EspP2 Regulates the Adhesion of Glaesserella parasuis via Rap1 Signaling Pathway.

Different levels of EspP2 expression are seen in strains of Glaesserella parasuis with high and low pathogenicity. As a potential virulence factor for G. parasuis, the pathogenic mechanism of EspP2 in infection of host cells is not clear. To begin to elucidate the effect of EspP2 on virulence, we used G. parasuis SC1401 in its wild-type form and SC1401, which was made EspP2-deficient. We demonstrated that EspP2 causes up-regulation of claudin-1 and occludin expression, thereby promoting the adhesion of G. parasuis to host cells; EspP2-deficiency resulted in significantly reduced adhesion of G. parasuis to cells. Transcriptome sequencing analysis of EspP2-treated PK15 cells revealed that the Rap1 signaling pathway is stimulated by EspP2. Blocking this pathway diminished occludin expression and adhesion. These results indicated that EspP2 regulates the adhesion of Glaesserella parasuis via Rap1 signaling pathway.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

CRISPR/Cas9-Mediated Knockout of the HuR Gene in U251 Cell Inhibits Japanese Encephalitis Virus Replication.

Human antigen R (HuR) is an RNA-binding protein that regulates the post-transcriptional reaction of its target mRNAs. HuR is a critical factor in cancer development and has been identified as a potential target in many cancer models. It participates in the viral life cycle by binding to viral RNAs. In prior work, we used CRISPR/Cas9 screening to identify HuR as a prospective host factor facilitating Japanese encephalitis virus (JEV) infection. The HuR gene was successfully knocked out in U251 cell lines using the CRISPR/Cas9 gene-editing system, with no significant difference in cell growth between U251-WT and U251-HuR-KO2 cells. Here, we experimentally demonstrate for the first time that the knockout of the HuR gene inhibits the replication ability of JEV in U251 cell lines. These results play an essential role in regulating the replication level of JEV and providing new insights into virus-host interactions and potential antiviral strategies. It also offers a platform for investigating the function of HuR in the life cycle of flaviviruses.

HSP90AB1 is a host factor that promotes porcine deltacoronavirus replication.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus. It causes mortality in neonatal piglets and is of growing concern because of its broad host range, including humans. To date, the mechanism of PDCoV infection remains poorly understood. Here, based on a genome-wide CRISPR screen of PDCoV-infected cells, we found that HSP90AB1 (heat shock protein 90 alpha family class B1) promotes PDCoV infection. Knockdown or KO of HSP90AB1 in LLC-PK cells resulted in a significantly suppressed PDCoV infection. Infected cells treated with HSP90 inhibitors 17-AAG and VER-82576 also showed a significantly suppressed PDCoV infection, although KW-2478, which does not affect the ATPase activity of HSP90AB1, had no effect on PDCoV infection. We found that HSP90AB1 interacts with the N, NS7, and NSP10 proteins of PDCoV. We further evaluated the interaction between N and HSP90AB1 and found that the C-tail domain of the N protein is the HSP90AB1-interacting domain. Further studies showed that HSP90AB1 protects N protein from degradation via the proteasome pathway. In summary, our results reveal a key role for HSP90AB1 in the mechanism of PDCoV infection and contribute to provide new host targets for PDCoV antiviral research.

Phylogenetic Analysis and Serological Investigation of Porcine Circovirus Indicates Frequent Infection with Various Subtypes.

Porcine circoviruses (PCVs) are notorious for triggering severe diseases in pigs and causing serious economic losses to the swine industry. In the present study, we undertook a comprehensive approach for the investigation of PCV prevalence, including the phylogenetic analysis of obtained PCV sequences, the determination of major circulating genotypes and serological screening based on different recombinant Cap proteins with specific immunoreactivity. Epidemiological surveillance data indicate that PCV2d and PCV3a are widely distributed in Southwest China, while PCV4 has only sporadic circulation. Meanwhile, serological investigations showed high PCV2 antibody positivity in collected serum samples (>50%), followed by PCV4 (nearly 50%) and PCV3 (30-35%). The analysis supports different circulation patterns of PCV2, PCV3 and PCV4 and illustrates the PCV2/PCV3 genetic evolution characteristics on a nationwide basis. Taken together, our findings add up to the current understanding of PCV epidemiology and provide new tools and insight for PCV antiviral intervention.

HSP90AB1 Is a Host Factor Required for Transmissible Gastroenteritis Virus Infection.

Transmissible gastroenteritis virus (TGEV) is an important swine enteric coronavirus causing viral diarrhea in pigs of all ages. Currently, the development of antiviral agents targeting host proteins to combat viral infection has received great attention. The heat shock protein 90 (HSP90) is a critical host factor and has important regulatory effects on the infection of various viruses. However, its roles in porcine coronavirus infection remain unclear. In this study, the effect of HSP90 on TGEV infection was evaluated. In addition, the influence of its inhibitor VER-82576 on proinflammatory cytokine (IL-6, IL-12, TNF-α, CXCL10, and CXCL11) production induced by TGEV infection was further analyzed. The results showed that the knockdown of HSP90AB1 and HSP90 inhibitor VER-82576 treatment resulted in a reduction in TGEV M gene mRNA levels, the N protein level, and virus titers in a dose-dependent manner, while the knockdown of HSP90AA1 and KW-2478 treatment had no significant effect on TGEV infection. A time-of-addition assay indicated that the inhibitory effect of VER-82576 on TGEV infection mainly occurred at the early stage of viral replication. Moreover, the TGEV-induced upregulation of proinflammatory cytokine (IL-6, IL-12, TNF-α, CXCL10, and CXCL11) expression was significantly inhibited by VER-82576. In summary, these findings indicated that HSP90AB1 is a host factor enhancing TGEV infection, and the HSP90 inhibitor VER-82576 could reduce TGEV infection and proinflammatory cytokine production, providing a new perspective for TGEV antiviral drug target design.

Porcine deltacoronavirus enters ST cells by clathrin-mediated endocytosis and does not require Rab5, Rab7, or Rab11.

IMPORTANCE: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus threatening pig industries worldwide. Our previous work showed that PDCoV enters porcine kidney (PK-15) cells through a caveolae-dependent pathway, but the entry mechanism for PDCoV into swine testicle (ST) cells remains unclear. Mechanisms of virus entry can be different with different virus isolates and cell types. Here, we determined that PDCoV enters ST cells via clathrin-mediated endocytosis. Additionally, we found that PDCoV entry does not require Rab5, Rab7, or Rab11. These findings provide additional understanding of the entry mechanisms of PDCoV and possible antiviral targets.

Metabolome and Transcriptome Combinatory Profiling Reveals Fluconazole Resistance Mechanisms of Trichosporon asahii and the Role of Farnesol in Fluconazole Tolerance.

Trichosporon asahii is a basidiomycete yeast that is pathogenic to humans and animals, and fluconazole-resistant strains have recently increased. Farnesol secreted by fungi is a factor that causes variations in fluconazole resistance; however, few studies have explored the underlying mechanisms. Therefore, this study aims to delineate the fluconazole resistance mechanisms of T. asahii and explore farnesol's effects on these processes. A comparative metabolome-transcriptome analysis of untreated fluconazole-sensitive (YAN), fluconazole-resistant (PB) T. asahii strains, and 25 µM farnesol-treated strains (YAN-25 and PB-25, respectively) was performed. The membrane lipid-related genes and metabolites were upregulated in the PB vs. YAN and PB-25 vs. PB comparisons. Farnesol demonstrated strain-dependent mechanisms underlying fluconazole tolerance between the YAN and PB strains, and upregulated and downregulated efflux pumps in PB-25 and YAN-25 strains, respectively. Membrane lipid-related metabolites were highly correlated with transporter-coding genes. Fluconazole resistance in T. asahii was induced by membrane lipid bio-synthesis activation. Farnesol inhibited fluconazole resistance in the sensitive strain, but enhanced resistance in the resistant strain by upregulating efflux pump genes and membrane lipids. This study offers valuable insights into the mechanisms underlying fungal drug resistance and provides guidance for future research aimed at developing more potent antifungal drugs for clinical use.

CD4 is an important host factor for Japanese encephalitis virus entry and replication in PK-15 cells.

Japanese encephalitis virus (JEV) is a flavivirus that is spread through mosquito bites and is the leading cause of viral encephalitis in Asia. JEV can infect a variety of cell types; however, crucial receptor molecules remain unclear. The purpose of this study was to determine whether porcine CD4 protein is a receptor protein that impacts JEV entry into PK15 cells and subsequent viral replication. We confirmed the interaction between the JEV E protein and the CD4 protein through Co-IP, virus binding and internalization, antibody blocking, and overexpression and created a PK-15 cell line with CD4 gene knockdown by CRISPR/Cas9. The results show that CD4 interacts with JEV E and that CD4 knockdown cells altered virus adsorption and internalization, drastically reducing virus attachment. The level of viral transcription in CD4 antibody-blocked cells, vs. control cells, was decreased by 49.1%. Based on these results, we believe that CD4 is a receptor protein for JEVs. Furthermore, most viral receptors appear to be associated with lipid rafts, and colocalization studies demonstrate the presence of CD4 protein on lipid rafts. RT‒qPCR and WB results show that virus replication was suppressed in PK-15-CD4KD cells. The difference in viral titer between KD and WT PK-15 cells peaked at 24 h, and the viral titer in WT PK-15 cells was 5.6 × 106, whereas in PK-15-CD4KD cells, it was only 1.8 × 106, a 64% drop, demonstrating that CD4 deficiency has an effect on the process of viral replication. These findings suggest that JEV enters porcine kidney cells via lipid raft-colocalized CD4, and the proliferation process is positively correlated with CD4.

Upregulation of occludin by cytolethal distending toxin facilitates Glaesserella parasuis adhesion to respiratory tract cells.

Virulent Glaesserella parasuis may engender systemic infection characterized by fibrinous polyserositis and pneumonia. G. parasuis causes systemic disease through upper respiratory tract infection, but the mechanism has not been fully characterized. Tight junction (TJ) proteins maintain the integrity and impermeability of the epithelial barriers. In this work, we applied the recombinant cytolethal distending toxin (CDT) holotoxin and cdt-deficient mutants to assess whether CDT interacted with TJ proteins of airway tract cells. Our results indicated that CDT induced the TJ occludin (OCLN) expression in newborn pig tracheal epithelial cells within the first 3 hours of bacterial infection, followed by a significant decrease. Overexpression of OCLN in target cells made them more susceptible to G. parasuis adhesion, whereas ablation of OCLN expression by CRISPR/Cas 9 gene editing technology in target cells decreased their susceptibility to bacterial adhesion. In addition, CDT treatment could upregulate the OCLN levels in the lung tissue of C57/BL6 mice. In summary, highly virulent G. parasuis strain SC1401 stimulated the tight junction expression, resulting in higher bacterial adhesion to respiratory tract cells, and this process is closely related to CDT. Our results may provide novel insights into G. parasuis infection and CDT-mediated pathogenesis.

IFN-γ promotes PANoptosis in Pasteurella multocida toxin-induced pneumonia in mice.

Interferon-γ (IFN-γ) is a pleiotropic cytokine that regulates diverse biological functions, including modulation of inflammatory response and innate and adaptive immunity. In our study, we found that IFN-γ plays an important role in the regulation of Pasteurella multocida toxin-associated pneumonia. In work described here, we demonstrated that rPMT induced a lethal pneumonia in WT mice and the severity of the pneumonia was substantially alleviated in IFN-γ-deficient mice, IFN-γ deficiency significantly elevated the survival rate and reduced the pathological lesions of the lungs after rPMT challenged. Notably, IFN-γ deficiency significantly decreased myeloperoxidase (MPO) expression abundance in the lung tissue, and the MPO was mainly expressed in the lung tissue injury region of WT mice. More importantly, IFN-γ deficiency impaired the activation of PANoptosis specific markers, including the caspase 3, GSDMD, and MLKL, and reduced the expression of IL-1ß. Cumulatively, this study demonstrates that IFN-γ promotes PANoptosis in PMT induced pneumonia in mice, providing a basis for studying the pathogenic mechanism of PMT.

Porcine reproductive and respiratory syndrome virus upregulates SMPDL3B to promote viral replication by modulating lipid metabolism.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat to the health of pigs globally. Host factors play a critical role in PRRSV replication. Using PRRSV as a model for genome-scale CRISPR knockout (KO) screening, we identified a host factor critical to PRRSV infection: sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B). Our findings show that SMPDL3B restricted PRRSV attachment, entry, replication, and secretion and that its depletion significantly inhibited PRRSV proliferation, indicating that SMPDL3B plays a positive role in PRRSV replication. Our data also show that SMPDL3B deficiency resulted in an accumulation of intracellular lipid droplets (LDs). The expression level of key genes (ACC, SCD-1, and FASN) involved in lipogenesis was increased, whereas the fundamental lipolysis gene, ATGL, was inhibited when SMPDL3B was knocked down. Overall, our findings suggest that SMPDL3B deficiency can effectively inhibit viral infection through the modulation of lipid metabolism.

Genomic traits of multidrug resistant enterotoxigenic Escherichia coli isolates from diarrheic pigs.

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) infections poses a significant challenge in global pig farming. To address this issue, the study was conducted to identify and characterize 19 ETEC isolates from fecal samples of diarrheic pigs sourced from large-scale farms in Sichuan Province, China. Whole-genome sequencing and bioinformatic analysis were utilized for identification and characterization. The isolates exhibited substantial resistance to cefotaxime, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, ampicillin, tetracycline, florfenicol, and sulfadiazine, but were highly susceptible to amikacin, imipenem, and cefoxitin. Genetic diversity among the isolates was observed, with serotypes O22:H10, O163orOX21:H4, and O105:H8 being dominant. Further analysis revealed 53 resistance genes and 13 categories of 195 virulence factors. Of concern was the presence of tet(X4) in some isolates, indicating potential public health risks. The ETEC isolates demonstrated the ability to produce either heat-stable enterotoxin (ST) alone or both heat-labile enterotoxin (LT) and ST simultaneously, involving various virulence genes. Notably, STa were linked to human disease. Additionally, the presence of 4 hybrid ETEC/STEC isolates harboring Shiga-like toxin-related virulence factors, namely stx2a, stx2b, and stx2e-ONT-2771, was identified. IncF plasmids carrying multiple antimicrobial resistance genes were prevalent, and a hybrid ETEC/STEC plasmid was detected, highlighting the role of plasmids in hybrid pathotype emergence. These findings emphasized the multidrug resistance and pathogenicity of porcine-origin ETEC strains and the potential risk of epidemics through horizontal transmission of drug resistance, which is crucial for effective control strategies and interventions to mitigate the impact on animal and human health.

Research Note: A recombinant duck-derived H6N2 subtype avian influenza virus can replicate and shed in young chickens and cause disease.

The H6N2 subtype avian influenza virus (AIV) is commonly detected in the migratory waterfowl reservoirs. Previously, H6N2 AIV was believed to be nonpathogenic to young chickens and could not infect or shed in their respiratory tract under experimental conditions. However, in present study, a highly recombinant strain of duck-derived H6N2 AIV was discovered and isolated for pathogenicity tests. The results revealed that H6N2 could induce seroconversion in chickens and high morbidity of over 86.7%, along with evident upper respiratory tract hemorrhage. Moreover, 5 substitutions were detected in the upper respiratory tract shedding reisolated virus, with a high viral load in the target organs of infected chickens. In contrast, ducks failed to exhibit any symptoms, pathological lesions, or viral shedding, while demonstrated seroconversion and high viral load in the livers. These findings indicate that H6N2 AIV could also show pathogenicity to chickens under experimental conditions, thereby effectively replicating and shedding in chickens. Therefore, the study provides further elucidations on the pathogenicity of H6N2 AIV.

Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G.

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3-86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26-506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G.

Evolution of prevalent H9N2 subtype of avian influenza virus during 2019 to 2022 for the development of a control strategy in China.

The H9N2 subtype of avian influenza virus (H9N2 AIV) has caused significant losses in chicken flocks throughout China. At present, consensus has been reached that field isolates of H9N2 underwent antigenic drift to evolve into distinct groups with significant antigenic divergence from the commercially available vaccines in China. This project continues to monitor the evolution characteristics of H9N2 hemagglutinin (HA) genes in China over the past 3 yr. The results showed that the current circling H9N2 viruses were diversified into h9.4.2.5 subclade, which was genetically distant from commonly used commercial vaccine strains. Compared with vaccine strains or 2014 strains, more than 42.1% of the variable antigenic sites in recent 3 yr' strains have shown significant changes and these stacked changes have caused significant differences in antigenicity. We constructed a recombinant vaccine strain rCQY-GHHA, which uses A/Chicken/China/SichuanCQY/2014 as the framework and A/Chicken/China/SichuanGH/2020 strain, which meets the recent viral antigenic characteristics, as the HA gene donor. The recombinant strain was prepared as an oil-adjuvant inactivated vaccine following an industrial process. The results of the immune protection experiment showed that the rCQY-GHHA vaccine was better than the commercial vaccine strain SS in reducing the morbidity, pathological lesion, virus shedding, and viral load. These results provide a reference for the control of H9N2 AIV in China.

Identification of potential SLA-I-specific T-cell epitopes within the structural proteins of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that mainly threatens newborn piglets and poses a potential broad cross-species transmission risk. The antigenic epitopes of PDCoV are currently unidentified, and no information about T cell epitopes is available. Here, T-cell epitopes of PDCoV structural proteins were predicted using computational methods. 17 epitope peptides were synthesized and then screened using ELIspot, intracellular cytokine staining (ICS), and RT-qPCR detection of IFN-γ mRNA to evaluate their ability to elicit interferon-gamma (IFN-γ) responses in peripheral blood mononuclear cells (PBMCs) from PDCoV-challenged pigs. Five peptides (M1, M2, M3, N6, and S4) elicited high levels of IFN-γ and were investigated further as potential T-cell epitope candidates. All five peptides were cytotoxic T lymphocyte (CTL) epitopes, and two peptides (M3, N6) were recognized simultaneously by CD8 + and CD4 + T cells. A multi-epitope peptide combining the five epitopes (designated "5T") was synthesized and its immune response and protection efficacy was evaluated in a piglet model. ELISpot assay results indicated that 5T induces robust epitope-specific cellular immune responses. Four epitopes (M1, M2, N6, S4) elicited IFN-γ responses in 5T-vaccinated piglets. No obvious protection efficacy was detected in piglets vaccinated with 5T alone. Our results provide valuable information concerning PDCoV-related antigenic epitopes and will be useful in the design of epitope-based vaccines.

Identification of an immunodominant neutralizing epitope of porcine Deltacoronavirus spike protein.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that, because of its broad host range, poses a potential threat to public health. Here, to identify the neutralizing B-cell epitopes within the S1-CTD protein, we generated three anti-PDCoV monoclonal antibodies (mAbs). Of these, the antibody designated 4E-3 effectively neutralized PDCoV with an IC50 of 3.155 µg/mL. mAb 4E-3 and one other, mAb 2A-12, recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 4E-3 was mapped to 280FYSDPKSAV288 and designated S280-288, the minimal fragment recognized by mAb 2A-12 was mapped to 506TENNRFTT513, and designated S506-513. Subsequently, alanine (A)-scanning mutagenesis indicated that Asp283, Lys285, and Val288 were the critical residues recognized by mAb 4E-3. The S280-288 epitope induces PDCoV specific neutralizing antibodies in mice, demonstrating that it is a neutralizing epitope. Of note, the S280-288 coupled to Keyhole Limpet Hemocyanin (KLH) produces PDCoV neutralizing antibodies in vitro and in vivo, in challenged piglets it potentiates interferon-γ responses and provides partial protection against disease. This is the first report about the PDCoV S protein neutralizing epitope, which will contribute to research of PDCoV-related pathogenic mechanism, vaccine design and antiviral drug development.

Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody.

Glaesserella parasuis (G. parasuis.) is the etiological pathogen of Glässer's disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in G. parasuis. In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against recombinant HbpA (rHbpA) of G. parasuis SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of G. parasuis. None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among G. parasuis strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for G. parasuis. Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.

BAK-Mediated Pyroptosis Promotes Japanese Encephalitis Virus Proliferation in Porcine Kidney 15 Cells.

As a zoonotic virus, Japanese Encephalitis virus (JEV) poses a serious threat to human health and the breeding industry. Regarding the mechanism and complications of tissue inflammation caused by JEV, such as encephalitis and orchitis, there is no effective drug treatment currently, and the mechanism of occurrence has not been thoroughly studied. Therefore, it is necessary to study the mechanism of the inflammatory pathway caused by JEV. As one of the key proteins regulating cell death, BCL2 antagonist/killer (BAK) is also a necessary prerequisite for the release of cellular inflammatory factors. We found that after JEV infection, BAK-knockdown cells died less than normal cells, and the transcription levels of inflammatory factors such as TNF, IFNα, and IL-1ß and their corresponding regulatory genes were also significantly reduced. By further verifying protein expression on the cell death pathway, it was found that pyroptotic activation and virus titer were also significantly reduced in BAK.KD cells, suggesting that JEV proliferation might be related to BAK-induced cell death. From our data, we could conclude that JEV utilized the BAK-promoted pyroptotic pathway to release more virions after the final Gasdermin D-N (GSDMD-N) protein pore formation for the purpose of JEV proliferation. Therefore, the study of the endogenous cell death activator protein BAK and the final release pathway of JEV, is expected to provide some new theoretical basis for future research on the screening of targeted drugs for the treatment of inflammatory diseases caused by JEV.