A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

Cynoglossus semilaevis papillomavirus (CsPaV) is an emerging viral pathogen associated with high mortality in farmed Chinese tongue soles. A sensitive, specific, and quantitative method is needed for CsPaV surveillance and epidemiological studies. In this study, we designed primers and a probe that targeted the CsPaV L1 gene. We then established a TaqMan probe-based quantitative PCR (qPCR) assay. The assay detected as few as 5.8 × 10¹ copies/µL and was 1000-fold more sensitive than conventional PCR. The assay did not cross-react with the other aquatic pathogens tested. The intra-assay coefficients of variation ranged from 0.23% to 0.84%, and the inter-assay coefficients ranged from 0.56% to 1.48%. These results showed that the assay had good repeatability. We used the assay to study the tissue distribution and temporal dynamics of CsPaV after experimental infection. Healthy Chinese tongue soles received an intraperitoneal injection of 300 µL of inoculum containing 3.66 × 10⁵ CsPaV copies/µL. Three fish were sampled at 1, 2, 3, 5, and 7 days post-infection. CsPaV DNA was detected in all tissues examined. The kidney had the highest viral DNA load, which reached 2.47 × 10⁷ copies/mg at 7 days post-infection. Among 176 clinically diseased Chinese tongue soles collected between 2023 and 2026, 80.68% tested positive for CsPaV. CsPaV DNA was also detected in several other aquatic species. These findings provide a sensitive tool for CsPaV surveillance and molecular epidemiological studies. The assay may also support disease prevention and control in Chinese tongue sole aquaculture.