VP3 protein of Senecavirus A promotes viral IRES-driven translation and attenuates innate immunity by specifically relocalizing hnRNPA2B1.

Viruses deploy sophisticated strategies to hijack the host's translation machinery to favor viral protein synthesis and counteract innate cellular defenses. However, little is known about the mechanisms by which Senecavirus A (SVA) controls the host's translation. Using a series of sophisticated molecular cell manipulation techniques, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as an essential host factor involved in translation control in SVA-infected cells. It was also determined that the SVA structural protein, VP3, binds to and relocalizes hnRNPA2B1, which interferes with the host's protein synthesis machinery to establish a cellular environment that facilitates viral propagation via a two-pronged strategy: first, hnRNPA2B1 serves as a potent internal ribosome entry site (IRES) trans-acting factor, which is selectively co-opted to promote viral IRES-driven translation by supporting the assembly of translation initiation complexes. Second, a strong repression of host cell translation occurs in the context of the VP3-hnRNPA2B1 interaction, resulting in attenuation of the interferons response. This is the first study to demonstrate the interaction between SVA VP3 and hnRNPA2B1, and to characterize their key roles in manipulating translation. This novel dual mechanism, which regulates selective mRNA translation and immune evasion of virus-infected cells, highlights the VP3-hnRNPA2B1 complex as a potential target for the development of modified antiviral or oncolytic reagents. IMPORTANCE: Viral reproduction is contingent on viral protein synthesis, which relies entirely on the host's translation machinery. As such, viruses often need to control the cellular translational apparatus to favor viral protein production and avoid host innate defenses. Senecavirus A (SVA) is an important virus, both as an emerging pathogen in the pork industry and as a potential oncolytic virus for neuroendocrine cancers. Here, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a critical regulator of the translational landscape during SVA infection. This study supports a model whereby the VP3 protein of SVA efficiently subverts the host's protein synthesis machinery through its ability to bind to and relocalize hnRNPA2B1, not only selectively promoting viral internal ribosome entry site-driven translation but also resulting in global translation shutdown and immune evasion. Together, these data provide new insights into how the complex interactions between translation machinery, SVA, and innate immunity contribute to the pathogenicity of the SVA.

NEDD8 enhances Hippo signaling by mediating YAP1 neddylation.

The Hippo-YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo-YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling.

Pseudorabies virus tegument protein US2 antagonizes antiviral innate immunity by targeting cGAS-STING signaling pathway.

Background: The cGAS-STING axis-mediated type I interferon pathway is a crucial strategy for host defense against DNA virus infection. Numerous evasion strategies developed by the pseudorabies virus (PRV) counteract host antiviral immunity. To what extent PRV-encoded proteins evade the cGAS-STING signaling pathway is unknown. Methods: Using US2 stably expressing cell lines and US2-deficient PRV model, we revealed that the PRV tegument protein US2 reduces STING protein stability and downregulates STING-mediated antiviral signaling. Results: To promote K48-linked ubiquitination and STING degradation, US2 interacts with the LBD structural domain of STING and recruits the E3 ligase TRIM21. TRIM21 deficiency consistently strengthens the host antiviral immune response brought on by PRV infection. Additionally, US2-deficient PRV is less harmful in mice. Conclusions: Our study implies that PRV US2 inhibits IFN signaling by a new mechanism that selectively targets STING while successfully evading the host antiviral response. As a result, the present study reveals a novel strategy by which PRV evades host defense and offers explanations for why the Bartha-K61 classical vaccine strain failed to offer effective defense against PRV variant strains in China, indicating that US2 may be a key target for developing gene-deficient PRV vaccines.

A thorough investigation into the correlation between migraines and the gut microbiome: an in-depth analysis using Mendelian randomization studies.

Objective: A growing body of evidence underscores a significant association between neurological disorders, particularly migraines, and the gut microbiota. However, a research gap persists in understanding the cause-and-effect dynamics between these elements. Therefore, we employed robust methodologies aimed at thoroughly exploring the causal relationship between the gut microbiome and migraines. Methods: Employing bidirectional Two Sample Mendelian Randomization (TSMR) analysis, we investigated the causal association between the composition of the gut microbiota and migraines. Data summarizing the relationship between gut microbiota and migraines were extracted from one or more genome-wide association studies. The TSMR analysis employed five methods to assess the correlation between the gut microbiota and migraines, with the inverse variance-weighted method serving as the primary approach for analyzing causal links. Sensitivity analyses were applied to address horizontal pleiotropy and heterogeneity. Simultaneously, a meta-analysis was performed to strengthen the robustness of the findings. Additionally, a reverse TSMR was carried out to explore potential occurrences of reverse causal relationships. Results: The ongoing TSMR analysis identified a collection of 14 bacterial taxa connected to migraines. Among these, 8 taxa exhibited a protective effect, while 5 taxa had a detrimental impact, and 1 taxon maintained a neutral relationship. The reverse Mendelian randomization analysis highlighted stable outcomes for only one bacterial taxonomic group. Conclusion: The study confirms a causal relationship between the gut microbiota and migraines, offering a new perspective for migraine research. Strategically targeting specific bacterial taxa with dysregulation may be effective in both preventing and treating migraines, thus opening new avenues for therapeutic strategies.

Preparation and epitope analysis of monoclonal antibodies against African swine fever virus DP96R protein.

BACKGROUND: Many proteins of African swine fever virus (ASFV, such as p72, p54, p30, CD2v, K205R) have been successfully expressed and characterized. However, there are few reports on the DP96R protein of ASFV, which is the virulence protein of ASFV and plays an important role in the process of host infection and invasion of ASFV. RESULTS: Firstly, the prokaryotic expression vector of DP96R gene was constructed, the prokaryotic system was used to induce the expression of DP96R protein, and monoclonal antibody was prepared by immunizing mice. Four monoclonal cells of DP96R protein were obtained by three ELISA screening and two sub-cloning; the titer of ascites antibody was up to 1:500,000, and the monoclonal antibody could specifically recognize DP96R protein. Finally, the subtypes of the four strains of monoclonal antibodies were identified and the minimum epitopes recognized by them were determined. CONCLUSION: Monoclonal antibody against ASFV DP96R protein was successfully prepared and identified, which lays a foundation for further exploration of the structure and function of DP96R protein and ASFV diagnostic technology.

Advancing vaccine development: Evaluation of a mannose-modified lipid nanoparticle-based candidate for African swine fever p30 mRNA vaccine eliciting robust immune response in mice.

The African swine fever virus (ASFV) continues to pose significant economic and pandemic risks. Consequently, discovering new, efficient vaccines is crucial. Messenger RNA (mRNA) vaccines have emerged as promising candidates, providing minimal risk of insertional mutagenesis, high safety profiles, effectiveness, rapid scalability in production, and cost-effectiveness. In this study, we have developed an ASF p30 mRNA vaccine candidate (mRNA/Man-LNP) employing mannose-modified lipid nanoparticles (LNPs). The mRNA/Man-LNP exhibited effective antigen presentation and facilitated dendritic cells (DCs) maturation. Notably, it elicited strong IgG titers and activated CD4+ and CD8+ T-cells in immunized mice, all while adhering to stringent biosafety standards. This investigation demonstrates that mRNA/Man-LNP can trigger both humoral and cellular immune responses, suggesting its potential as a potent and promising vaccine candidate for controlling African swine fever (ASF).

Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1ß to promote viral replication by regulating K48 ubiquitination.

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1ß (Nsp1ß) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1ß protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (Nsp1ß) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1ß, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1ß, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

Genomic analysis of almost 8,000 Salmonella genomes reveals drivers and landscape of antimicrobial resistance in China.

IMPORTANCE: We established the largest Salmonella genome database from China and presented the landscape and spatiotemporal dynamics of antimicrobial resistance genes. We also found that economic, climatic, and social factors can drive the rise of antimicrobial resistance. The Chinese local Salmonella genome database version 2 was released as an open-access database (https://nmdc.cn/clsgdbv2) and thus can assist surveillance studies across the globe. This database will help inform interventions for AMR, food safety, and public health.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Development and characterization of monoclonal antibody against the critical loop structure of african swine fever virus P72 protein.

African swine fever (ASF) is a highly infectious and lethal viral disease caused by the African swine fever virus (ASFV). The four prominent loop structures on the surface of the primary structural protein P72 are considered to be key protective epitopes. In this study, the four critical loops (ER1-4) of the ASFV p72 protein were individually fused to hepatitis B virus core particles (HBc) and self-assembled into nanoparticles to preserve the natural conformation of the loop structure and enhance its immunogenicity. Then, four recombinant proteins were obtained in E. coli expression system and monoclonal antibodies (mAbs) were developed and characterized. All 10 mAbs obtained were able to react with P72 protein and ASFV with potencies up to 1:204 800. Amino acids 250-274, 279-299 and 507-517 of the P72 protein were identified as linear epitopes and highly conserved. The mAb 4G8 showed the highest inhibition rate of 84% against ASFV positive sera. Importantly, neutralization experiments illustrated that mAb 4G8 has a 67% inhibition rate, indicating that its corresponding epitopes are potential candidates for ASFV vaccine. In conclusion, highly immunogenic nanoparticles of the ASFV P72 key loop were constructed to induce the production of highly effective mAbs and clarify their epitope information for the diagnosis and prevention of ASFV.

Identification of a Linear B Cell Epitope on p54 of African Swine Fever Virus Using Nanobodies as a Novel Tool.

African swine fever (ASF) has received great attention from the swine industry due to the pandemic and the lack of vaccines or effective treatments. In the present study, 13 African swine fever virus (ASFV) p54-specific nanobodies (Nbs) were successfully screened based on Bactrian camel immunization of p54 protein and phage display technology, and their reactivity with the p54 C-terminal domain (p54-CTD) was determined; however, only Nb8-horseradish peroxidase (Nb8-HRP) exhibited the best reactivity. Immunoperoxidase monolayer assay (IPMA) and immunofluorescence assay (IFA) results indicated that Nb8-HRP specifically reacted with ASFV-infected cells. Then, the possible epitopes of p54 were identified using Nb8-HRP. The results showed that Nb8-HRP could recognize p54-CTD truncated mutant p54-T1. Then, 6 overlapping peptides covering p54-T1 were synthesized to determine the possible epitopes. Dot blot and peptide-based enzyme-linked immunosorbent assay (ELISA) results suggested that one novel minimal linear B cell epitope, 76QQWVEV81, which had never been reported before, was identified. Alanine-scanning mutagenesis revealed that 76QQWV79 was the core binding site for Nb8. Epitope 76QQWVEV81 was highly conserved among genotype II ASFV strains and could react with inactivated ASFV antibody-positive serum from naturally infected pigs, indicating that it was a natural linear B cell epitope. These findings provide valuable insights for vaccine design and p54 as an effective diagnostic tool. IMPORTANCE The ASFV p54 protein plays an important role in inducing neutralization antibodies in vivo after viral infection and is often used as a candidate protein for subunit vaccine development. The full understanding of the p54 protein epitope provides a sufficient theoretical basis for p54 as a vaccine candidate protein. The present study uses a p54-specific nanobody as a probe to identify a highly conserved antigenic epitope, 76QQWVEV81, among different ASFV strains, and it can induce humoral immune responses in pigs. This is the first report using virus-specific nanobodies as a tool to identify some special epitopes that cannot be recognized by conventional monoclonal antibodies. This study opens up nanobodies as a new tool for identifying epitopes and also provides a theoretical basis for understanding p54-induced neutralizing antibodies.

A Novel Linear B-Cell Epitope on the P54 Protein of African Swine Fever Virus Identified Using Monoclonal Antibodies.

The African swine fever virus (ASFV) is a highly infectious viral pathogen that presents a major threat to the global pig industry. No effective vaccine is available for the virus. The p54 protein, a major structural component of ASFV, is involved in virus adsorption and entry to target cells and also plays a key role in ASFV vaccine development and disease prevention. Here, we generated species-specific monoclonal antibodies (mAbs), namely 7G10A7F7, 6E8G8E1, 6C3A6D12, and 8D10C12C8 (subtype IgG1/kappa type), against the ASFV p54 protein and characterized the specificity of these mAbs. Peptide scanning techniques were used to determine the epitopes that are recognized by the mAbs, which defined a new B-cell epitope, TMSAIENLR. Amino acid sequence comparison showed that this epitope is conserved among all reference ASFV strains from different regions of China, including the widely prevalent, highly pathogenic strain Georgia 2007/1 (NC_044959.2). This study reveals important signposts for the design and development of ASFV vaccines and also provides critical information for the functional studies of the p54 protein via deletion analysis.

TAT Nanobody Exerts Antiviral Effect against PRRSV In Vitro by Targeting Viral Nucleocapsid Protein.

Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV), which has brought huge economic losses to the pork industry worldwide since its first discovery in the late 1980s in North America. To date, there are no effective commercial vaccines or therapeutic drugs available for controlling the spread of PRRSV. Due to their unique advantages of high affinity and high specificity, nanobodies (Nbs) have received increasing attention in the process of disease diagnosis and treatment. Trans-activator transcription (TAT) can serve as a vector to carry specific proteins into cells by passing through cell membranes. In our previous study, a specific Nb against the PRRSV nucleocapsid (N) protein was screened using phage display technology. For this study, we developed a novel recombinant protein constituting a TAT-conjugated Nb, which we call TAT-Nb1. The target cell entry efficiency of TAT-Nb1 and its effect on PRRSV infection and replication were then investigated. Our results indicate that TAT delivered Nb1 into Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner. Furthermore, TAT-Nb1 dose-dependently suppressed PRRSV infection and replication, where this antiviral effect was independent of PRRSV strain. Co-immunoprecipitation results revealed that Nb1 efficiently interacted with the N protein of PRRSV. Taken together, the presented results suggest that TAT-Nb1 can effectively suppress PRRSV replication, and it may be considered as a new anti-PRRSV candidate drug.

Identification of a linear B-cell epitope on the African swine fever virus CD2v protein.

African swine fever virus (ASFV) poses a serious threat to domestic pigs and wild boars, which is responsible for substantial production and economic losses. A dominant ASFV specific linear B cell epitope that reacted with the convalescent serum was explored and identified with the help of immune informatics techniques. It is essential in understanding the host immunity and in developing diagnostic technical guidelines and vaccine design. The confirmation of dominant epitopes with a positive serological matrix is feasible. To improve the immunogenicity of the epitope, we designed the dominant epitope of CD2v in the form of 2 branch Multiple-Antigen peptide (MAPs-2), CD2v-MAPs-2. Notably, CD2v peptide can be taken up by dendritic cells (DCs) to activate T lymphocytes and induce highly effective valence antibodies in BALB/c mice. The specific CD8+ T cell response were observed. The dominant epitope peptide identified in this study was able to effectively activate humoral and cellular immunity in mice model.

Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

African swine fever virus (ASFV), a DNA double-stranded virus with high infectivity and mortality, causing a devastating blow to the pig industry and the world economy. The CD2v protein is an essential immunoprotective protein of ASFV. In this study, we expressed the extracellular region of the CD2v protein in the 293F expression system to achieve proper glycosylation. Monoclonal antibodies (mAbs) were prepared by immunizing mice with the recombinant CD2v protein. Eventually, four mAbs that target the extracellular region of the ASFV CD2v protein were obtained. All four mAbs responded well to the ASFV HLJ/18 strain and recognized the same linear epitope, 154SILE157. The specific shortest amino acid sequence of this epitope has been accurately identified for the first time. Meaningfully, the 154SILE157 epitope was highly conformed in the ASFV Chinese epidemic strain and Georgia2008/1 strains according to the analysis of the conservation and have a fair protective effect. These findings contribute to further understanding of the protein function of CD2v and provide potential support for the development of diagnostic tools and vaccines for ASFV.

African Swine Fever Virus MGF110-7L Induces Host Cell Translation Suppression and Stress Granule Formation by Activating the PERK/PKR-eIF2α Pathway.

African swine fever (ASF) is a highly contagious and often lethal disease of pigs caused by ASF virus (ASFV) and recognized as the biggest killer in global swine industry. Despite exhibiting incredible self-sufficiency, ASFV remains unconditionally dependent on the host translation machinery for its mRNA translation. However, less is yet known regarding how ASFV-encoded proteins regulate host translation machinery in infected cells. Here, we examined how ASFV interacts with the eukaryotic initiation factor 2α (eIF2α) signaling axis, which directs host translation control and adaptation to cellular stress. We found that ASFV MGF110-7L, a previously uncharacterized member of the multigene family 110, remarkably enhanced the phosphorylation level of eIF2α. In porcine alveolar macrophage 3D4/21 and porcine kidney-15 cells, MGF110-7L triggered eIF2α signaling and the integrated stress response, resulting in the suppression of host translation and the formation of stress granules (SGs). Mechanistically, MGF110-7L-induced phosphorylation of eIF2α was mediated via protein kinase R (PKR) and PKR-like endoplasmic reticulum (ER) kinase (PERK), and this process was essential for host translation repression and SG formation. Notably, our subsequent analyses confirmed that MGF110-7L was overwhelmingly retained in the ER and caused a specific reorganization of the secretory pathway. Further proteomic analyses and biochemical experiments revealed that MGF110-7L could trigger ER stress and activate the unfolded protein response, thus contributing to eIF2α phosphorylation and translation reprogramming. Overall, our study both identifies a novel mechanism by which ASFV MGF110-7L subverts the host protein synthesis machinery and provides further insights into the translation regulation that occurs during ASFV infection. IMPORTANCE African swine fever (ASF) has become a socioeconomic burden and a threat to food security and biodiversity, but no commercial vaccines or antivirals are available currently. Understanding the viral strategies to subvert the host translation machinery during ASF virus (ASFV) infection could potentially lead to new vaccines and antiviral therapies. In this study, we dissected how ASFV MGF110-7L interacts with the eIF2α signaling axis controlling translational reprogramming, and we addressed the role of MGF110-7L in induction of cellular stress responses, eIF2α phosphorylation, translation suppression, and stress granule formation. These results define several molecular interfaces by which ASFV MGF110-7L subverts host cell translation, which may guide research on antiviral strategies and dissection of ASFV pathogenesis.

Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.

Identification of three conserved linear B cell epitopes on the SARS-CoV-2 spike protein.

Spike (S) glycoprotein is the most significant structural protein of SARS-CoV-2 and a key target for neutralizing antibodies. In light of the on-going SARS-CoV-2 pandemic, identification and screening of epitopes of spike glycoproteins will provide vital progress in the development of sensitive and specific diagnostic tools. In the present study, NTD, RBD, and S2 genes were inserted into the pcDNA3.1(+) vector and designed with N-terminal 6× His-tag for fusion expression in HEK293F cells by transient transfection. Six monoclonal antibodies (4G, 9E, 4B, 7D, 8F, and 3D) were prepared using the expressed proteins by cell fusion technique. The characterization of mAbs was performed by indirect -ELISA, western blot, and IFA. We designed 49 overlapping synthesized peptides that cover the extracellular region of S protein in which 6 amino acid residues were offset between adjacent (S1-S49). Peptides S12, S19, and S49 were identified as the immunodominant epitope regions by the mAbs. These regions were further truncated and the peptides S12.2 286TDAVDCALDPLS297, S19.2 464FERDISTEIYQA475, and S49.4 1202ELGKYEQYIKWP1213 were identified as B- cell linear epitopes for the first time. Alanine scans showed that the D467, I468, E471, Q474, and A475 of the epitope S19.2 and K1205, Q1208, and Y1209 of the epitope S49.4 were the core sites involved in the mAbs binding. The multiple sequence alignment analysis showed that these three epitopes were highly conserved among the variants of concern (VOCs) and variants of interest (VOIs). Taken together, the findings provide a potential material for rapid diagnosis methods of COVID-19.

Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect.

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1-69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.