Co-adaptation between viruses and autophagy has equipped viruses with diverse strategies to regulate host redox homeostasis, thereby facilitating viral replication. However, the mechanisms by which viruses manipulate PRDX1 (peroxiredoxin 1), a key antioxidative enzyme, via autophagy remain poorly understood. Here, we demonstrate that infection by Senecavirus A (SVA), an emerging picornavirus, induces PRDX1 degradation, and that PRDX1 negatively regulates viral replication. Decreased PRDX1 expression impairs cellular antioxidant defenses, leading to enhanced reactive oxygen species generation that facilitates SVA replication. Screening of viral proteins revealed that SVA VP1, VP2, and 3A induce PRDX1 degradation through vesicle formation-dependent macroautophagy. Notably, viral VP2 can also recruit HSPA8/HSC70 to specifically target PRDX1, directing it for degradation via LAMP2A-mediated chaperone-mediated autophagy (CMA). Collectively, these findings demonstrate that the SVA VP2 protein plays a central role in orchestrating both macroautophagy- and CMA-mediated PRDX1 degradation, establishing PRDX1 as a potential intervention target for countering SVA infection.Abbreviations: AKT/protein kinase B: AKT serine/threonine kinase; ATP: adenosine triphosphate; BHK-21: baby hamster kidney-21; CAT: catalase; CCCP: BMDMs: bone marrow-derived macrophages; CMA: chaperone-mediated autophagy; co-IP: co-immunoprecipitation; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CQ: chloroquine; DCFH-DA: 2',7'-dichlorodihydrofluorescein diacetate; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; GPX: glutathione peroxidase; GSH: glutathione; HEK-293T: human embryonic kidney 293T; hpi: hours post-infection; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KO: knockout; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mdivi-1: mitochondrial division inhibitor-1; mM: millimole; MMP: mitochondrial membrane potential; mPTP: mitochondrial permeability transition pore; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetylcysteine; PI3K: phosphoinositide 3-kinase; PRDX1: peroxiredoxin 1; RT-qPCR: real-time quantitative reverse transcription polymerase chain reaction; ROS: reactive oxygen species; SD: standard deviation; SOD: superoxide dismutase; SQSTM1: sequestosome 1; SVA: Senecavirus A; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type; μg: microgram; μm: micrometer; μM: micromolar.

Co-evolution between viruses and autophagy has led to the emergence of viral strategies that manipulate host endoplasmic reticulum (ER) homeostasis, ultimately promoting viral replication. ER turnover is achieved through selective autophagy, also referred to as ER-phagy, which is regulated by the RETREG1/FAM134B (reticulophagy regulator 1) family of reticulon proteins. Nevertheless, how viruses target RETREG1, a receptor for ER-phagy, remains largely unclear. In this study, we demonstrate that infection with Senecavirus A (SVA), an emerging picornavirus, triggers the cleavage of RETREG1, which functions as a negative regulator of viral replication. By screening viral proteins, we identified the SVA 3C protease (3C[pro]) as the executor of this cleavage event. Detailed mapping revealed that residues Q428, E430, and G431 of RETREG1 are involved in its cleavage by the 3C[pro], and the resulting two fragments fail to suppress viral replication. Furthermore, proteolytic cleavage of RETREG1 by 3C[pro] impairs its ability to relieve ER stress and mediate ITPR1 degradation via RETREG1-dependent ER-phagy. This disruption leads to increased ER calcium (Ca2+) release and subsequent activation of autophagy through the CAMKK2-PRKAA2-MTOR axis, which ultimately facilitates SVA replication. Taken together, these findings indicate that SVA antagonizes the antiviral function of RETREG1-mediated ER-phagy via its 3C[pro], highlighting RETREG1 as a potential therapeutic target for combating SVA infection.Abbreviations: 2-APB: 2-aminoethyl diphenylborinate; PRKAA2/AMPK: protein kinase AMP-activated catalytic subunit alpha 2; ATL3: atlastin GTPase 3; BHK-21: baby hamster kidney-21; CAMKK2: calcium/calmodulin dependent proteinkinase kinase2; CCPG1: cell cycle progression 1; CKAP4/CLIMP63: cytoskeleton associated protein 4; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DM: double mutant; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; eGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; GFP: green fluorescent protein; HSPA5/GRP78/BiP: heat shock protein family A (Hsp70) member 5; HA: hemagglutinin; HDAC4: histone deacetylase 4; HEK-293T: human embryonic kidney 293T; hpi: hours post-infection; IFA: indirect immunofluorescence assay; ITPR1/IP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LIR: LC3-interacting region; mCherry: monomeric cherry; MTOR: mechanistic target of rapamycin kinase; REEP5: receptor accessory protein 5; RETREG1/FAM134B: reticulophagy regulator 1; RTN3: reticulon 3; SD: standard deviation; SEC61B: SEC61 translocon subunit beta; SEC62: SEC62 preprotein translocation factor; SERP1/RAMP4: stress associated endoplasmic reticulum protein 1; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; ST: swine testis; SVA: Senecavirus A; TEM: transmission electron microscopy; TEX264: testis expressed 264, ER-phagy receptor; Tm: tunicamycin; U2OS: human osteosarcoma epithelial cells; UV: ultraviolet; ZVAD-FMK: benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; μg: microgram; μm: micrometer; μM: micromole.

Porcine circovirus type 2 (PCV2), a major immunosuppressive pathogen, causes substantial economic losses to the global swine industry. PCV2d is the dominant genotype in multiple regions, but its immunopathological mechanisms, particularly virus‒host immune crosstalk, remain unclear. Here, we evaluated immune responses to a rescued replication-competent PCV2d in C57BL/6 mice, focusing on lymphocyte activation, thymic T-cell apoptosis and dynamics, T-cell immunophenotype shifts, and splenic regulatory T (Treg) cell dynamics. CD69-based assessment of lymphocyte activation revealed time-specific patterns: CD8⁺ T cells were activated throughout infection, with stronger activation at 7–14 days post-infection (dpi); γδ T cells were activated only early; CD4⁺ T cells and natural killer (NK) cells were activated late (14–28 dpi); and B cells were persistently activated. Thymic analysis revealed subset-specific apoptosis; for example, CD8⁺ T cells underwent early apoptosis across all stages, immature CD4⁻CD8⁻ T cells were vulnerable late, and altered dynamics were detected: CD4⁺ and CD8⁺ T cells first increased and then decreased from early to late infection, while CD4⁺CD8⁺ T cells showed the opposite trend. T-cell immunophenotyping with CD44 and CD62L revealed reduced numbers of naive CD4⁺/CD8⁺ T cells, increased numbers of memory T cells, and upregulated the expression of PD-1, a marker of T-cell exhaustion. Splenic Treg cells (CD4⁺CD25⁺Foxp3⁺) expanded, with a peak at 21 dpi. These findings demonstrate the multifaceted immunomodulatory effects of PCV2d, including innate activation, adaptive immune suppression, thymic dysfunction, T-cell exhaustion, and Treg-mediated immunosuppression. This work improves our understanding of PCV2d pathogenesis, aiding the development of targeted control strategies and vaccines for swine PCV2.

Porcine circovirus type 2 (PCV2), a major pathogen of PCV2-associated diseases, causes immense economic losses. Understanding the cellular metabolic reprogramming induced by viruses provides novel clues for screening antiviral drugs. However, until now, no reports have described the cellular metabolic profile during PCV2 infection. In this study, we performed untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis of PCV2-infected PK-15 cells, and found that PCV2 infection upregulated significantly arginine and its metabolite spermidine. Further, we found that arginine downstream metabolites spermidine and putrescine markedly promoted PCV2 replication and played a proviral role during the replication stage of PCV2 infection. Difluoromethylornithine (DFMO)-an ornithine decarboxylase 1 (ODC1) inhibitor-decreases endogenous polyamine levels, and its treatment suppressed PCV2 replication. Supplementation with exogenous spermidine or putrescine, but not spermine, strikingly facilitated PCV2 replication, and inhibition of polyamine biogenesis significantly curtailed PCV2 replication in vitro and in the murine model. Taken together, the study revealed that spermidine plays an important proviral role in PCV2 replication and suggests that cellular polyamine metabolism is a suitable target for therapeutic intervention against PCV2 infection.

Porcine circovirus type 2 (PCV2), a major causative agent of PCV2-associated diseases, poses a serious threat to the global swine industry. The stability of PCV2 capsid (Cap) protein is critical for viral replication; however, the underlying mechanisms regulating Cap stability during PCV2 infection remain poorly understood. In this study, co-immunoprecipitation assays were used to demonstrate that PCV2 Cap protein can maintain its own stability by binding to the cellular Y-box-binding protein 1 (YBX1) during viral infection. Furthermore, binding domain mapping experiments revealed two specific regions essential for this interaction: the N-terminal arginine-rich motif (ARM) of PCV2 Cap (amino acid sequence: 1MTYPRRRYRRRRHRPRSHLG20) and the amino acid residues 189RRRR192 within the C-terminal domain (CTD) of YBX1. Virus rescue experiments further confirmed that substituting all arginine residues with alanine in the N-terminal ARM of Cap completely abolished its binding to YBX1. Analysis of viral replication capacity showed that YBX1 promotes PCV2 replication by stabilizing the viral Cap protein. Additionally, knockdown of YBX1 significantly reduced the protein levels of PCV2 Cap, and this effect was reversed either by treatment with the proteasome inhibitor MG132 or by restoring YBX1 expression. Collectively, these findings demonstrate that YBX1 promotes PCV2 replication by directly interacting with the ARM of PCV2 Cap, thereby inhibiting the proteasomal degradation of Cap during viral infection.

Abstract Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV), a member of the Coronaviridae family that comprises two genotypes, FCoV-I and FCoV-II. From the ascitic fluid of a British shorthair cat diagnosed with FIP, we isolated and identified an FCoV strain designated AH2023. Western blotting, RT‒PCR, and transmission electron microscopy were employed for characterization. According to phylogenetic analyses of the full viral genome and the spike, nucleocapsid, and 3C-like protease genes, strain AH2023 consistently fell within the clade of previously reported Chinese FCoV-II strains and exhibited greater evolutionary proximity to transmissible gastroenteritis virus (TGEV) and canine coronavirus (CCoV) than to FCoV-I did. Recombination analysis using SimPlot further revealed strain AH2023 to be a recombinant virus. Strain AH2023 is genetically distinct, carrying four amino acid substitutions in the spike protein. Substitutions at positions 387, 577, and 829 are shared with strains SH2021 and DQ2023, whereas the substitution at position 1375 is shared with strain DF2. Pathogenicity tests in 12‑week‑old cats confirmed the virulence of AH2023. All the cats died between 12 and 18 days post-challenge (dpc) and presented severe intestinal, hepatic, and renal lesions. The virus exhibited broad tissue tropism, with the highest viral load (10 4 copies/mg) found in the intestine. Fecal shedding began at 7 dpc and reached 10 4 copies by 10 dpc. These results advance our understanding of FCoV evolution and pathogenesis, especially for type II strains in China, and aid in the development of effective FIP prevention strategies.

The objective of this study is to evaluate the effects of crude protein (CP) levels and antibiotics in feed on the growth performance, body composition, nutrient digestion and absorption, gut health, and microbiota of weaned piglets. A total of 100 piglets (Duroc × Landrace × Yorkshire, weaned at day 21) with an initial weight of 6.39 ± 0.03 kg were randomly assigned to four treatments and fed with 18% CP antibiotic-free diets, 18% CP with antibiotics diets, 24% CP antibiotic-free diets, and 24% CP with antibiotics diets for 0–14 d (S1). Furthermore, the piglets were fed with an identical diet (19% CP and without antibiotics) starting day 15 until they reached 25 kg (S2). Results indicated that the average daily gain (ADG) and gain feed ratio (G:F) during the S1 phase as well as BW at day 14, were significantly decreased (p < 0.05) in the 18% CP group compared with the 24% CP group. Similarly, piglets in the 18% CP group showed a significantly decreased body protein content and body water, protein deposition rates at the S1 phase, and significantly improved the body lipid content and deposition rate but not (p > 0.05) at the S2 phase. The 18% CP group demonstrated significantly increased (p < 0.05) serum concentrations of Threonine, Valine, Isoleucine, and Lysine in weaned piglets during the S1 phase, while Glycine concentration significantly decreased, and no significant (p > 0.05) effects were observed in the S2 phase. Furthermore, dietary antibiotic supplementation had no effect on (p > 0.05) piglet growth performance, body composition, nutrient digestion, and metabolism throughout the entire trial period. In the absence of antibiotics, compared to the 24% CP diet group, the piglets fed with an 18% CP diet significantly increased the relative abundance of Lactobacillus in the colon of weaned piglets upon reaching 25 kg BW (p < 0.05). Compared to the diet without added antibiotics, the addition of antibiotics to the diet significantly decreased the relative abundance of Pseudoramibacter in the colon of weaned piglets on day 14. No significant (p > 0.05) difference was observed in intestinal morphology or gastrointestinal pH among the groups. In conclusion, lower dietary protein levels in the early post-weaning period reduced growth performance and promoted gut microbiota balance, and compensatory growth occurred after returning to higher protein levels two weeks after weaning.

ABSTRACT Our previous research has shown that maternal dietary 40 mg/kg isoflavone‐S (ISO‐S) improves sow reproductive and offspring growth during late gestation and lactation. However, the mechanism by which maternal ISO‐S improves intestinal homeostasis in offspring remains unknown. The results showed that maternal ISO‐S reduced the depth of the jejunal crypt and increased the villous height to crypt depth ratio in both the ileum and jejunum of the offspring. Furthermore, we found that maternal ISO‐S increased the levels of T‐SOD, CAT, and GSH‐Px in the jejunum, as well as the levels of T‐SOD and CAT in the ileum, and the expression of SOD1 and SOD2 mRNA in the offspring's jejunum. Metabolomics analysis identified 56 differentially expressed metabolites in colostrum, with significant changes observed in antioxidant metabolites. Further in vitro studies showed that ISO‐S decreased ROS levels in PMECs under 4‐HNE‐induced oxidative stress. ISO‐S increased the mRNA expression of VADC1 and Atg5 and the protein expression of mitophagy‐related genes. Maternal ISO‐S not only enhanced SIgA production and mRNA expression of SIgA‐related factors in the offspring jejunum, but also affected the gut microbiota composition of the offspring piglets. The relative abundance of the Tenericutes (phylum) and the Bradymonadales (order) was significantly increased. In conclusion, the results indicate that supplementing sows with ISO‐S during late gestation and lactation helps improve offspring intestinal health, likely due to its enhancement of antioxidant enzymes, antioxidant metabolites, and immunoglobulins in colostrum. Furthermore, ISO‐S alleviates 4‐HNE‐induced oxidative stress in mammary epithelial cells by promoting mitophagy.

Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes porcine dermatitis, and reproductive failure. PCV3 Cap interacts with DExD/H-box helicase 36 (DHX36), a protein that functions primarily through regulating interferon (IFN)-β production. However, how the interaction between DHX36 and PCV3 Cap regulates viral replication remains unknown. Herein, we observed impaired PCV3 proliferation after DHX36 overexpression as indicated by decreased Rep protein expression and virus production. In contrast, PCV3 replication increased upon small interfering RNA-mediated DHX36 depletion. Furthermore, DHX36 positively regulated IFN-β production and interferon-stimulated genes (ISGs) expression. Mechanistically, PCV3 Cap interacted with DHX36, and the PCV3 Cap-NLS and DHX36-NTD were essential for the interaction. Furthermore, DHX36 may get degraded because its binding cellular partners became ubiquitinated and then reduced, and PCV3 Cap-(35-100aa) also promoted the degradation of DHX36 through the K48-linked ubiquitination. Taken together, these results show that DHX36 antagonizes PCV3 replication by interacting with PCV3 Cap and activating IFN-β response, which provides important insight on the prevention and controlling of PCV3 infection. IMPORTANCE: Porcine circovirus type 3 (PCV3) is a newly discovered pathogen associated with multiple clinicopathological signs. Clarifying the mechanisms that host factors modulate PCV3 replication helps understanding of the viral pathogenesis. The PCV3 capsid (Cap) protein has been shown to interact with DExD/H-box helicase 36 (DHX36) (Zhou et al., 2022b), a crucial protein that regulates virus replication. Herein, we further demonstrated that DHX36 protein is degraded in PCV3-infected cells and antagonizes the replication of PCV3 and that DHX36 increases interferon-β and interferon-stimulated gene levels by binding to PCV3 Cap. In addition, PCV3 infection could decrease DHX36 expression levels to antagonize its antiviral activity. These results reveal a molecular mechanism by which DHX36 antagonizes PCV3 replication by binding to PCV3 Cap protein and activating IFN signals, thereby providing important targets for preventing and controlling PCV3 infection.

This study evaluated the effect of mixed meal replacement of soybean meal on growth conditions, carcass traits, and meat quality of finishing pigs by partially and entirely replacing soybean meal with equal proportions of rapeseed, cotton, and sunflower meal. A total of fifty-four pigs with an average initial weight of 97.60 ± 0.30 kg were selected and randomly divided into three groups according to their initial weight, with six pens in each group and three pigs in each pen. The experimental groups were as follows: control group (CON), fed corn–soybean meal type basal diet; corn–soybean mixed meal group (CSM), using equal proportions of rapeseed meal, cotton meal, and sunflower meal (3.52% each) to replace 9.06% of soybean meal in the basal diet; and corn mixed meal group (CMM), using equal proportions of rapeseed meal, cotton meal, and sunflower meal (6.46% each) to replace soybean meal in the basal diet completely. According to the results, the use of mixed meal as a replacement for soybean meal did not have a significant impact (p > 0.05) on the average daily weight gain, average daily feed intake, feed-to-weight ratio, body size, carcass traits, and meat quality of finishing pigs. The entire replacement of soybean meal with a mixed meal resulted in a significant increase (p < 0.05) in leaf fat weight. The use of mixed meal as a substitute for soybean meal had no significant effect (p > 0.05) on the antioxidant capacity and fatty acid composition of the longissimus thoracis in finishing pigs. However, longissimus thoracis muscle fiber diameter was reduced in the mixed meal partially replaced soybean meal group compared to the mixed meal completely replaced soybean meal group (p < 0.05). In addition, mixed meal replacing soybean meal did not significantly affect (p > 0.05) the expression of the longissimus thoracis muscle fiber type genes MYHC1 and MYHC2. Mixed meal replacement of soybean meal did not significantly affect (p > 0.05) the expression of ACACA, FASN, and PPARG genes in the longissimus thoracis. This study showed that mixed meal as an alternative to soybean meal in diets did not have significant negative effects on the growth performance and meat quality of finishing pigs. These results can help develop further mixed meals as a functional alternative feed ingredient for soybean meals in pig diets.

ABSTRACT Seneca Valley virus (SVV), also known as Senecavirus A, a porcine pathogen that causes vesicular diseases, is prevalent in pig herds worldwide. SVV infection induces endoplasmic reticulum (ER) stress in PK-15 and BHK-21 cells, accompanied by activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) pathways, which in turn facilitates SVV replication. ER stress is associated with the regulation of Ca 2+ homeostasis and mitochondrial apoptosis. However, the precise role of Ca 2+ in SVV-induced apoptosis remains unclear. In this study, western blotting, flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) detection revealed that either ER stress or the PERK pathway is involved in the apoptosis of SVV-infected cells treated with specific inhibitors. Furthermore, SVV-mediated ER stress markedly contributed to the transfer of Ca 2+ from the ER to mitochondria. The subsequent increase in mitochondrial Ca 2+ content was accompanied by an increased number of ER membranes near the mitochondria. Finally, the inhibition of mitochondrial Ca 2+ overload, ER stress, and the PERK pathway substantially attenuated SVV-mediated mitochondrial dysfunction, as evidenced by analyzing mitochondrial membrane potential (MMP), mitochondrial permeability transition poremPTP, reactive oxygen speciesROS, and adenosine 5′-triphosphate ATP, and the levels of mitochondrial apoptosis. These findings demonstrate that SVV induces mitochondrial apoptosis, which is dependent on ER stress-mediated transmission of Ca 2+ from the ER to the mitochondria. IMPORTANCE Viruses have developed multiple mechanisms to facilitate their proliferation or persistence through manipulating various organelles in cells. Seneca Valley virus (SVV), as a novel emerging pathogen associated with vesicular disease, is clinically and economically important infections that affect farm animals. Previously, we had confirmed that SVV-induced endoplasmic reticulum (ER) stress benefited for viral replication. Ca 2+ , as an intracellular signaling messenger mainly stored in the ER, is regulated by ER stress and then involved in apoptosis. However, the precise mechanism that Ca 2+ transfer induced by SVV infection triggered apoptosis remained unclear. Here, we found that SVV infection triggered the Ca 2+ transform from ER to mitochondria, resulting in mitochondrial dysfunction, and finally induced mitochondrial apoptosis. Our study shed light on a novel mechanism revealing how ER stress manipulates Ca 2+ homeostasis to induce mitochondrial apoptosis and regulate viral proliferation.

Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes porcine dermatitis, nephropathy syndrome-like symptoms, multisystemic inflammation, and reproductive failure. The PCV3 capsid (Cap) protein interacts with DDX21, which functions mainly through controlling interferon (IFN)-β levels. However, how the interaction between DDX21 and PCV3 Cap regulates viral replication remains unknown. In the present study, upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV3 proliferation via a lentivirus-delivered system, as indicated by reduced replicase (Rep) protein levels and viral titers. Furthermore, DDX21 negatively regulated IFN-β and interferon-stimulated gene (ISG) levels, promoting PCV3 replication. Mechanistically, PCV3 Cap co-localized and interacted with DDX21, and the nuclear localization signal (NLS) of PCV3 Cap and 763GSRSNRFQNK772 at the C-terminal domain (CTD) of DDX21 were indispensable to the interaction. Moreover, PCV3 infection prevented the repression of DDX21 to facilitate its pro-viral activity. Taken together, these results show that DDX21 promotes PCV3 replication by binding to the PCV3 Cap protein and prohibiting IFN-β response, which provides important insight on the prevention and control of PCV3 infection.

Senecavirus A (SVA) is an emerging pathogen responsible for vesicular lesions and neonatal mortality in swine. In the absence of effective vaccines or therapeutics, early and accurate diagnosis is essential for controlling SVA outbreaks. Although nucleic acid-based detection methods are commonly employed, there remains a pressing need for rapid, convenient, highly sensitive, and specific diagnostic tools. Here, we developed a two-pot assay combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a containing crRNA targeting canonical protospacer adjacent motifs (PAMs) for simple, rapid, and visual identification of SVA in clinical samples. Subsequently, we successfully streamlined this system into a one-pot assay by selecting a specially designed crRNA targeting suboptimal PAM and integrating RPA amplification reagents and CRISPR/Cas12a detection components into a single reaction system in one tube. The developed methods exhibited diagnostic specificity, showing no cross-reactivity with four major swine viruses, while showing remarkable sensitivity with a lower detection limit of just two copies. Clinical validation in field samples using these two methods revealed perfect agreement (100% concordance) with conventional quantitative PCR (qPCR) results (sample size, n = 28), with both assays completing detection within 30 min. These results demonstrate that both the one-pot and two-pot RPA-CRISPR/Cas12a assays offer a reliable and efficient method for detecting SVA in this pilot study. Despite the limited sample size, the assays combine rapid reaction time with high sensitivity and specificity, showing great potential for future diagnostic applications.

Senecavirus A (SVA), an emerging vesicular pathogen, poses a significant threat to the global pig industry. STIP1 homology and U-box-containing protein 1 (STUB1), a chaperone-dependent E3 ubiquitin ligase, plays a pivotal role in protein quality control by mediating target protein degradation. However, its precise role of STUB1 in regulating SVA replication remains undefined. In this study, we combined liquid chromatography-mass spectrometry, confocal imaging, and Western blotting to demonstrate that STUB1 interacts with the SVA VP1 protein and negatively regulates SVA replication. Mechanistically, STUB1 promotes the ubiquitination-dependent degradation of VP1 by specifically targeting lysine residues at positions 177 and 260 (K177 and K260). This degradation process is significantly enhanced by heat shock protein 70 (HSP70) and heat shock cognate protein 70 (HSC70), which strengthen the STUB1-VP1 interaction. Notably, the SVA 3C protease (3Cpro) counteracts this antiviral defense by enzymatically reducing STUB1 expression. In vivo studies using a mouse model showed that a VP1 mutant virus lacking STUB1-targeted ubiquitination sites replicates more efficiently than the wild-type strain, resulting in significantly higher viral loads across multiple tissues and more severe pulmonary pathology. Together, these findings reveal that STUB1 inhibits SVA replication through ubiquitination-dependent degradation of VP1, a process that is antagonized by viral 3C protease via suppression of STUB1 expression. IMPORTANCE:Viruses have evolved diverse strategies to enhance their replication efficiency. Senecavirus A (SVA), an emerging porcine pathogen associated with vesicular disease outbreaks, has become increasingly prevalent in swine populations worldwide. As a chaperone-dependent E3 ubiquitin ligase, STUB1 plays a crucial role in maintaining cellular protein homeostasis. In this study, we elucidated the functional interplay between STUB1 and SVA replication. Our results demonstrate that STUB1 directly interacts with the viral protein VP1 and mediates its ubiquitination-dependent degradation through specific targeting of lysine residues at positions 177 and 260 (K177 and K260), thereby significantly inhibiting viral replication. However, SVA has evolved a countermeasure, whereby its 3C protease (3Cpro) downregulates STUB1 expression, effectively blocking VP1 degradation and subverting this host antiviral defense to promote viral propagation. These findings not only reveal novel host-virus interaction mechanisms but also provide valuable molecular targets for developing innovative strategies to control SVA infection.

ABSTRACT Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes porcine dermatitis and nephropathy syndrome-like symptoms, multisystemic inflammation, and reproductive failure. The PCV3 capsid (Cap) protein interacts with DEAD-box RNA helicase 10 (DDX10), a protein that functions primarily through regulating interferon (IFN)-β production to exert its antiviral activity. However, how the interaction between DDX10 and PCV3 Cap regulates viral replication remains unknown. We used Western blotting, interaction assays, and knockdown analyses to observe impaired PCV3 proliferation in transiently DDX10-overexpressing cells, as indicated by decreased viral protein expression levels and virus production. In contrast, PCV3 replication increased upon small interfering RNA-mediated DDX10 depletion. Furthermore, DDX10 positively regulated IFN-β production and interferon-stimulated gene expression, inhibiting PCV3 replication. Mechanistically, PCV3 Cap co-localized and interacted with DDX10, and the N-terminal nuclear localization signal of PCV3 Cap and the helicase domain of DDX10 were essential for the Cap-DDX10 interaction. Furthermore, PCV3 infection decreases DDX10 expression to antagonize its antiviral activity. These results show that DDX10 antagonizes PCV3 replication by interacting with the PCV3 Cap protein and activating IFN-β responses, which provides important insight into the prevention and control of PCV3 infection. IMPORTANCE Clarifying how host factors contribute to infection with PCV3, a newly discovered pathogen associated with multiple clinicopathological signs in swine, helps elucidate viral pathogenesis. The PCV3 Cap protein has been shown to interact with DDX10, a crucial protein that regulates RNA virus replication. Herein, we further demonstrated that DDX10 expression is downregulated in PCV3-infected cells and antagonizes the replication of PCV3 and that DDX10 increases interferon-β and interferon-stimulated gene levels to inhibit PCV3 replication by binding to the PCV3 Cap. In addition, PCV3 infection decreases DDX10 expression to antagonize its antiviral activity. These results reveal a molecular mechanism by which DDX10 antagonizes PCV3 replication by binding to the PCV3 Cap protein and activating IFN signals, thereby providing important targets for preventing and controlling PCV3 infection.

Avian metapneumovirus (aMPV) is a viral pathogen that mainly causes respiratory signs and drops in egg production in turkeys, chickens, and ducks. Here, an aMPV subgroup C (aMPV/C) strain, designated GX22-01, was isolated and identified from severe respiratory disease in broiler breeder chickens in 2022 in Jiangsu, China, as evidenced by indirect immunofluorescence and western blotting using specific anti-viral protein antibodies and by sequence analysis of viral nucleoprotein (N) gene. N gene sequencing indicated that the GX22-01 strain shares a high identity (94.3%-99.8 %) with aMPV/C isolates, especially with Chinese aMPV/C isolates from ducks and chickens, which are divided into aMPV/C cluster through N gene-based phylogenetic analysis. The aMPV/C GX22-01 strain was continuously passaged in Vero cells and the viral titers approximately reached 106.0 TCID50/0.1 mL. Pathogenic analysis showed that aMPV/C GX22-01 strain inoculation caused respiratory signs in 2-week-old specific-pathogen-free (SPF) chickens and resulted in pathogenic damage in tracheae and lung tissues, accompanied by positive viral signals using indirect immunohistochemistry. These results provide epidemiological and pathogenic data for developing effective measures against aMPV/C infection in China.

IntroductionSince their identification in 1974, circoviruses have caused clinicopathological diseases in various animal species, including humans. However, their origin, transmission, and genetic evolution remain poorly understood.MethodsIn this study, the genome sequences of circovirus were obtained from GenBank, and the Bayesian stochastic search variable selection algorithm was employed to analyzed the evolution and origin of circovirus.ResultsHere, the evolutionary origin, mode of transmission, and genetic recombination of the circovirus were determined based on the available circovirus genome sequences. The origin of circoviruses can be traced back to fish circovirus, which might derive from fish genome, and human contributes to transmission of fish circovirus to other species. Furthermore, mosquitos, ticks, bats, and/or rodents might play a role as intermediate hosts in circovirus intra- and inter-species transmission. Two major lineages (A and B) of circoviruses are identified, and frequent recombination events accelerate their variation and spread. The time to the most recent common ancestor of circoviruses can be traced back to around A.D. 600 and has been evolving at a rate of 10-4 substitutions site-1 year-1 for a long time.DiscussionThese comprehensive findings shed light on the evolutionary origin, population dynamics, transmission model, and genetic recombination of the circovirus providing valuable insights for the development of prevention and control strategies against circovirus infections.

Avian metapneumovirus subgroup C (aMPV/C), an important pathogen causing acute respiratory infection in chickens and turkeys, contributes to substantial economic losses in the poultry industry worldwide. aMPV/C has been reported to induce autophagy, which is beneficial to virus replication. Sequestosome 1 (SQSTM1/P62), a selective autophagic receptor, plays a crucial role in viral replication by clearing ubiquitinated proteins. However, the relationship between SQSTM1-mediated selective autophagy and aMPV/C replication is unclear. In this study, we found that the expression of SQSTM1 negatively regulates aMPV/C replication by reducing viral protein expression and viral titers. Further studies revealed that the interaction between SQSTM1 and aMPV/C M2-2 protein is mediated via the Phox and Bem1 (PB1) domain of the former, which recognizes a ubiquitinated lysine at position 67 of the M2-2 protein, and finally degrades M2-2 via SQSTM1-mediated selective autophagy. Collectively, our results reveal that SQSTM1 degrades M2-2 via a process of selective autophagy to suppress aMPV/C replication, thereby providing novel insights for the prevention and control of aMPV/C infection.IMPORTANCEThe selective autophagy plays an important role in virus replication. As an emerging pathogen of avian respiratory virus, clarification of the effect of SQSTM1, a selective autophagic receptor, on aMPV/C replication in host cells enables us to better understand the viral pathogenesis. Previous study showed that aMPV/C infection reduced the SQSTM1 expression accompanied by virus proliferation, but the specific regulatory mechanism between them was still unclear. In this study, we demonstrated for the first time that SQSTM1 recognizes the 67th amino acid of M2-2 protein by the interaction between them, followed by M2-2 degradation via the SQSTM1-mediated selective autophagy, and finally inhibits aMPV/C replication. This information supplies the mechanism by which SQSTM1 negatively regulates viral replication, and provides new insights for preventing and controlling aMPV/C infection.

IntroductionThis study was carried out to investigate the effects of mixed meal (rapeseed meal, cotton meal, and sunflower meal) replacement soybean meal on growth performance, nutrient apparent digestibility, serum inflammatory factors and immunoglobulins, serum biochemical parameters, intestinal permeability, short-chain fatty acid content, and gut microbiota of finishing pigs.MethodsA total of 54 pigs with an average initial weight of 97.60 ± 0.30 kg were selected and randomly divided into 3 groups according to their initial weight, with 6 replicates in each group and 3 pigs in each replicate. The trial period was 26 days. The groups were as follows: control group (CON), fed corn-soybean meal type basal diet; Corn-soybean-mixed meal group (CSM), fed corn-soybean meal-mixed meal diet with a ratio of rapeseed meal, cotton meal, and sunflower meal of 1:1:1 to replace 9.06% soybean meal in the basal diet; Corn-mixed meal group (CMM), fed a corn-mixed meal diet with a ratio of Rapeseed meal, Cotton meal and Sunflower meal of 1:1:1 to replace soybean meal in the basal diet completely. The crude protein level of the three diets was maintained at 12.5%.ResultsOur findings revealed no significant impact of replacing soybean meal with the mixed meal (rapeseed meal, cotton meal, and sunflower meal) on the ADG (Average daily gain), ADFI (Average daily feed intake), and F/G (Feed gain ratio) (P > 0.05), or crude protein, crude fat, and gross energy (P > 0.05) in the diet of finishing pigs. Compared with the CON group, the serum interleukin 6 (IL-6) and interleukin 10 (IL-10) concentrations were significantly decreased in the CMM group (P < 0.05). However, there is no significant effect of the mixed meal (rapeseed meal, cotton meal, and sunflower meal) replacing soybean meal in the diet on the serum interleukin 1β (IL-1β), interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-α), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations (P > 0.05). Concordantly, there is no significant effect of mixed meal (rapeseed meal, cotton meal, and sunflower meal) replacing soybean meal in the diet on the serum antioxidant capacity, such as total antioxidant capacity (T-AOC), catalase (CAT), and malondialdehyde (MDA) levels of finishing pigs. Moreover, compared with the CON group, serum low-density lipoprotein (LDL-C) levels were significantly lower in the CSM group (P < 0.05) and their total bilirubin (TBIL) levels were significantly lower in the CMM group (P < 0.05). There is not a significant effect on serum D-lactate and diamine oxidase (DAO) concentrations (P > 0.05). The next section of the survey showed that the replacement of soybean meal with a mixed meal (rapeseed meal, cotton meal, and sunflower meal) in the diet did not significantly influence the acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid in the colon contents (P > 0.05). Furthermore, compared with the CON group, the CMM group diet significantly increased the abundance of Actinobacteria at the phylum level (P < 0.05), U_Actinobacteria at the class level (P < 0.05), and U_Bacteria at the class level (P < 0.05). The result also showed that the CMM group significantly reduced the abundance of Oscillospirales at the order level (P < 0.05) and Streptococcaceae at the family level (P < 0.05) compared with the CON group. The Spearman correlation analysis depicted a statistically significant positive correlation identified at the class level between the relative abundance of U_Bacteria and the serum T. BILI concentrations (P < 0.05). Moreover, a significant negative correlation was detected at the order level between the relative abundance of Oscillospirales and the levels of acetic and propionic acids in the colonic contents (P < 0.05). Additionally, there was a significant positive correlation between the serum concentrations of IL-6 and IL-10 and the relative abundance of the family Streptococcaceae (P < 0.05).DiscussionThis study demonstrated that the mixed meal (rapeseed meal, cotton meal, and sunflower meal) as a substitute for soybean meal in the diet had no significant negative effects on the growth performance, nutrient apparent digestibility, serum immunoglobulins, serum antioxidant capacity, intestinal permeability, short-chain fatty acid content, and diversity of gut microbiota of finishing pigs. These results can help develop further mixed meals (rapeseed meal, cotton meal, and sunflower meal) as a functional alternative feed ingredient for soybean meals in pig diets.