BACKGROUND:Peste des petits ruminants (PPR) is a highly contagious and economically devastating disease affecting sheep and goats. The performance and cost of serological diagnostic kits remain a major challenge for PPR eradication. OBJECTIVE:The nucleocapsid (N) protein of PPR virus and its monoclonal antibody (mAb) were engineered for development of a blocking ELISA. METHODS:The N protein was truncated and expressed by the eukaryotic expression system. The mAb 65A8 was recovered, followed by sequencing and cloning of its variable regions into expression vector. The recombinant mAb was expressed in 293F cells. Key characteristics of the recombinant mAb were evaluated. A blocking ELISA was developed and validated. Comparison between the blocking ELISA and a commercial blocking ELISA kit was performed. RESULTS:The truncated N protein exhibited a higher yield compared to the full-length N protein. Recombinant mAb 65A8 demonstrated the same blocking efficacy as that of ascites-derived mAb. The yield of recombinant mAb reached 5.3 mg/100 mL. The affinity constant of recombinant mAb 65A8 was comparable to that of the ascites-derived 65A8 (3.48 × 109 L/mol versus 6.76 × 109 L/mol). Based on the truncated N and recombinant mAb, a blocking ELISA was established. The detection limit of blocking ELISA was 1:128 and 1:32 based on strong positive serum and weak positive serum, respectively. Inter- and intra-assay coefficients of variation were <10%, indicating robust reproducibility. The blocking ELISA kit showed high coincidence with the commercial ELISA kit recommended by the World Organization for Animal Health reference laboratory. CONCLUSIONS:This study successfully developed a reliable and cost-effective blocking ELISA by protein engineering of the antigen and mAb. HIGHLIGHTS:The mAb of PPR Virus was recombinantly expressed in 239F cells, which was employed in the development of a reliable blocking ELISA. This work provided novel ideas and methods for the development of the blocking ELISA kits.

Porcine Epidemic Diarrhea Virus (PEDV) causes highly contagious intestinal disease in swine and results in severe economic losses worldwide. PEDV Nsp3, a nonstructural protein produced in the early stage of viral replication, is responsible for cleaving polyproteins to generate nonstructural proteins and the replication-transcription complex, as well as inducing the formation of coronavirus replication organelles known as double-membrane vesicles (DMVs). Nsp3 is a key protein for viral replication; however, its specific mechanism of action remains unclear. To investigate the role of Nsp3 during viral infection, we performed LC-MS/MS analysis and identified 29 host proteins that potentially interact with Nsp3, among which RBM10 was selected for further study. This study confirms that RBM10 is a host factor that restricts PEDV replication, as validated by functional assays: RBM10 inhibits PEDV replication, while knockdown of RBM10 abolishes this inhibitory effect. This study proposes for the first time an RBM10-MARCHF9-Nsp3-p62 axis, which mediates the selective autophagic degradation of PEDV Nsp3. Mechanistically, RBM10 recruits the E3 ligase MARCHF9 to catalyze K33-linked ubiquitination of Nsp3. Subsequently, the ubiquitinated cargo is recognized by the selective autophagy receptor p62 and delivered to autophagosomes for degradation. Blocking autophagic flux, knocking down p62, or silencing key autophagy-related proteins restores Nsp3 protein levels. In summary, this study confirms RBM10 as a novel anti-PEDV host factor, providing new theoretical insights into host-directed regulatory mechanisms of PEDV replication and suggesting that RBM10 may serve as a potential target for the development of novel anti-PEDV strategies.

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that causes severe gastrointestinal disease in neonatal piglets and poses a potential risk of cross-species transmission. Although viral strategies that counteract host immune responses have been widely studied, host factors that restrict PDCoV replication remain poorly characterized. In this study, we identify ANKFY1 as a previously unrecognized host factor that restricts PDCoV replication. PDCoV infection markedly upregulated ANKFY1 expression in LLC-PK1 cells. Using gain- and loss-of-function approaches, we demonstrated that ANKFY1 significantly suppresses PDCoV replication, whereas depletion of ANKFY1 restored both viral RNA synthesis and progeny virus production. Mechanistically, ANKFY1 specifically interacts with the viral nonstructural protein nsp8 and promoted its degradation. We further show that ANKFY1 recruits the E3 ubiquitin ligase Cullin 3 to catalyze K63/K33-linked ubiquitination of nsp8, primarily at lysine 58. The ubiquitinated nsp8 is subsequently recognized by the selective autophagy receptor p62 and delivered to autolysosomes for degradation. Disruption of p62 or autophagy flux abolished ANKFY1-mediated nsp8 degradation and antiviral activity, underscoring the essential role of the ANKFY1-Cullin3-p62 axis. Collectively, our results reveal a novel host defense mechanism in which ANKFY1 mediates selective autophagic degradation of PDCoV nsp8 to restrict viral replication. This study not only advances our understanding of PDCoV-host interactions but also identifies the ANKFY1-Cullin3-p62 pathway as a potential target for developing of broad-spectrum antiviral strategies.

Porcine deltacoronavirus (PDCoV) is a major enteric pathogen that inflicts lethal diarrhea in sucking piglets. In this study, we constructed an infectious cDNA clone of PDCoV strain JS2021-LX using a bacterial artificial chromosome (BAC) system and generated a recombinant virus (rPDCoV-ΔNS6-EGFP) by replacing NS6 gene with an EGFP reporter via CRISPR/Cas9. In vitro analyses revealed that rPDCoV-ΔNS6-EGFP exhibited attenuated replication kinetics and formed smaller plaque compared to both the parental PDCoV and rescued PDCoV (rPDCoV). Furthermore, we observed that infection with rPDCoV-ΔNS6‑EGFP restored the host innate immune response, which was otherwise suppressed by PDCoV. Critically, piglet challenge experiments demonstrated that NS6 deletion significantly reduced PDCoV pathogenicity, as evidenced by the absence of clinical symptoms, diminished intestinal lesions, and markedly lower viral shedding and antigen load. Additionally, immunological experiment in Mice confirmed that rPDCoV-ΔNS6-EGFP retained robust immunogenicity, inducing virus-specific IgG, neutralizing antibodies, and cytokines (IL-4 and IFN-γ). These findings identify NS6 as a critical virulence factor for PDCoV and support the potential of rPDCoV-ΔNS6-EGFP as a safe and immunogenic live-attenuated vaccine candidate.

Functional monoclonal antibodies (mAbs) are crucial for the development of effective control and therapeutic strategies against lumpy skin disease (LSD), a cattle disease caused by Lumpy skin disease virus (LSDV) that results in significant economic losses. In this study, the soluble recombinant ORF117 protein of LSDV was expressed in 293F cells. It was then employed as an immunogen to generate two mAbs, designated 11C4 and 28B5. Bio-layer interferometry measurements indicated high binding affinities, with equilibrium dissociation constants (Kd) values of 0.05 nM for 11C4 and 0.8 nM for 28B5. Indirect ELISA revealed half-maximal effective concentration (EC50) value of 6.69 × 10-3μg/mL for 11C4 and 11.22 × 10-3μg/mL for 28B5. Immunofluorescence assays confirmed that both mAbs recognized the native viral ORF117. Competition ELISA demonstrated that the mAbs preferentially binded to viral particles rather than to recombinant ORF117 in a co-incubation system. Neutralization assays showed that 11C4 achieved 45.1 % neutralization of LSDV infection, while 28B5 exhibited comparable activity. Epitope mapping indicated both mAbs recognized novel distinct epitopes. 11C4 targeted the epitope (IFPGDDDETNERNINHREKT) with loop structure, whereas 28B5 bound to a novel distinct region (KKIINERYSNYISIDDDEISDILKDSFISNEEMQI) with helix structure. Structural docking simulations revealed that 11C4 recognized a conserved segment IFPGDDDET among Chordopoxvirinae subfamily, with Asp12 and Glu14 identified as critical residues forantibody binding. This study provided valuable tools to advance the development of diagnostic and therapeutic measures against LSDV and related poxviruses.

BACKGROUND:Bovine coronavirus (BCoV) is an economically significant pathogen that causes respiratory and enteric infections in cattle and wild ruminants. Although multiple diagnostic methods are available, there is still an urgent need for a sensitive real-time reverse transcription quantitative PCR (RT-qPCR) assay capable of detecting continuously emerging novel strains of BCoV. OBJECTIVE:This study aimed to develop a sensitive and broad-spectrum RT-qPCR assay for the efficient detection of BCoV. METHODS:Following optimization and comparison of two primer-probe sets targeting the M gene, an RT-qPCR assay was established. Using Decaro's method as a reference, gradient dilutions of transcribed RNA solutions and BCoV-derived cDNA were prepared to test sensitivity, repeatability, and reproducibility. Additionally, four other bovine viruses were used to evaluate specificity. Finally, the diagnostic sensitivity and specificity of both assays were analyzed using 46 field samples. RESULTS:A sensitive and broad-spectrum RT-qPCR assay was developed in this study. Its analytical sensitivity was 10 copies/μL with transcribed RNA as the template, comparable to that of Decaro's method. While the cDNA of an emerging Chinese viral isolate was used as the template, the analytical sensitivity was one order of magnitude higher than that of Decaro's method. Reproducibility testing revealed intra-assay coefficients of variation (CV) ranging from 1.75 to 3.56%, and inter-assay CV values between 3.13 and 4.91%. When evaluating the diagnostic sensitivity and specificity with 46 field samples, our assay exhibited higher diagnostic sensitivity compared to Decaro's method, a difference explained by two mutations within the primer and probe regions of Decaro's method. CONCLUSION:A highly sensitive and broad-spectrum RT-qPCR assay was successfully developed for the detection of BCoV, especially for the detection of emerging novel strains. HIGHLIGHTS:The RT-qPCR assay developed in this study exhibited high sensitivity, particularly in detecting BCoV field strains and newly emerging variants circulating in recent years.

ObjectivesThe aim of the present study was to evaluate the sedative and echocardiographic effects of dexmedetomidine (DEX) administered via intranasal (IN) and intramuscular (IM) routes in cats.MethodsThis randomised, blinded crossover study involved eight healthy adult cats. Cats were randomly allocated to receive DEX 10 μg/kg via either the IN or IM routes. Sedation, mechanical nociception and muscle relaxation were subjectively assessed and physiological variables recorded at baseline and at 5 min intervals for up to 40 mins after drug delivery. Echocardiography was performed 15 mins after delivery.ResultsIn both treatment groups, sedation assessment scores significantly increased compared with baseline values (P <0.05). At 25-35 mins after delivery, only the IN group exhibited a significant decrease in mechanical nociception scores compared with baseline (P = 0.041, P = 0.042, P = 0.026). DEX delivery via both routes resulted in significant reductions in pulse rate (P <0.05). In the IM group, mean arterial blood pressure measurements 35-40 mins after delivery were significantly lower than baseline (P = 0.012, P = 0.012). Fractional shortening significantly decreased in both the IN and IM groups compared with baseline (P = 0.016 and P = 0.049, respectively). Both routes caused reductions in cardiac systolic function, with no significant difference between the two routes. Vomiting occurred in half of the IN group (4/8) and in all cats of the IM group (8/8), with a significantly lower incidence in the IN group (P = 0.046).Conclusions and relevanceIN delivery of-DEX provided comparable sedation, increased tolerance to mechanical nociception and muscle relaxation effects while causing fewer adverse effects than IM-DEX. Both routes similarly reduced cardiac contractile function. Thus, IN-DEX at a dose of 10 μg/kg is a viable alternative to IM-DEX for sedation in healthy cats.

Programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), function as pivotal immune checkpoints. Numerous studies have demonstrated the association between the malignant progression of various swine diseases and aberrant expression of PD-1 and PD-L1, hence the development and screening of high-affinity porcine PD-1 and PD-L1 monoclonal antibodies (mAbs) holds substantial significance for advancing research and therapeutic interventions. In this study, we produced porcine PD-1 and PD-L1 mAbs which exhibited robust reactivity in western blot (WB), indirect immunofluorescence assay (IFA), and flow cytometry (FCM). The newly identified B-cell epitope 90GRDPRFHVTPL100 and 185REEKLFNVTST195 of PD-1 and PD-L1 mAbs were linear and surface-exposed as illustrated by WB and structure analysis. Comparative sequence analysis demonstrated that the PD-L1 epitope is highly conserved across species, whereas the PD-1 epitope exhibits lower interspecies conservation. In addition, the blocking efficacy of the two PD-1 mAbs and six PD-L1 mAbs was predicted low via molecular docking. To further evaluate the blocking efficacy, we generated a flow cytometry-based assay by using a porcine PD-L1-rabbit Fc fusion protein, expressed via a eukaryotic system. In agreement with the prediction, our in vitro data demonstrated a blocking rate below 4% compared with the IgG group for PD-1 mAbs and PD-L1 mAbs. In summary, we herein generated porcine PD-1 and PD-L1 mAbs recognizing unreported B-cell epitopes, and established a reliable method for identifying the nonblocking mAb and epitopes, which may facilitate the development of novel diagnostic approaches and therapeutic agents.

Porcine circovirus type 3 (PCV3) has been identified worldwide and is associated with reproductive and systemic diseases, yet the dynamics of PCV3 within pig farms remain unclear. Building upon our previous study, which initialised comparisons of different sample types for the detection of PCV3 in a sow farm, this study expanded both the range of sample types and the timeline of sampling in piglets and sows to better understand the PCV3 dynamics. This study collected two additional sample types—oropharyngeal swab (OS) and oral fluid (OF) along with placental umbilical cord (PUC) blood and processing fluid (PF) that were used in the previous study. Data were collected from July to August and October 2022; the aforementioned four sample types from 51 litters were collected, and additional OS samples were collected from two to three identified piglets per litter on days 1, 7, 14, and 21 post‐farrowing. Besides, blood swabs were taken from 135 sows subject to both PCR test and oestrogen measurement. PF showed the highest detection rates (50/51), while OS and OF revealed 33/51 (95% confidence interval [CI]: 51.2%–76.8%) and 37/51 (95% CI: 59.5%–83.5%) detection rates; both were higher than that of PUC blood (22/51, 95% CI: 30.2%–56.8%). Despite the similarity between OS and OF samples, they did not identify the same population as infected, as the agreement between the samples was only fair at 90% level. The Bayesian generalised linear mixed model suggested PCV3 was more likely to be detected in both OS and OF compared to PUC blood, and PCV3 was present in the farrowing room throughout the pre‐weaning period using an OS. Finally, we observed higher PCV3 detection rates in sows after farrowing; however, no evidence was found that such a pattern was associated with the decreased concentration of oestrogen.

Tibet orbivirus (TIBOV) is an orbivirus transmitted by mosquitoes and Culicoides, despite specific neutralizing antibodies being detected in pigs, but the molecular genetic characteristics of TIBOV strains in infected pigs are completely uncharted, and their pathogenicity in piglets is poorly elucidated. This study aimed to investigate the genetic characteristics of TIBOV in infected pigs and evaluate the pathogenicity of TIBOV in weaned piglets. Through viral metagenomic sequencing, seven segments (VP1-VP4, VP6, NS1, and NS2) of TIBOV were obtained from swine tissues, and the sequences showed high identity with TIBOVs isolated from Culicoides, mosquitos, and cattle. After infection with TIBOV, the body temperature, appetite, and behavior of the piglets were normal, whereas hemorrhage nodes were observed on the hooves of all piglets and on the abdominal skin of one pig. Viremia was first detected at 2 days postinfection (dpi), peaked at 6 dpi, and remained high until 21 dpi. The virus was distributed in multiple organs, and the highest viral load and strongest viral nucleic acid signals were observed in the spleen. The most severe lesion was observed in the spleen with white pulp atrophy, a decreased number of lymphocytes, and widened septa of the medullary cord, indicating that the spleen was the most important target organ of TIBOV infection. The levels of inflammatory cytokines, including interleukin (IL)-18, tumor necrosis factor-α (TNF-α), interferon (IFN)-α, and IFN-λ3 in peripheral blood lymphocytes decreased significantly from 2 to 6 dpi, and interferon-stimulated gene-15 (ISG-15) and IFN regulatory factor 7 (IRF-7) expression levels declined significantly from 2 to 9 dpi, suggesting that the host immune response was inhibited within 6 dpi. Our findings confirmed that TIBOV elicited long-term viremia with mild clinical symptoms in piglets, the spleen was the target organ of TIBOV proliferation, and the host immune response may be slightly inhibited in the early stage of viral infection.

Porcine epidemic diarrhea virus (PEDV), a highly virulent enteric coronavirus, induces severe watery diarrhea and mortality in suckling piglets. The spike (S) protein, a critical mediator of viral entry, undergoes extensive N-linked glycosylation. To elucidate the functional significance of these post-translational modifications, we employed a reverse genetics system to generate 19 recombinant PEDV strains with single-site mutations at predicted N-glycosylation sites. In vitro experiments revealed that mutations at residues N118, N216, N726, N1232, and N1249 significantly attenuated viral replication and reduced plaque size. Our data demonstrated that these mutations impaired viral attachment and internalization. Importantly, in vivo pathogenicity assays in piglets indicated that the N1232Q and N1249Q mutants presented minimal faecal viral shedding and no clinical symptoms, suggesting their potential as live attenuated vaccine candidates. These findings underscore the critical role of S protein glycosylation in PEDV infectivity and virulence, providing a molecular basis for rational vaccine design.

Porcine Sapelovirus (PSV) is widely prevalent in pig herds throughout the world and induces diarrhea, encephalomyelitis, respiratory tract symptoms, and reproductive disorders. However, the epidemiological and genetic evolution characteristics of PSV remain unclear in Yunnan Province. In this study, 1622 fecal samples were collected from pig farms in Yunnan Province. PSV and its co-infection rates with other pathogens were detected; then, the PSV VP1 gene was amplified and sequenced; and the genetic evolution characteristics of the VP1 gene were analyzed. The overall infection rate of PSV in Yunnan Province was 36.50%, and the differences among regions were significant (p < 0.05). The positive rates among different seasons were significantly different (p < 0.01), ranging from 73.33% (autumn) to 19.00% (summer). The PSV positive rate in diarrhea samples (47.26%) was significantly higher (p < 0.001) than that of non-diarrhea samples (31.77%). The co-infection rates of PSV with porcine rotavirus (PoRV) and PSV with porcine epidemic diarrhea virus (PEDV) were 5.07% and 3.04%. A total of 36 VP1 sequences were obtained, and the average identity among the 36 sequences was 85.3%, which was higher than that with other reference strains. Phylogenetic analysis revealed that all 36 PSV strains belonged to the PSV-1 genotype. The VP1 gene was under strong negative selection pressure (average dN/dS = 0.0838); however, the 95th amino acid was under positive selection pressure. Our study revealed the epidemiological, co-infection, and genetic evolution characteristics of PSV in pig herds of Yunnan Province, providing more data for preventing and controlling diarrhea pathogens in pigs.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that causes significant economic losses in many Asian and European countries. It is characterized by lethal watery diarrhea and high mortality rate in piglets. Serum amyloid P component (SAP), a member of acute response phase protein (APP) family, has been reported to play a crucial role in innate immune response against various microbial pathogens. However, its antiviral activities are little known. In this study, the antiviral activity of SAP during PEDV infection was investigated. In virus-infected IPEC cells, it was found that SAP expression was significantly upregulated. To study the role of SAP in PEDV replication, the expression of SAP was regulated in cells using eukaryotic expression plasmids expressing the SAP protein and sgRNA. PEDV replication was then assessed through real-time PCR, Western blotting, and TCID50 assays. The result showed that PEDV replication was inhibited in cells overexpressing SAP and promoted in cells with SAP knocking out. To further investigate the mechanism by which SAP inhibits PEDV replication, Interferon Beta (IFN-β) and its related signaling pathway proteins were detected. The results demonstrated that SAP activates the promoter of (IFN-β) and IFN regulatory factor 3 (IRF3) mediated by Toll-Like Receptor 4 (TLR4) signaling. During PEDV infection, SAP enhances TLR4-mediated IFN-β signaling, leading to increased IFN-β expression, which subsequently suppresses PEDV replication. By using TBK1/IKBKE inhibitor MRT67307 in PEDV-infected cells, the antiviral activity of SAP was inhibited. This suggests that the antiviral effect of SAP may rely on the activation of the TBK1/IKBKE signaling pathway, which is critical for the induction of type I interferons and other antiviral responses. Moreover, the interaction between SAP and PEDV N protein and the functional domain of SAP were investigated. From the results of this study, it can be concluded that the interaction between SAP and PEDV N protein activates the TLR4-mediated IFN signaling pathway, thereby inhibiting PEDV replication.

Bordering cities and farms serve as potential hotspots for the transboundary spread of animal infectious agents. This study aimed to investigate the presence and genetic variability of porcine circovirus types 2 (PCV2) and 3 (PCV3) in live markets across six border cities (Dandong, Ji'an, Hunchun, Mishan, Fuyuan, and Heihe) in northeast China. Samples from pork (n = 44), cutting boards (n = 46), and meat stall floors (n = 42) were collected in 46 meat stalls. Quantitative PCR analysis detected PCV2 in 75.0% (95% CI: 59.7%-86.8%) of pork samples, 73.9% (95% CI: 58.9%-85.7%) of cutting board swabs, and 64.3% (95% CI: 48.0%-78.4%) of meat stall floor swabs. For PCV3, the detection rates in pork, cutting board swabs, and meat stall floor swabs were 31.8% (95% CI: 18.6%-47.6%), 67.4% (95% CI: 52.0%-80.5%), and 54.7% (95% CI: 38.7%-70.2%), respectively. Subsequent sequencing of positive samples identified five open reading frame (ORF)2 sequences of PCV2 from markets in Dandong, Fuyuan, and Hunchun, with one sequence from Dandong shared 99.4% homology with a Russia sequence. Similarly, five ORF2 sequences of PCV3 were obtained from samples in Hunchun, Heihe, and Ji'an, including a sequence from Hunchun showing 99.6% homology to a sequence from a pig farm in Changchun in Jilin Province. These findings suggest that border market pork trade may contribute to the introduction and dissemination of PCV2 and PCV3. The observed genetic similarities highlight potential transboundary transmission routes, emphasizing the need for active surveillance and control measures to mitigate the risks associated with the transboundary transmission of emerging swine pathogens.

ABSTRACT Porcine epidemic diarrhea virus (PEDV), a highly pathogenic enteric coronavirus, has caused significant economic losses worldwide in recent years. The PEDV spike (S) protein has been reported to undergo extensive N-glycosylation, suggesting that glycosylation plays a crucial role in PEDV replication. In this study, we demonstrated that the N-glycosylation pathway promotes PEDV replication by facilitating the glycosylation of the S protein. First, we observed that pharmacological inhibition of host N-glycosylation using specific inhibitors significantly reduces viral replication. Furthermore, genetic ablation of STT3A or STT3B, the catalytically active subunits of the oligosaccharyltransferase (OST) complex, revealed that the STT3B-OST complex, but not STT3A, is preferentially required for PEDV replication. Notably, we showed that the N-glycosylation of the PEDV S protein depends on the oligosaccharyltransferase activity of STT3B. Together, the study demonstrated the critical role of the N-glycosylation pathway in PEDV replication by elucidating the relationship between the N-glycosylation of the PEDV S protein and STT3B, thereby presenting a potential new target for the prevention and control of PEDV. IMPORTANCE The highly N-glycosylated spike protein of porcine epidemic diarrhea virus (PEDV) is a multifunctional protein that plays a crucial role in the viral replication cycle. In this study, using pharmacological inhibitors, we demonstrated the importance of the N-glycosylation pathway in PEDV replication. Genetic analysis revealed that STT3B, one of the catalytically active subunits of the oligosaccharyltransferase complex, promotes viral proliferation by regulating the N-glycosylation of the PEDV spike protein. Our findings enhance the understanding of the role of the N-glycosylation pathway in viral infection and identify STT3B as a potential therapeutic target for controlling PEDV infection.

Objectives The aim of the study was to evaluate the effects of trazodone on sedation, and physiological and echocardiographic variables in healthy cats. Methods This randomised, blinded, crossover study involved eight healthy adult cats receiving either a placebo or oral doses of trazodone (50 mg, 75 mg, 100 mg), with a washout period of at least 1 week between doses. Sedation, muscle relaxation and analgesia scores were assessed, along with physiological variables including systolic blood pressure (SBP), pulse rate (PR) and respiratory rate (RR) at baseline (T0) and at 30-min intervals after administration (T30–T240). Echocardiographic variables were measured at T0 and T90. Results In the trazodone groups, cats’ sedation scores significantly increased compared with T0, with no significant changes in muscle relaxation or analgesia scores. A significant mean reduction of 22 ± 7 mmHg in SBP was observed only at T150 after oral administration of 100 mg trazodone compared with the placebo, but the SBP still remained within the reference interval. Across all trazodone doses, PR showed no significant changes, while RR significantly decreased compared with T0. There were no significant changes in echocardiographic variables after administration of three different doses of trazodone. Conclusions and relevance Oral administration of 50 mg, 75 mg or 100 mg of trazodone in cats produces mild sedation but there is a lack of muscle relaxation and analgesic effects. Trazodone has minimal effects on SBP, PR and RR in cats, although the 100 mg dose may cause a slight decrease in SBP within the physiological interval. Furthermore, oral trazodone at the tested doses has no impact on echocardiographic variables.

Porcine circovirus type 3 (PCV3) has been detected in wild boars across many countries in Europe, Asia, and South America. However, data regarding the presence of porcine circoviruses in wild boars and ticks remain limited. In this study, we investigated the presence and genetic characteristics of PCV3 in wild boars and parasitizing ticks in Jiangsu, China. Samples, including whole blood, serum, tissues, feces, and oral fluids from wild boars, as well as ticks collected from 47 wild boars, were obtained between March 2021 and November 2022. PCR results indicated that 34.0% (16/47) of wild boars tested positive for PCV3, while ELISA detected 41.9% (18/43) seropositivity. RT-qPCR results showed that 7.2% (6/83) were positive for PCV3 in 83 analyzed tick samples, with all positive samples identified as Amblyomma testudinarium. The PCV3 genome obtained from wild boars was classified as PCV3a and was closely related to the strain identified in domestic pigs in Nanjing, Jiangsu Province. Collectively, these findings confirm the presence of PCV3 in wild boars in Jiangsu and suggest a possible link of PCV3 infection among domestic pigs, wild boars, and ticks, providing new insights into the transmission risk of PCV3 at wildlife–livestock–human interfaces and highlighting the genetic homology between strains from wild and domestic pigs.

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus that causes severe intestinal disease in piglets, employs sophisticated strategies to subvert host antiviral immunity. While type III interferons (IFN-III) play a pivotal role in mucosal defense at intestinal epithelial barriers, the mechanisms underlying PEDV evasion of IFN-III signaling remain poorly understood. Given that peroxisomes serve as critical platforms for IFN-III signaling and that their abundance influences immune activation, we investigated the role of pexophagy in PEDV-mediated immune evasion. We demonstrated that the PEDV nonstructural protein NSP8 functions as a potent inhibitor of mitochondrial antiviral-signaling protein (MAVS)-dependent IFN-III production. Functional analyses revealed that NSP8 significantly reduces peroxisomal protein levels and promotes pexophagy. Mechanistically, mass spectrometry identified a direct interaction between NSP8 and PEX13. NSP8 also induces dose-dependent degradation of PEX13 via the autophagy-lysosomal pathway. This downregulation of PEX13 triggers ubiquitination of the peroxisomal import receptor PEX5, facilitating its recognition by the autophagy receptor NBR1 and the ubiquitin ligase PEX2, thereby promoting autophagic clearance of peroxisomes. Collectively, our findings reveal a novel immune evasion strategy in which PEDV exploits NSP8 to disrupt peroxisome homeostasis by targeting PEX13, thereby dismantling MAVS-dependent IFN-III antiviral signaling through pexophagy. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) NSP8 is a highly conserved protein that plays a crucial role in viral replication. Investigating the functional mechanisms of NSP8 contributes to a deeper understanding of PEDV pathogenesis and supports the development of antiviral strategies against coronaviruses. In this study, we elucidate how NSP8 suppresses type Ⅲ interferon (IFN-Ⅲ) production by promoting pexophagy through the downregulation of PEX13. We demonstrate that NSP8 directly interacts with PEX13 and enhances the ubiquitination of PEX5, leading to reduced peroxisome abundance and impaired mitochondrial antiviral-signaling protein (MAVS)-mediated IFN-Ⅲ signaling. These findings suggest that NSP8 hijacks the PEX13-dependent pexophagy pathway as a means of evading host antiviral defenses. This work provides critical insights into the interplay between viral proteins and host cellular machinery and highlights the NSP8-PEX13 axis as a promising target for therapeutic interventions aimed at enhancing antiviral immunity against PEDV and related coronaviruses.

ObjectiveTo investigate the effect of three different doses of oral pregabalin on minimum alveolar concentration of isoflurane (MACISO) in cats.Study designProspective, randomized, placebo controlled, blinded, crossover trial.AnimalsA group of eight healthy adult cats (24–48 months old).MethodsCats were randomly assigned to three oral doses of pregabalin (low dose: 2.5 mg kg-1, medium dose: 5 mg kg-1, high dose: 10 mg kg-1) or placebo 2 hours before MACISO determination, with the multiple treatments administered with a minimum 7 day washout period. Anesthesia was induced and maintained with isoflurane in oxygen until endotracheal intubation was achieved, and maintained with isoflurane with volume-controlled ventilation. MACISO was determined in triplicate using the bracketing technique and tail clamp method 120 minutes after pregabalin or placebo administration. Physiologic variables (including heart rate and blood pressure) recorded during MACISO determination were averaged and compared between the pregabalin and placebo treatments. One-way analysis of variance and the Friedman test were used to assess the difference for normally and non-normally distributed data, respectively. The Tukey test was employed as a post hoc analysis. Values of p < 0.05 were considered significant.ResultsThe MACISO with the medium and high dose pregabalin treatments were 1.33 ± 0.21% and 1.23 ± 0.17%, respectively. These were significantly lower than MACISO after placebo treatment (1.62 ± 0.13%; p = 0.014, p < 0.001, respectively), representing a decrease of 18 ± 9% and 24 ± 6%. The mean plasma pregabalin concentration was negatively correlated with MACISO values. Physiologic variables did not differ significantly between treatments.Conclusion and clinical relevanceDoses of 5 or 10 mg kg-1 pregabalin, given orally 2 hours before determining MACISO, had a significant isoflurane-sparing effect in cats.

Objectives The aim of this study was to evaluate the efficacy of a single dose of oral pregabalin (PGB) for sedation and its impact on physiological and echocardiographic variables in healthy cats.Methods This study was a randomised, blinded, crossover trial. Eight cats were randomly assigned to receive PGB or placebo, with a 1-week washout period between each administration. Cats in the treatment group received oral PGB at varying doses (low dose: 2.5 mg/kg, medium dose: 5 mg/kg, high dose: 10 mg/kg). Systolic blood pressure (SBP), pulse rate (PR), respiratory rate (RR) and sedation score were measured at intervals of 30 mins after administration. Echocardiography was performed 120 mins after administration.Results Oral administration of PGB 2.5 mg/kg and 5 mg/kg significantly increased sedation scores starting at 150 mins, while 10 mg/kg PGB showed a significant increase in sedation scores starting at 120 mins compared with placebo. PGB 5 mg/kg and 10 mg/kg resulted in a significant reduction in SBP compared with placebo, with minimal impact on PR and RR. In addition, PGB 10 mg/kg resulted in significant changes in the peak velocity of late diastolic transmitral flow (A) and the ratio of peak velocity of early diastolic transmitral flow and A; however, these changes were of marginal clinical significance.Conclusions and relevance A single dose of oral PGB could cause mild to moderate sedation. Hypotension was more prevalent in the PGB 5 mg/kg and 10 mg/kg groups among the majority of cats, but it was less frequently observed in the PGB 2.5 mg/kg group.