Frog Virus Research Articles

Ranavirus frog virus 3 infection in Nile tilapia: evidence of pathogenesis and host susceptibility

Ranavirus frog virus 3 infection in Nile tilapia: evidence of pathogenesis and host susceptibility

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.

Exploring the Effectiveness of Acyclovir against Ranaviral Thymidine Kinases: Molecular Docking and Experimental Validation in a Fish Cell Line.

The effectiveness of acyclovir, a selective anti-herpesvirus agent, was tested both in silico and in vitro against two ranaviruses, namely the European catfish virus (ECV) and Frog virus 3 (FV3). ECV can cause significant losses in catfish aquaculture, while FV3 poses a risk to vulnerable amphibian populations. The genome of ranaviruses encodes thymidine kinases (TKs) similar to those of herpesviruses. Molecular docking simulations demonstrated that the acyclovir molecule can bind to the active sites of both investigated viral TKs in an orientation conducive to phosphorylation. Subsequently, the antiviral effect of acyclovir was tested in vitro in Epithelioma Papulosum Cyprini (EPC) cells with endpoint titration and qPCR. Acyclovir was used at a concentration of 800 µM, which significantly reduced the viral loads and titers of the ranaviruses. A similar reduction rate was observed with Ictalurid herpesvirus 2, which was used as a positive control virus. These promising results indicate that acyclovir might have a wider range of uses; besides its effectiveness against herpesviruses, it could also be used against ranavirus infections.

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.

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning.

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.

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

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

Genomic characterization of two ranavirus isolates identified from a gopher frog (Lithobates capito) and a striped newt (Notophthalmus perstriatus) during a mass mortality event in Florida.

Two ranavirus isolates were recovered from anuran and salamander samples collected during an amphibian mass mortality event in North-Central Florida in 2021. Phylogenetic analyses of the full genomes confirmed that the two isolates were nearly identical and strains of the species Frog virus 3.

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

IntroductionRanavirus 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).MethodsFor 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.ResultsWe 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.DiscussionOur 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.

Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning.

A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, a limit of detection (LoD) of 100 aM (60.2 copies/μL) is achieved by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, smartphone microscopy is implemented, and an LoD of 10 aM is achieved in 40 min. The proposed system detects four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32. In addition, deep learning models are deployed for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100 and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, the proposed RPA-CRISPR/Cas12a combined with smartphone readouts and artificial-intelligence-assisted classification showcases the great potential for FV3 detection, specifically POC detection of DNA virus.

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.

The wood frog (Rana sylvatica): An emerging comparative model for anuran immunity and host-ranavirus interactions

The wood frog (Rana sylvatica) is widely distributed across North America and is the only amphibian found north of the Arctic Circle due to its remarkable ability to tolerate whole-body freezing. Recent mass mortalities attributable to Ranavirus spp. (family Iridoviridae) in wild juvenile wood frogs, coupled with the apparent high susceptibility of wood frogs to experimental infection with frog virus 3 (FV3), the type species of the Ranavirus genus, or FV3-like isolates underscore the serious threat ranaviruses poses to wood frog populations. Despite the ecological relevance and unique life history of wood frogs, our understanding of the wood frog immune system and antiviral response to ranaviral infections is in its infancy. Here we aim to (1) synthesize the limited knowledge of wood frog immune defences, (2) review recent progress in establishing the wood frog as a study system for ranavirus infection, and (3) highlight the future use of wood frogs as a model anuran to provide insight into the evolution of anuran immune systems and antiviral responses.

Systemic Nannizziopsis arthrosporioides in an African Side-neck Turtle (Pelomedusa subrufa)

A 2-yr-old female African side-neck turtle (Pelomedusa subrufa) housed at a pet store in Illinois, USA presented for evaluation of nasal discharge and an ulcerative skin lesion. Molecular diagnostics for Mycoplasma spp., herpesviruses and frog virus 3 (ranavirus) were negative and cytologic evaluation of the ulceration showed non-septic inflammation without fungal elements. A presumptive diagnosis of a bacterial upper respiratory infection was treated with ceftazidime (20 mg/kg SC every 72 hrs) and husbandry improvements were recommended. One month later, the turtle re-presented and physical examination revealed diffuse skin sloughing and severe dehydration, and the animal was euthanized. Necropsy and histopathology revealed multiple fungal granulomas throughout the liver, gastrointestinal tract, and lungs. Heavy growth of a white powdery fungal organism was obtained from culture. Four gene targets (D1/D2 domains of the 28S ribosomal DNA, 18S-28S ribosomal internal transcribed spacer region, actin and β-tubulin) were sequenced from the fungal isolate and each was >98% identical to Nannizziopsis arthrosporioides. A maximum likelihood phylogenetic tree constructed from concatenated D1/D2, ITS, actin, and β-tubulin sequences from Nannizziopsis spp. placed the new fungal isolate in a highly supported (99% bootstrap support) monophyletic group with two N. arthrosporioides isolates. This case report describes the first known N. arthrosporioides infection in an aquatic turtle species and adds to the growing literature surrounding N. arthrosporioides infections in non-squamate reptilian patients.

HEALTH ASSESSMENT OF PAINTED TURTLES (CHRYSEMYS PICTA) IN A RESTORED WETLAND HABITAT IN NORTHWESTERN INDIANA, USA.

Freshwater turtles are sensitive to environmental changes and can serve as sentinel species for ecosystem health assessments. The Efroymson Restoration at Kankakee Sands in northwestern Indiana, USA has been restored in the past 25 yr from primarily agricultural land to a mosaic of prairie and wetland habitats. Health assessments of 40 free-ranging painted turtles (Chrysemys picta) at Kankakee Sands were performed in May 2021 to evaluate overall health status, screen for infectious diseases, and obtain baseline clinical pathology values in this population. Assessment of each turtle included a physical examination, complete blood count, plasma biochemistry panel, blood lactate level, venous blood gas analysis, serum trace mineral panel, serum vitamin D3 level, and plasma protein electrophoresis. Oral and cloacal swabs were tested for adenoviruses, herpesviruses, frog virus 3, and Mycoplasmopsis species by PCR in 39 painted turtles. Four turtles were positive for adenovirus, which shared 100% homology to Sulawesi tortoise adenovirus. Two turtles were herpesvirus-positive with 100% homology to emydid herpesvirus 1. No Mycoplasmopsis spp. or frog virus 3 was detected. Female turtles had significantly higher manganese, prealbumin, uric acid, triglycerides, and ionized calcium levels, while male turtles had significantly higher cholesterol, glutamate dehydrogenase, and CO2 levels. These baseline data can be used for future research into freshwater turtle health in restored wetland habitats.

Is Xenopus laevis introduction linked with Ranavirus incursion, persistence and spread in Chile?

Ranaviruses have been associated with amphibian, fish and reptile mortality events worldwide and with amphibian population declines in parts of Europe. Xenopus laevis is a widespread invasive amphibian species in Chile. Recently, Frog virus 3 (FV3), the type species of the Ranavirus genus, was detected in two wild populations of this frog near Santiago in Chile, however, the extent of ranavirus infection in this country remains unknown. To obtain more information about the origin of ranavirus in Chile, its distribution, species affected, and the role of invasive amphibians and freshwater fish in the epidemiology of ranavirus, a surveillance study comprising wild and farmed amphibians and wild fish over a large latitudinal gradient (2,500 km) was carried out in 2015-2017. In total, 1,752 amphibians and 496 fish were tested using a ranavirus-specific qPCR assay, and positive samples were analyzed for virus characterization through whole genome sequencing of viral DNA obtained from infected tissue. Ranavirus was detected at low viral loads in nine of 1,011 X. laevis from four populations in central Chile. No other amphibian or fish species tested were positive for ranavirus, suggesting ranavirus is not threatening native Chilean species yet. Phylogenetic analysis of partial ranavirus sequences showed 100% similarity with FV3. Our results show a restricted range of ranavirus infection in central Chile, coinciding with X. laevis presence, and suggest that FV3 may have entered the country through infected X. laevis, which appears to act as a competent reservoir host, and may contribute to the spread the virus locally as it invades new areas, and globally through the pet trade.

Climate warming triggers the emergence of native viruses in Iberian amphibians.

The number of epizootics in amphibian populations caused by viruses of the genus Ranavirus is increasing worldwide. Yet, causes for pathogen emergence are poorly understood. Here, we confirmed that the Common midwife toad virus (CMTV) and Frog virus 3 (FV3) are responsible for mass mortalities in Iberia since the late 1980s. Our results illustrate the Iberian Peninsula as a diversity hotspot for the highly virulent CMTV. Although this pattern of diversity in Europe is consistent with spread by natural dispersal, the exact origin of the emergence of CMTV remains uncertain. Nevertheless, our data allow hypothesizing that the Iberian Peninsula might harbor the ancestral population of CMTVs that could have spread into the rest of Europe. In addition, we found that climate warming could be triggering the CMTV outbreaks, supporting its endemic status in the Iberian Peninsula.

PREVALENCE OF MULTIPLE REPTILIAN PATHOGENS IN THE OROPHARYNGEAL MUCOSA, CLOACAL MUCOSA, AND BLOOD OF DIAMONDBACK TERRAPIN (MALACLEMYS TERRAPIN) POPULATIONS FROM MARYLAND AND GEORGIA, USA.

The diamondback terrapin (Malaclemys terrapin) is a coastal turtle with a range from Massachusetts to Texas and is the only exclusively brackish water turtle in North America. Two populations of wild terrapins from Maryland (n=55) and Georgia (n=7) were examined and tested for potential reptile pathogens. Whole blood and a mucosal (combined oropharyngeal and cloacal) swab from each animal were evaluated by quantitative PCR for 15 potential pathogens including frog virus 3, box turtle Mycoplasmopsis, Mycoplasma agassizii, Mycoplasma testudineum, Salmonella Enteritidis, Salmonella Typhimurium, Borrelia burgdorferi, Anaplasma phagocytophilum, tortoise intranuclear coccidia, testudinid alphaherpesvirus 2, terrapene herpesvirus 1, and terrapene adenovirus. Swabs were positive for a DNA segment 100% homologous to M. testudineum in both populations, with Maryland animals 87% (48 of 55) positive and Georgia animals 86% (6 of 7) positive. Although Mycoplasmopsis spp. are important respiratory pathogens for members of the order Testudines, none of the animals in the study showed any sign of upper respiratory disease. Our data suggest that M. testudineum may survive in non-Testudinidae turtles without causing clinical sigs of disease and suggesting appropriate precautions should be taken in facilities that house multiple species of turtles simultaneously.

Non-Lethal Detection of Frog Virus 3-Like (RUK13) and Common Midwife Toad Virus-Like (PDE18) Ranaviruses in Two UK-Native Amphibian Species.

Ranaviruses have been involved in amphibian mass mortality events worldwide. Effective screening to control this pathogen is essential; however, current sampling methods are unsuitable for the detection of subclinical infections. Non-lethal screening is needed to prevent both further spread of ranavirus and losses of at-risk species. To assess non-lethal sampling methods, we conducted two experiments: bath exposing common frogs to RUK13 ranavirus at three concentrations, and exposing common toads to RUK13 or PDE18. Non-lethal sampling included buccal, digit, body and tank swabs, along with toe clips and stool taken across three time-points post-exposure. The presence/load of ranavirus was examined using quantitative PCR in 11 different tissues obtained from the same euthanised animals (incl. liver, gastro-intestinal tract and kidney). Buccal swab screening had the highest virus detection rate in both species (62% frogs; 71% toads) and produced consistently high virus levels compared to other non-lethal assays. The buccal swab was effective across multiple stages of infection and differing infection intensities, though low levels of infection were more difficult to detect. Buccal swab assays competed with, and even outperformed, lethal sampling in frogs and toads, respectively. Successful virus detection in the absence of clinical signs was observed (33% frogs; 50% toads); we found no difference in detectability for RUK13 and PDE18. Our results suggest that buccal swabbing could replace lethal sampling for screening and be introduced as standard practice for ranavirus surveillance.

Using long, sequence-specific dsRNA to knockdown inducible protein expression and virus production via an RNAi-like mechanism

RNA interference (RNAi) is a powerful innate immune mechanism to knock down translation of specific proteins whose machinery is conserved from plants to mammals. The template used to determine which mRNA's translation is inhibited is dsRNA, whose origin can range from viruses (long dsRNA, ∼100–1000s bp) to host (micro(mi)RNA, ∼20mers). While miRNA-mediated RNAi is well described in vertebrates, the ability of long dsRNA to guide RNAi-mediated translation inhibition in vertebrates is controversial. Indeed, as long dsRNA is so effective at inducing type I interferons (IFNs), and IFNs down-regulate RNAi machinery, it is believed that IFN-competent cells are not capable of using long dsRNA for RNAi. In the present study the ability of long, sequence specific dsRNA to knock down both host protein expression and viral replication is investigated in IFN-competent rainbow trout cells. Before exploring RNAi effects, the optimal dsRNA concentration that would funnel into RNAi without triggering the IFN response was determined. After which, the ability of sequence specific long dsRNA to target knockdown via RNAi was evaluated in: (1) uninfected host cells using inducible luciferase gene expression and (2) host cells infected with chum salmon reovirus (CSV), frog virus 3 (FV3) or viral hemorrhagic septicemia virus genotype IVa (VHSV-IVa). Induced expression studies utilized RTG-P1, a luciferase reporter cell line, and dsRNA containing luciferase sequence (dsRNA-Luc) or a mis-matched sequence (dsRNA-GFP), and subsequent luminescence intensity was measured. Anti-CSV studies used dsRNA-CSVseg7 and dsRNA-CSVseg10 to target CSV segment 7 and CSV segment 10 respectively. Inhibition of virus replication was measured by viral titration and RT-qPCR. Taking advantage of the fact that long dsRNA can accommodate more sequences than miRNAs, the antiviral capability of dsRNA molecules containing both CSV segment 7 and segment 10 simultaneously was also measured. Target sequence appears important, as dsRNA-FV3MCP did not knock down FV3 titres, and while dsRNA-VHSV-N knocked down VHSV-IVa, dsRNA-VHSV-G and dsRNA-VHSV-M did not. This is the first study in fish to provide evidence that sequence specific long dsRNA induces potent gene expression silencing and antiviral responses in vitro via an RNAi-like mechanism instead of an IFN-dependent response.