Dynamics of Amphibian Pathogen Detection Using Extended Museum Specimens.

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection.

Cytokinins Reduce Viral Replication and Alter Plaque Morphology of Frog Virus 3 In Vitro.

Cytokinins (CKs) are a group of N6-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, N6-isopentenyladenine (iP), N6-isopentenyladenosine (iPR), N6-isopentenyladenosine-5'monophosphate (iPMP), and 2-methylthiol-N6-isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations.

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.

No Ranaviral DNA Found in Australian Freshwater Turtles, 2014-19, Despite Previous Serologic Evidence.

Ranaviruses are pathogens of ectothermic vertebrates (fish, amphibians, and reptiles). Turtles are the most common group of reptiles reported with ranaviral infections. However, there have been no surveys for wild ranaviral infection in any turtles from the suborder Pleurodira, despite ranaviral distributions and experimentally susceptible pleurodiran turtle populations overlapping in several areas, including Australia. We assayed 397 pooled blood samples from six Australian freshwater turtle species collected from five different sites in northern Australia between 2014 and 2019. Historical serologic surveys in the area had found antiranaviral antibodies; however, we did not detect any ranaviral DNA in our samples. Discrepancies between historical serologic and our molecular results may be explained by low viral prevalence during the years that these samples were collected, survivorship bias, or possibly an age class bias in sampling.

Ranavirus and helminth parasite co-infection in invasive American bullfrogs in the Atlantic forest, Brazil.

Emerging infectious diseases threaten amphibian species across the globe. In Brazil, the American bullfrog (Aquarana catesbeiana) is a highly invasive species that can potentially transmit parasites and pathogens to native amphibians. This is the first assessment of co-infection of Ranavirus and helminth macroparasites in invasive populations of bullfrogs in South America. We collected, measured, and euthanized 65 specimens of A. catesbeiana sampled from 9 sites across three states of Brazil in the Atlantic Forest biome. We collected and identified helminth macroparasites and sampled host liver tissue to test for the presence and load of Ranavirus with quantitative PCR. We documented patterns of prevalence, parasite load, and co-infection with generalized linear mixed models, generalized logistic regressions, and randomization tests. Most individual bullfrogs did not exhibit clinical signs of infection, but the overall Ranavirus prevalence was 27% (95% confidence interval, [CI 17-38]). Bullfrogs were infected with helminth macroparasites from 5 taxa. Co-infection of helminth macroparasites and Ranavirus was also common (21% CI [12-31]). Bullfrog size was positively correlated with total macroparasite abundance and richness, and the best-fitting model included a significant interaction between bullfrog size and Ranavirus infection status. We observed a negative correlation between Ranavirus viral load and nematode abundance (slope = -0.22, P = 0.03). Invasive bullfrogs (A. catesbeiana) in Brazil were frequently infected with both Ranavirus and helminth macroparasites, so adult bullfrogs could serve as reservoir hosts for both pathogens and parasites. However, many macroparasites collected were encysted and not developing. Coinfection patterns suggest a potential interaction between Ranavirus and macroparasites because helminth abundance increased with bullfrog size but was lower in Ranavirus infected individuals. Future studies of bullfrogs in the Atlantic Forest should investigate their potential role in pathogen and parasite transmission to native anurans.

The Binding, Infection, and Promoted Growth of Batrachochytrium dendrobatidis by the Ranavirus FV3.

Increasing reports suggest the occurrence of co-infection between Ranaviruses such as Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) in various amphibian species. However, the potential direct interaction of these two pathogens has not been examined to date. In this study, we investigated whether FV3 can interact with Bd in vitro using qPCR, conventional microscopy, and immunofluorescent microscopy. Our results reveal the unexpected ability of FV3 to bind, promote aggregation, productively infect, and significantly increase Bd growth in vitro. To extend these results in vivo, we assessed the impact of FV3 on Xenopus tropicalis frogs previously infected with Bd. Consistent with in vitro results, FV3 exposure to previously Bd-infected X. tropicalis significantly increased Bd loads and decreased the host's survival.

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.

Vitamin D3 can effectively and rapidly clear largemouth bass ranavirus by immunoregulation.

Largemouth bass ranavirus (LMBV) is a highly destructive pathogen that causes significant mortality rates among largemouth bass populations. Unfortunately, there is a dearth of drug development efforts specifically aimed at treating LMBV. To address this, our study sought to investigate the potential effectiveness of incorporating varying doses of VD3 into the diet as a treatment for LMBV. Through qRT-PCR and semi-qPCR, we observed significant suppression and clearance of LMBV pathogens in largemouth bass fed with 15000 IU/Kg and 20000 IU/Kg of VD3 within 14 days. In addition, VD3 treatment significantly increased the expression levels of key immune-related genes such as IL-1ß, IFN-γ, Mx, and IgM. Encouragingly, we observed that VD3 significantly increased antioxidant and immune activities such as TSOD, TAOC and C3 in serum and maintained total protein levels. Additionally, tissue pathology sections highlighted a dose-dependent relationship between VD3 supplementation and tissue damage, with the 15000 IU and 20000 IU groups exhibiting minimal damage. In conclusion, a reasonable concentration of VD3 effectively reduced LMBV replication and tissue damages, while improved immune-related genes expression and serum biochemical indices. These findings declare the considerable therapeutic potential of VD3 supplementation for combating LMBV disease and provide an alternative treatment option for fish farming.

The oral antigen-adjuvant fusion vaccine P-MCP-FlaC provides effective protective effect against largemouth bass ranavirus infection.

Largemouth bass ranavirus (LMBV) is highly contagious and lethal to largemouth bass, causing significant economic losses to the aquaculture industry. Oral vaccination is generally considered the most ideal strategy for protecting fish from viral infection. In this study, the fusion protein MCP-FlaC, consisting of the main capsid protein (MCP) as the antigen and flagellin C (FlaC) as the adjuvant, was intracellularly expressed in Pichia pastoris. Subsequently, the recombinant P. pastoris was freeze-dried to prepare the oral vaccine P-MCP-FlaC. Transmission electron microscopy and scanning electron microscopy analysis showed that the morphology and structure of the freeze-dried recombinant P. pastoris vaccine remained intact. The experiment fish (n = 100) was divided into five groups (P-MCP-FlaC, P-MCP, P-FlaC, P-pPIC3.5K, control) to evaluate the protective efficacy of the recombinant vaccine. Oral P-MCP-FlaC vaccine effectively up-regulated the serum enzymes activity (total superoxide dismutase, lysozyme, total antioxidant capacity, and complement component 3). The survival rate of P-MCP-FlaC group was significantly higher than that of the other groups. The mRNA expression of crucial immune genes (IL-1ß, TNF-α, MHC-II, IFN-γ, Mx, IgM, IgT) was also signally elevated in P-MCP-FlaC group. Vaccine P-MCP-FlaC markedly inhibited the replication of LMBV in the spleen, head kidney, and intestine, while reducing the degree of lesion in the spleen. These results suggest that the oral P-MCP-FlaC vaccine could effectively control LMBV infection, proving an effective strategy for viral diseases prevention in aquaculture.

Age- and dose-dependent susceptibility of axolotls (Ambystoma mexicanum) by bath exposure to Ambystoma tigrinum virus (ATV).

Ranaviruses are large, dsDNA viruses that have significant ecological and economic impact on cold-blooded vertebrates. However, our understanding of the viral proteins and subsequent host immune response(s) that impact susceptibility to infection and disease is not clear. The ranavirus Ambystoma tigrinum virus (ATV), originally isolated from the Sonoran tiger salamander (Ambystoma mavortium stebbinsi), is highly pathogenic at low doses of ATV at all tiger salamander life stages and this model has been used to explore the host-pathogen interactions of ATV infection. However, inconsistencies in the availability of laboratory reared larval tiger salamanders required us to look at the well characterized axolotl (A. mexicanum) as a model for ATV infection. Data obtained from five infection experiments over different developmental timepoints suggest that axolotls are susceptible to ATV in an age- and dose-dependent manner. These data support the use of the ATV-axolotl model to further explore the host-pathogen interactions of ranavirus infections.

Relationship between two pathogens in an amphibian community that experienced mass mortalities.

Because host species tend to harbor multiple parasitic species, coinfection in a host is common. The chytrid fungus Batrachochytrium dendrobatidis (Bd) and the viruses in the genus Ranavirus (Rv) are responsible for the decline of amphibians worldwide. Despite wide geographical co-occurrence and the serious conservation problem that coinfection with these pathogens could represent, little is known about their possible synergistic interactions and effects in a host community. We investigated the occurrence and associations between these two pathogens in an amphibian community after Rv-driven disease outbreaks were detected in four populations of the Iberian ribbed newt (Pleurodeles waltl) in northwestern Spain. We collected tissue samples from amphibians and fish and estimated Bd and Rv infection loads by qPCR. A few months after the most recent mass mortality event, Rv infection parameters at the affected sites decreased significantly or were lower than such registered at the sites where no outbreaks were recorded. Both pathogens were simultaneously present in almost all sites, but coinfection in a single host was rare. Our findings suggest that the co-occurrence of Bd and Rv does not predict adverse outcomes (e.g., enhanced susceptibility of hosts to one pathogen due to the presence or infection intensity of the other) following an outbreak. Other variables (such as species identity or site) were more important than infection with a pathogen in predicting the infection status and severity of infection with the other pathogen. Our results highlight the importance of host-specific and environmental characteristics in the dynamics of infections, coinfection patterns, and their impacts.

Relaciones entre dos patógenos en una comunidad anfibia que experimentó mortalidad masiva Resumen La coinfección es común en especies hospederas ya que estas especies tienden a albergar muchas especies parasíticas. El hongo quitridio Batrachochytrium dendrobatidis (Bd) y los virus del género Ranavirus (Rv) son responsables de la declinación mundial de anfibios. A pesar de la amplia co-ocurrencia geográfica y el problema serio de conservación que podría representar la coinfección con estos patógenos, se conoce muy poco sobre sus posibles interacciones sinérgicas y sus efectos en una comunidad hospedera. Investigamos la incidencia y las asociaciones entre estos dos patógenos en una comunidad anfibia después de que se detectaron brotes de enfermedades causados por Rv en cuatro poblaciones del tritón estriado ibérico (Pleurodeles waltl) en el noroeste de España. Recolectamos muestras de tejido de anfibios y peces y estimamos la carga infecciosa de Bd y Rv con una qPCR. Unos meses después del evento de mortalidad masiva más reciente, los parámetros de infección de Rv en los sitios afectados disminuyeron significativamente o fueron más bajos que los registrados en sitios sin brotes. Ambos patógenos estuvieron presentes de forma simultánea en casi todos los sitios, pero fue raro encontrar la coinfección en un solo hospedero. Nuestros descubrimientos sugieren que la coocurrencia de Bd y Rv no pronostica resultados adversos (aumento en la susceptibilidad de los hospederos a un patógeno debido a la presencia o intensidad de infección del otro patógeno) después de un brote. Otras variables, como la identidad de la especie o el sitio, fueron más importantes que la infección con un patógeno en la predicción del estado de infección y la severidad de la infección con otro patógeno. Nuestros resultados resaltan la importancia de las características ambientales y aquellas específicas del hospedero en las dinámicas de infección, los patrones de coinfección y sus impactos.

Advances on genomes studies of large DNA viruses in aquaculture: A minireview.

Genomic studies of viral diseases in aquaculture have received more and more attention with the growth of the aquaculture industry, especially the emerging and re-emerging viruses whose genome could contain recombination, mutation, insertion, and so on, and may lead to more severe diseases and more widespread infections in aquaculture animals. The present review is focused on aquaculture viruses, which is belonged to two clades, Varidnaviria and Duplodnaviria, and one class Naldaviricetes, and respectively three families: Iridoviridae (ranaviruses), Alloherpesviridae (fish herpesviruses), and Nimaviridae (whispoviruses). The viruses possessed DNA genomes nearly or larger than 100 kbp with gene numbers more than 100 and were considered large DNA viruses. Genome analysis and experimental investigation have identified several genes involved in genome replication, transcription, and virus-host interactions. In addition, some genes involved in virus genetic variation or specificity were also discussed. A summary of these advances would provide reference to future discovery and research on emerging or re-emerging aquaculture viruses.

Components of the Nucleotide Salvage Pathway Increase Frog Virus 3 (FV3) Replication.

Viruses are obligate intracellular parasites that alter host metabolic machinery to obtain energy and macromolecules that are pivotal for replication. Ranavirus, including the type species of the genus frog virus 3 (FV3), represent an ecologically important group of viruses that infect fish, amphibians, and reptiles. It was established that fatty acid synthesis, glucose, and glutamine metabolism exert roles during iridovirus infections; however, no information exists regarding the role of purine metabolism. In this study, we assessed the impact of exogenously applied purines adenine, adenosine, adenosine 5'-monophosphate (AMP), inosine 5'-monophosphate (IMP), inosine, S-adenosyl-L-homocysteine (SAH), and S-adenosyl-L-methionine (SAM) on FV3 replication. We found that all compounds except for SAH increased FV3 replication in a dose-dependent manner. Of the purines investigated, adenine and adenosine produced the most robust response, increasing FV3 replication by 58% and 51%, respectively. While all compounds except SAH increased FV3 replication, only adenine increased plaque area. This suggests that the stimulatory effect of adenine on FV3 replication is mediated by a mechanism that is at least in part independent from the other compounds investigated. Our results are the first to report a response to exogenously applied purines and may provide insight into the importance of purine metabolism during iridoviral infection.

Development and characterization of a spleen cell line from yellowfin seabream Acanthopagrus latus and its susceptibility to Mandarinfish ranavirus.

Yellowfin seabream (Acanthopagrus latus) is one of the most commercially important marine fish in China. In this study, a new continuous cell line, named ALS cells, was developed from the spleen tissue of A. latus. The cell line was maintained in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and successfully cultured up to 50 passages. The cell line was authenticated by amplifying and sequencing mitochondrial cytochrome C oxidase subunit-I (coi-I) gene. The ALS cell line had the maximum growth rate in DMEM/F-12 medium containing 20% FBS at 27°C. Chromosome number analysis showed that the ALS cells have a modal diploid chromosome number of 34. The ALS cell line was transfected with the pEGFP-N1 plasmid, and green fluorescence was observed. The ALS cell line was used for testing Mandarinfish ranavirus (MRV) susceptibility, and the cytopathic effects in the cell line were observed at 4 days post-infection (dpi). Furthermore, the susceptibility of the ALS cell line to MRV and the levels of MRV mRNA and viral loads were found to be significantly increased at 1-7 dpi. This study revealed that the ALS cell line could be useful for molecular, virological, and biotechnological studies on yellowfin seabream.

Fusing a TurboID tag with the Andrias davidianus ranavirus 2L reduced virus adsorption efficiency.

Andrias davidianus ranavirus (ADRV) is a member of the genus ranavirus (family Iridoviridae). ADRV 2L is an envelope protein that could be essential in viral infection. In the present study, the function of ADRV 2L was investigated by fusion with the biotin ligase TurboID tag. A recombinant ADRV with a V5-TurboID tag fused in the N-terminal of 2L (ADRVT-2L) and a recombinant ADRV expressing V5-TurboID (ADRVT) were constructed, respectively. Infection of the recombinant viruses and wild-type ADRV (ADRVWT) in the Chinese giant salamander thymus cell line (GSTC) showed that ADRVT-2L had reduced cytopathic effect and lower virus titers than the other two viruses, indicating the fusion of a big tag affected ADRV infection. Analysis of the temporal expression profile showed that the expression of V5-TurboID-2L was delayed than wild-type 2L. However, electron microscopy found that the virion morphogenesis was not affected in ADRVT-2L-infected cells. Furthermore, the virus binding assay revealed that the adsorption efficiency of ADRVT-2L was considerably decreased compared to the other two viruses. Therefore, these data showed that linking the TurboID tag to ADRV 2L affected virus adsorption to the cell membrane, which suggested an important role of 2L in virus entry into cells.

Diet- and salinity-induced modifications of the gut microbiota are associated with differential physiological responses to ranavirus infection in Rana sylvatica.

Greater knowledge of how host-microbiome interactions vary with anthropogenic environmental change and influence pathogenic infections is needed to better understand stress-mediated disease outcomes. We investigated how increasing salinization in freshwaters (e.g. due to road de-icing salt runoff) and associated increases in growth of nutritional algae influenced gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). Elevating salinity and supplementing a basic larval diet with algae increased larval growth and also increased ranavirus loads. However, larvae given algae did not exhibit elevated kidney corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. Thus, algal supplementation reversed a potentially maladaptive stress response to infection observed in prior studies in this system. Algae supplementation also reduced gut bacterial diversity. Notably, we observed higher relative abundances of Firmicutes in treatments with algae-a pattern consistent with increased growth and fat deposition in mammals-that may contribute to the diminished stress responses to infection via regulation of host metabolism and endocrine function. Our study informs mechanistic hypotheses about the role of microbiome mediation of host responses to infection that can be tested in future experiments in this host-pathogen system. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass.

Ranaviruses are promiscuous pathogens that threaten lower vertebrates globally. In the present study, two ranaviruses (SCRaV and MSRaV) were isolated from two fishes of the order Perciformes: mandarin fish (Siniperca chuatsi) and largemouth bass (Micropterus salmoides). The two ranaviruses both induced cytopathic effects in cultured cells from fish and amphibians and have the typical morphologic characteristics of ranaviruses. Complete genomes of the two ranaviruses were then sequenced and analyzed. Genomes of SCRaV and MSRaV have a length of 99, 405, and 99, 171 bp, respectively, and both contain 105 predicted open reading frames (ORFs). Eleven of the predicted proteins have differences between SCRaV and MSRaV, in which only one (79L) possessed a relatively large difference. A comparison of the sequenced six ranaviruses from the two fish species worldwide revealed that sequence identities of the six proteins (11R, 19R, 34L, 68L, 77L, and 103R) were related to the place where the virus was isolated. However, there were obvious differences in protein sequence identities between the two viruses and iridoviruses from other hosts, with more than half lower than 55%. Especially, 12 proteins of the two isolates had no homologs in viruses from other hosts. Phylogenetic analysis revealed that ranaviruses from the two fishes clustered in one clade. Further genome alignment showed five groups of genome arrangements of ranaviruses based on the locally collinear blocks, in which the ranaviruses, including SCRaV and MSRaV, constitute the fifth group. These results provide new information on the ranaviruses infecting fishes of Perciformes and also are useful for further research of functional genomics of the type of ranaviruses.

Ranavirus infection does not reduce heat tolerance in a larval amphibian.

Extreme heat events and emerging infectious diseases negatively impact wildlife populations, but the interacting effects of infection and host heat tolerance remain understudied. The few studies covering this subject have demonstrated that pathogens lower the heat tolerance of their hosts, which places infected hosts at a greater risk experiencing lethal heat stress. Here, we studied how ranavirus infection influenced heat tolerance in larval wood frogs (Lithobates sylvaticus). In line with similar studies, we predicted the elevated costs of ranavirus infection would lower heat tolerance, measured as critical thermal maximum (CTmax), compared to uninfected controls. Ranavirus infection did not reduce CTmax and there was a positive relationship between CTmax and viral loads. Our results demonstrate that ranavirus-infected wood frog larvae had no loss in heat tolerance compared to uninfected larvae, even at viral loads associated with high mortality rates, which contradicts the common pattern for other pathogenic infections in ectotherms. Larval anurans may prioritize maintenance of their CTmax when infected with ranavirus to promote selection of warmer temperatures during behavioral fever that can improve pathogen clearance. Our study represents the first to examine the effect of ranavirus infection on host heat tolerance, and because no decline in CTmax was observed, this suggests that infected hosts would not be under greater risk of heat stress.

Oral Vaccination of Largemouth Bass (Micropterus salmoides) against Largemouth Bass Ranavirus (LMBV) Using Yeast Surface Display Technology.

Largemouth bass ranavirus (LMBV) infects largemouth bass, leading to significant mortality and economic losses. There are no safe and effective drugs against this disease. Oral vaccines that directly target the intestinal mucosal immune system play an important role in resisting pathogens. Herein, the B subunit of Escherichia coli heat-labile enterotoxin (LTB, a mucosal immune adjuvant) and the LMBV main capsid protein (MCP) were expressed using Saccharomyces cerevisiae surface display technology. The yeast-prepared oral vaccines were named EBY100-OMCP and EBY100-LTB-OMCP. The candidate vaccines could resist the acidic intestinal environment. After 7 days of continuous oral immunization, indicators of innate and adaptive immunity were measured on days 1, 7, 14, 21, 28, 35, and 42. High activities of immune enzymes (T-SOD, AKP, ACP, and LZM) in serum and intestinal mucus were detected. IgM in the head kidney was significantly upregulated (EBY100-OMCP group: 3.8-fold; BY100-LTB-OMCP group: 4.3-fold). IgT was upregulated in the intestines (EBY100-OMCP group: 5.6-fold; EBY100-LTB-OMCP group: 6.7-fold). Serum neutralizing antibody titers of the two groups reached 1:85. Oral vaccination protected against LMBV infection. The relative percent survival was 52.1% (EBY100-OMCP) and 66.7% (EBY100-LTB-OMCP). Thus, EBY100-OMCP and EBY100-LTB-OMCP are promising and effective candidate vaccines against LMBV infection.