Ranaviruses and reptiles

Introduction

Ranaviruses (family Iridoviridae) are emerging lethal pathogens of ectothermic vertebrates. First discovered in 1965 (Granoff, Came & Rafferty, 1965), ranaviruses were initially studied for their interesting molecular biology but rose to reportable pathogen status as more epizootics were discovered (Schloegel et al., 2010; Gray & Chinchar, 2015). The vast majority of research on the genus Ranavirus has been conducted in amphibians (Rana is Latin for frog), but despite their name, ranaviruses do not occur only in amphibians (Chinchar & Waltzek, 2014). This group of viruses infects over 175 species of ectothermic vertebrates; including reptile species from at least 12 different families (Duffus et al., 2015). Temperature appears to be the major factor preventing ranaviral infection outside of ectothermic vertebrates; these viruses can replicate in mammalian cell lines, but only when the temperature is below 32 °C (Gray & Chinchar, 2015).

Many advances in the field of ranavirology have been made since the discovery of ranaviruses; however, for the most part, this research is specific to amphibians. Reptiles and amphibians are very different physiologically and although they sometimes share habitats, their ecology is different. Some results from one host group can translate to the other; however, there is no substitute for host-specific research. As ranavirus research continues, it is important to focus efforts on all Classes of hosts, including reptiles.

Since the initial report of ranaviruses in reptiles in the early 1980s (Heldstab & Bestetti, 1982), infections have been reported in wild and captive reptiles, and the number of reports continues to grow, representing an increasing problem for reptiles (Marschang, Stöhr & Allender, 2016). In this review, we summarise findings in all areas of reptilian ranavirus research. We identify major gaps in this field of knowledge and include recommendations for future research directions.

Survey Methodology

To ensure this review included as many publications focusing on ranaviruses and reptiles as possible, an extensive search of multiple databases using broad search queries was conducted. Databases used in the search strategy included: Web of Science, PubMed, and Google Scholar. The search strategy included keywords such as ‘ranavirus’ and ‘reptiles’ and their conjugations as well as more specific terms such as ‘turtle’, ‘lizard’, and ‘snake’. To broaden the search further, references for articles found in the initial database search were then assessed for content relating to ranaviruses and reptiles. As a baseline for general ranavirus literature, relevant references were extracted from the 2015 Ranavirus book (Gray & Chinchar, 2015).

Taxonomy

Ranaviruses are large (∼150 nm), nucleocytoplasmic viruses with icosahedral virions and double-stranded DNA genomes that contain approximately 100 genes (Jancovich et al., 2015). Ranavirus is a genus in the family Iridoviridae: a group of five related viral genera. Of the five Iridoviridae genera, only ranaviruses cause significant disease in wild reptiles.

The taxonomy of the genus Ranavirus is changing; as more viruses are isolated and sequenced a clearer picture of the phylogenetic distribution of this group is developing. The international committee on taxonomy of viruses (ICTV) currently recognises eight species in the genus Ranavirus (Lefkowitz et al., 2018), none of which were originally isolated from reptiles. The official ICTV process of species recognition takes time and coordination within the scientific community. Many isolates, including isolates from reptiles, remain unclassified (Chinchar et al., 2017).

The current phylogeny of the genus Ranavirus can be subdivided into five major lineages based on comparison of conserved genes (Claytor et al., 2017; Jancovich et al., 2015; Stöhr et al., 2015; Price et al., 2017). No Ranavirus lineage exclusively infects reptiles, and the majority of reptile infections appear to originate from putative amphibian specialist viruses (Price et al., 2017). The factors that control the host specificity of these viruses remain unknown. Phylogenetic analyses of sequences from different reptilian and amphibian viruses have revealed that viruses found in reptiles are often more closely related to amphibian ranaviruses from the same geographical region than to each other (Stöhr et al., 2015). This provides support for the hypothesis that the jump into reptile hosts is relatively recent and has occurred multiple times (Jancovich et al., 2010, Stöhr et al., 2015).

Bibliometrics

Despite a lack of host specificity, the vast majority of ranaviral literature is on amphibians. As of February 2018, 449 references were returned when the Web of Science ^TM database was queried for the topic ‘ranavirus’. Of these, over 200 used the term ‘amphibian’ in their title or abstract while fewer than 60 used the term ‘reptile’. However, plotting the usage of these terms over the last 10 years shows a steady increase in the ratio of ‘reptile’ to ‘amphibian’ terms, possibly reflecting an increase in reptilian ranavirus research or an increased awareness of the role of reptiles in this disease (Fig. 1).

Diagnostics and Surveillance

The World Organization for Animal Health (OIE) provides guidelines for diagnostic methods in their Diagnostic Manual for Aquatic Animal Health (OIE, 2012), and Miller et al. (2015) summarised the diagnostic techniques used in ranaviral research. The most commonly used methods to confirm the presence of a ranavirus in host samples have included electron microscopy, enzyme-linked immunosorbent assays (ELISAs), viral isolation, immunohistochemistry (IHC), DNA amplification using polymerase chain reaction (PCR), and more recently, next generation sequencing. All of these techniques have been used at some stage in the study of ranaviruses from reptiles; however, the selection of diagnostic technique is highly dependent on the resident expertise in the laboratory, the data required, and the type of study (Miller et al., 2015). Before their application in new hosts or against new pathogens, all diagnostic techniques should be thoroughly tested and optimised with appropriate controls (e.g., different species of reptiles or ranaviruses, or both) (Wobeser, 2007).

Most ranaviruses can be grown using commercially available fish cell lines (Miller et al., 2015). Reptile cell lines such as Russell’s viper heart cells, gecko lung cells, turtle heart, and iguana heart cells have also been used successfully to isolate ranaviruses from reptiles (Hyatt et al., 2002; Johnson et al., 2008; Alves de Matos et al., 2011).

Distribution, Host Range and Impact

Ranaviruses capable of infecting reptiles have been found on all continents, except Antarctica (Duffus et al., 2015). Ranaviruses have been detected in over 12 families of the orders Testudines (turtles, tortoises and terrapins) and Squamata (lizards and snakes). It is important to note that given reptile populations often share habitat with susceptible fish and amphibian species, it may be possible to infer reptile ranavirus distributions based on amphibian ranaviral prevalence patterns and vice versa (Duffus et al., 2015). It is not clear if reptilian ecology influences patterns of ranavirus host range or susceptibility. Aquatic reptiles may be more likely to be exposed to ranaviruses; however, ranaviruses are still detected in terrestrial reptiles (Duffus et al., 2015). There is some evidence aquatic turtles are less susceptible to ranaviral disease; however, this is far from settled and should be investigated further (Brunner et al., 2015). Recently, Adamovicz et al. (2018) reported that the use of moist microhabitats is correlated with ranavirus detection in eastern box turtles.

Pathology

The clinical signs and pathogenesis of natural ranaviral infection in reptiles can be extremely variable. Mortality during an epizootic can range from 0–100% and the effect on a host can vary from quite mild to extremely severe, requiring immediate veterinary attention or euthanasia (Miller et al., 2015). There is evidence that reptiles can also be asymptomatic carriers of ranaviruses (Stohr et al., 2013; Goodman, Hargadon & Carter, 2018). Quiescent viral reactivation in amphibians that have recovered from infection is possible; however, the same is not known for reptiles (Robert et al., 2014). The complex presentation and inconsistency in the pathogenesis of ranaviral infection in reptiles may occur because of the influence of host physiology and life history, and varying degrees of viral virulence, stressors, and temperatures acting on the course and outcome of infection (see Susceptibility section).

Descriptions of pathogenesis in reptiles infected with a variety of ranaviral strains in several host species under experimental, wild, and captive conditions are presented in Table 1. Despite differences in descriptions of pathogenesis and the fact the reports are often confounded with co-infections (Sim et al., 2016; Adamovicz et al., 2018), some common patterns of ranaviral pathogenesis have emerged.

General lethargy and inappetence are associated with many cases of ranaviral infection in reptiles; however, such clinical signs are common to many diseases and are not pathognomonic for ranaviral infection. Turtles often present with respiratory signs, including nasal and oral discharge (Johnson, Pessier & Jacobson, 2007; Johnson et al., 2008; Allender et al., 2013b; Kimble et al., 2017). Oedema, especially of the eyes or neck, is also commonly associated with this infection in the order Testudines (Chen, Zheng & Jiang, 1999; Johnson, Pessier & Jacobson, 2007; Johnson et al., 2008; Allender et al., 2013b). The clinical signs of ranaviral infection in Squamates are scarcely described. This is partially due to the lack of experimental infection trials in this group, which would help describe pathogenesis markers. Maclaine et al. (2018) recently demonstrated the susceptibility of an Australian lizard species (Intellagama lesueurii lesueurii) to ranaviral infection, documenting that clinical signs and histopathological changes varied with inoculation route. With increasing descriptions of ranaviral infected lizards over the last decade, an emerging trend suggests that skin lesions may be a common occurrence (Behncke et al., 2013; Stohr et al., 2013; Tamukai et al., 2016).

Ranaviral infections are systemic, and there is often extensive damage to multiple organs during infection, especially the liver and spleen in reptiles. Liver lesions are also very common in the pathogenesis of ranaviruses in amphibian and fish species (Miller et al., 2015). Histopathology is frequently characterised by inflammation and multifocal necrosis in multiple organs, and is often associated with hematopoietic tissue (Ariel et al., 2015). Reptilian hosts of ranaviruses experience a range of histological changes including necrosis and inflammation of the respiratory tract, pneumonia, conjunctivitis, stomatitis, esophagitis, tracheitis, necrosis of endothelial cells and the submucosa of the gastrointestinal tract, glomerulonephritis, multifocal hepatic necrosis, splenitis, intracytoplasmic inclusion bodies in many tissues, and necrotizing myositis (see Table 1). Evidence from epizootics in reptiles indicates that ranaviral infection can be accompanied by secondary pathogens that may exacerbate the disease and mask clinical signs of ranaviral infection (Stohr et al., 2013; Sim et al., 2016; Archer et al., 2017).

Transmission

The natural route of transmission of ranaviruses in wild populations of reptiles is still debated, although experimental data suggest multiple transmission routes are possible (Brunner et al., 2015). During an experimental challenge of adult red-eared sliders (Trachemys scripta elegans), Johnson, Pessier & Jacobson (2007) found that the orally exposed animals were refractory to infection while animals challenged with the same dose via intramuscular injection developed severe disease. In another study, exposure to ranavirus in water via cohabitation resulted in subclinical infection in some red-eared slider hatchlings (T. scripta elegans), although the route of infection was not determined, and the concentration of virus in the water was not quantified (Brenes et al., 2014a). Ariel et al. (2015) found that adult freshwater turtles (Emydura krefftii and Elseya latisternum), freshwater crocodiles (C. johnstoni), and several species of snakes were refractory to infection irrespective of the route of exposure. The hatchlings of both species of freshwater turtles were susceptible to infection via intra-coelomic exposure although oral inculcation was not attempted. Juvenile Australian eastern water dragons (Intellagama lesueurii lesueurii) developed ranaviral disease from all exposure routes tested (oral, intramuscular, and cohabitation) (Maclaine et al., 2018).

Differences in susceptibility via different routes of exposure may reflect real differences in natural transmission routes between reptiles and other Classes. More experimental studies using a variety of species, life stages, and routes of transmission are needed to resolve this.

Amphibians are highly susceptible to ranaviral infection via all tested forms of inoculation (water bath, skin contact, oral inoculation or injection) (Miller et al., 2015). Fish are also susceptible via multiple inoculation routes, although it appears to be species-dependent (Bang Jensen, Ersbøll & Ariel, 2009; Gobbo et al., 2010; Jensen et al., 2011). Differences in viable transmission routes result in different epidemiologies, and thus research from other host classes with different viable transmission routes may not accurately reflect risks and susceptibility of reptilian populations. It is therefore important to account for variation in transmission routes among reptile species when developing statistical models for reptilian disease.

Vectors

Humans are contributing to the global spread of ranaviruses, primarily though global animal trade (Kolby et al., 2014; Duffus et al., 2015; Stöhr et al., 2015). Although there are reports of ranaviral infection in traded reptiles (Hyatt et al., 2002; Stohr et al., 2013), no systematic survey of ranaviral infection in traded reptiles has been conducted. There have been some ranaviral disease outbreaks in private reptile collections and zoos (Marschang, Braun & Becher, 2005; Sim et al., 2016), but the full extent of disease prevalence is hard to assess, both because of inapparent infections, and lack of reporting of dead animals amongst reptile breeders and collectors.

Ranaviral DNA sequences have been identified in mosquitoes associated with a ranavirus outbreak in box turtles, providing evidence for vector transmission (Kimble et al., 2014). Ranaviral DNA and antigens have been detected in blood and blood-associated tissues of reptiles (Allender et al., 2013a; Ariel et al., 2015; Miller et al., 2015). Leeches are common ectoparasites of aquatic reptile species and can act as vectors for blood-borne diseases (Siddall & Desser, 1992; Watermolen, 1996; Readel, Phillips & Wetzel, 2008). There has been at least one report of a ranavirus-positive leech (PCR for MCP) associated with an infected amphibian host, although there are no reports for leeches of reptiles (Hardman et al., 2013). Some low density reptile populations that experience ranaviral epizootics do not appear to be capable of propagating ranaviral disease through physical contact alone (Brunner et al., 2015). Despite these indicators of the possible involvement of vectors in ranavirus transmission, no experimental studies have been published that support or refute this hypothesis in reptiles.

Reservoirs

Ranaviral virions are extremely stable in controlled settings, they are capable of withstanding high and low pH and temperatures and are resistant to desiccation, remaining viable for days to years (Granoff, Came & Rafferty, 1965; Langdon et al., 1986; Langdon, 1989; Munro et al., 2016; Nazir, Spengler & Marschang, 2012). These qualities of stability may not to carry to ecological settings as interactions with the aquatic biotic communities can reduce the longevity of infectious ranaviral particles (Brunner et al., 2015). Reinauer, Bohm & Marschang (2005), found that tortoise ranaviruses remain infectious in lake water samples and in soil for many days; however, biotic communities were not quantified. It also appears that moisture is important for persistence in soil environments (Brunner et al., 2015; Nazir, Spengler & Marschang, 2012). Animals, both live and dead, are also probably reservoirs for reptilian ranavirus infections (Gray, Miller & Hoverman, 2009). Reptiles are known to consume frogs, fish, and even other reptiles as a part of their natural diet (Kischinovsky, Raftery & Sawmy, 2017). Dead and decaying animals continue to release virions and might be consumed by susceptible reptiles (Brunner et al., 2015; Gray & Chinchar, 2015). Asymptomatic amphibians are sometimes reservoirs; they can spread virus to other susceptible species, and possibly reptiles, in multispecies ranavirus epizootics (Brenes et al., 2014a; Brenes et al., 2014b; Brunner et al., 2015).

Correlates of Susceptibility

Reptiles are ectotherms and so their physiology is strongly influenced by the temperature of their surrounding environment. By extension, the innate and adaptive immune response of reptiles is also linked to available environmental temperatures (Zimmerman, Vogel & Bowden, 2010). Ranavirus-infected reptiles, such as turtles, exhibit temperature-dependent pathogenesis (Allender et al., 2013b; Allender et al., 2018) similar to that observed in fish and amphibians (Brunner et al., 2015; Brand et al., 2016); however, the replication efficacy of the virus is also linked to temperature (Ariel et al., 2009). Thus, it is difficult to determine the degree to which temperature-dependent pathogenesis is a result of the effect of temperature on the replication of the virus or on the immune system of the turtles. Several studies have quantified the temperature-dependent activity of the innate immune system of reptiles (Merchant et al., 2006; Ferronato et al., 2009; Merchant et al., 2012). In experimental infections of ranaviruses, temperature is often uncontrolled (reported as ‘room temperature’).

Allender et al. (2013b) suggested that an environmental temperature increase of 6 °C is enough to significantly reduce ranavirus loads and halve morbidity in infected adult turtles. However, in a follow up study, in juvenile turtles, it was found that increased temperature reduced median survival time of all four species tested (Allender et al., 2018). Similar patterns of reduced time until death but lower mortality rates with increasing temperature have been seen with other environmental temperature-dependent host-pathogen systems such as amphibians with chytridiomycosis (Berger et al., 2004). This pattern of temperature-related susceptibility in reptiles is important for future studies to quantify.

The effects of stressors on reptilian ranaviral disease are poorly understood (Polakiewicz & Goodman, 2013). Several studies in amphibians have examined the effects of stressors on disease in experimental infections (Echaubard et al., 2010; Forson & Storfer, 2006; Haislip et al., 2012; Kerby, Hart & Storfer, 2011; Reeve et al., 2013), and epidemiological studies have looked for correlations between environmental stressors and ranaviral prevalence (St-Amour et al., 2008; Brunner et al., 2015). The immunosuppressive effects of some anthropogenic stressors (e.g., pesticides, herbicides, and heavy metals) on the reptile immune system suggest a possible mechanism of environmental influence on susceptibility. Future epidemiological studies should consider these factors (Keller et al., 2006; Soltanian, 2016).

Immunology

Studies of ranaviral host immunity and immune evasion in amphibians are extensive, while similar work in reptiles is limited (Grayfer et al., 2015). The immunology section in the 2015 Ranavirus book, although comprehensive on amphibians, only mentions reptiles in passing (Grayfer et al., 2015). Immunology is an area in which a great number of unknowns remain for ranaviruses and reptiles.

Treatment

For treatment of acute ranaviral infection, several antivirals have been considered and tested (Allender, 2012; Li et al., 2015; Sim et al., 2016). However, there are few examples of their successful use to treat clinical cases (Johnson et al., 2010; Allender, 2012; Miller et al., 2015). Many in vitro antiviral studies that show promising results do not carry to in vivo models or have not been thoroughly tested in vivo.

Acyclovir, the most extensively studied antiviral in reptiles, does not appear to be an effective anti-ranaviral agent. Viral thymidine kinase (present in some herpesviruses and ranaviruses) is required for activation of acyclovir, which then blocks viral DNA replication through competitive inhibition of the viral DNA polymerase (Beutner, 1995). In vitro results have been mixed; Johnson (2006) found that acyclovir provided a dose-dependent partial inhibition of a FV3-like ranavirus, and Ferguson et al. (2014) found no statistically significant effect of acyclovir on FV3 replication. Plasma concentrations of orally dosed acyclovir do not reach levels in turtles that have been suggested as sufficient for ranavirus inhibition (Allender, 2012; Gaio et al., 2007). It is difficult to interpret the results of the use of this drug in uncontrolled clinical settings; however, it is clear that in several cases acyclovir has not stopped the progression of reptilian ranaviral disease (DeVoe et al., 2004; Johnson et al., 2008).

Pharmacological studies of the effectiveness of different antivirals at different severities and durations of ranaviral infection in reptiles have not been conducted, but would be extremely useful for guiding the treatment of acute ranaviral infection in reptiles.

Iridoviral vaccine development has been limited to the aquaculture industry (Miller et al., 2015). Frogs can produce long-lasting FV3-specific neutralising antibodies on second exposure (Maniero et al., 2006), suggesting it would be possible to develop vaccines for them. Reptiles can produce anti-ranaviral antibodies during infection (Ariel et al., 2017; Johnson et al., 2010), and vaccines have been developed for other reptilian pathogens with varying success (Horner, 1988; Jacobson et al., 1991; Mohan et al., 1997; Marschang, Milde & Bellavista, 2001; Yang, Pan & Sun, 2007). Vaccine research and development are extremely costly, and more epidemiological research is required to determine if the development of a ranaviral vaccine would be efficacious for wild reptilian populations. However, there are several instances where a vaccine could be useful for small scale use. For example: in zoo collections, for valuable broodstock, and for endangered or at risk populations. Epidemiological studies may feasibly identify and prophylactically treat animals most at risk.

Environmental temperature can have a substantial effect on the humoral immune system (e.g., antibody production) of ectotherms (Tait, 1969), which opens up the possibility of influencing the outcome of an infection via control of environmental temperatures (see Susceptibility section). Increased ambient temperature has been suggested as a treatment method for ranavirus infection in reptiles (Hyndman & Marschang, 2017). However, Allender et al. (2018) recently reported that increased temperature (22 °C to 27 °C) resulted in reduced median survival time of ranaviral infected Testudines. It is likely that there is a threshold temperature, which dramatically improves survival as occurs with chytridiomycosis in amphibians (Berger et al., 2009). Further investigation is required to determine the optimal temperature for increasing survival of ranaviral infected reptiles, which may also be viral and host species dependent.

Future Research and Conclusions

The field of ranavirus research is dominated by studies on fish and amphibians, these studies can serve as a guide for the tremendous number of directions ranaviral research in reptiles could take. An increase in the number of epidemiological studies and surveys of ranaviruses in reptile populations is required to understand the distribution of these viruses in the class Reptilia, and to identify at-risk populations. Pathogenesis and transmission of ranaviruses in reptiles are still poorly understood and will require elucidation before this disease can be correctly modelled and appropriately managed in reptile populations. Reptile ranaviral host immunity and immune evasion strategies of the virus are also under-represented in the literature. From predator to pollinator to prey, reptiles play vital roles in the ecosystems they inhabit, but like amphibians, reptiles are experiencing global declines (Gibbons et al., 2000). It is, therefore, imperative that research continues to expand our understanding of reptiles and ranaviruses to help protect this valuable part of biodiversity.