Patterns of infection, origins, and transmission of ranaviruses among the ectothermic vertebrates of Asia

Abstract Ranaviral infections, a malady of ectothermic vertebrates, are becoming frequent, severe, and widespread, causing mortality among both wild and cultured species, raising odds of species extinctions and economic losses. This increase in infection is possibly due to the broad host range of ranaviruses and the transmission of these pathogens through regional and international trade in Asia, where outbreaks have been increasingly reported over the past decade. Here, we focus attention on the origins, means of transmission, and patterns of spread of this infection within the region. Infections have been recorded in both cultured and wild populations in at least nine countries/administrative regions, together with mass die‐offs in some regions. Despite the imminent seriousness of the disease in Asia, surveillance efforts are still incipient. Some of the viral strains within Asia may transmit across host–taxon barriers, posing a significant risk to native species. Factors such as rising temperatures due to global climate change seem to exacerbate ranaviral activity, as most known outbreaks have been recorded during summer; however, data are still inadequate to verify this pattern for Asia. Import risk analysis, using protocols such as Pandora+, pre‐border pathogen screening, and effective biosecurity measures, can be used to mitigate introduction of ranaviruses to uninfected areas and curb transmission within Asia. Comprehensive surveillance using molecular diagnostic tools for ranavirus species and variants will help in understanding the prevalence and disease burden in the region. This is an important step toward conserving native biodiversity and safeguarding the aquaculture industry.


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HERATH ET Al. international trade, ranaviruses could contribute to species declines and pose a significant threat to cultured species and wildlife (Brunner et al., 2015).
Here, we first outline the general background of the disease in terms of host range, major outbreaks and severity, population declines and recovery, optimal climate, and diagnostic techniques.
We then assess patterns of infection, origins, and transmission of ranavirus infections in Asia while concentrating specifically on the following: (1) We compile recorded ranavirus infections by country, highlighting clinical presentation in hosts, seasonality, incidences of mass die-off events, whether hosts are wild or cultured species, native or alien, their likely origins, and the diagnostic tools used; (2) we evaluate potential routes of entry of ranavirus to Asia through cultured species; (3) we infer how climate change may affect ranavirus transmission dynamics in Asia; and (4) we present a set of recommendations for inhibiting further ranavirus transmissions to hitherto unaffected parts of Asia, and measures to strengthen biosecurity. We hope thereby to enhance understanding of ranavirus species, evaluate the disease burden, help quantifying risks, and identify host-pathogen interactions, epidemiology, and conservation concerns, in the context of the economic importance of the disease in Asia.

| D ISCOVERY OF R ANAVIRUS E S , HOS T R ANG E , AND OUTB RE AK S
Ranavirus is one of the seven genera within the family Iridoviridae (International Committee on Taxonomy of Viruses, 2021a), a pathogen that infects ectothermic vertebrates such as bony fish, amphibians, and reptiles. The diseases resulting from infections by species of Ranavirus (hereafter, ranaviruses) are characterized by a short incubation period and high mortality (Allender, 2018), affecting the health of both free-ranging and captive populations of hosts. Seven  (Jancovich et al., 2012). Among these, Frog virus 3, the type species of Ranavirus, was discovered originally in northern leopard frogs (Lithobates pipiens; Granoff et al., 1965) and ranaviruses are now known to infect at least 175 species across 52 families of ectothermic vertebrates on all continents except Antarctica (Duffus et al., 2015). The Frog virus 3 group is restricted to amphibian hosts, whereas ATVs are predominantly fish specialists that made a single switch to caudate amphibians (Price et al., 2017). Further, "CMTV-like" ranaviruses may circulate independently in amphibian and fish communities. By 2015, ranaviruses had been reported in at least 105 species of amphibians in 18 families across 25 countries (Duffus et al., 2015).
Most cases of ranavirosis have been recorded in North America, Europe, and Australia, with relatively few reported from Asia and Africa (Duffus et al., 2015).  amphibians have been detected in many different species of anurans and urodeles around the world: 42 amphibians from 9 species (an overall prevalence of 16.6%) tested positive for ranaviruses in Costa Rica (Whitfield et al., 2013); FV3-like ranaviruses in five amphibian species in Europe (Gray & Chinchar, 2015); and FV3 infection in wild three-spine stickleback fish in the eastern continental USA . Despite the seeming host specificity outlined here, ranaviruses can cross taxon barriers.
The ability of ranaviruses to cross species barriers can give rise to catastrophic consequences (Price et al., 2014), especially in small populations where recovery is slow (Earl & Gray, 2014;Price et al., 2014). Ranavirus die-off sites indicate up to 80% declines in common frog abundance in England (Teacher et al., 2010). In addition, at sites where ranavirus die-offs occurred, amphibian recruitment attenuated in consecutive years (Petranka et al., 2003), signifying poor recovery following population declines. Another intriguing pattern in ranavirosis is the variation in mortality ranging from absence to massive die-offs (Gray et al., 2009), where stressors can play an important role.

| ANTHROP OG ENIC EFFEC TS
Environmental and anthropogenic stressors are important in disease outbreaks leading to mass mortality, with the latter playing a dominant role. For instance, the prevalence of ranaviral infection in green frog (Lithobates clamitans) populations increases with proximity to industry and housing, but the underlying mechanisms are unclear (Daszak et al., 2001;St-Amour et al., 2008). Immunosuppressive corticosterone (CORT), wetland water quality deterioration due to cattle, water pollutants from unconventional oil and gas extraction (UOG), and salinity-related stress may also reduce tolerance to infection, thus increasing mortality in amphibians Gray et al., 2007;Hall et al., 2020;Robert et al., 2019).

| S E A SONALIT Y
Another factor influencing ranavirosis is seasonality. The present understanding of the seasonality of ranavirus diseases derives mostly from studies outside of Asia. In amphibians and fish, there is usually a rapid onset of ranaviral epidemics in the mid-to-late Northern Hemisphere summer, while outbreaks among reptiles seem to be irregular. Two distinct patterns of die-offs in farmed and wild populations of amphibians, fish, and reptiles attributed to ranavirus have been observed (Brunner et al., 2015). The first shows rapid onset of die-offs, usually occurring in fish and amphibians during summer months. The second shows variable vulnerability of host populations, as mentioned earlier, ranging from absence of mortality to near-complete die-offs.
An environmental DNA-based study in Wood frogs (L. sylvatica) found that the timing of pathogen introduction did not affect the timing of epidemics or the resulting die-offs, and instead, timing appears to be driven by development and/or temperature-dependent changes in pathogenicity (Hall et al., 2018). Outbreaks of EHNV infecting redfin perch (Perca fluviatilis) in northeastern Victoria, Australia, were recorded in November-December (early summer in Southern Hemisphere) (Langdon et al., 1986). Incidence of LMBV in Largemouth bass die-offs was associated with increased summer temperatures (Grizzle & Brunner, 2003). However, when ranavirus epidemics occur during late spring or summer, they are of shorter duration-only lasting for a few weeks (Green et al., 2002). Broadly, these studies indicate greater disease incidence during the summer months.
Only a few studies report seasonality in ranavirus outbreaks in Asia. However, the few outbreaks recorded so far involved Asian amphibians during summer, mostly in cultured species when mass mortality occurred. These include the following: in exotic North American bullfrog (Lithobates catesbeianus, formerly Rana catesbeiana) larvae in Japan from early September to mid-October ; in cultured Chinese giant salamanders (Andrias davidianus) between February and November Cunningham et al., 2016;Du et al., 2016;Geng et al., 2011); in black-spotted pond frogs (Rana nigromaculata) in March; and in cultured Chinese tiger frog (Hoplobatrachus cf. rugulosus, formerly R. tigrina rugulosa) between May and June (Mu et al., 2018;Weng et al., 2002). However, generalizing seasonality of ranavirosis based on a few studies is inadequate as there may be significant geographic variation and some of these patterns may already be altering due to climatic change.

| R ANAVIRUS INFEC TI ON S AND CLIMATIC CHANG E
Since temperature can influence both the kinetics of host-parasite interactions and act as a host stressor (Altizer et al., 2013), climate change may already be playing a significant role in ranavirosis.
It has been noted that most die-off events caused by ranaviruses begin (and often end) during the summer months (Brunner et al., 2015) and that various ranaviral strains respond differently to temperature. Under laboratory conditions, Short-finned eel ranavirus (SERV) replicated optimally at 20°C (Ariel & Jensen, 2009), while LMBV grew slightly faster at 30°C than at 25°C (Grant et al., 2003).
Higher mortality rates due to ranavirosis are in many cases associated with higher temperatures. For example, Redfin perch and rainbow trout infected with EHNV (Ariel & Jensen, 2009) and common frog tadpoles exposed to ranaviruses (Gray et al., 2009) showed increased mortality with higher temperatures. Although there are a few exceptions (Allender et al., 2018;Rojas et al., 2005), most studies suggest that higher temperatures stress host species allowing ranavirus infections to proliferate (Ariel & Jensen, 2009;Bayley et al., 2013;Echaubard et al., 2014;Price et al., 2019).
Climate projections suggest that global warming will likely play a significant role in shaping future ranavirus disease dynamics in amphibians, altering both geographic extent and length of temporal window for disease risk and severity (Price et al., 2019). Furthermore, unprecedented urbanization in Asia, together with associated temperature increases (Song et al., 2003;Wang, Zhang, et al., 2014;Wang, Ji, et al., 2014), may exacerbate ranaviral outbreaks in the region.

| D IAG NOS TI C TE S TS
Earlier methods for detection of ranaviruses included electron microscopy (EM), histopathology, and cytology, and newer approaches include virus isolation, antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA), immunohistochemistry (IHC), and PCR (Miller et al., 2015). In both conventional and real-time PCR (qPCR), the major capsid protein (MCP) gene, neurofilament triplet H1 protein, DNA polymerase, and an intergenic variable region can be used as targets (Holopainen et al., 2009;Hyatt et al., 2000;Jancovich et al., 2005;Mao et al., 1997). PCR is currently the commonly used approach for ranavirus detection.

| R ANAVIRUS INFEC TI ON S IN A S IA
Ranaviruses have been recorded in several countries in the region.
The first case of ranavirus infection in Asia was reported in the cultured pig frog (Lithobates grylio, formerly Rana grylio) in China (Zhang et al., 1996(Zhang et al., , 2001. Only a few countries carry out surveillance, and the preponderance of monitoring has been confined to species that are cultured either for food or for the pet industry. The limited intensity of surveillance implies a high probability of ranaviruses infecting wild species undetected (Kwon et al., 2017;Park et al., 2021;Zhu & Wang, 2016). So far, the region harbors four out of seven ranavirus species (or ranavirus isolates considered members  was recorded among farmed populations in Shanxi Province (Geng et al., 2011), where all specimens collected tested positive for ranavirus. The Chinese giant salamander is one of the largest amphibians in the world, and it is Critically Endangered (IUCN Red List, 2012) because of threats such as habitat loss and overharvesting. Emerging infectious diseases such as the Chinese giant salamander iridovirus (CGSIV) can also cause population declines, with mass mortality events increasing their extinction risk (Dong et al., 2011;Meng et al., 2014;Wang, Zhang, et al., 2014;Wang, Ji, et al., 2014). Adult Chinese giant salamanders have been shown to be infected in natural habitats and/or farms in Hunan, Jiangxi, and Henan Provinces during 2011-2012 . The isolate responsible for this infection was provisionally designated as Andrias davidianus ranavirus (ADRV).

| Mainland China
In terms of species, anurans are the group that is mostly infected with ranavirus in China. A study of wild Dybowski's frogs (Rana dybowskii) in Heilongjiang Province showed that the overall infection prevalence is 5.7% in adults and 42.5% in tadpoles (Xu et al., 2010).
Overall prevalence of infection in adults was shown to be low compared with tadpoles; neither showed clinical signs. Another study highlighted ten strains of FV3-like ranavirus in wild, native R. dybowskii and R. amurensis in northeastern China (Zhu & Wang, 2016). Eight of these strains were isolated from R. dybowskii from Heihe, Hebei, and Dongfanghong, while the remaining two were detected in R. amurensis from Hebei. These 10 strains were homologous to FV3, though genetic differences were noted among the isolates. "Frog Virus 3 (FV3)"-like iridoviruses can cause widespread, severe disease and mortality among cultured pig frogs (L. grylio), and farms in Hubei Province and Hunan Province have experienced mortality rates above 90% (Zhang et al., 2001). Another outbreak was recorded in cultured Chinese edible frogs (Hoplobatrachus rugulosus) in culture facilities in Guangdong and Hainan Provinces, which was also closely related to FV3 . Tiger frog virus in a captive population of H. F I G U R E 1 Regional distribution of Asian ranavirus cases by country. Hosts of ranaviruses (salamanders/newts, anurans, fish, agamid lizards, and testudines) are indicated by the respective silhouettes of hosts rugulosus in Nanhai, Guangdong, in Southern China, caused high mortality of tadpoles Yuan et al., 2016). Further, tadpoles of a cultured population of black-spotted pond frogs (R. nigromaculata) in Shuangliu County, China, were diagnosed with a FV3-like ranavirus infection where substantial mortality was reported (90%; Mu et al., 2018). Similar outbreak among black-spotted pond frogs (R. nigromaculata) was recorded in tadpoles from a frog farm located in Shuangliu (Sichuan Province) in 2017 (Yu et al., 2020).

Fishes of economic value have been affected by ranaviruses.
High mortality in cultured largemouth bass (Micropterus salmoides) was observed in Foshan, Guangdong Province, in 2008, and the virus was identified as identical to doctor fish virus (DFV), a pathogen closely related to largemouth bass virus (LMBV) (Deng et al., 2011).
Another high mortality event occurred due to "Common midwife toad virus (CMTV)"-like ranavirus in a cultured loach (Triplophysa siluorides) in Sichuan Province, which affected about 75% of the stock (Deng et al., 2020). Isolates of Santee-Cooper ranaviruses from diseased Chinese perch (Siniperca chuatsi) and snakehead fish (Channa maculata) from Guangdong Province suggest that infection may be widespread in China (Fu et al., 2017).

F I G U R E 2 Asian ranaviruses on a phylogeny: Phylogenetic perspective of Asian ranaviruses (highlighted in red) in the context of broad virus type (Frog virus (FV3)-like, Common midwife toad virus (CMTV)-like, and Ambystoma tigrinum virus (ATV)-like). Hosts of Asian
ranaviruses (salamanders/newts, anurans, fish, and testudines) are indicated by the respective silhouettes of hosts. The overall topology of the tree was obtained by a two-stage Bayesian approach PASTIS (Thomas et al., 2013), which uses a backbone topology based on molecular data, a set of taxonomic postulates and user-defined priors on branch lengths and topologies to generate a posterior distribution of complete ultrametric trees that capture uncertainty under a homogeneous birth-death prior model of diversification and placement constraints. Twenty-five complete genomes (~100,000 bp) deposited in GenBank (last accessed on February 2021) were aligned in MAFFT and were analyzed in several tree-building programs such as MAFFT, IQtree, NDtree, and CSI phylogeny, using the best-fitting model as TVMe+ R2, which provided topologies congruent with those of Chinchar et al. (2017). The tree output given by the MAFFT (i.e., the ultrametric tree) was used as the backbone tree in the analysis. To incorporate isolates with short sequence data into the backbone tree, molecular data for several loci (MCP, DPG, RDRASPG, and RDRBS) for all taxa included in the tree were downloaded from GenBank. They were aligned in MEGA using MUSCLE, were softly constrained into their specific broad virus type based on the taxonomic information available in the literature using PASTIS, and were analyzed in MrBayes by running for 60 million generations. As soft constraints were allowed to move freely on branches, they were able to fix at places with maximum-likelihood values, revealing possible relationships of taxa whose phylogenetic positions were previously unknown or doubtful. Branches with posterior probability values >95% are marked by asterisks. Lymphocystis disease virus from China was used as the outgroup. The world map in the subfigure shows known locations of ranaviruses, in which the highlighted extent depicts the Asian region considered in this paper. Note: For detailed descriptions of ranavirus isolates in Asia, see Table 1. Inland bearded dragon ranavirus, Koi ranavirus, Oxyeleotris ranavirus, Goldfish ranavirus, Asian grass frog ranavirus, and East Asian bull frog ranavirus are not indicated in the phylogeny as their phylogenetic relationships were unspecified or doubtful In contrast to fishes, only two species of reptile have been found to be infected so far. Soft-shelled turtle iridovirus (STIV) causing "red neck disease" in farmed soft-shelled turtle (Trionyx sinensis) has been recorded in Shenzhen (Chen et al., 1999). Phylogenetic analyses suggested that STIV and FV3 are closely related and may transmit between reptiles and amphibians (Huang et al., 2009). A ranavirus in testudines was recorded in snapping turtles (Macrochelys temminckii) in Sichuan Province (Yu et al., 2015).

| Hong Kong SAR, China
Ranaviruses have been detected in 105 of 185 (56.8%) of the number of individual amphibians tested, which were exported from Hong Kong to the USA (Kolby et al., 2014). Ranaviruses were identified in oriental fire-bellied toad (Bombina orientalis), oriental fire-bellied newt (Cynops orientalis), and Hong Kong newt (Paramesotriton hongkongensis). However, it is not known whether the infection is only found among traded species or whether native amphibians in Hong Kong are already infected.

| Taiwan, China
Only fish have been recorded with ranaviruses so far in Taiwan.
Grouper iridovirus (GIV) was isolated from yellow groupers (Epinephelus awoara) in a fish farm in Hsiau Liouchiou Island (Lai et al., 2000;Murali et al., 2002), where mortality reached 100% within 11 to 25 days post-infection (Murali et al., 2002). Furthermore, twenty- isolates were divided into six groups within the genera Ranavirus and Megalocytivirus. (Huang et al., 2011). The phylogenetic analysis of viral genomic DNA based on the MCP genes showed that the genotypes of these isolates were closely related to SGIV and GIV. Given the wide range of habitats and high number of potential host species (76 reptiles, 30 amphibians, and 130 freshwater fishes) present in Taiwan (WWF, 2020), the propensity for the pathogen to spread should be closely monitored.

| Japan
Biogeographically isolated from the rest of Asia by the Sea of Japan, Japan harbors a high amphibian diversity. These amphibian species have a high risk of acquiring ranaviruses from imported aquatic organisms. Wild North American bullfrog (L. catesbeiana) larvae suffered a mass die-off in a pond in western Japan during the autumn of 2008, attributed to RCV-JP (Une, Sakuma, et al., 2009). Another outbreak was reported in a captive collection, which involved several species of poison dart frogs (Dendrobates spp. and Phyllobates spp.); this infection was triggered following an introduction of imported Dendrobates spp. from the Netherlands in 2012 (Une et al., 2014).
An outbreak occurred in a captive colony of Japanese clouded salamander (Hynobius nebulosus), and the entire colony was destroyed in 20 days (Une, Nakajima, et al., 2009).
The most recent reported ranavirus outbreak was detected in a population of inland bearded dragons (Pogona vitticeps; n = 100) at a breeding facility in Japan. This was named as the inland bearded dragon ranavirus (IBDRV), and MCP gene sequence analysis showed it to be similar to the three ranaviruses described in infected amphibians in Japan, Korea, and Taiwan. Reptilian ranaviruses, which often cluster closely with amphibian ranaviruses ("FV3-like," "TFV-like," or "CMTV-like"), were also detected in the vicinity of the breeding facility, from which horizontal transmission may have occurred (Tamukai et al., 2016).

| South Korea
There are five cases of ranaviral infections reported from South Korea. The first of these was a mass mortality event in a natural population of huanren frog (R. huanrenensis) tadpoles by a ranavirus closely related to the Rana catesbeiana virus JP MCP, isolated from invasive bullfrog tadpoles in Japan (Kwon et al., 2017). This is perhaps the best-known case of ranavirosis implicated in a mass mortality event of an endemic wild amphibian in Asia. Another mass mortality event involving an adult population of Dybowski's brown frogs (R. dybowskii) was detected in 2017, from a stream Moksangdong (Park et al., 2021). The MCP sequence resembled the Frog virus 3 (FV3) that had been collected earlier from huanren brown frog (R. huanrenensis) tadpoles in South Korea. Dead adults of boreal digging frog (Kaloula borealis) from Eoeun-dong, Daejeon-si, were confirmed to be infected with ranavirus, while tadpoles of the Japanese tree frog (Hyla japonica) from Ugok-ri, Yongju-myeon, were also confirmed to be infected . Last case from South Korea was reported from gold-spotted pond frogs (Pelophylax chosenicus, formerly Rana plancyi chosenica) (Kim et al., 2009). Both the tadpoles and adults were infected in a culture facility, and the virus resembled Frog Virus 3 (FV3).

| Singapore
Groupers are the only group detected with ranaviruses in Singapore. A newly isolated grouper virus from a diseased brown-spotted grouper (Epinephelus tauvina), related to largemouth bass virus (LMBV), FV3, and Regina ranavirus (RRV), was named as Singapore grouper iridovirus (SGIV; Qin et al., 2003). SGIV was shown to cause serious systemic disease capable of killing 96% of grouper fry. Mariculture of Epinephelus species is rapidly developing in Singapore and other Southeast Asian countries, to which this virus poses a serious threat. Outbreaks of a novel viral disease called "sleepy grouper disease" (SGD) was first observed in E. tauvina in Singapore in 1994 (electron microscopic analyses), causing economic losses (Chua et al., 1994). However, the virus strain was not verified at the time by cell culture techniques. Later, it was identified as SGIV. Another outbreak of the same disease occurred in fry and adults of brown-spotted groupers in 1998. These fry were imported from other Southeast Asian countries, and the outbreak lasted several weeks, resulting in more than 90% mortality.

| India
There are two instances of ranavirus infections recorded from India.
The first was in freshwater fishes, following a mass mortality event.
A virus resembling Santee-Cooper ranavirus was detected from koi carp (Cyprinus rubrofuscus) in ornamental fish farms of South India (George et al., 2015). The second case was reported from farmreared similar damselfish (Pomacentrus similis) with frequent mortality events reported from marine ornamental fish farms of South India (Sivasankar et al., 2017). The name "Similar damselfish virus" (SRDV) was proposed, and the MCP gene showed a close relationship to largemouth bass virus (LMBV).

| Cambodia and Vietnam
The only study conducted so far in Cambodia and Vietnam failed to detect ranavirus in these countries (Gilbert et al., 2012). The screening was based on qPCR and histopathology of liver and other tissue samples collected from 74 frogs, with most samples being from Cambodia (n = 70).

| The Philippines
A study carried out to investigate ranaviruses along with Batrachochytrium dendrobatidis among wild amphibians (n = 304, from seven sites) from the Philippine islands of Luzon, Negros, Calayan, and Camiguin Norte failed to detect ranaviruses (Smith et al., 2019).

| Malaysia
Groupers are the only group detected with ranavirusses in Malaysia.

| S E VERIT Y OF R ANAVIRUS INFEC TIONS AT G LOBAL SC ALE
Some host species are highly susceptible to ranaviruses, and experimental studies suggest that novel strains of ranaviruses introduced into native populations could have devastating consequences (Duffus et al., 2015). Despite limited monitoring efforts, the alarming increase in recent reports of ranavirus emergence in Asia may be an underlying reason for unexplained population declines such as in the case of Chinese giant salamanders (Dong et al., 2011;Meng et al., 2014;Wang, Zhang, et al., 2014;Wang, Ji, et al., 2014). High mortality rates of hosts and a diverse host range (and hence the potential to affect numerous novel species) have prompted the World Organization for Animal Health (OIE) to list ranavirus as a notifiable disease (i.e., transmissible diseases that have the potential for profoundly serious and rapid spread, irrespective of national borders, that entail serious socio-economic or public health consequence and that are of major importance in the international trade of animals and animal products). Epizootic hematopoietic necrosis virus (EHNV) is listed as a fish disease, while infection by ranavirus species is listed as amphibian disease in the OIE's listed diseases (World Organization for Animal Health, 2021). This designation requires countries (which have subscribed to OIE policies) to screen a sample of ranavirus hosts that cross international borders for ranaviruses . Quarterly Aquatic Animal Disease Report

| INTRODUCING R ANAVIRUS E S TO UNINFEC TED ARE A S AND TR AN S MISS I ON WITHIN A S IA
Growing international trade of live amphibians, reptiles, and fish, taken from the wild or bred in captivity, and sold commercially as food or ornamental species/pets, appears to increase the risk of introducing and dispersing ranaviruses across Asia. Several studies suggest that many species imported to Asia could potentially host ranaviruses (Table 1).
It is possible that ranavirus-caused diseases may have existed undetected in Asia over an extended period, though now coming to be better understood as a result of the widespread application of molecular diagnostic techniques. Alternatively, the detected outbreaks could signal a recent emerging infection spreading rapidly across the world because of the ever-increasing mobility of pathogens due to global trade of live animals. Application of molecular diagnostic techniques and phylogenomic analyses is important for disease detection and for understanding recombinants that can be more virulent.
Phylogenomic analyses using full genomes would explain disease dynamics better. For instance, putative recombinants between FV3, a pathogen widely distributed within wild populations, and CMTV, have caused high pathogenicity. While CMTV-derived genes associated with virulence are reported in wild strains in Canada, FV3 has been linked to amphibian die-offs in North America (Vilaca et al., 2019).
The latter study provides an insight on how pathogen surveillance and viral dynamics using full genomes can be used to understand the mechanisms of disease origin and spread more clearly. New recombinants arriving through animal trade can pose a high risk to Asia.
It is possible that ranaviruses can be transmitted between ectothermic vertebrate classes through water (Brenes et al., 2014).
Further, fish and reptiles might serve as reservoirs for ranavirus, given their ability to live with subclinical infections, which may contribute to the pathogen's persistence, especially when highly susceptible hosts such as amphibians are absent due to seasonal population fluctuations.

Studies from Asia indicate that ranavirus infections cross species
barriers, allowing the virus to infect previously uninfected indigenous hosts. Further, given the high mutability of ranaviruses, new strains can emerge . Some of the introduced host species are known to be infected with ranaviruses in their original locations. There are a few records from other countries in the region as well.

One of the best examples is in
The barcoo grunter fish (S. barcoo), which is imported from Australia and cultured in Thailand, was infected with a ranavirus similar to Largemouth bass virus (LMBV) (Kayansamruaj et al., 2017). Inland bearded dragons (P. vitticeps), a pet species imported to Japan, was found to be infected with Inland bearded dragon ranavirus (IBDRV), though the details of origin are not available (Tamukai et al., 2016).
In addition, some of the ranavirus infections may already have been transmitted among Asian countries. Asian grass frog ranavirus (AGFRV) has been recorded in Asian grass frogs (F. limnocharis) imported from Cambodia to Thailand's culture facilities (Sriwanayos et al., 2020). Meanwhile, grouper fry imported from other Southeast Asian countries might have carried the Singapore grouper iridovirus (SGIV) into Singapore and Malaysia (Hazeri et al., 2016(Hazeri et al., , 2017Qin et al., 2003).

| P OTENTIAL ENTRY OF R ANAVIRUS TO A S IA THROUG H CULTURED FROG S -AMERIC AN BULLFROG (Lithobate s cate sbeia nus) AND PI G FROG (Lithobate s gr ylio) A S RE S ERVOIR S
One of the species highlighted so far in this regard is the American bullfrog (L. catesbeianus), which seems to have played a key role in spreading the pathogen to new locations (Both et al., 2011;Mazzoni et al., 2009;Ruggeri et al., 2019;Schloegel et al., 2009Schloegel et al., , 2010. Cultured American bullfrogs often carry ranavirus infection FV3  and may have served as a vector transmitting the disease to native amphibians and fish in Brazil (Mazzoni et al., 2009;Ruggeri et al., 2019). It is striking those American bullfrogs have been introduced and now occur in nearly 40 countries in Africa, Asia, and North, Central, and South America, and islands of the Mediterranean, South Pacific, and Caribbean (Kraus, 2009). These frogs are of particular concern as vectors of the disease as they are China (Ningbo and Tianjin cities and Guangdong province) between TA B L E 1 Regional distribution of Asian ranavirus infections or mortality in wild and captive ectothermic vertebrates (Continues)

Detection method/phylogenetic analysis Reference
Severely hemorrhagic lesions High mortality Virus isolation, electron microscopy, challenge experiments, and genome annotation and analysis Chen et al. (2013) Swelling and bleeding of the head (known locally as big head disease) or feet (big foot disease), necrosis, and bleeding of the oral mucosa (bad mouth disease) or tail (bad tail disease), and skin bleeding.
isolates from southern China were similar to the North American bullfrog isolates from Japan, which indicates the long-term trade exchange.
Interestingly, a ranavirus named RCV-JP has been identified in cultured North American bullfrogs (L. catesbeianus) in Japan (Une, Sakuma, et al., 2009 North American bullfrogs in a frog farm in the USA (Majji et al., 2006), which suggests that the infection may have persisted in these frogs when they were introduced to Japan from the USA.
Further, L. grylio, which is native to the south-eastern USA, has been imported into China for farming purposes. There is a possibility that these imported frogs harbored the virus and were the focus of the initial infection (Zhang et al., 2001). The evidence of recording the Rana grylio virus (RGV), which is similar to the FV3 virus among cultured pig frogs (L. grylio) in China, could provide more evidence of introduction of the disease from USA, as this species too had been imported.

| P OTENTIAL TR AN S MISS I ON TO NATIVE/ENDEMIC S PECIE S: J UMPING S PECIE S BARRIER S
Several cases indicate that the ranaviral infections in native/endemic host species could potentially jump species barriers of cultured species. Ranavirus infections that have been recorded in Chinese Giant salamander may have been transmitted from pig frogs (L. grylio) that are routinely fed to farmed Chinese Giant Salamanders (Cunningham et al., 2016). This is supported by the fact that ranavirus from Chinese giant salamanders in Sichuan Province shows a close relationship to ranavirus in pig frogs (Cunningham et al., 2016;Zhou et al., 2013).
This hypothesis is further supported by ADRV having been shown to be more closely related to frog (anuran)-infecting ranaviruses such as CMTV, RGV, FV3, and TFV than ATV, which is the salamander (urodele)-infecting ranavirus . The phylogeny we constructed from existing data supports this hypothesis ( Figure 2).  IRAs can also be used to establish or revise trade or translocation guidelines for wildlife that could be subclinically infected with a pathogen (Smith et al., 2009). Risk analysis on wildlife species in trade, pre-border pathogen screening, and voluntary support should help reduce costs associated with species invasion, as well as protecting the public and enhancing environmental and animal health (Smith et al., 2009 to the sustainability of the aquaculture industry (Gudding, 2012).

| B I OS ECURIT Y AND PRE VENTING FURTHER ENTRY OF R ANAVIRUS E S TO
Meanwhile, existing national, regional, and international laws and regulations can be successfully used to improve biosecurity mea- infection could also be transmitted via other media such as contaminated waste. Given that ranaviruses are often reported during summer months, rising temperatures associated with climate change may facilitate ranavirus spread. Mass die-offs have been recorded in several countries such as China and Japan. So far, no effective treatment to reduce mortality or morbidity has been found. Therefore, control measures, such as limiting international trade of animals and screening for disease, must be strictly followed. Molecular diagnostic techniques can be successfully used to observe the phylogenetic relationships and their host ranges, to detect the possible original sources of introduction. Well-planned, widely distributed systematic screening is essential to understand the prevalence and impact in Asia, to conserve the biodiversity and to safeguard the widely established and economically important aquaculture industry.

ACK N OWLED G M ENTS
We thank Rohan Pethiyagoda (Australian Museum, Sydney) for suggestions to improve the manuscript and the Guangxi University for MM's startup grant and facilities provided to conduct disease ecology research.

CO N FLI C T O F I NTE R E S T
None to declare. Project administration (equal); Resources (lead); Supervision (lead); Writing-original draft (equal); Writing-review & editing (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing: not applicable. All data and sources used in the study are provided within the framework of the study.