Cross-Species Spillover of Tiger Frog Virus Caused Lethal Systemic Disease in Captive Geochelone sulcata: Etiology, Pathology, and Genomic Characterization

In 2025, a severe mass mortality outbreak struck captive Geochelone sulcata at a zoo in Guangdong Province, China. To trace the causative agent, identify the viral strain and characterize associated pathological lesions, respiratory tract samples collected from diseased tortoises were subjected to a combined technical workflow. Metagenomic high-throughput sequencing was first applied to screen for potential pathogens, followed by virus isolation via cell culture. Transmission electron microscopy (TEM) was used to observe viral morphology. The major capsid protein (MCP) gene was amplified by PCR for molecular identification, and whole-genome sequencing together with phylogenetic analysis was performed to clarify the genetic features of the isolate. The results verified that ranavirus was the primary pathogen responsible for the mortality. Pathological examination demonstrated acute necrosis, hemorrhage and inflammatory infiltration in multiple organs including the liver, spleen, lung, and pancreas. TEM observation revealed typical iridovirus-like particles with an average diameter of approximately 70 nm. Molecular and genomic analyses confirmed the pathogen as Tiger Frog Virus (TFV) of the genus Ranavirus, designated TFV-CN2025, which shared 99.8% nucleotide sequence homology with known TFV reference strains. To our knowledge, this is the first report of lethal TFV infection in G. sulcata, which provides detailed pathological evidence for this cross-species transmission event from amphibian hosts to terrestrial chelonians. Our findings indicate that TFV poses considerable risks to the tortoise breeding industry and ecological security in China. We therefore suggest incorporating TFV detection into routine quarantine and disease surveillance programs for captive tortoises.