A recombinant pseudorabies virus surface - displaying the classical swine fever E2 protein induces specific antibodies rapidly.

Pseudorabies virus (PRV) and classical swine fever virus (CSFV) are both economically important pathogens threatening the pig industry in many countries. The triple-gene-deleted variant of PRV, herein referred to as rPRVTJ-delgE/gI/TK, has exhibited pronounced efficacy and safety profiles. This underscores its viability as a prospective vaccine vector. However, the generation of specific anti-E2 antibodies necessitates elevated immunization doses and extended durations when the extracellular domain of the E2 protein of CSFV is secreted via the recombinant rPRVTJ-delgE/gI/TK vector. To enhance the presentation of exogenous antigens by antigen-presenting cells (APCs), we engineered the E2 protein expressed on the surface of PRV particles in this study. The recombinant virus expressing the E2 protein with a heterogonous transmembrane domain was generated in the backbone of rPRVTJ-delgE/gI/TK and designated as rPRVTJ-UL44-E2. The E2 gene was fused to the 3' terminus of the UL44 gene utilizing P2A, a self-cleaving peptide sequence. The electron microscopy showed that the E2 protein was anchored on the surface of the viral particles of rPRVTJ-delgE/gI/TK-E2. The insertion of the E2 gene did not alter the native biological characteristics of the viral vector. Rabbits immunized with 107 median tissue culture infective doses (TCID50) of rPRVTJ-UL44-E2 exhibited a rapid seroconversion to anti-E2 specific antibodies within 7 days post-immunization (dpi). All the rabbits immunized with the rPRVTJ-UL44-E2 had generated antibodies specific to E2 prior to the administration of the booster immunization. However, the immunized rabbits were not protected from the CSFV C-strain challenge. Nevertheless, this strategy has notably achieved rapid induction of E2-specific non-neutralizing antibodies. These findings provide insights that the design of rPRVTJ-UL44-E2 requires optimization, thereby indicating a promising avenue for augmenting vaccine-induced immune responses.

Immune responses induced by a recombinant C-strain of classical swine fever virus expressing the F317L protein of African swine fever virus.

African swine fever (ASF), a highly infectious and devastating disease affecting both domestic pigs and wild boars, owes its etiology to African swine fever virus (ASFV). ASFV encodes more than 165 proteins. However, novel immunogenic proteins remain unknown. This study aimed to determine the antigenicity of the F317L protein (pF317L) of ASFV. The results revealed that pF317L was able to react with convalescent pig sera, indicating that pF317L could be a candidate antigen. The antigenic potential of pF317L expressed by rHCLV-F317L, a recombinant virus in the backbone of C-strain (a lapinized live attenuated classical swine fever virus) was further investigated in rabbits and pigs. The results revealed that antibodies and cell-mediated immune responses against pF317L were induced in either rabbits or pigs inoculated with rHCLV-F317L. Importantly, anti-pF317L antibodies from rabbits or pigs immunized with rHCLV-F317L significantly inhibited ASFV replication in vitro. In conclusion, pF317L demonstrates favorable immunogenic properties, positioning it as a promising candidate for the development of protective antigens in the ongoing endeavor to formulate efficacious ASF vaccine strategies.

Crosstalk between Dysfunctional Mitochondria and Proinflammatory Responses during Viral Infections.

Mitochondria play pivotal roles in sustaining various biological functions including energy metabolism, cellular signaling transduction, and innate immune responses. Viruses exploit cellular metabolic synthesis to facilitate viral replication, potentially disrupting mitochondrial functions and subsequently eliciting a cascade of proinflammatory responses in host cells. Additionally, the disruption of mitochondrial membranes is involved in immune regulation. During viral infections, mitochondria orchestrate innate immune responses through the generation of reactive oxygen species (ROS) and the release of mitochondrial DNA, which serves as an effective defense mechanism against virus invasion. The targeting of mitochondrial damage may represent a novel approach to antiviral intervention. This review summarizes the regulatory mechanism underlying proinflammatory response induced by mitochondrial damage during viral infections, providing new insights for antiviral strategies.

African Swine Fever Virus Modulates the Endoplasmic Reticulum Stress-ATF6-Calcium Axis to Facilitate Viral Replication.

ABSTRACTAfrican swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. The endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with the unfolded protein response (UPR) (downstream the ER stress) are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

Structural basis for difunctional mechanism of m-AMSA against African swine fever virus pP1192R.

The African swine fever virus (ASFV) type II topoisomerase (Topo II), pP1192R, is the only known Topo II expressed by mammalian viruses and is essential for ASFV replication in the host cytoplasm. Herein, we report the structures of pP1192R in various enzymatic stages using both X-ray crystallography and single-particle cryo-electron microscopy. Our data structurally define the pP1192R-modulated DNA topology changes. By presenting the A2+-like metal ion at the pre-cleavage site, the pP1192R-DNA-m-AMSA complex structure provides support for the classical two-metal mechanism in Topo II-mediated DNA cleavage and a better explanation for nucleophile formation. The unique inhibitor selectivity of pP1192R and the difunctional mechanism of pP1192R inhibition by m-AMSA highlight the specificity of viral Topo II in the poison binding site. Altogether, this study provides the information applicable to the development of a pP1192R-targeting anti-ASFV strategy.

Viral replication organelles: the highly complex and programmed replication machinery.

Viral infections usually induce the rearrangement of cellular cytoskeletal proteins and organelle membrane structures, thus creating independent compartments [termed replication organelles (ROs)] to facilitate viral genome replication. Within the ROs, viral replicases, including polymerases, helicases, and ligases, play functional roles during viral replication. These viral replicases are pivotal in the virus life cycle, and numerous studies have demonstrated that the viral replicases could be the potential targets for drugs development. Here, we summarize primarily the key replicases within viral ROs and emphasize the advancements of antiviral drugs targeting crucial viral replicases, providing novel insights into the future development of antiviral strategies.

A Standardized Pipeline for Assembly and Annotation of African Swine Fever Virus Genome.

Obtaining a complete good-quality sequence and annotation for the long double-stranded DNA genome of the African swine fever virus (ASFV) from next-generation sequencing (NGS) technology has proven difficult, despite the increasing availability of reference genome sequences and the increasing affordability of NGS. A gap analysis conducted by the global African swine fever research alliance (GARA) partners identified that a standardized, automatic pipeline for NGS analysis was urgently needed, particularly for new outbreak strains. Whilst there are several diagnostic and research labs worldwide that collect isolates of the ASFV from outbreaks, many do not have the capability to analyze, annotate, and format NGS data from outbreaks for submission to NCBI, and some publicly available ASFV genomes have missing or incorrect annotations. We developed an automated, standardized pipeline for the analysis of NGS reads that directly provides users with assemblies and annotations formatted for their submission to NCBI. This pipeline is freely available on GitHub and has been tested through the GARA partners by examining two previously sequenced ASFV genomes; this study also aimed to assess the accuracy and limitations of two strategies present within the pipeline: reference-based (Illumina reads) and de novo assembly (Illumina and Nanopore reads) strategies.

Immune Responses Induced by a Recombinant Lactiplantibacillus plantarum Surface-Displaying the gD Protein of Pseudorabies Virus.

Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (109 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (104 TCID50) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3+CD4+ T cells, but CD3+CD8+ T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.

The multifaceted roles of selective autophagy receptors in viral infections.

Selective autophagy is a protein clearance mechanism mediated by evolutionarily conserved selective autophagy receptors (SARs), which specifically degrades misfolded, misassembled, or metabolically regulated proteins. SARs help the host to suppress viral infections by degrading viral proteins. However, viruses have evolved sophisticated mechanisms to counteract, evade, or co-opt autophagic processes, thereby facilitating viral replication. Therefore, this review aims to summarize the complex mechanisms of SARs involved in viral infections, specifically focusing on how viruses exploit strategies to regulate selective autophagy. We present an updated understanding of the various critical roles of SARs in viral pathogenesis. Furthermore, newly discovered evasion strategies employed by viruses are discussed and the ubiquitination-autophagy-innate immune regulatory axis is proposed to be a crucial pathway to control viral infections. This review highlights the remarkable flexibility and plasticity of SARs in viral infections.

The antibodies against the A137R protein drive antibody-dependent enhancement of African swine fever virus infection in porcine alveolar macrophages.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly contagious disease that can kill up to 100% of domestic pigs and wild boars. It has been shown that the pigs inoculated with some ASF vaccine candidates display more severe clinical signs and die earlier than do pigs not immunized. We hypothesize that antibody-dependent enhancement (ADE) of ASFV infection may be caused by the presence of some unidentified antibodies. In this study, we found that the ASFV-encoded structural protein A137R (pA137R) can be recognized by the anti-ASFV positive sera, indicating that the anti-pA137R antibodies are induced in the ASFV-infected pigs. Interestingly, our results demonstrated that the anti-pA137R antibodies produced in rabbits or pigs enhanced viral replication of different ASFV strains in primary porcine alveolar macrophages (PAMs), the target cells of ASFV. Mechanistic investigations revealed that anti-pA137R antibodies were able to promote the attachment of ASFV to PAMs and two types of Fc gamma receptors (FcγRs), FcγRII and FcγRIII, mediated the ADE of ASFV infection. Taken together, anti-pA137R antibodies are able to drive ASFV ADE in PAMs. These findings shed new light on the roles of anti-ASFV antibodies and have implications for the pathophysiology of the disease and the development of ASF vaccines.

The Distal Promoter of the B438L Gene of African Swine Fever Virus Is Responsible for the Transcription of the Alternatively Spliced B169L.

The B169L protein (pB169L) of African swine fever virus (ASFV) is a structural protein with an unidentified function during the virus replication. The sequences of the B169L gene and the downstream B438L gene are separated by short intergenic regions. However, the regulatory mode of the gene transcription remains unknown. Here, we identified two distinct promoter regions and two transcription start sites (TSSs) located upstream of the open reading frame (ORF) of B438L. Using the promoter reporter system, we demonstrated that the cis activity of the ORF proximal promoter exhibited significantly higher levels compared with that of the distal promoter located in the B169L gene. Furthermore, transfection with the plasmids with two different promoters for B438L could initiate the transcription and expression of the B438L gene in HEK293T cells, and the cis activity of the ORF proximal promoter also displayed higher activities compared with the distal promoter. Interestingly, the B438L distal promoter also initiated the transcription of the alternatively spliced B169L mRNA (B169L mRNA2) encoding a truncated pB169L (tpB169L) (amino acids 92-169), and the gene transcription efficiency was increased upon mutation of the initiation codon located upstream of the alternatively spliced B169L gene. Taken together, we demonstrated that the distal promoter of B438L gene initiates the transcription of both the B438L mRNA and B169L mRNA2. Comprehensive analysis of the transcriptional regulatory mode of the B438L gene is beneficial for the understanding of the association of B438L protein and pB169L and the construction of the gene-deleted ASFV.

Advances in the immunoescape mechanisms exploited by alphaherpesviruses.

Alphaherpesviruses, categorized as viruses with linear DNA composed of two complementary strands, can potentially to induce diseases in both humans and animals as pathogens. Mature viral particles comprise of a core, capsid, tegument, and envelope. While herpesvirus infection can elicit robust immune and inflammatory reactions in the host, its persistence stems from its prolonged interaction with the host, fostering a diverse array of immunoescape mechanisms. In recent years, significant advancements have been achieved in comprehending the immunoescape tactics employed by alphaherpesviruses, including pseudorabies virus (PRV), herpes simplex virus (HSV), varicella-zoster virus (VZV), feline herpesvirus (FeHV), equine herpesvirus (EHV), and caprine herpesvirus type I (CpHV-1). Researchers have unveiled the intricate adaptive mechanisms existing between viruses and their natural hosts. This review endeavors to illuminate the research advancements concerning the immunoescape mechanisms of alphaherpesviruses by delineating the pertinent proteins and genes involved in virus immunity. It aims to furnish valuable insights for further research on related mechanisms and vaccine development, ultimately contributing to virus control and containment efforts.

Genetic Variations of African Swine Fever Virus: Major Challenges and Prospects.

African swine fever (ASF) is a contagious viral disease affecting pigs and wild boars. It typically presents as a hemorrhagic fever but can also manifest in various forms, ranging from acute to asymptomatic. ASF has spread extensively globally, significantly impacting the swine industry. The complex and highly variable character of the ASFV genome makes vaccine development and disease surveillance extremely difficult. The overall trend in ASFV evolution is towards decreased virulence and increased transmissibility. Factors such as gene mutation, viral recombination, and the strain-specificity of virulence-associated genes facilitate viral variations. This review deeply discusses the influence of these factors on viral immune evasion, pathogenicity, and the ensuing complexities encountered in vaccine development, disease detection, and surveillance. The ultimate goal of this review is to thoroughly explore the genetic evolution patterns and variation mechanisms of ASFV, providing a theoretical foundation for advancement in vaccine and diagnostic technologies.

The MGF300-2R Protein of African Swine Fever Virus Promotes IKKß Ubiquitination by Recruiting the E3 Ubiquitin Ligase TRIM21.

African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease in pigs caused by African swine fever virus (ASFV). Our previous study identified that the ASFV MGF300-2R protein functions as a virulence factor and found that MGF300-2R degrades IKKß via selective autophagy. However, the E3 ubiquitin ligase responsible for IKKß ubiquitination during autophagic degradation still remains unknown. In order to solve this problem, we first pulled down 328 proteins interacting with MGF300-2R through immunoprecipitation-mass spectrometry. Next, we analyzed and confirmed the interaction between the E3 ubiquitin ligase TRIM21 and MGF300-2R and demonstrated the catalytic role of TRIM21 in IKKß ubiquitination. Finally, we indicated that the degradation of IKKß by MGF300-2R was dependent on TRIM21. In summary, our results indicate TRIM21 is the E3 ubiquitin ligase involved in the degradation of IKKß by MGF300-2R, thereby augmenting our understanding of the functions of MGF300-2R and offering insights into the rational design of live attenuated vaccines and antiviral strategies against ASF.

Ferritin Nanoparticle Delivery of the E2 Protein of Classical Swine Fever Virus Completely Protects Pigs from Lethal Challenge.

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), results in significant economic losses to the swine industry in many countries. Vaccination represents the primary strategy to control CSF and the CSFV E2 protein is known as the major protective antigen. However, the E2 protein expressed or presented by different systems elicits distinct immune responses. In this study, we established a stable CHO cell line to express the E2 protein and delivered it using self-assembled ferritin nanoparticles (NPs). Subsequently, we compared the adaptive immune responses induced by the E2-ferritin NPs and the monomeric E2 protein produced by the CHO cells or a baculovirus expression system. The results revealed that the NP-delivered E2 protein elicited higher titers of neutralizing antibodies than did the monomeric E2 protein in pigs. Importantly, only the NP-delivered E2 protein significantly induced CSFV-specific IFN-γ-secreting cells. Furthermore, all the pigs inoculated with the E2-ferritin NPs were completely protected from a lethal CSFV challenge infection. These findings demonstrate the ability of the E2-ferritin NPs to protect pigs against the lethal CSFV challenge by eliciting robust humoral and cellular immune responses.

The African swine fever virus MGF300-4L protein is associated with viral pathogenicity by promoting the autophagic degradation of IKKß and increasing the stability of IκBα.

African swine fever (ASF) is a highly contagious, often fatal viral disease caused by African swine fever virus (ASFV), which imposes a substantial economic burden on the global pig industry. When screening for the virus replication-regulating genes in the left variable region of the ASFV genome, we observed a notable reduction in ASFV replication following the deletion of the MGF300-4L gene. However, the role of MGF300-4L in ASFV infection remains unexplored. In this study, we found that MGF300-4L could effectively inhibit the production of proinflammatory cytokines IL-1ß and TNF-α, which are regulated by the NF-κB signaling pathway. Mechanistically, we demonstrated that MGF300-4L interacts with IKKß and promotes its lysosomal degradation via the chaperone-mediated autophagy. Meanwhile, the interaction between MGF300-4L and IκBα competitively inhibits the binding of the E3 ligase ß-TrCP to IκBα, thereby inhibiting the ubiquitination-dependent degradation of IκBα. Remarkably, although ASFV encodes other inhibitors of NF-κB, the MGF300-4L gene-deleted ASFV (Del4L) showed reduced virulence in pigs, indicating that MGF300-4L plays a critical role in ASFV pathogenicity. Importantly, the attenuation of Del4L was associated with a significant increase in the production of IL-1ß and TNF-α early in the infection of pigs. Our findings provide insights into the functions of MGF300-4L in ASFV pathogenicity, suggesting that MGF300-4L could be a promising target for developing novel strategies and live attenuated vaccines against ASF.

Development and application of an indirect ELISA for detection of antibodies against emerging atypical porcine pestivirus.

BACKGROUND: Atypical porcine pestivirus (APPV) is a newly discovered swine pestivirus, which can cause congenital tremor and high mortality in newborn piglets and subclinical infection in adult pigs, leading to significant impacts on the pig industry. Currently, there is no approved serological method to assess APPV infection status in pig farms. METHODS: In this study, the envelope glycoprotein E2 of APPV was highly expressed in suspension HEK293 cells, and further an indirect enzyme-linked immunosorbent assay based on the recombinant E2 protein (E2-iELISA) was developed and evaluated. RESULTS: The reaction parameters of the E2-iELISA were optimized, and the cutoff value was determined to be 0.2 by analyzing S/P values of 165 negative sera against APPV that were confirmed by virus neutralization test (VNT). Specificity test showed that the method had no cross-reaction with other common swine viruses. The E2-iELISA was evaluated using a panel of swine sera, and showed high sensitivity (113/120, 94.2%) and specificity (65/70, 92.9%), and the agreement rate with VNT was 93.7% (178/190). Subsequently, the E2-iELISA was utilized to investigate the seroprevalence of APPV in pig herds of China. When detecting 1368 pig serum samples collected from nine provinces in China, the overall seroprevalence of APPV was 73.9% (1011/1368). CONCLUSION: Our findings suggest that the E2-iELISA is specific and sensitive, and could be a valuable tool for serological surveillance of APPV infection in pigs.

Clinical Efficacy, Analgesic Efficacy, and Effects of Cocktail Analgesic Regimens in Patients Undergoing Total Knee Arthroplasty: A Systematic Review and Meta-Analysis.

Objective: Multimodal cocktail analgesic injection (CAI) is widely used as an adjunct pain-reliever in the postoperative phase of patients undergoing total knee arthroplasty (TKA) due to intense postoperative pain accompanying the procedure leading to complications, thereby extending hospital stays. The aim of this study is to establish the clinical efficacy and effects of utilizing CAI regimens during the TKA procedure and the corresponding postoperative patient outcomes. Methods: A database search for pertinent articles literature search was performed in Embase, PubMed, Cochrane Library, Web of Science, and MEDLINE databases. RevMan version 5.4 was used to perform a meta-analysis on the included studies. Results: Data screening and selection produced 15 relevant articles that met the eligibility criteria of this study. The meta-analysis revealed insignificant difference between cocktail injected and control groups in VAS postoperative pain scores both at rest and during activity (OR 0.79, 95% CI 0.59 to 1.05; I2 = 0%; P = .93) and (OR 0.79, 95% CI 0.57 to 1.10; I2 = 0%; P = .75), respectively. Similarly, there was insignificant differences in postoperative knee flexion ROM, postoperative narcotic consumption, and length of stays between the two groups, (OR 1.20, 95% CI 1.03 to 1.40; P = .53), (OR 0.62, 95% CI 0.36 to 1.07; P = .09), and (OR 0.45, 95% CI 0.29 to 0.70; P = .21), respectively. However, the postoperative complications reveal statistical significance between the cocktail injected and the control group (OR 0.45, 95% CI 0.29 to 0.70; P = .004). Conclusion: It is concluded that CAI can play a crucial role in minimizing post-operative complications for patients undergoing TKA.

[Advances in methodologies for evaluating cell-mediated immune responses].

Cell-mediated immune response is an important part of machinery in maintaining the body's homeostasis. After the innate immune system selectively activates the adaptive immune system, the cell-mediated immunity exerts its killing and clearance functions. Therefore, evaluating the level of cell-mediated immune response is crucial in the diagnosis and treatment of cancer, monitoring the immune status after organ transplantation, diagnosing and preventing viral diseases, and evaluating the effectiveness of vaccines and other areas. From the initial overall assessment of the immune effects in vivo to the precise detection of the number and function of multiple immune cells, the evaluation methods of cell-mediated immune response have greatly advanced. However, cell-mediated immune response involves multiple levels in the body, and it's difficult to choose the numerous detection methods available. The article systematically compares the evaluation methods of cell-mediated immune response at four different levels: the organism, the tissue and organ, the immune cells and the immune molecules, with the aim to facilitate the applications of related technologies.

Autophagy as a dual-faced host response to viral infections.

Autophagy selectively degrades viral particles or cellular components, either facilitating or inhibiting viral replication. Conversely, most viruses have evolved strategies to escape or exploit autophagy. Moreover, autophagy collaborates with the pattern recognition receptor signaling, influencing the expression of adaptor molecules involved in the innate immune response and regulating the expression of interferons (IFNs). The intricate relationship between autophagy and IFNs plays a critical role in the host cell defense against microbial invasion. Therefore, it is important to summarize the interactions between viral infections, autophagy, and the host defense mechanisms against viruses. This review specifically focuses on the interactions between autophagy and IFN pathways during viral infections, providing a comprehensive summary of the molecular mechanisms utilized or evaded by different viruses.