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.

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.

Porcine epidemic diarrhea virus (PEDV) variant strains lead to vaccine failures and significant economic losses in swine farming, highlighting the need for new natural small-molecule antiviral candidates. In this study, we screened a library of 45 small molecules using a cytopathic effect (CPE)-based cytoprotective assay. Saikosaponin B2 (SSB2), a triterpenoid saponin derived from Bupleuri Radix, emerged as the most promising candidate, boasting a selectivity index of 19.77, which surpasses that of most natural compounds. Dose–response tests confirmed SSB2’s concentration-dependent anti-PEDV activity, with an inhibitory plateau reached at concentrations above 60 μmol/L (μM). The 50 half-maximal effective concentration (EC₅₀) values differed between CPE reduction and viral titer readouts, a discrepancy commonly due to varying detection sensitivities. Mechanistic assays revealed that SSB2 does not possess direct virucidal properties; instead, its antiviral efficacy depends on sustained treatment to inhibit viral adsorption and internalization. Time-of-addition experiments further demonstrated that SSB2 effectively suppresses both early viral entry and mid-stage intracellular genome replication, without affecting viral release. Notably, post-infection treatment resulted in stronger inhibition compared to continuous administration, possibly due to disrupted cellular proliferation homeostasis from prolonged exposure. Additionally, SSB2 maintained strong inhibitory effects against prevalent G2a (JL-06) and G2b (JL-08) field variants, though its efficacy was reduced for both strains and slightly weaker against JL-08 than JL-06. Overall, this study systematically characterizes SSB2’s dual-stage anti-PEDV profile and its broad-spectrum activity against spike-mutant strains, supporting SSB2 as a promising herbal lead for developing veterinary antivirals targeting variant PEDV.