Abstract Chuviruses (family Chuviridae ), which are in the recently discovered order Jingchuvirales , were first identified in arthropods in 2015 and have been detected through metagenomics in numerous invertebrates, but only a few vertebrates. With only few metagenomically based detections in vertebrates, their replication competency in vertebrates remained questioned, let alone their pathological significance. This study identified three novel chuviruses as the etiology of lymphocytic meningoencephalomyelitis in three wild aquatic turtles: an alligator snapping turtle ( Macrochelys sp.), a Kemp’s ridley turtle ( Lepidochelys kempii ), and a loggerhead turtle ( Caretta caretta ). The application of random, deep sequencing successfully assembled the complete snapping turtle chuvirus-1 [STCV-1], Kemp’s ridley turtle chuvirus-1 [KTCV-1] genome, and loggerhead turtle chuvirus-1 [LTCV-1]) genome. The STCV-1 and KTCV-1 sequences were used to create custom RNAscope ™ probes for in situ hybridization, which confirmed STCV-1, KTCV-1, and LTCV-1 (cross reactivity to the KTCV-1 probe) RNA within the inflamed region of the brain and spinal cord. STCV-1 and KTCV-1 were isolated on several turtle-origin cell lines. Phylogenetic analysis illustrated that all three viruses clustered with other vertebrate chuviruses, all within the genus Piscichuvirus . With more than 91% pairwise amino acid identity of the polymerase proteins, STCV-1, KTCV-1, and LTCV-1 belong to the same novel species, putatively named Piscichuvirus testudinae. This study demonstrates the first in situ evidence of chuviral pathogenicity in animals and only the second instance of jingchuviral isolation. The association of these chuviruses in three different chelonians with neurologic disease suggests the possibility that chuviruses are a significant, previously unrecognized cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles. Additional studies of these viruses are needed to elucidate their role in chelonians and the possibility of related viruses in other related hosts. Importance Chuviruses have been identified in multiple animal species, including humans. However, most were identified metagenomically, and detection was not strongly associated with disease. This study provides the first evidence of chuviral disease in animals in diseased tissue: mononuclear meningoencephalomyelitis in three chelonians from three different genera, two distinct families. These pathogenic turtle chuviruses belong to the genus Piscichuvirus containing other non-mammalian vertebrate chuviruses and were classified together within a novel chuviral species. This study supports the need for further investigations into chuviruses to understand their biology, pathogenic potential, and their association with central nervous system inflammation in chelonians, other reptiles, and other vertebrates.

Piscichuviral encephalitis in marine and freshwater chelonians: first evidence of jingchuviral disease