First Report of Endemic Frog Virus 3 (FV3)-like Ranaviruses in the Korean Clawed Salamander (Onychodactylus koreanus) in Asia.

Frog virus 3 (FV3) in the genus Ranavirus of the family Iridoviridae causes mass mortality in both anurans and urodeles worldwide; however, the phylogenetic origin of FV3-like ranaviruses is not well established. In Asia, three FV3-like ranaviruses have been reported in farmed populations of amphibians and reptiles. Here, we report the first case of endemic FV3-like ranavirus infections in the Korean clawed salamander Onychodactylus koreanus, caught in wild mountain streams in the Republic of Korea (ROK), through whole-genome sequencing and phylogenetic analysis. Two isolated FV3-like ranaviruses (Onychodactylus koreanus ranavirus, OKRV1 and 2) showed high similarity with the Rana grylio virus (RGV, 91.5%) and Rana nigromaculata ranavirus (RNRV, 92.2%) but relatively low similarity with the soft-shelled turtle iridovirus (STIV, 84.2%) in open reading frame (ORF) comparisons. OKRV1 and 2 formed a monophyletic clade with previously known Asian FV3-like ranaviruses, a sister group of the New World FV3-like ranavirus clade. Our results suggest that OKRV1 and 2 are FV3-like ranaviruses endemic to the ROK, and RGV and RNRV might also be endemic strains in China, unlike previous speculation. Our data have great implications for the study of the phylogeny and spreading routes of FV3-like ranaviruses and suggest the need for additional detection and analysis of FV3-like ranaviruses in wild populations in Asian countries.

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.

Prevalence of Ranavirus Infection in Three Anuran Species across South Korea.

To cope with amphibian die-offs caused by ranavirus, it is important to know the underlying ranavirus prevalence in a region. We studied the ranavirus prevalence in tadpoles of two native and one introduced anuran species inhabiting agricultural and surrounding areas at 49 locations across eight provinces of South Korea by applying qPCR. The local ranavirus prevalence and the individual infection rates at infected locations were 32.6% and 16.1%, respectively, for Dryophytes japonicus (Japanese tree frog); 25.6% and 26.1% for Pelophylax nigromaculatus (Black-spotted pond frog); and 30.5% and 50.0% for Lithobates catesbeianus (American bullfrog). The individual infection rate of L. catesbeianus was significantly greater than that of D. japonicus. The individual infection rate of P. nigromaculatus was related to the site-specific precipitation and air temperature. The individual infection rate gradually increased from Gosner development stage 39, and intermittent infection was confirmed in the early and middle developmental stages. Our results show that ranavirus is widespread among wild amphibians living in agricultural areas of South Korea, and mass die-offs by ranavirus could occur at any time.

The Effect of Largemouth Bass Virus on Bass Populations in Kansas Impoundments.

Largemouth Bass virus (LMBV) first became a concern in Kansas when it was identified as a potential cause of decreased catch rates at Crawford State Fishing Lake in 2007. The discovery of LMBV in eight additional impoundments from 2008 to 2017 increased concern about the prevalence and effects of LMBV in Kansas. In response, the Kansas Department of Wildlife, Parks, and Tourism tested 25 Largemouth Bass Micropterus salmoides impoundments for the presence of LMBV. The objectives of this study were to quantify the incidence of LMBV and examine differences in population metrics (i.e., body condition, relative abundance, and growth). A total of 1,260 Largemouth Bass were collected by using standard spring electrofishing surveys, and sagittal otoliths were collected from all of the sampled fish to estimate growth rates. Of the 25 study impoundments, 14 tested positive for LMBV. There was no evidence of LMBV effects on body condition, relative abundance of quality-length fish, or growth rates. The initial dates of LMBV infection of Largemouth Bass in these impoundments are unknown. The LMBV-positive populations in Kansas may have been exposed to the virus many years ago, and the fish may be in the process of rebounding from any potential negative effects.

High pathogen prevalence in an amphibian and reptile assemblage at a site with risk factors for dispersal in Galicia, Spain.

Ranaviruses are agents of disease, mortality and population declines in ectothermic vertebrates and emergences have been repeatedly linked to human activities. Ranaviruses in the common midwife toad ranavirus lineage are emerging in Europe. They are known to be severe multi-host pathogens of amphibians and can also cause disease in reptiles. Recurrent outbreaks of ranavirus disease and mortality affecting three species have occurred at a small reservoir in north-west Spain but no data were available on occurrence of the pathogen in the other amphibian and reptile species present or at adjacent sites. We sampled nine species of amphibians and reptiles at the reservoir and nearby sites and screened for ranavirus presence using molecular methods. Our results show infection with ranavirus in all nine species, including first reports for Hyla molleri, Pelophylax perezi, Rana iberica, and Podarcis bocagei. We detected ranavirus in all four local sites and confirmed mass mortality incidents involving Lissotriton boscai and Triturus marmoratus were ongoing. The reservoir regularly hosts water sports tournaments and the risks of ranavirus dispersal through the translocation of contaminated equipment are discussed.

GEOGRAPHIC AND INDIVIDUAL DETERMINANTS OF IMPORTANT AMPHIBIAN PATHOGENS IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS) IN TENNESSEE AND ARKANSAS, USA.

Wildlife diseases are a major threat for species conservation and there is a growing need to implement disease surveillance programs to protect species of concern. Globally, amphibian populations have suffered considerable losses from disease, particularly from chytrid fungi (Batrachochytrium dendrobatidis [Bd] and Batrachochytrium salamandrivorans [Bsal]) and ranavirus. Hellbenders (Cryptobranchus alleganiensis) are large riverine salamanders historically found throughout several watersheds of the eastern and midwestern US. Populations of both subspecies (Ozark hellbender, Cryptobranchus alleganiensis bishopi; eastern hellbender, Cryptobranchus alleganiensis alleganiensis) have experienced precipitous declines over at least the past five decades, and emerging pathogens are hypothesized to play a role. We surveyed Ozark hellbender populations in Arkansas (AR) and eastern hellbender populations in Middle Tennessee (MTN) and East Tennessee (ETN) for both chytrid fungi and ranavirus from swabs and tail tissue, respectively, from 2011 to 2017. Overall, we detected Bd on hellbenders from nine out of 15 rivers, with total prevalence of 26.7% (54/ 202) that varied regionally (AR: 33%, 28/86; MTN: 11%, 4/36; ETN: 28%, 22/80). Ranavirus prevalence (9.0%, 18/200) was comparatively lower than Bd, with less regional variation in prevalence (AR: 6%, 5/ 85; MTN: 11%, 4/36; ETN: 10%, 8/79). We did not detect Bsal in any hellbender populations. We detected a significant negative correlation between body condition score and probability of ranavirus infection (ß=-0.13, SE=0.06, 95% confidence interval: -0.24, -0.02). Evaluation of infection load of positive individuals revealed different trends than prevalence alone for both ranavirus and Bd, with MTN having a significantly greater average ranaviral load than both other regions. We documented a variety of lesions that likely have multiple etiologies on hellbenders located within all geographic regions. Our data represent a multiyear pathogen dataset across several regions of C. alleganiensis, and we emphasize the need for continued pathogen surveillance.

Detection of the Amphibian Pathogens Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in West Texas, USA, Using Environmental DNA.

Environmental DNA (eDNA) methods provide novel options for the detection of pathogens. The amphibian pathogens Batrachochytrium dendrobatidis (Bd) and Ranavirus have been relatively understudied in Texas, US, so we applied eDNA assays for the surveillance of these pathogens in the upper Brazos River basin near the Texas panhandle. We collected water samples from five urban playa lakes and one reservoir in and around Lubbock, Texas. Quantitative PCR detected both Bd and Ranavirus at one playa lake, representing novel detection of both pathogens in the region. Based on these results, we recommend increased monitoring for the pathogens and symptoms of amphibian disease throughout the region.

Pathogen Surveillance and Detection of Ranavirus (Frog virus 3) in Translocated Gopher Tortoises (Gopherus polyphemus).

Emerging pathogens may pose additional threats to already vulnerable populations of chelonians, such as gopher tortoises (Gopherus polyphemus). In response to a mortality event on a translocation site in northwest Florida, US during 2013-15, 13 gopher tortoises were necropsied and their tissues were screened for 12 pathogens, including Mycoplasma agassizii, Mycoplasma testudineum, and Frog virus 3-like ranavirus (FV3). The DNA of FV3 was detected via quantitative PCR in the gastrointestinal tract of three tortoises. Subsequently, pathogen surveillance was performed on whole blood and oral-cloacal swab samples of live translocated tortoises from two different enclosures within the site (n=68), rehabilitated tortoises from the site (n=18), and tortoises prior to release on site (n=35) during 2015-17. Mycoplasma spp. were present in all groups and years of live tortoises tested. The DNA of FV3 was detected in 15 individuals both with and without clinical signs of disease in 2016. We recaptured 20 tortoises and captured an additional 20 tortoises in 2017 for surveillance, yet FV3 DNA was no longer detected, even in those that had previously tested positive (n=7). The results of this study contribute to the epidemiology of ranavirus in chelonians and suggests that gopher tortoises could be reservoirs for FV3. We recommend that the status of Ranavirus infection should be included for health screens for gopher tortoises in translocation programs.

Characterization of ranaviruses isolated from lumpfish Cyclopterus lumpus L. in the North Atlantic area: proposal for a new ranavirus species (European North Atlantic Ranavirus).

The commercial production of lumpfish Cyclopterus lumpus L. is expanding with the increased demand for their use as cleaner fish, to control sea-lice numbers, at marine Atlantic salmon Salmo salar L. aquaculture sites throughout Northern Europe. A new ranavirus has been isolated from lumpfish at multiple locations in the North Atlantic area. First isolated in 2014 in the Faroe Islands, the virus has subsequently been found in lumpfish from Iceland in 2015 and from Scotland and Ireland in 2016. The Icelandic lumpfish ranavirus has been characterized by immunofluorescent antibody test, optimal growth conditions and transmission electron microscopy. Partial sequences of the major capsid protein gene from 12 isolates showed 99.79-100% nt identity between the lumpfish ranaviruses. Complete genome sequencing from three of the isolates and phylogenetic analysis based on the concatenated 26 iridovirus core genes suggest these lumpfish ranavirus isolates form a distinct clade with ranaviruses from cod Gadus morhua L. and turbot Scophthalmus maximus L. isolated in Denmark in 1979 and 1999, respectively. These data suggest that these viruses should be grouped together as a new ranavirus species, European North Atlantic Ranavirus, which encompasses ranaviruses isolated from marine fishes in European North Atlantic waters.

CHARACTERIZING THE EPIDEMIOLOGY OF HISTORIC AND NOVEL PATHOGENS IN BLANDING'S TURTLES (EMYDOIDEA BLANDINGII).

Pathogens such as herpesviruses, Mycoplasma spp., and frog virus 3-like ranavirus have contributed to morbidity and mortality in many species of free-living and zoo-maintained chelonians. However, their prevalence is understudied in Blanding's turtles (Emydoidea blandingii) across North America. To assess the presence of these pathogens, Blanding's turtles were sampled in Lake County, Illinois, in 2017 (N = 213) and 2018 (N = 160). DNA from cloacal-oral swabs was assayed for four ranaviruses, three Mycoplasma spp., two Salmonella spp., Emydoidea herpesvirus 1 (EBHV1), and tortoise intranuclear coccidiosis (TINC) using a multiplex quantitative polymerase chain reaction (qPCR). Pathogens were most frequently detected in adult turtles (n = 25) and rarely in subadults (n = 2) or juveniles (n = 1). EBHV1 was detected in 22 individuals with no clinical signs of illness, most (n = 20) occurring in the month of May (P < 0.0001). EBHV1 cases at one study site significantly clustered within the same 0.64-km area from 17 to 22 May 2017 (P < 0.0001) and 14 to 15 May 2018 (P = 0.0006). Individuals were rarely positive for Salmonella typhimurium (n = 6). A novel Mycoplasma sp. sharing high homology with other emydid Mycoplasma spp. was detected in one turtle with nasal discharge. Neither TINC nor any ranaviruses were detected. Continued monitoring of this population and habitat may facilitate identification of risk factors for pathogen occurrence and clarify the impact of infectious diseases on Blanding's turtle conservation outcomes.

Genome analysis of Ranavirus frog virus 3 isolated from American Bullfrog (Lithobates catesbeianus) in South America.

Ranaviruses (family Iridoviridae) cause important diseases in cold-blooded vertebrates. In addition, some occurrences indicate that, in this genus, the same virus can infect animals from different taxonomic groups. A strain isolated from a Ranavirus outbreak (2012) in the state of Sao Paulo, Brazil, had its genome sequenced and presented 99.26% and 36.85% identity with samples of Frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV) ranaviruses, respectively. Eight potential recombination events among the analyzed sample and reference FV3 samples were identified, including a recombination with Bohle iridovirus (BIV) sample from Oceania. The analyzed sample presented several rearrangements compared to FV3 reference samples from North America and European continent. We report for the first time the complete genome of Ranavirus FV3 isolated from South America, these results contribute to a greater knowledge related to evolutionary events of potentially lethal infectious agent for cold-blooded animals.

Isolation and Characterization of a Ranavirus Associated with Disease Outbreaks in Cultured Hybrid Grouper (♀ Tiger Grouper Epinephelus fuscoguttatus × â™‚ Giant Grouper E. lanceolatus) in Guangxi, China.

An outbreak of suspected iridovirus disease in cultured hybrid grouper (♀Tiger Grouper Epinephelus fuscoguttatus × â™‚ Giant Grouper Epinephelus lanceolatus) occurred in the Guangxi Province in July, 2018. In this study, grouper iridovirus Guangxi (SGIV-Gx) was isolated from diseased hybrid grouper that were collected from Guangxi. Cytopathic effects were observed and identified in grouper spleen cells that were incubated with diseased tissue homogenates after 24 h, and the effects increased at 48 h postinfection. The transmission electron microscopy results showed that viral particles that were about 200 nm in diameter with hexagonal profiles were present in the cell cytoplasm of suspected virus-infected cells. The presence of SGIV-Gx (accession number: MK107821) was identified by polymerase chain reaction (PCR) and amplicon sequencing, which showed that this strain was most closely related to Singapore grouper iridovirus (AY521625.1). The detection of SGIV-Gx infection was further supported by novel aptamer (Q2c)-based detection technology. The effects of temperature and pH on viral infectivity were analyzed by using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and cell culture. The results indicated that SGIV-Gx was resistant to exposure to pH levels 5, 7, and 7.5 for 1 h, but its infectivity was remarkably lower at pH levels 3 and 10 after 1 h. The analyses showed that SGIV-Gx was stable for 1 h at 4°C and 25°C but was inactivated after 1 h at 40, 50, and 60°C.

Dose-dependent morbidity of freshwater turtle hatchlings, Emydura macquarii krefftii, inoculated with Ranavirus isolate (Bohle iridovirus, Iridoviridae).

Ranaviral infections cause mass die-offs in wild and captive turtle populations. Two experimental studies were performed to first determine the susceptibility of an Australian turtle species (Emydura macquarii krefftii) to different routes of infection and second examine the effect of viral titre on the morbidity in hatchlings. All inoculation routes (intracoelomic, intramuscular and oral) produced disease, but the clinical signs, histopathology and time to onset of disease varied with the route. The median infectious and lethal doses for intramuscularly inoculated hatchlings were 102.52 (1.98-2.93) and 104.43 (3.81-5.19) TCID50 ml-1, respectively. Clinical signs began 14 to 29 days post-inoculation and the median survival time was 22 days (16-25) across all dose groups. For every 10-fold increase in dose, the odds of developing any clinical signs or severe clinical signs increased by 3.39 [P<0.01, 95 % confidence interval (CI): 1.81-6.36] and 3.71 (P<0.01, 95 % CI: 1.76-7.80), respectively. Skin lesions, previously only reported in ranaviral infection in lizards, were observed in the majority of intramuscularly inoculated hatchlings that developed ranaviral disease. The histological changes were consistent with those in previous reports for reptiles and consisted of necrosis at or near the site of injection, in the spleen, liver and oral cavity. Systemic inflammation was also observed, predominantly affecting necrotic organs. The estimates reported here can be used to model ranaviral disease and quantify and manage at-risk populations.

Rapid detection of giant salamander iridovirus by cross-priming amplification.

Giant salamander iridovirus (GSIV) belongs to the epizootic genus Ranavirus, and is the cause of epidemic diseases associated with high mortality and great losses to artificial breeding and farming. Here, we established a simple, accurate, and reliable cross-priming amplification (CPA) method to detect GSIV. The CPA assay targets the major caspid protein gene of the GSIV genome to design crossing primer pairs, and the reaction conditions were optimized, including optimal concentrations of the primers, betaine, dNTPs, Mg2+, and Bst DNA polymerase, and reaction conditions. The sensitivity was shown to be 10 times higher than that of conventional polymerase chain reaction (PCR), and the specificity was 100%. The results were identified on nucleic acid strips within 3-5 min. Application of the CPA and PCR to 54 samples of giant salamander showed a positive rate of 72.22% and 74.07%, respectively, demonstrating high coincidence (94.44%, kappa = 8.7, P < 0.0001). The sensitivity of the CPA assay was 97.50% and the specificity was 92.86%. Thus, the CPA assay is as effective as conventional PCR, but with added practical advantages of simplicity and an almost instrument-free platform, which will be useful for both laboratories and giant salamander farms.

Modelling Ranavirus Transmission in Populations of Common Frogs (Rana temporaria) in the United Kingdom.

Ranaviruses began emerging in common frogs (Rana temporaria) in the United Kingdom in the late 1980s and early 1990s, causing severe disease and declines in the populations of these animals. Herein, we explored the transmission dynamics of the ranavirus(es) present in common frog populations, in the context of a simple susceptible-infected (SI) model, using parameters derived from the literature. We explored the effects of disease-induced population decline on the dynamics of the ranavirus. We then extended the model to consider the infection dynamics in populations exposed to both ulcerative and hemorrhagic forms of the ranaviral disease. The preliminary investigation indicated the important interactions between the forms. When the ulcerative form was present in a population and the hemorrhagic form was later introduced, the hemorrhagic form of the disease needed to be highly contagious, to persist. We highlighted the areas where further research and experimental evidence is needed and hope that these models would act as a guide for further research into the amphibian disease dynamics.

eDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species' activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Discovery of Wild Amphibians Infected with Ranavirus in Brazil.

Ranavirus is a double-stranded DNA virus associated with amphibian, fish and reptile die-offs worldwide. International trade of live animals farmed for human consumption, such as the American bullfrog (Lithobates catesbeianus), plays a key role in spreading the pathogen. In Brazil, ranavirus has only been reported in captive bullfrog farms. We found infected tadpoles of both native species and the American bullfrog in the wild, and a case of mass mortality of amphibians and fish potentially associated with ranavirus. Dead animals presented skin ulcerations, hemorrhages, and edemas. We also found an overall prevalence of 37% of the amphibian chytrid in the area, and two bullfrog tadpoles were co-infected with both pathogens. We suggest that the interaction between the two pathogens should be investigated to improve global conservation of ectothermic vertebrates.

Geographic Distribution of Epizootic haematopoietic necrosis virus (EHNV) in Freshwater Fish in South Eastern Australia: Lost Opportunity for a Notifiable Pathogen to Expand Its Geographic Range.

Epizootic haematopoietic necrosis virus (EHNV) was originally detected in Victoria, Australia in 1984. It spread rapidly over two decades with epidemic mortality events in wild redfin perch (Perca fluviatilis) and mild disease in farmed rainbow trout (Oncorhynchus mykiss) being documented across southeastern Australia in New South Wales (NSW), the Australian Capital Territory (ACT), Victoria, and South Australia. We conducted a survey for EHNV between July 2007 and June 2011. The disease occurred in juvenile redfin perch in ACT in December 2008, and in NSW in December 2009 and December 2010. Based on testing 3622 tissue and 492 blood samples collected from fish across southeastern Australia, it was concluded that EHNV was most likely absent from redfin perch outside the endemic area in the upper Murrumbidgee River catchment in the Murray⁻Darling Basin (MDB), and it was not detected in other fish species. The frequency of outbreaks in redfin perch has diminished over time, and there have been no reports since 2012. As the disease is notifiable and a range of fish species are known to be susceptible to EHNV, existing policies to reduce the likelihood of spreading out of the endemic area are justified.

Genomic sequence of a Bohle iridovirus strain isolated from a diseased boreal toad (Anaxyrus boreas boreas) in a North American aquarium.

Genomic sequence analysis of zoo ranavirus (ZRV) suggests it is a strain of Bohle iridovirus (BIV), a virus that was first detected in, and thought to be confined to, Australia. Furthermore, marked sequence similarity and genomic co-linearity among ZRV, BIV, and German gecko ranavirus (GGRV) are consistent with the view that all three are strains  of Frog virus 3, the type species of the genus Ranavirus, family Iridoviridae.

Survey of Ranavirus and Batrachochytrium dendrobatidis in Introduced Frogs in Hawaii, USA.

Ranaviruses and the fungus Batrachochytrium dendrobatidis are globally important agents of emerging infectious amphibian diseases. Amphibians on Oahu, the Hawaiian Island with the greatest potential for disease introduction through the movement of goods and people, have never been surveyed for ranaviruses or B. dendrobatidis. We surveyed all five species of frogs on Oahu, Hawaii, US for these pathogens. Of 325 individuals sampled from six sites, none were positive for ranavirus. However, we found B. dendrobatidis in a total of four individuals of three species, the cane toad (Bufo marinus), the American bullfrog (Rana catesbeiana), and the greenhouse frog (Eleutherodactylus planirostris), but not in the green and black poison dart frog (Dendrobates auratus) or the Japanese wrinkled frog (Rana rugosa). The apparent lack of ranavirus and low prevalence of B. dendrobatidis are noteworthy given how widespread these pathogens are in terms of both global distribution and host range. Surveillance should continue to document any changes in B. dendrobatidis prevalence or the arrival of ranaviruses in Hawaii.