Japanese encephalitis virus (JEV) remains a major global zoonotic threat to both animal and human health. Viral attachment to host cells is an early and targetable step in infection. Here, we investigated the role of the C-type lectin-like receptor CD161 (encoded by Killer cell lectin-like receptor B1, KLRB1) in JEV infection using PK15 cells. The stable CD161-knockout PK15 cell lines (PK15-K.O.) and CD161-overexpressing PK15 cell lines (PK15-O.E.) were generated using CRISPR/Cas9 genome editing and lentiviral transduction, respectively. Following JEV infection, PK15-K.O. cells showed markedly reduced viral titer, intracellular viral RNA levels, and JEV envelope (E) protein expression. Conversely, PK15-O.E. showed increases in all these viral indicators. Further analyses revealed that CD161 functions during the early entry stage, promoting both viral attachment and subsequent internalization. Taken together, these findings firstly identify porcine CD161 as a key host factor that facilitates JEV entry into PK15 cells, suggesting that CD161 is a potential host-directed antiviral target.

Porcine deltacoronavirus (PDCoV) causes severe diarrhea in piglets and poses a potential public health risk due to cross-species transmission. Antiviral drugs are urgently needed. We designed peptides HR1P and HR2P from the viral spike (S) protein and evaluated their antiviral efficacy. HR2P exhibited potent in vitro activity (EC50 = 6.65 μM), maintained efficacy against HR1-mutated variants, and showed broad cross-species inhibitory activity across avian, nonhuman primate, and human-derived cells. Molecular docking analysis predicted that HR2P targets conserved structural sites shared with PEDV and TGEV, avoiding observed HR1 mutation sites. In piglets, early-stage HR2P intervention effectively reduced viral loads in the jejunum and ileum, alleviating clinical diarrhea. In addition, pharmacokinetic (PK) analysis further confirmed that intramuscular (IM) administration of HR2P (10 mg/kg) achieved stable systemic exposure, with a peak plasma concentration (Cmax = 194.6 μg/mL) substantially exceeding the in vitro EC50. These findings suggest HR2P as a potential broad-spectrum therapeutic candidate.

Japanese Encephalitis Virus (JEV) is a mosquito-borne zoonotic pathogen that triggers severe inflammatory responses upon infection. Previous research has demonstrated that the interaction between Ras-associated GTP-binding protein 4B (Rab4b) and the JEV E protein is essential for the virus's entry into early endosomes. However, the precise mechanism by which Rab4b contributes to JEV-induced inflammatory responses remains inadequately understood. In this study, we developed a Rab4b-knockout U251 cell model and found that Rab4b deficiency markedly inhibited viral replication and concurrently reduced levels of key inflammatory mediators, including IL-1β, IL-6, TNF-α, and IFN-β. Transcriptomic analysis further indicated that the loss of Rab4b disrupted the Toll-like receptor 3 (TLR3)-related gene network. Subsequent co-immunoprecipitation experiments confirmed an interaction between Rab4b and TLR3, and treatment with a TLR3 inhibitor recapitulated the anti-inflammatory phenotype observed in Rab4b-deficient cells, suggesting the involvement of Rab4b in TLR3-related signaling. In conclusion, these findings support an association between Rab4b and a TLR3-related inflammatory signaling axis during JEV infection, although the precise mechanistic relationship requires further investigation.

Porcine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine deltacoronavirus (PDCoV), cause severe watery diarrhea, vomiting, dehydration, and high mortality in piglets, leading to enormous economic losses in the swine industry worldwide. They have the capability to infect a variety of cell lines from pigs, humans, and other animals, with high risks of interspecies transmission and potential threats to public health. These viruses employ their spike glycoproteins to engage with various receptors, coreceptors, cofactors, and other host factors that further mediate membrane fusion to accomplish the entry process. This review summarizes the recent findings regarding the pathways, receptors, coreceptors, cofactors, and other host factors utilized by TGEV, PEDV, SADS-CoV, and PDCoV for cellular entry. Several important targets for antiviral therapeutics and some key aspects of the entry process for these viruses that await discovery are highlighted. A comprehensive understanding of the entry mechanisms of porcine enteric coronaviruses will provide new insight into the development of novel antiviral therapeutic strategies.

Background:Cytolethal Distending Toxin (CDT) is the only exotoxin that Glaesserella parasuis (G. parasuis) can secrete. G. parasuis CDT (GpCDT) triggers DNA damage responses, leading to irreversible cell cycle arrest and apoptosis, playing an important role in the pathogenic process of G. parasuis. Currently, research on the host cell receptors of GpCDT remains limited. Screening and identification of host cell receptors that interact with GpCDT are crucial for systematically elucidating the cytotoxic mechanisms induced by this toxin. Methods:This study employed Co-immunoprecipitation (Co-IP) combined with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to identify potential host proteins interacting with GpCDT in PK15 cells. Nine Proteins were selected for further evaluation based on subcellular localization and Gene Ontology classification. Eukaryotic expression and Co-IP validated four interacting proteins. Subsequently, heterozygous knockout PK15 cell lines for these genes were generated via CRISPR/Cas9, and CCK-8 assays identified TM9SF4 as having the most significant impact on GpCDT virulence. Therefore, a homozygous TM9SF4 knockout PK15 cell line (KO) was generated via limited dilution, and a stable TM9SF4-overexpressing PK15 cell line (OE) was established through lentiviral packaging. Western blotting and qRT-PCR confirmed protein and gene expression, and CCK-8 assays combined with cytopathic effect (CPE) observation determined the role of TM9SF4 in GpCDT-induced cytotoxicity. Finally, indirect immunofluorescence was performed to assess co-localization of TM9SF4 with GpCDT. Results:We identified 287 proteins in PK15 cells that potentially interact with GpCDT, among which 58 were localized to the plasma membrane or extracellular. Nine proteins were selected for further investigation. Among them, EPHB4, LITAF, TM9SF4, SLC12A4 interacted with GpCDT, but only the deficiency of TM9SF4 significantly inhibited the virulence of the GpCDT. Results from CCK-8 and CPE showed that KO cells exhibited significantly higher survival rates and suppressed GpCDT-induced cellular distention and cell death, whereas OE cells showed decreased survival rates and typical cytopathic change. Finally, indirect immunofluorescence confirmed strong co-localization between TM9SF4 and GpCDT. Conclusion:We initially proposed TM9SF4 as a receptor for GpCDT in PK15 cells, essential for GpCDT binding and cytotoxicity. This study may provide a new theoretical basis for targeted prevention and treatment of swine Glässer's disease.

Aflatoxin B1 (AFB1) is a prevalent food- and feed-borne mycotoxin, and growing evidence indicates that the renal epithelium is a vulnerable target. However, host determinants that modify epithelial susceptibility remain poorly defined. Here, we investigated AFB1-triggered injury in porcine kidney epithelial PK15 cells and examined the contribution of CXCL8. PK15 cells were exposed to AFB1 (0-32 μM) to define dose-dependent cytotoxicity, and sub-IC50 conditions (4 and 8 μM for 24 h) were used for mechanistic analyses; RNA sequencing at 4 μM for 24 h was used as an exploratory screen to prioritize candidate susceptibility factors. CXCL8 emerged as the most strongly induced transcript and was subsequently evaluated using CRISPR/Cas9-mediated knockout. CXCL8 deficiency attenuated AFB1-induced loss of viability, reduced Annexin V/PI-positive cells, and alleviated mitochondrial ultrastructural injury. In parallel, CXCL8 knockout decreased ROS accumulation, partially restored intracellular GSH and the BCL2/BAX transcriptional ratio, and reduced caspase-3 induction and cleavage. Collectively, these data support CXCL8 as an AFB1-inducible susceptibility factor associated with oxidative stress amplification and caspase-3 activation in PK15 cells, while indicating that the upstream regulatory axis and the precise downstream signaling route require further validation in physiologically relevant renal models.

Pseudorabies virus (PRV) is an important swine pathogen that causes significant economic losses in the pig industry. In addition to its swine host, PRV also infects a wide variety of animals as well as humans, posing a threat to veterinary public health. Thus, the development of potent antiviral drugs against PRV is required. Bovine lactoferrin (BLF) is widely known as an iron-binding glycoprotein in the transferrin family, which gains great attention for biomedical applications due to its beneficial physiological functions to human health as an antioxidant, antimicrobial, antiviral, anticancer, and immunomodulatory agent. In the work described here, we identified BLF as a novel and potent antiviral agent against PRV. BLF exhibited strong antiviral activity against PRV infection in multiple pig, human, and mouse permissive cells. Systematic analysis of the effect of BLF on PRV inhibition revealed that BLF blocks PRV attachment to target cells. Through surface plasmon resonance (SPR) analysis, BLF was demonstrated to directly interact with heparan sulfate (HS) proteoglycan (HSPG), a primary attachment receptor for PRV. Addition of exogenous HS completely abolished BLF inhibition of PRV attachment, demonstrating that BLF represses PRV attachment by interacting with cell-surface HS. However, BLF failed to inhibit the attachment of a PRV mutant virus with the deletion of glycoprotein C (gC), the primary viral envelop component for HS binding, suggesting that PRV gC is required for BLF inhibition of PRV infection. Cumulatively, these findings demonstrated that BLF inhibits PRV attachment by binding cell-surface HS to block the interaction between viral gC and HS.

The Getah virus (GETV) is a mosquito-borne pathogen that infects diverse hosts, including pigs, horses, and humans, which can cause swine reproductive disorders such as abortion and stillbirth, posing a potential threat to animal and public health. Therefore, there is an urgent need for efficient and accurate serological diagnostic methods for surveillance and control of GETV. However, commercial diagnostic kits for swine GETV infection remain unavailable. In this study, we developed a novel enzyme-linked immunosorbent assay (ELISA) based on a GETV-specific epitope peptide (E2EP3) for serological detection. The N-terminally biotinylated E2EP3 peptide was synthesized, and the reaction conditions were systematically optimized, resulting in a cut-off value of 0.363. The assay exhibited no cross-reactivity with Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), pseudorabies virus (PRV), or classical swine fever virus (CSFV). It demonstrated good reproducibility and high sensitivity, detecting GETV-positive serum diluted up to 1:640. The overall agreement rate reached 95%, consistent with a conventional recombinant GETV E2 protein-based ELISA. Benefiting from the biotin-streptavidin system, this assay achieved strong signal amplification and low background. Moreover, the procedure is simple, cost-effective, and stable, making it suitable for GETV large-scale serological surveillance and vaccine evaluation.

The Tembusu virus (TMUV) is a mosquito-borne pathogen affecting the birds, causing economic losses in poultry and potential public health risks. Research mainly focuses on virus’s characteristics and its interaction with birds, especially the ducks, while entomological studies monitor mosquito populations for epidemiological insights. TMUV’s transmission dynamics are not fully understood, with varying abilities of mosquito species to transmit the virus. There is limited research on the vector competence and vertical transmission potential of different mosquito species, particularly in China. We compared the vector competence of Culex quinquefasciatus and Aedes albopictus for TMUV. The vector competence of Culex quinquefasciatus and Aedes albopictus for TMUV was assessed through intrathoracic microinjection and artificial membrane feeding techniques. Mosquitoes were sampled at 1-, 3-, 6-, and 9-days post-inoculation (dpi) following intrathoracic microinjection of TMUV, and at 4-, 8-, and 14-dpi following artificial membrane feeding. Samples of heads, midguts, and salivary glands were collected for subsequent analysis. Furthermore, the potential for vertical transmission of TMUV over two gonotrophic cycles was evaluated using the artificial membrane feeding approach. The distribution of TMUV within various organs and tissues of both mosquito species at 4-, 8-, and 14-dpi was investigated by immunofluorescence assays. The findings suggest that both Culex quinquefasciatus and Aedes albopictus are effective carriers of TMUV, with Aedes albopictus showing greater vector competence. Vertical transmission of TMUV was observed in both species across two successive oviposition cycles, with Aedes albopictus displaying slightly higher efficiency. This study represents the inaugural comparative assessment of vector competence between Sichuan-native Culex quinquefasciatus and Aedes albopictus (Guangzhou) for TMUV, thereby addressing critical research gaps and providing novel insights for the development of TMUV biocontrol strategies.

Pseudorabies virus (PRV) is an important herpesvirus that infects pigs and many other animals. PRV can also infect humans, but the relevant reports are still very rare. PRV is not only a serious concern in veterinary medicine, but also a potential threat to public health. Thus, the development of effective antiviral agents against PRV is needed for its prevention and control. PRV utilizes viral glycoprotein C (gC) to bind heparan sulfate (HS) on target cells to complete virus attachment. Here, we identified aminoquinoline surfen, a small-molecule antagonist of HS, as a novel and potent antiviral agent against PRV. We demonstrated that surfen not only effectively inhibited PRV infection in multiple permissive pig, human, and mouse cell lines, but also significantly elevated mice survival after lethal PRV challenge. Surfen pre-treatment significantly reduced PRV attachment and infectious virus production in target cells. Enzymatic removal of HS chains by heparinase or addition of exogenous HS counteracted PRV inhibition by surfen, demonstrating that its antiviral activity is dependent on HS. Notably, surfen lost its ability to inhibit the attachment of PRV gC-deficient virus, which enters cells in an HS-independent manner, indicating that the binding of surfen to HS interferes with the interaction between PRV gC and HS on the cell surface. Cumulatively, these findings demonstrate that surfen inhibits PRV attachment by preventing the binding of PRV gC to HS on target cells and reveal that targeted disruption of PRV gC-HS interaction is an effective strategy to develop antiviral drugs to defend PRV infection.IMPORTANCEPseudorabies virus (PRV) causes severe respiratory, reproductive, and neurological disorders in pigs, and its infection in humans is also reported occasionally. PRV is not only a serious concern in veterinary medicine, but also a potential threat to public health. Therefore, the development of effective antiviral agents against PRV is needed to prevent its infection and spread. Here, we demonstrated that aminoquinoline surfen, a small-molecule antagonist of HS, effectively inhibited PRV infection in multiple permissive cell lines in vitro and significantly elevated mice survival after lethal PRV challenge in vivo, revealing it as a novel and potent antiviral agent against PRV. Mechanistic studies demonstrated that surfen inhibited PRV attachment by preventing the binding of PRV viral glycoprotein gC to heparan sulfate on target cells. Cumulatively, these findings reveal that targeted disruption of PRV gC-HS interaction is an effective strategy to develop antiviral drugs to defend PRV infection.

Abstract Parainfluenza virus type 5 (PIV5) can infect a variety of animals and can lead mainly to respiratory symptoms. In recent years, PIV5 has been frequently detected in swine fecal samples with diarrhea symptoms, but its role in pig diseases remains unclear. In this study, a swine PIV5 strain, CHN-SCMY2025, was isolated from diarrheal piglets in Sichuan Province, Southwest China. The cytopathic effect of the isolate was systematically characterized by electron microscopy and immunofluorescence. The results demonstrated that CHN-SCMY2025 is approximately 150–250 nm in diameter and has a morphology typical of parainfluenza virus. In addition to Vero cells, CHN-SCMY2025 exhibits broad cellular tropism and can replicate in BHK-21, LLC-PK, IPEC-J2, ST, and 293 T cells. Sequence alignment based on the NP (PX583268.1), HN (PX583267.1), F (PX583266.1), M (PX963773.2), and L (PZ011079.1) of CHN-SCMY2025 was determined, which shares 95.83–99.82%, 96.53–99.88%, 96.67–100%, 95.51–99.21%, and 97.89–99.78% nucleotide identity with other PIV5 reference strains in GenBank, respectively. Phylogenetic analysis revealed that CHN-SCMY2025 is closely related to the swine PIV5 isolate (KC237064.1). In vivo infection demonstrated that CHN-SCMY2025 could induce mild to moderate respiratory symptoms and mild diarrhea in suckling piglets. The virus was shed daily in the feces of infected suckling piglets and upon necropsy. The virus was distributed in multiple organs, but relatively high viral loads were detected mainly in the lung, duodenum, and jejunum. CHN-SCMY2025 is the first systematically characterized swine PIV5 strain from southwestern China. Our results provide information on epidemiology, pathogenicity, and interspecies transmission associated with swine PIV5.

BackgroundThe cytolethal distending toxin (CDT), a significant exotoxin, is closely linked to the pathogenicity of Glaesserella parasuis (GPS), but its pathogenic not yet fully elucidated. Previously, we identified Rab4b as a potential host factor contributing to the cytotoxicity of GpCDT through a whole-genome CRISPR/Cas9 screen technology, and subsequently confirmed its association with GpCDT cytotoxicity in PK-15 cells.AimsIn this study, our data first indicated that Rab4b could interact with the active subunit of the Glaesserella parasuis cytolethal distending toxin.MethodsInvestigating the relationship between Rab4b and GpCDT subunits as confirmed by coimmunoprecipitation assay. Next, the porcine alveolar macrophage cell line 3D4/21 was used to establish an infected cell model. Using CRISPR/Cas9 gene editing, we established Rab4b and EEA1-expression-deficient 3D4/21 cell lines. 3D4/21 cells, Rab4b-KO cells and EEA1-KO cells were treated with GpCDT. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Western blotting and qRT-PCR were used to measure the expression of related proteins and genes, and cell morphology observation and indirect immunofluorescence were performed to evaluate the GpCDT-mediated cytotoxicity. Then utilise transcriptome sequencing analysis to investigate its specific mechanisms.ResultIn this study, our data first indicated that Rab4b could interact with the active subunit of the GpCDT. Next, we demonstrated that Rab4b also influences GpCDT-induced cytotoxicity and vesicle trafficking in 3D4/21 cells. To investigate the Rab4b-mediated cytotoxicity of GpCDT in 3D4/21 cells, we screened for EEA1, a gene critical in this process, by transcriptome sequencing analysis. 3D4/21 cells exposed to GpCDT exhibit upregulated EEA1 expression, an event that is lost in the absence of Rab4b. Using CRISPR/Cas9 gene editing, we established EEA1 expression-deficient 3D4/21 cell lines that fail to internalize GpCdtB, resulting in resistance to GpCDT-induced toxic effects.ConclusionsWe suggest that Rab4b facilitates the cellular uptake of GpCDTby upregulating EEA1 protein expression, thereby facilitating the vesicular transport of GpCDT in 3D4/21 cells. Our findings may provide new insights into the pathogenicity of GpCDT and lay the experimental foundation for a deeper understanding of the role of Rab4b proteins

Glaesserella parasuis cytolethal distending toxin (GpCDT) is a bacterial genotoxin whose main action is to activate DNA damage responses, induce cell cycle arrest, and induce the apoptosis of host cells. In our previous studies, we reported that cells incubated with GpCDT exhibited changes in the expression of ferroptosis-related proteins; thus, we hypothesized that, in addition to apoptosis, GpCDT may also cause ferroptosis, a novel mode of cell death. Here, we observed that treatment of 3D4/21 cells with GpCDT resulted in cytoplasmic iron overload, depletion of GSH (reduced glutathione), and overproduction of reactive oxygen species (ROS) and malondialdehyde (MDA), indicating that GpCDT disrupted iron metabolism and redox homeostasis in these cells. These phenomena were counteracted by the specific ferroptosis inhibitor ferrostatin-1 and the iron chelator deferoxamine mesylate. In vitro infection with the Glaesserella parasuis field isolate strain SC1401 (CDT positive) induced changes in the expression of ferroptosis biomarkers and proteins. Infection of C57BL/6 mice yielded similar results. Our results suggest that ferroptosis may play a substantial role in GpCDT-induced cellular injury.

Swine acute diarrhoea syndrome coronavirus (SADS-CoV), a novel HKU2-related coronavirus of bat origin, is a newly emerged swine enteropathogenic coronavirus that causes severe diarrhoea in piglets. SADS-CoV has a broad cell tropism with the capability to infect a wide variety of cells from human and diverse animals, which implicates its ability to hold high risks of cross-species transmission. The intracellular antiviral immunity, comprised of the intrinsic and innate immunity, represents the first line of host defence against viral infection prior to the onset of adaptive immunity. To date, there are no vaccines and drugs approved to prevent or treat SADS-CoV infection. Understanding of the mutual relationship between SADS-CoV infection and host immunity is crucial for the development of novel vaccines and drugs against SADS-CoV. Here, we review recent advancements in our understanding of the interplay between SADS-CoV infection and the host intrinsic and innate immunity. The extensive and in-depth investigation on their interactive relationship will contribute to the identification of new targets for developing intervention strategies to control SADS-CoV infection.

Pseudorabies virus (PRV) is an important swine herpesvirus that causes fatal encephalitis in newborn piglets and severe reproductive failure in pregnant sows, resulting in enormous economic losses in the pig industry worldwide. It has broad cell tropisms with the capability to infect a wide range of animals, including humans, thus posing a potential threat to human health. Neddylation is an important protein posttranslational modification that is catalyzed by an E1-E2-E3 enzyme cascade to covalently conjugate the ubiquitin-like molecule neural precursor cell expressed developmentally downregulated 8 (NEDD8) to substrate proteins. It has been demonstrated to play a key role in regulating numerous important biological processes, including cell proliferation, gene expression, signal transduction, and viral infection. However, the specific function of the neddylation pathway during PRV infection remains largely unknown. In the work described here, we identified a critically important role for neddylation in PRV replication by utilizing short hairpin RNA (shRNA)-mediated depletion of NEDD8 or the NEDD8-activating enzyme E1 subunit 1 (NAE1). Through systematic investigation of E2-E3 neddylation partners, we further demonstrated that silencing of the ubiquitin conjugating enzyme E2 F (UBE2F)-RING-box protein 2 (RBX2) axis significantly decreased PRV replication. Furthermore, knockdown of the neddylation substrate Cullin5 (CUL5) or pharmacological inhibition of CUL5 neddylation significantly attenuated PRV replication. Cumulatively, these findings demonstrate that the UBE2F-RBX2-mediated neddylation of CUL5 facilitates PRV replication. This study provides a new theoretical basis for in-depth understanding of PRV-host interaction and reveals neddylation as a novel target for antiviral strategies against PRV.

BACKGROUND:The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is known for its capacity to cause severe neurological disease in Asia. Neurotropic flaviviruses within the Japanese encephalitis (JE) serogroup possess the distinctive feature of expressing a unique nonstructural protein, NS1'. The NS1' protein consists of the full NS1 protein with an additional 52 amino acid extension at the C-terminus and has been demonstrated to exhibit virulence in mammalian hosts upon infection. However, the precise role of the NS1' protein in the mosquito vectors has yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS:In this study, an NS1'-defective virus (rG66A) was engineered, and its effect on the infection of mosquito cells was investigated. The results demonstrated a significant reduction in the infectivity of the rG66A virus in mosquito cells by RT-qPCR, indicating that the absence of the NS1' protein impedes JEV replication in Culex mosquitoes. Additionally, this research elucidated the underlying mechanism by which the NS1' protein enhances viral infection in mosquitoes by RNA-Seq analysis. Specifically, the NS1' protein was found to facilitate infection through the suppression of antimicrobial peptides (AMPs) regulated by the Toll pathway. CONCLUSIONS/SIGNIFICANCE:Our research demonstrated that the JEV NS1' protein contributes to immune escape, thereby enhancing viral infection in mosquitoes. This finding offers new insights into the transmission mechanisms of JEV, elucidating novel aspects of viral propagation.

IntroductionAfter being discovered for the first time in China in 2017, porcine reproductive and respiratory syndrome virus (PRRSV) NADC34-like strains have become the prevalent strain of PRRSV in certain regions of China. Our previous study showed that reduced Ingelvac PRRS MLV vaccination dosages against NADC30-like CF PRRSV had a better protection effect than the normal dosage. However, the protective effect of reduced dosages vaccination of Ingelvac PRRS MLV against NADC34-like PRRSV is unclear. Therefore, this study compared the effectiveness of 0.1 and 1 dosages against a NADC34-like PRRSV infection using commercial PRRSV vaccines, Ingelvac PRRS MLV, which have been widely utilized in China.MethodsIn this study, we immunized piglets with two different dosages of the MLV vaccine and infected piglets within a nasal way with NADC34-like CF PRRSV at 42 days post-vaccination. We observed the changes in growth performance before and after the NADC34-like PRRSV DX strain challenge and the protective effect of different vaccine dosages through multiple assays.ResultsAfter the challenge, the piglets from the challenge control group displayed clinical signs typical of PRRSV infection, including transient fever, high viremia, mild clinical symptoms, and histopathological changes in the lungs and lymph nodes, which indicates DX is a virulent virus. Without the challenge, the average daily gain of the non-immunized group at 5 weeks after the vaccination is greater than that of the 0.01 dosage group than that of the 1 dosage group, which proved that the commercial MLV vaccine has a negative effect on the growth performance of pigs and this effect may be dose-dependent. After the NADC34-like PRRSV challenge, there was no difference in average daily gain between the immunized pigs and pigs from the challenge control group. From the perspective of clinical score, gross lung lesions, and microscopic lesions, immunization with MLV vaccine can indeed relieve symptoms and lesions caused by the virus, and 0.1 dosage vaccination has a better effect in these aspects. Also, both dosages of MLV immunization shortened viremia with similar effects.DiscussionOur research suggests that the MLV vaccine can provide piglets with some protection against NADC34-like PRRSV and the 0.1 dosage Ingelvac PRRS MLV vaccination showed greater benefits in our study. Therefore, considering the cost, side effects, and subsequent protective effects, we can adjust the immune dosage appropriately after further investigation to ensure safety, improve production efficiency, and reduce immunization costs.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. PCV3 capsid protein (Cap) can self-assemble into viruslike particles (VLPs), and is an ideal candidate for vaccines and diagnostic reagents.In this study, the recombinant PCV3 Cap protein was successfully expressed in E. coli by deleting the nuclear localization sequence (NLS). The PCV3 VLPs were observed by transmission electron microscopy, and its immunogenicity was evaluated in sixweek-old female BALB/c mice. A monoclonal antibody was named mAb 2D6, and demonstrated strong reactivity and specificity to PCV3 Cap. The purified mAb 2D6 was further used for bio-panning to select phage expressing specific epitopes from phage-displayed 7 mer-peptide library. A novel linear B-cell epitope, recognized by mAb 2D6, was identified at the amino acid region 47-53 of Cap. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. These results provide insights for developing diagnostic tools and potential vaccines for PCV3.

Aflatoxin B1 (AFB1), recognized as a highly toxic and carcinogenic mycotoxin, contaminates more than 25 % of the global grain supply, thereby presenting a substantial public health threat and posing significant risks to renal health. However, the host factors that mediate the associated damage remain inadequately understood. This study aimed to identify key host factors in AFB1-induced cytotoxicity using a genome-wide CRISPR/Cas9 screen and elucidate the underlying molecular mechanisms. We developed a porcine kidney epithelial (PK15) cell model, followed by knockout validation, CCK-8 assays, qRT-PCR, western blotting, AO-EB staining, flow cytometry, and co-immunoprecipitation to dissect mechanistic pathways. Receptor-Interacting Protein Kinase 4 (RIPK4) was identified as a critical pro-apoptotic factor. RIPK4 knockout increased PK15 cell viability by ~50 % (P < 0.001) and reduced apoptosis by ~44 %(P < 0.001), accompanied by downregulation of APAF1, Cyt-c, cleaved-Caspase-9/-3, and p53 Ser15 phosphorylation, and upregulation of Bcl-2. Mechanistically, RIPK4 directly interacted with p53 via its N-terminal 1-490 aa region, enhancing its phosphorylation and pro-apoptotic activity. In conclusion, RIPK4 promotes AFB1 nephrotoxicity by activating p53-mediated mitochondrial apoptosis, identifying it as a novel therapeutic target. Future studies should validate these findings in vivo models and explore the potential of RIPK4-specific inhibitors for mitigating nephrotoxicity.

Porcine deltacoronavirus (PDCoV) is coronavirus mainly causing piglets diarrhea and posing potential cross-species transmission risk, the development of novel anti-PDCoV drugs is critically important. Poria cocos polysaccharide (PCP) has been proved to pose antiviral bioactivity, but its inhibitory effect on PDCoV remains unclear. Here, the antiviral effect of PCP on PDCoV was systematically evaluated and the potential mechanism was further explored. The PCP structure was first analyzed by using Fourier transform infrared (FT-IR), zeta potential detection, and Scanning electron microscopy (SEM) morphology analysis. Antiviral results showed PCP could significant inhibit PDCoV on attachment stage and replication stage. Network pharmacology combined with transcriptomic analysis and verification indicated that PCP could significantly inhibit the activation of PI3K-AKT/Nf-κB pathway induced by PDCoV, alleviating PDCoV-induced cell apoptosis. Additionally, PCP showed broad-spectrum antiviral effect against Porcine epidemic diarrhea virus (PEDV) and Transmissible Gastroenteritis Virus (TGEV) in vitro. Taken together, we report here for the first time the antiviral effects of PCP against PDCoV and the potential antiviral mechanism of regulating PI3K Akt/NF- κB pathway to affect apoptosis, our findings will contribute to the development of polysaccharides based antiviral drugs against PDCoV and other porcine coronaviruses.