Coinfection caused by various genotypes of porcine epidemic diarrhea virus (PEDV) is a new disease situation. We previously reported the coexistence of PEDV strains containing different ORF3 genotypes in China. In this study, the PEDV strains 17GXCZ-1ORF3d and 17GXCZ-1ORF3c were isolated and plaque-purified from the same piglet, which had a natural large deletion at the 172–554 bp position of the ORF3 gene or possessed a complete ORF3 gene, respectively. Meanwhile, 17GXCZ-1ORF3d had >99% nt identity with 17GXCZ-1ORF3c in the 5′UTR, ORF1a/1b, S, E, M, N and 3′UTR regions but only demonstrated low nucleotide identities (80.5%) in the ORF3 gene. To elucidate the pathogenicity, 7-day-old piglets were infected. Piglets infected with these two PEDV strains exhibited severe clinical signs and shed the virus at the highest level within 96 hpi. Compared with the piglets inoculated with the 17GXCZ-1ORF3c strain, the piglets inoculated with the 17GXCZ-1ORF3d strain had higher mortality rates (75% vs. 50%), an earlier onset of clinical signs with a significantly higher diarrhea score, lower VH:CD ratios and a higher percentage of PEDV-positive enterocytes. This study is the first to report PEDV coinfections with different ORF3 genotypes, and a PEDV strain with a large deletion in the ORF3 gene might have the advantage of a potential genetic marker, which would be useful during vaccine development.

Background: Clonorchiasis, an infectious disease caused by the liver fluke Clonorchis sinensis, may lead to the development of liver and gallbladder diseases, and even cholangiocarcinoma (CCA). However, the pathogenesis, host-pathogen interaction, and diagnostic markers for clonorchiasis remain unclear. Methods: Eighteen rabbits were randomly divided into control group (n = 9) and C. sinensis-infected group (n = 9), and their plasma samples were collected at 7, 14, 28, and 63 days post-infection (dpi). Biochemical indices and metabolites in different infection periods were detected. A non-targeted ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was employed to investigate the metabolic profiles of plasma in rabbits, and related metabolic pathways of differential metabolites and correlation between candidate biochemical indices and differential metabolites were analyzed. Finally, the candidate biomarkers were verified with human samples using a targeted metabolomics method. Results: The result of biochemical indices indicated C. sinensis infection would affect the liver function biochemical indices, especially alanine aminotransferase, aspartate transaminase (AST), glutamyl transpeptidase (GGT), total bile acid, high-density lipoprotein, and cholinesterase. The metabonomic results showed that 58, 212, 23, and 21 differential metabolites were identified in different phases of the infection. Multivariate statistical analysis of differential metabolites revealed distinct metabolic signatures during different phases of infection, with most of these signatures being observed at 14 dpi, which mainly influences the amino acid metabolisms. For metabolites and biochemical indices, AST, GGT, hypoxanthine, l-pipecolic acid, and d-glucuronate represented potential noninvasive biomarkers for the diagnosis of C. sinensis (P < 0.05 and AUC > 0.8). Furthermore, GGT and d-glucuronate levels were positively correlated with the infection (r(28) = 0.98, P < 0.0001) and showed excellent diagnostic performance (AUC = 0.972; 95% confidence interval, 0.921 to 1.000). Conclusions: The present results provide new insights into plasma metabolic changes in rabbits during C. sinensis infection, and the potential biomarker may be used for developing an effective method to diagnose clonorchiasis in the future. Graphical

Background: Fish-borne zoonotic clonorchiasis, caused by Clonorchis sinensis, is an emerging public health problem in several countries with more than 15 million people infected globally. However, a lack of accurate point-of-care (POC) diagnostic tests in resource-limited areas is still a critical barrier to effective treatment and control of clonorchiasis. The development of the recombinase polymerase amplification(RPA) assay, a POC diagnostic test based on the amplification of pathogen DNA, has provided a new, simple and inexpensive tool for disease detection with high sensitivity and specificity. Methods: A novel RPA method was developed based on specific primers and probes, and combined with the dipstick, to allow for the rapid and intuitive detection of C. sinensis through the amplification of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. The lower limit of detection for the combined RPA/lateral flow dipstick (RPA-LFD) assay was evaluated using dilutions of the target DNA sequence. Cross-reactivity was evaluated using genomic DNA from 10 additional control parasites. Forty human clinical stool samples were tested to verify its performance. Results: The evaluated primers designed from the C. sinensisCOX1 region can be used to detect adult worms, metacercariae, and eggs at 39 °C within 20 min, and the results can be visually observed using the LFD. The detection limit of pathogen genomic DNA was as low as 10 fg, and the number of metacercaria(e) in fish and egg(s) in faeces were both as low as one. This improved the sensitivity of low-infection detection tremendously. The test is species-specific, and no other related control parasites were detected. In human stool samples with eggs per gram (EPG) > 50, the RPA-LFD assay was performed consistent with conventional Kato-Katz (KK) and PCR methods. Conclusion: The established RPA-LFD assay provides a powerful tool for the diagnosis and epidemiological survey of C. sinensis from human and animal samples, and has important implications for the effective control of clonorchiasis. Graphical

Background: Myocardial ischemia–reperfusion (IR) injury is a critical complication in patients with myocardial infarction following the restoration of coronary blood flow. Echinococcus granulosus cyst fluid protein antigen B ( Eg AgB) is known to modulate host immune response. This study investigates whether the recombinant antigen B subunit 2 (r Eg AgB8/2) can mitigate IR injury by suppressing inflammation and promoting myocardial tissue repair. Methods: An IR injury model was induced by ligating the left anterior descending (LAD) coronary artery for 1 h, followed by 24 h reperfusion. Recombinant Eg AgB8/2 protein expressed in E. coli was administered intraperitoneally at the onset of reperfusion to evaluate its therapeutic potential. After 24 h of reperfusion, cardiac function and injury were assessed using echocardiography, TTC/Evans Blue staining, and histopathological analysis. Protein and mRNA levels of cytokines (TNF-α, IL-6, IL-10, and TGF-β) in sera and cardiac tissue were quantified using ELISA and RT-qPCR, respectively. In vitro studies, cytokine secretion in bone marrow-derived macrophages (BMDMs) culture supernatants was measured by ELISA. Macrophage polarization was evaluated via flow cytometry by identifying surface markers for M1 (CD86⁺) and M2 (CD206⁺) phenotypes. Expression levels of proteins in the cGAS/STING/p-TBK1/p-IRF3 signaling pathway in both myocardial tissues and BMDMs were detected by Western blotting. Results: Treatment with r Eg AgB8/2 significantly improved cardiac function and reduced myocardial injury markers (LDH, CK-MB) and infarct size in mice with IR injury. Compared to untreated IR controls, r Eg AgB8/2 treatment significantly reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in both serum and cardiac tissue, and downregulated the M1 macrophage marker iNOS in cardiac tissue. Concurrently, it increased the expression of anti-inflammatory cytokines (IL-10 and TGF-β) in serum and cardiac tissue, along with upregulating the M2 macrophage marker Arg-1 in cardiac tissue. This reduction in the inflammatory response in IR-injured cardiac tissue was associated with enhanced polarization toward the M2 macrophage phenotype. In vitro studies further showed that r Eg AgB8/2 promotes M2 macrophage polarization associated with reduced activation of the cGAS/STING/TBK1/IRF3 signaling pathway. Conclusions: r Eg AgB8/2 promotes macrophage polarization toward M2 phenotype in a manner associated with suppression of the cGAS/STING/TBK1/IRF3 signaling pathway to attenuate myocardial IR injury and associated inflammation. It provides a novel therapeutic strategy for ischemia–reperfusion injury and related inflammatory diseases.