Porcine epidemic diarrhea virus (PEDV) induces enteritis and diarrhea in piglets. Mitochondrial DNA (mtDNA) contributes to virus-induced inflammatory responses; however, the involvement of inflammasomes in PEDV infection responses remains unclear. We investigated the mechanism underlying inflammasome-mediated interleukin (IL)-1β secretion during the PEDV infection of porcine intestinal epithelial (IPEC-J2) cells. IL-1β production and caspase-1 activity were assessed by quantitative PCR and enzyme-linked immunosorbent assay. NLRP3 inflammasome activation was assessed using immunoprecipitation experiments. Mitochondrial damage was evaluated by analyzing the mitochondrial membrane potential and ATP levels and by the flow cytometry examination of mitochondrial reactive oxygen species (mtROS). Mitochondria and mtDNA localization were observed using immunofluorescence. The inhibition of mtROS and mtDNA production allowed NLRP3 inflammasome and IL-1β expression detection and the evaluation of the pathway underlying NLRP3 inflammasome activation in PEDV-infected IPEC-J2 cells. IPEC-J2 cells upregulated IL-1β upon PEDV infection, where mature IL-1β secretion depended on caspase-1 activity, triggered NLRP3 inflammasome expression and assembly, and caused mitochondrial dysfunction, leading to mtDNA release and NLRP3 inflammasome activation, while mtROS contributed to NF-κB pathway activation, enhancing IL-1β secretion. This is the first demonstration of the mechanism underlying mtDNA release and NLRP3 inflammasome activation facilitating IL-1β secretion from PEDV-infected IPEC-J2 cells. These data enhance our understanding of the inflammatory mechanisms triggered by PEDV.

Feline panleukopenia is an acute, highly contagious, and fatal infectious disease, posing a serious threat to the health and conservation of felids. It is necessary to develop diagnostic tools suitable for point-of-care testing (POCT). Herein, a colorimetric loop-mediated isothermal amplification (LAMP) assay targeting the FPV VP2 gene was developed using cresol red as a colorimetric indicator. The colorimetric LAMP enables result interpretation by visual observation of a color change from violet to yellow after amplification at 64 °C for 40 min. The limit of detection was 18.38 copies/µL for pMD-VP2 plasmid and 10^1.62 TCID₅₀/mL for crudely extracted nucleic acids. This method exhibits no cross-reactivity with other common feline pathogens, and shows good repeatability. Nevertheless, this method is unable to differentiate FPV from canine parvovirus (CPV) owing to their high genetic similarity. Testing of 153 clinical samples revealed a 40.52% (62/153) positive rate, with 98.04% agreement compared to a commercial qPCR kit (42.48% positive rate). Collectively, the developed colorimetric LAMP assay offers a specific, sensitive, simple and visual approach for FPV detection, demonstrating great potential for field point-of-care applications.

Feline herpesvirus type 1 (FHV-1) is one of the major pathogens causing upper respiratory tract disease (URTD) in cats, and is widely prevalent among domestic cats and other felids worldwide. Herein, we developed a closed one-tube colorimetric and fluorescent LAMP assay targeting the FHV-1 TK gene, using cresol red as the colorimetric indicator and EvaGreen as the fluorescent dye. The assay was completed within 40 min at 63 °C, with results readable either by colorimetric observation or fluorescence amplification curve. FHV-1-positive reaction could be confirmed by a color change from violet to yellow with the naked eye, and also could be determined by fluorescence amplification curve. The assay showed no cross-reactivity with other related pathogens, and had a detection limit of 100 copies. Among 87 clinical nasal swabs tested, 44 were positive, achieving an overall agreement of 97.7% compared to TaqMan quantitative PCR. The newly developed LAMP assay is simple, efficient, result-visual and reliable, which can be applied in the clinical diagnosis and epidemiological investigation of FHV-1, especially for point-of-care testing of FHV-1 in primary veterinary clinics and field settings.