A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida, USA

Abstract

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

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

“…However, a large proportion of suspected viral encephalitis cases remain idiopathic, which prevents understanding the true impact on animal populations and collections (4, 16). Random sequencing has provided an unbiased mechanism to detect viruses, resulting in the identification of arenavirus encephalitis in human (17), astrovirus encephalitis in cattle (18), and the recently discovered turtle fraservirus-1 encephalitis in freshwater turtles (19).…”

Piscichuvirus-Associated Severe Meningoencephalomyelitis in Aquatic Turtles, United States, 2009–2021

“…The associated neuroparenchyma was vacuolated, and some neurons exhibited central chromatolysis ( Figure 2 , panel C). Subsequent results from ancillary testing (i.e., Ziehl-Neelsen staining [turtles 2–4] or PCR for herpesvirus [turtle 1] [ 24 ] and turtle fraservirus 1 [turtle 4] [ 4 ]) were negative. Turtles 1–3 were used for metagenomic sequencing; turtles 1–6, along with 6 control turtles, were used for ISH.…”

“…In addition to anthropogenic threats, infectious agents also negatively affect free-ranging turtles. For example, chelonid herpesvirus 5 ( Scutavirus chelonidalpha5 : Alphaherpesvirinae ) is associated with transmissible fibropapillomatosis in sea turtles around the world ( 3 ), and epizootic outbreaks of meningoencephalitis in Florida freshwater turtles have been attributed to the recently discovered turtle fraservirus 1 ( Fraservirus testudinis : Tosoviridae ) ( 4 ). However, learning about infectious agents in such turtles is complicated by their aquatic nature and cryptic lifestyles, which prevents full appreciation of the threat posed by viruses to free-ranging turtles.…”

ICTV Virus Taxonomy Profile: Tosoviridae 2023

“…The family Tosoviridae includes the genus Fraservirus and the species Fraservirus testudinis for viruses that infect turtles. Tosovirids are most closely related to, but distinct from, viruses in the subphyla Haploviricotina and Polyploviricotina [1,4] (Fig. 2).…”

“…Each segment contains two ORFs in ambisense orientation (). The S segment encodes an NP and a GPC; the L segment encodes an L protein with an RdRP domain, and a Z protein [1]. Superficially, this organization is reminiscent of that of arenavirid genomes [3].…”