Antioxidant anthocyanin synergistic immune enhancer nanopeptide C-I20 remarkably enhances the protective effect of largemouth bass against largemouth bass ranavirus.

Largemouth bass ranavirus (LMBV) infection results in huge economic losses in largemouth bass (Micropterus salmoides) industry. Nanopeptide C-I20 and anthocyanins have a positive effect on promoting immune responses and antioxidant mechanisms in several aquatic organisms, and are therefore used to inhibit LMBV infection. In this study, we developed an LMBV immersion challenge model using three different viral concentrations (1×104 copies/mL, 1×105 copies/mL, and 1×106 copies/mL) to infect largemouth bass, and LMBV-MCP mRNA expression was detected in infected fish. Following infection, the fish exhibited severe external ulceration, redness swelling, and darkening of the skin. Histopathological examination revealed significant necrosis and inflammation in muscle tissue, epithelial cell shedding in renal tubules, macrophage aggregation centers and cellular vacuolization in spleen and head kidney, and cellular hypertrophy in liver. To mitigate LMBV infection, we explored the protective effects of a combined treatment strategy involving C-I20 and anthocyanin. Overall, the combination of anthocyanin and C-I20 demonstrated the highest protective efficacy, significantly reducing viral loads in muscle, liver, spleen, and head kidney. Moreover, this treatment regimen enhanced antioxidant enzyme activities (T-AOC, TSOD, GSH-Px, CAT) and modulated important immune genes (IL-1, IL-8, TNF-α, IL-10, Mx, and IgM) expression. In conclusion, the synergistic application of anthocyanin and C-I20 demonstrates significant efficacy in mitigating LMBV infection. This research introduces a novel and promising approach to managing infectious diseases in aquaculture settings.

Lactobacillus casei displaying MCP2α and FlaC delivered by PLA microspheres effectively enhances the immune protection of largemouth bass (Micropterus salmoides) against LMBV infection.

Largemouth bass ranavirus (LMBV) seriously affects the development of largemouth bass (Micropterus salmoides) industry and causes huge economic losses. Oral vaccine can be a promising method for viral disease precaution. In this study, MCP2α was identified as a valuable epitope region superior to MCP and MCP2 of LMBV by neutralizing antibody experiments. Then, recombinant Lactobacillus casei expressing the fusion protein MCP2αC (MCP2α as antigen, C represents flagellin C from Aeromonas hydrophila as adjuvant) on surface was constructed and verified. Further, PLA microsphere vaccine loading recombinant MCP2αC L. casei was prepared. The PLA microspheres vaccine were observed by scanning electron microscopy and showed a smooth, regular spherical surface with a particle size distribution between 100 and 200 µm. Furthermore, we evaluated the tolerance of PLA-MCP2αC vaccine in simulated gastric fluid and simulated intestinal fluid, and the results showed that PLA-MCP2αC can effectively resist the gastrointestinal environment. Moreover, the protective effect of PLA-MCP2αC against LMBV was evaluated after oral immunization and LMBV challenge. The results showed that PLA-MCP2αC effectively up-regulated the activity of serum biochemical enzymes (T-SOD, T-AOC, LZM, complement C3) and induced the mRNA expression of representative immune genes (IL-1ß, TNF-α, IFN-γ, MHC-IIα, Mx, IgM) in spleen and head kidney tissues. The survival rate of largemouth bass vaccinated with PLA-MCP2αC increased from 24 % to 68 %. Meanwhile, PLA-MCP2αC inhibited the LMBV burden in spleen, head kidney and liver tissues and attenuated tissue damage in spleen. These results suggested that PLA-MCP2αC can be used as a candidate oral vaccine against LMBV infection in aquaculture.

Isolation and Characterization of a Frog Virus 3 Strain from a Wood Frog (Rana sylvatica) in Wood Buffalo National Park.

Members of the Iridoviridae family, genus Ranavirus, represent a group of globally emerging pathogens of ecological and economic importance. In 2017, an amphibian die-off of wood frogs (Rana sylvatica) and boreal chorus frogs (Pseudacris maculata) was reported in Wood Buffalo National Park, Canada. Isolation and complete genomic sequencing of the tissues of a wood frog revealed the presence of a frog virus 3 (FV3)-like isolate, Rana sylvatica ranavirus (RSR), with a genome size of 105,895 base pairs, 97 predicted open reading frames (ORFs) bearing sequence similarity to FV3 (99.98%) and a FV3-like isolate from a spotted salamander in Maine (SSME; 99.64%). Despite high sequence similarity, RSR had a unique genomic composition containing ORFs specific to either FV3 or SSME. In addition, RSR had a unique 13 amino acid insertion in ORF 49/50L. No differences were found in the in vitro growth kinetics of FV3, SSME, and RSR; however, genomic differences between these isolates were in non-core genes, implicated in nucleic acid metabolism and immune evasion. This study highlights the importance of viral isolation and complete genomic analysis as these not only provide information on ranavirus spatial distribution but may elucidate genomic factors contributing to host tropism and pathogenicity.

Genome-Wide Association Study of Resistance to Largemouth Bass Ranavirus (LMBV) in Micropterus salmoides.

The disease caused by Largemouth bass ranavirus (LMBV) is one of the most severe viral diseases in largemouth bass (Micropterus salmoides). It is crucial to evaluate the genetic resistance of largemouth bass to LMBV and develop markers for disease-resistance breeding. In this study, 100 individuals (45 resistant and 55 susceptible) were sequenced and evaluated for resistance to LMBV and a total of 2,579,770 variant sites (SNPs-single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels)) were identified. A total of 2348 SNPs-InDels and 1018 putative candidate genes associated with LMBV resistance were identified by genome-wide association analyses (GWAS). Furthermore, GO and KEGG analyses revealed that the 10 candidate genes (MHC II, p38 MAPK, AMPK, SGK1, FOXO3, FOXO6, S1PR1, IL7R, RBL2, and GADD45) were related to intestinal immune network for IgA production pathway and FoxO signaling pathway. The acquisition of candidate genes related to resistance will help to explore the molecular mechanism of resistance to LMBV in largemouth bass. The potential polymorphic markers identified in this study are important molecular markers for disease resistance breeding in largemouth bass.

A naturaly attenuated largemouth bass ranavirus strain provided protection for Micropterus salmoides by immersion immunization.

Largemouth bass ranavirus (LMBV) causes disease outbreaks and high mortality at all stages of largemouth bass farming. Therefore, live vaccine development is critical for largemouth bass prevention against LMBV by immersion immunization. Herein, an attenuated LMBV strain with good immunogenicity, designated as LMBV-2007136, was screened from the natural LMBV strains bank through challenge assay and immersion immunization experiment. After determing the safe concentration range of LMBV-2007136, the minimum immunizing dose of immersion immunization was verified. When largemouth bass were vaccinated by immersion at the lowest concentration of 102.0 TCID50/mL, all of fish were survival post virulent LMBV challenge, and the relative percent survival (RPS) was 100 %. And the immune gene expression levels of IL-10, IL-12, IFN-γ, and IgM in the spleen and kidney post-vaccination were significantly up-regulated compared to the control group, but TNF-α expression showed no significant changes. The safety and efficacy of LMBV-2007136 at passages P8, P13, and P18 were futher assessed, and no death of largemouth bass was observed within 21 days post-immunization and RPS of three vaccination groups was 100 %, suggesting that the safety and efficacy of the attenuated strain at different passages was stable. Furthermore, in the virulence reversion test, the attenuated strain was propagated through 5 times in largemouth bass by intraperitoneal injection and no abnormality and mortality were observed, further proving the attenuated vaccine candidate LMBV-2007136 was safe. These results proved that LMBV-2007136 could be a promising candidate for a live vaccine to protect largemouth bass from LMBV disease.

Exploring Codon Usage Patterns and Influencing Factors in Ranavirus DNA Polymerase Genes.

Ranaviruses, members of the genus Ranavirus within the family Iridoviridae, have become a significant concern for amphibian populations globally, along with other cold-blooded vertebrates, due to their emergence as a significant threat. We employed bioinformatics tools to examine the codon usage patterns in 61 DNA pol genes from Ranavirus, Lymphocystivirus, Megalocytivirus, and two unclassified ranaviruses, as no prior studies had been conducted on this topic. The results showed a slight or low level of codon usage bias (CUB) in the DNA pol genes of Ranavirus. Relative synonymous codon usage (RSCU) analysis indicated that the predominant codons favored in Ranavirus DNA pol genes terminate with C or G. Correlation analysis examining nucleotide content, third codon position, effective number of codons (ENC), correspondence analysis (COA), Aroma values, and GRAVY values indicated that the CUB across DNA pol genes could be influenced by both mutation pressure and natural selection. The neutrality plot indicated that natural selection is the primary factor driving codon usage. Furthermore, the analysis of the codon adaptation index (CAI) illustrated the robust adaptability of Ranavirus DNA pol genes to their hosts. Analysis of the relative codon deoptimization index (RCDI) suggested that Ranavirus DNA pol genes underwent greater selection pressure from their hosts. These findings will aid in comprehending the factors influencing the evolution and adaptation of Ranavirus to its hosts.

Mandarin fish von Hippel-Lindau protein regulates the NF-κB signaling pathway via interaction with IκB to promote fish ranavirus replication.

The von Hippel-Lindau tumor suppressor protein (VHL), an E3 ubiquitin ligase, functions as a critical regulator of the oxygen-sensing pathway for targeting hypoxia-inducible factors. Recent evidence suggests that mammalian VHL may also be critical to the NF-κB signaling pathway, although the specific molecular mechanisms remain unclear. Herein, the roles of mandarin fish ( Siniperca chuatsi) VHL ( scVHL) in the NF-κB signaling pathway and mandarin fish ranavirus (MRV) replication were explored. The transcription of scVHL was induced by immune stimulation and MRV infection, indicating a potential role in innate immunity. Dual-luciferase reporter gene assays and reverse transcription quantitative PCR (RT-qPCR) results demonstrated that scVHL evoked and positively regulated the NF-κB signaling pathway. Treatment with NF-κB signaling pathway inhibitors indicated that the role of scVHL may be mediated through scIKKα, scIKKß, scIκBα, or scp65. Co-immunoprecipitation (Co-IP) analysis identified scIκBα as a novel target protein of scVHL. Moreover, scVHL targeted scIκBα to catalyze the formation of K63-linked polyubiquitin chains to activate the NF-κB signaling pathway. Following MRV infection, NF-κB signaling remained activated, which, in turn, promoted MRV replication. These findings suggest that scVHL not only positively regulates NF-κB but also significantly enhances MRV replication. This study reveals a novel function of scVHL in NF-κB signaling and viral infection in fish.

Abiotic and biotic factors jointly influence the contact and environmental transmission of a generalist pathogen.

The joint influence of abiotic and biotic factors is important for understanding the transmission of generalist pathogens. Abiotic factors such as temperature can directly influence pathogen persistence in the environment and will also affect biotic factors, such as host community composition and abundance. At intermediate spatial scales, the effects of temperature, community composition, and host abundance are expected to contribute to generalist pathogen transmission. We use a simple transmission model to explain and predict how host community composition, host abundance, and environmental pathogen persistence times can independently and jointly influence transmission. Our transmission model clarifies how abiotic and biotic factors can synergistically support the transmission of a pathogen. The empirical data show that high community competence, high abundance, and low temperatures correlate with high levels of transmission of ranavirus in larval amphibian communities. Discrete wetlands inhabited by larval amphibians in the presence of ranavirus provide a compelling case study comprising distinct host communities at a spatial scale anticipated to demonstrate abiotic and biotic influence on transmission. We use these host communities to observe phenomena demonstrated in our theoretical model. These findings emphasize the importance of considering both abiotic and biotic factors, and concomitant direct and indirect mechanisms, in the study of pathogen transmission and should extend to other generalist pathogens with the capacity for environmental transmission.

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.

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.

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.