RAA technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of PCV2. Primers and probes targeting the PCV2 ORF2 gene were designed according to the RAA technology principles. The PCV2 RAA-strip established in this study could detect as low as 103 copies/µL of recombinant plasmids containing the PCV2 ORF2 gene fragment. The lowest detection limit about viral DNA and virus titers was 6.7 × 10-6 ng/µL and 10 TCID50/mL, respectively. Furthermore, no cross-reaction with other porcine viruses occurred at 37°C and within 15 min. We used 42 clinical samples to assess the performance of our established method. The positive rate of clinical samples detected by PCV2 RAA-strip was 50.00%. This was similar to that detected by PCV2 PCR (45.24%). In conclusion, due to the advantages of strong specificity, high sensitivity, excellent reproducibility, and simple operation method, our PCV2 RAA-strip is suitable for the rapid clinical detection of PCV2 on-site.

Anti-Classical Swine Fever Virus Strategies.

Classical swine fever (CSF), caused by CSF virus (CSFV), is a highly contagious swine disease with high morbidity and mortality, which has caused significant economic losses to the pig industry worldwide. Biosecurity measures and vaccination are the main methods for prevention and control of CSF since no specific drug is available for the effective treatment of CSF. Although a series of biosecurity and vaccination strategies have been developed to curb the outbreak events, it is still difficult to eliminate CSF in CSF-endemic and re-emerging areas. Thus, in addition to implementing enhanced biosecurity measures and exploring more effective CSF vaccines, other strategies are also needed for effectively controlling CSF. Currently, more and more research about anti-CSFV strategies was carried out by scientists, because of the great prospects and value of anti-CSFV strategies in the prevention and control of CSF. Additionally, studies on anti-CSFV strategies could be used as a reference for other viruses in the Flaviviridae family, such as hepatitis C virus, dengue virus, and Zika virus. In this review, we aim to summarize the research on anti-CSFV strategies. In detail, host proteins affecting CSFV replication, drug candidates with anti-CSFV effects, and RNA interference (RNAi) targeting CSFV viral genes were mentioned and the possible mechanisms related to anti-CSFV effects were also summarized.

Viral Infection Modulates Mitochondrial Function.

Mitochondria are important organelles involved in metabolism and programmed cell death in eukaryotic cells. In addition, mitochondria are also closely related to the innate immunity of host cells against viruses. The abnormality of mitochondrial morphology and function might lead to a variety of diseases. A large number of studies have found that a variety of viral infections could change mitochondrial dynamics, mediate mitochondria-induced cell death, and alter the mitochondrial metabolic status and cellular innate immune response to maintain intracellular survival. Meanwhile, mitochondria can also play an antiviral role during viral infection, thereby protecting the host. Therefore, mitochondria play an important role in the interaction between the host and the virus. Herein, we summarize how viral infections affect microbial pathogenesis by altering mitochondrial morphology and function and how viruses escape the host immune response.

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.

Development and Application of RAA Nucleic Acid Test Strip Assay and Double RAA Gel Electrophoresis Detection Methods for ASFV and CSFV.

African swine fever (ASF) is an acute, severe and hemorrhagic infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and wild boars. Since the outbreak of the disease in China in 2018, it has brought a great impact on China's pig industry. Classical swine fever (CSF) is an acute contact infectious disease of pigs caused by classical swine fever virus (CSFV) infection. Clinically, acute CSF usually shows persistent high fever, anorexia, extensive congestion and bleeding of the skin and mucosa, which are similar to ASF. It is of great significance to prevent, control and accurately detect ASF and CSF in pig farms. In this study, Recombinase aided amplification (RAA) technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of ASFV/CSFV. The sensitivity and preliminary clinical application results showed that the RAA test strip established in this study could detect recombinant plasmids containing ASFV/CSFV gene fragments as low as 103 copies/µL. The minimum detection limits of virus DNA/cDNA were 10 and 12 pg respectively, and there was no cross-reaction with other porcine viruses. The specificity of the method was good. We used 37-42 clinical samples to evaluate the performance of our established method, and the positive concordance rates with conventional PCR were 94.1 and 57.1%, respectively. In addition, ASFV and CSFV double RAA agarose gel electrophoresis detection methods were established. The results showed that the method had good specificity. The detection limit of this method is 106 copies for ASFV p72 gene recombinant plasmid and 105 copies for CSFV NS5B Gene recombinant plasmid. The use of this method for clinical material detection was consistent with the PCR method. In summary, the developed method of RAA-strip assay for ASFV and CSFV realized the visual detection of pathogens, and the developed method of dual RAA agarose gel electrophoresis assay for ASFV and CSFV realized the simultaneous detection of two pathogens in one reaction, with good specificity, high sensitivity and rapid reaction rate, which was expected to be clinically feasible for the differential diagnosis of ASF and CSF provided technical support.

The Network of Interactions Between Classical Swine Fever Virus Nonstructural Protein p7 and Host Proteins.

Classical swine fever (CSF) is a highly contagious viral disease causing severe economic losses to the swine industry. As viroporins of viruses modulate the cellular ion balance and then take over the cellular machinery, blocking the activity of viroporin or developing viroporin-defective attenuated vaccines offers new approaches to treat or prevent viral infection. Non-structural protein p7 of CSF virus (CSFV) is a viroporin, which was highly involved in CSFV virulence. Deciphering the interaction between p7 and host proteins will aid our understanding of the mechanism of p7-cellular protein interaction affecting CSFV replication. In the present study, seven host cellular proteins including microtubule-associated protein RP/EB family member 1 (MAPRE1), voltage-dependent anion channel 1 (VDAC1), proteasome maturation protein (POMP), protein inhibitor of activated STAT 1 (PIAS1), gametogenetin binding protein 2 (GGNBP2), COP9 signalosome subunit 2 (COPS2), and contactin 1 (CNTN1) were identified as the potential interactive cellular proteins of CSFV p7 by using yeast two-hybrid (Y2H) screening. Plus, the interaction of CSFV p7 with MAPRE1 and VDAC1 was further evaluated by co-immunoprecipitation and GST-pulldown assay. Besides, the p7-cellular protein interaction network was constructed based on these seven host cellular proteins and the STRING database. Enrichment analysis of GO and KEGG indicated that many host proteins in the p7-cellular protein interaction network were mainly related to the ubiquitin-proteasome system, cGMP-PKG signaling pathway, calcium signaling pathway, and JAK-STAT pathway. Overall, this study identified potential interactive cellular proteins of CSFV p7, constructed the p7-cellular protein interaction network, and predicted the potential pathways involved in the interaction between CSFV p7 and host cells.

Antiviral Role of Serine Incorporator 5 (SERINC5) Proteins in Classical Swine Fever Virus Infection.

Serine incorporator 5 (SERINC5), a multipass transmembrane protein, protects cells from viral infections. The mechanism by which SERINC5 protects against classical swine fever virus (CSFV) infection is unknown. In this study, overexpression of SERINC5 in PK-15 and 3D4/2 cells significantly inhibited the growth of CSFV, whereas SERINC5 silencing enhanced CSFV growth. Additionally, CSFV infection reduced SERINC5 production in cells and tissues. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and analyze protein and peptide molecules that potentially interact with SERINC5. A total of 33 cellular protein candidates were identified. Next, SERINC5 was shown to interact with melanoma differentiation-associated protein 5 (MDA5) by yeast two-hybrid, protein co-localization and co-immunoprecipitation assays. Furthermore, SERINC5 enhanced MDA5-mediated type I interferon (IFN) signaling in a dose-dependent manner. Our results suggest that the anti-CSFV effect of SERINC5 is dependent on the activation of the type I IFN, which may function along with MDA5. The inhibitory effect of SERINC5 on CSFV was disappeared when the endogenous expression of MDA5 was silenced using siRNA, suggesting that SERINC5 exerts an anti-CSFV effect in an MDA5-dependent manner. Our study demonstrated a novel link between SERINC5 and MDA5 in the inhibition of CSFV replication via the type I IFN signaling pathway.

Current State of Global African Swine Fever Vaccine Development under the Prevalence and Transmission of ASF in China.

African swine fever (ASF) is a highly lethal contagious disease of swine caused by African swine fever virus (ASFV). At present, it is listed as a notifiable disease reported to the World Organization for Animal Health (OIE) and a class one animal disease ruled by Chinese government. ASF has brought significant economic losses to the pig industry since its outbreak in China in August 2018. In this review, we recapitulated the epidemic situation of ASF in China as of July 2020 and analyzed the influencing factors during its transmission. Since the situation facing the prevention, control, and eradication of ASF in China is not optimistic, safe and effective vaccines are urgently needed. In light of the continuous development of ASF vaccines in the world, the current scenarios and evolving trends of ASF vaccines are emphatically analyzed in the latter part of the review. The latest research outcomes showed that attempts on ASF gene-deleted vaccines and virus-vectored vaccines have proven to provide complete homologous protection with promising efficacy. Moreover, gaps and future research directions of ASF vaccine are also discussed.

Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B.

Mx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione S-transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B.IMPORTANCE Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo, but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities.

Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells.

During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses.IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.