Phylogenomic Characterization of Ranavirus Isolated from Wild Smallmouth Bass (Micropterus dolomieu).

In September 2021, 14 smallmouth bass (SMB; Micropterus dolomieu) with skin lesions were collected from Green Bay waters of Lake Michigan and submitted for diagnostic evaluation. All the skin samples tested positive for largemouth bass virus (LMBV) by conventional PCR. The complete genome of the LMBV (99,328 bp) isolated from a homogenized skin sample was determined using an Illumina MiSeq sequencer. A maximum likelihood (ML) phylogenetic analysis based on the 21 core iridovirus genes supported the LMBV isolated from SMB (LMBV-WVL21117) as a member of the species Santee-Cooper ranavirus. Pairwise nucleotide comparison of the major capsid protein (MCP) gene showed that LMBV-WVL21117 is identical to other LMBV reported from the United States and nearly identical to doctor fish virus and guppy virus 6 (99.2%) from Southeast Asia, as well as LMBV isolates from China and Thailand (99.1%). In addition, ML phylogenetic analysis based on the MCP gene suggests three genotypes of LMBV separated by region: genotype one from the United States, genotype two from Southeast Asia, and genotype three from China and Thailand. Additional research is needed to understand the prevalence and genetic diversity of LMBV strains circulating in wild and managed fish populations from different regions.

Mycobacterium kansasii Infection in a Farmed White-Tailed Deer (Odocoileus virginianus) in Florida, USA.

A 7-year-old farmed white-tailed deer doe was transported to a Levy County, Florida property and began to decline in health, exhibiting weight loss and pelvic limb weakness. The doe prematurely delivered live twin fawns, both of which later died. The doe was treated with corticosteroids, antibiotics, gastric cytoprotectants, and B vitamins but showed no improvement. The doe was euthanized, and a post mortem examination was performed under the University of Florida's Cervidae Health Research Initiative. We collected lung tissue after the animal was euthanized and performed histological evaluation, using H&E and Ziehl-Neelsen (ZN) staining, and molecular evaluation, using conventional PCR, followed by Sanger sequencing. The microscopic observations of the H&E-stained lung showed multifocal granuloma, while the ZN-stained tissue revealed low numbers of beaded, magenta-staining rod bacteria inside the granuloma formation. Molecular analysis identified the presence of Mycobacterium kansasii. This isolation of a non-tuberculous Mycobacterium in a white-tailed deer emphasizes the importance of specific pathogen identification in cases of tuberculosis-like disease in farmed and free-ranging cervids. We report the first case of M. kansasii infection in a farmed white-tailed deer (Odocoileus virginianus) in Florida. Although M. kansasii cases are sporadic in white-tailed deer, it is important to maintain farm biosecurity and prevent farmed cervids from contacting wildlife to prevent disease transmission.

Genomic characterization of two ranavirus isolates identified from a gopher frog (Lithobates capito) and a striped newt (Notophthalmus perstriatus) during a mass mortality event in Florida.

Two ranavirus isolates were recovered from anuran and salamander samples collected during an amphibian mass mortality event in North-Central Florida in 2021. Phylogenetic analyses of the full genomes confirmed that the two isolates were nearly identical and strains of the species Frog virus 3.

Coding-complete genomic sequence of a rhinovirus C-32 in a human nasal swab sample that tested false positive in a SARS-CoV-2 antigen test.

Rhinovirus-A was previously shown to cause false-positive results in a Japanese SARS-CoV-2 antigen test. We report that a false-positive result was obtained in a specimen with rhinovirus C-32 that had been tested using an American SARS-CoV-2 antigen test.

Diagnosis, isolation and description of a novel amnoonvirus recovered from diseased fancy guppies, Poecilia reticulata.

The guppy, Poecilia reticulata, is one of the most common cultured ornamental fish species, and a popular pet fish highly desired by hobbyists worldwide due to its availability of many brilliantly coloured fish of many varieties. The susceptibility of guppies to diseases presents a remarkable concern for both breeders and hobbyists. In this study, we report the emergence of disease in fancy guppies caused by a previously uncharacterized virus in the USA. This virus was isolated from moribund guppies in two separate outbreaks in California and Alabama, from December 2021 to June 2023. The infected guppies presented with acute morbidity and mortality shortly after shipping, displaying nonspecific clinical signs and gross changes including lethargy, anorexia, swimming at the water surface, gill pallor, mild to moderate coelomic distension and occasional skin lesions including protruding scales, skin ulcers and hyperaemia. Histological changes in affected fish were mild and nonspecific; however, liver and testes from moribund fish were positive for Tilapia lake virus (TiLV), the single described member in the family Amnoonviridae, using immunohistochemistry and in situ hybridization, although the latter was weak. A virus was successfully recovered following tissue inoculation on epithelioma papulosum cyprini and snakehead fish cell lines. Whole genome sequencing and phylogenetic analyses revealed nucleotide and amino acid homologies from 78.3%-91.2%, and 78.2%-97.7%, respectively, when comparing the guppy virus genomes to TiLV isolates. Based on the criteria outlined herein, we propose the classification of this new virus, fancy tailed guppy virus (FTGV), as a member of the family Amnoonviridae, with the name Tilapinevirus poikilos (from the Greek 'poikilos', meaning of many colours; various sorts, akin to 'poecilia').

A novel bluetongue virus serotype 2 strain isolated from a farmed Florida white-tailed deer (Odocoileus virginianus) arose from reassortment of gene segments derived from co-circulating serotypes in the Southeastern USA.

Bluetongue disease is a reportable animal disease that affects wild and farmed ruminants, including white-tailed deer (WTD). This report documents the clinical findings, ancillary diagnostics, and genomic characterization of a novel reassortant bluetongue virus serotype 2 (BTV-2) strain isolated from a dead Florida farmed WTD in 2022. Our analyses support that this BTV-2 strain likely stemmed from the acquisition of genome segments from co-circulating BTV strains in Florida and Louisiana. In addition, our analyses also indicate that genetically uncharacterized BTV strains may be circulating in the Southeastern USA; however, the identity and reassortant status of these BTV strains cannot be determined based on the VP2 and VP5 genome sequences. Hence, continued surveillance based on complete genome characterization is needed to understand the genetic diversity of BTV strains in this region and the potential threat they may pose to the health of deer and other ruminants.

Ranavirus (Frog Virus 3) Infection in Free-Living Three-Toed Box Turtles (Terrapene mexicana triunguis) in Missouri, USA.

Frog virus 3 (FV3) and related ranaviruses are emerging infectious disease threats to ectothermic vertebrate species globally. Although the impact of these viruses on amphibian health is relatively well studied, less is understood about their effects on reptile health. We report two cases of FV3 infection, 11 mo apart, in three-toed box turtles (Terrapene mexicana triunguis) from a wildlife rehabilitation center. Case 1 had upper respiratory signs upon intake but had no clinical signs at the time of euthanasia 1 mo later. Case 2 presented for vehicular trauma, had ulcerative pharyngitis and glossitis, and died overnight. In case 1, we detected FV3 nucleic acid with qPCR in oral swabs, kidney, liver, spleen, and tongue. In case 2, we detected FV3 in an oral swab, an oral plaque, heart, kidney, lung, liver, spleen, and tongue. We also detected FV3 nucleic acid with in situ hybridization for case 2. For both cases, FV3 was isolated in cell culture and identified with DNA sequencing. Histopathologic examination of postmortem tissue from case 1 was unremarkable, whereas acute hemorrhagic pneumonia and splenic necrosis were noted in case 2. The difference in clinical signs between the two cases may have been due to differences in the temporal course of FV3 disease at the time of necropsy. Failure to detect this infection previously in Missouri reptiles may be due to lack of surveillance, although cases may also represent a novel spillover to box turtles in Missouri. Our findings reiterate previous suggestions that the range of FV3 infection may be greater than previously documented and that infection may occur in host species yet to be tested.

Emerging threat of ranavirus: prevalence, genetic diversity, and climatic drivers of Ranavirus (Iridoviridae) in ectothermic vertebrates of Asia.

Introduction: Ranavirus disease, caused by viruses within the genus Ranavirus (Iridoviridae), is considered a globally emerging infectious disease linked to mass mortality events in both wild and cultured ectothermic vertebrates. Surveillance work is, however, limited in Asia hence prevalence and the dynamics of the disease remain poorly understood. To understand disease burden and the potential biotic and abiotic drivers in southern China region, we conducted a systematic surveillance of the ranavirus across Guangxi Zhuang Autonomous region (GAR). Methods: For this, we used a multifaceted approach involving screening of amphibians and other potential hosts, diagnostic tests, phylogenetic analyses, prevalence estimation, co-infection assessments, and climatic niche analyses. Over one thousand individuals were sampled across 25 sampling sites. Results: We found ninety-two individuals from 18 species of ectothermic vertebrates to be infected with ranavirus. Two lineages were responsible - Rana nigromaculata ranavirus and tiger frog virus were identified using phylogenetic analysis based on the major capsid protein (MCP) gene fragment. Out of these two lineages, the presence of tiger frog virus is rare as we came across only one case. We also found evidence of a co-infection with ranavirus and Batrachochytrium dendrobatidis that can be highly detrimental to host populations; possibly the first such documentation in Asia. Our niche modelling analysis suggests that precipitation seasonality plays an important role in ranavirus prevalence in GAR - southwestern, southeastern, central and northeastern regions of GAR can be considered to be optimum habitats for ranaviruses. Infection rates in wild frog species have reached 100% in some areas, even in nature reserves. Discussion: Our research also indicates that culture facilities and pet markets are frequently infected, serving as likely vectors for the regional and global spread of ranaviruses. The knowledge generated suggests the need for systematic surveillance, stringent biosecurity measures, and control of international animal trade to prevent further transmission and protection of biodiversity and aquaculture industries across Asia.

Genome sequence of white sturgeon herpesvirus 1 isolated from farmed white sturgeon (Acipenser transmontanus).

The genome sequence of white sturgeon herpesvirus 1, which was isolated from farmed white sturgeon (Acipenser transmontanus), was determined. Comparative analyses suggest the classification of this virus as a new species in a new genus in the family Alloherpesviridae.

Characterization of two novel reassortant bluetongue virus serotype 1 strains isolated from farmed white-tailed deer (Odocoileus virginianus) in Florida, USA.

Hemorrhagic diseases caused by epizootic hemorrhagic disease virus or by bluetongue virus (BTV) are the most important orbivirus diseases affecting ruminants, including white-tailed deer (WTD). Bluetongue virus is of particular concern for farmed WTD in Florida, given its lethality and its wide distribution throughout the state. This study reports the clinical findings, ancillary diagnostics, and genomic characterization of two BTV serotype 1 strains isolated from two farmed WTD, from two different farms in Florida in 2019 and 2022. Phylogenetic and genetic analyses indicated that these two novel BTV-1 strains were reassortants. In addition, our analyses reveal that most genome segments of these strains were acquired from BTVs previously detected in ruminants in Florida, substantiating their endemism in the Southeastern U.S. Our findings underscore the need for additional research to determine the genetic diversity of BTV strains in Florida, their prevalence, and the potential risk of new BTV strains to WTD and other ruminants.

Alphaherpesvirus infection in a free-ranging narwhal Monodon monoceros from Arctic Canada.

We report the detection of an alphaherpesvirus infecting an adult female narwhal Monodon monoceros captured live during a tagging project in Tremblay Sound, Nunavut, Canada, in August 2018. The individual had 2 open wounds on the dorsum but appeared in good overall health. A blowhole swab was collected, and subsequent virus isolation was performed using a beluga whale primary cell line. Non-syncytial cytopathic effects were seen, in contrast to syncytial cytopathic effects described for monodontid alphaherpesvirus 1 (MoAHV1) isolates previously recovered from beluga whales Delphinapterus leucas from Alaska, USA, and the Northwest Territories, Canada. Next-generation sequencing was performed on a sequencing library generated from the DNA of the viral isolate and the analysis of the assembled contigs permitted the recovery of 6 genes, conserved in all members of the family Orthoherpesviridae, for downstream genetic and phylogenetic analyses. BLASTN (basic local alignment search tool, searching nucleotide databases using a nucleotide query) analyses of the narwhal herpesvirus conserved genes showed the highest nucleotide identities to MoAHV1, ranging between 88.5 and 96.8%. A maximum likelihood phylogenetic analysis based on concatenation of the 6 conserved herpesviruses amino acid alignments revealed the narwhal herpesvirus (NHV) to be the closest relative to MoAHV1, forming a clade within the subfamily Alphaherpesvirinae, genus Varicellovirus. NHV is the first alphaherpesvirus characterized from a narwhal and represents a new viral species, which we propose to be known as Varicellovirus monodontidalpha2. Further research is needed to determine the prevalence and potential clinical impacts of this alphaherpesvirus infection in narwhals.

Report of amoebic disease in a colony of Western honey bees (Apis mellifera).

The amoeba Malpighamoeba mellificae is the etiologic agent of amoebic (amoeba) disease of Western honey bees (Apis mellifera). M. mellificae damages the Malpighian tubules, which is believed to weaken and kill the host bee. Here, the authors describe the detection of this organism in a honey bee colony in the Yukon Territory, Canada. The Malpighian tubules of 14% (7/50) of the adult worker bees were discolored dark brown. Fifteen bees screened using conventional polymerase chain reaction for the 18S gene of M. mellificae were positive for the pathogen. Histologically, the lumens of Malpighian tubules were packed with amoebae, causing dilation of the tubules and attenuation and loss of the tubular epithelium. This phylogenetic analysis places M. mellificae in a new clade, a sister group to the Entamoebidae. This work provides a foundation for further investigation into the distribution, prevalence, and pathology associated with M. mellificae infection.

Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus.

Megalocytiviruses (MCVs) are double-stranded DNA viruses known to infect important freshwater and marine fish species in the aquaculture, food, and ornamental fish industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV) is the type species within the genus Megalocytivirus that causes red seabream iridoviral disease (RSIVD) which is a reportable disease to the World Animal Health Organization (WOAH). To better control the transboundary spread of this virus and support WOAH reporting requirements, we developed and partially validated a TaqMan real-time qPCR assay (ISKNV104R) to detect all three genotypes of ISKNV, including the two genotypes that cause RSIVD. Parameters averaged across 48 experiments used a 10-fold dilution series of linearized plasmid DNA (107-101 copies), carrying a fragment of the three-spot gourami iridovirus (TSGIV) hypothetical protein revealed that the assay was linear over 7 orders of magnitude (107-101), a mean efficiency of 99.97 ± 2.92%, a mean correlation coefficient of 1.000 ± 0.001, and a limit of detection (analytical sensitivity) of ≤10 copies of TSGIV DNA. The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was evaluated and compared to other published assays using a panel of 397 samples from 21 source populations with different prevalence of ISKNV infection (0-100%). The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was 91.99% (87.28-95.6; 95% CI) and 89.8% (83.53-94.84). The latent class analysis showed that the ISKNV104R qPCR assay had similar diagnostic sensitivities and specificities with overlapping confidence limits compared to a second TaqMan qPCR assay and a SYBR green assay. This newly developed TaqMan assay represents a partially validated qPCR assay for the detection of the three genotypes of the species ISKNV. The ISKNV104R qPCR assay once fully validated, will serve as an improved diagnostic tool that can be used for ISKNV surveillance efforts and diagnosis in subclinical fish to prevent further spread of MCVs throughout the aquaculture and ornamental fish industries.

Cutaneous ulcerative lesions of unknown etiology affecting lionfish Pterois spp. in the Gulf of Mexico.

OBJECTIVE: Cutaneous ulcerative skin lesions in a complex of invasive Gulf of Mexico lionfish (Red Lionfish Pterois volitans, Devil Firefish P. miles, and the hybrid Red Lionfish × Devil Firefish) became epizootic beginning in mid-August 2017. Herein, we provide the first pathological descriptions of these lesions and summarize our analyses to elucidate the etiology of the disease. METHODS: We examined ulcerated and normal fish through gross pathology and histopathology, bacterial sampling, and unbiased metagenomic next-generation sequencing. We tracked prevalence of the disease, and we used biological health indicators (condition factor, splenosomatic and hepatosomatic index) to evaluate impacts to health, while considering sex and age as potential risk factors. RESULT: Typical ulcerative lesions were deep, exposing skeletal muscle, and were bordered by pale or reddened areas often with some degree of scale loss. Only incidental parasites were found in our examinations. Most fish (86%; n = 50) exhibited wound healing grossly and histologically, confirmed by the presence of granulation tissues. A primary bacterial pathogen was not evident through bacterial culture or histopathology. Metagenomic next-generation sequencing did not reveal a viral pathogen (DNA or RNA) but did provide information about the microbiome of some ulcerated specimens. Compared with clinically healthy fish, ulcerated fish had a significantly lower condition factor and a higher splenosomatic index. Disease prevalence at monitored sites through July 2021 indicated that ulcerated fish were still present but at substantially lower prevalence than observed in 2017. CONCLUSION: Although some common findings in a number of specimens suggest a potential role for opportunistic bacteria, collectively our suite of diagnostics and analyses did not reveal an intralesional infectious agent, and we must consider the possibility that there was no communicable pathogen.

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

Viruses from a new species of piscichuvirus were strongly associated with severe lymphocytic meningoencephalomyelitis in several free-ranging aquatic turtles from 3 coastal US states during 2009-2021. Sequencing identified 2 variants (freshwater turtle neural virus 1 [FTuNV1] and sea turtle neural virus 1 [STuNV1]) of the new piscichuvirus species in 3 turtles of 3 species. In situ hybridization localized viral mRNA to the inflamed region of the central nervous system in all 3 sequenced isolates and in 2 of 3 additional nonsequenced isolates. All 3 sequenced isolates phylogenetically clustered with other vertebrate chuvirids within the genus Piscichuvirus. FTuNV1 and STuNV1 shared ≈92% pairwise amino acid identity of the large protein, which narrowly places them within the same novel species. The in situ association of the piscichuviruses in 5 of 6 turtles (representing 3 genera) with lymphocytic meningoencephalomyelitis suggests that piscichuviruses are a likely cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles.

Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Characterization of an alloherpesvirus from wild lake sturgeon Acipenser fulvescens in Wisconsin (USA).

In the spring of 2017, 2 adult lake sturgeon (LS) Acipenser fulvescens captured from the Wolf River, Wisconsin (USA), presented with multiple cutaneous plaques that, upon microscopic examination, indicated proliferative epidermitis. Ultrastructural examination of affected keratinocytes revealed particles in the nucleus having a morphology typical of herpesviruses. A degenerate PCR assay targeting the DNA polymerase catalytic subunit (pol) gene of large double-stranded DNA viruses generated amplicons of the anticipated size from skin samples, and sequences of amplicons confirmed the presence of a novel alloherpesvirus (lake sturgeon herpesvirus, LSHV) related to acipenserid herpesvirus 1 (AciHV1). The complete genome (202660 bp) of this virus was sequenced using a MiSeq System, and phylogenetic analyses substantiated the close relationship to AciHV1. A PCR assay targeting the LSHV DNA packaging terminase subunit 1 (ter1) gene demonstrated the presence of the virus in 39/42 skin lesion samples collected from wild LS captured in 2017-2019 and 2021 in 4/4 rivers in Wisconsin. Future efforts to isolate LSHV in cell culture would facilitate challenge studies to determine the disease potential of the virus.

Characterization of a Novel Reassortant Epizootic Hemorrhagic Disease Virus Serotype 6 Strain Isolated from Diseased White-Tailed Deer (Odocoileus virginianus) on a Florida Farm.

We report an outbreak of a novel reassortant epizootic hemorrhagic disease virus serotype 6 (EHDV-6) in white-tailed deer (WTD) on a Florida farm in 2019. At necropsy, most animals exhibited hemorrhagic lesions in the lung and heart, and congestion in the lung, liver, and spleen. Histopathology revealed multi-organ hemorrhage and congestion, and renal tubular necrosis. Tissues were screened by RT-qPCR and all animals tested positive for EHDV. Tissues were processed for virus isolation and next-generation sequencing was performed on cDNA libraries generated from the RNA extracts of cultures displaying cytopathic effects. Six isolates yielded nearly identical complete genome sequences of a novel U.S. EHDV-6 strain. Genetic and phylogenetic analyses revealed the novel strain to be most closely related to a reassortant EHDV-6 strain isolated from cattle in Trinidad and both strains received segment 4 from an Australian EHDV-2 strain. The novel U.S. EHDV-6 strain is unique in that it acquired segment 8 from an Australian EHDV-8 strain. An RNAscope® in situ hybridization assay was developed against the novel U.S. EHDV-6 strain and labeling was detected within lesions of the heart, kidney, liver, and lung. These data support the novel U.S. reassortant EHDV-6 strain as the cause of disease in the farmed WTD.

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

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.