The Roles of Amphibian (Xenopus laevis) Macrophages during Chronic Frog Virus 3 Infections

Infections by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to global amphibian decline. The Xenopus laevis frog is an ideal research platform upon which to study the roles of distinct frog leukocyte populations during FV3 infections. Frog macrophages (MΦs) are integrally involved during FV3 infection, as they facilitate viral dissemination and persistence but also participate in immune defense against this pathogen. In turn, MΦ differentiation and functionality depend on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by CSF-1 and iterleukin-34 (IL-34) cytokines. Our past work indicated that X. laevis CSF-1 and IL-34 give rise to morphologically and functionally distinct frog MΦ subsets, and that these CSF-1- and IL-34-MΦs respectively confer susceptibility and antiviral resistance to FV3. Because FV3 targets the frog kidneys and establishes chronic infections therein, presently we examined the roles of the frog CSF-1- and IL-34-MΦs in seeding and maintaining these chronic kidney infections. Our findings indicate that the frog CSF-1-MΦs result in more prominent kidney FV3 infections, which develop into greater reservoirs of lingering FV3 marked by infiltrating leukocytes, fibrosis, and overall immunosuppressive states. Moreover, the antiviral effects of IL-34-MΦs are short-lived and are lost as FV3 infections progress.

Genomic Sequencing of Ranavirus Isolates from a Three-Spined Stickleback ( Gasterosteus aculeatus ) and a Red-Legged Frog ( Rana aurora )

Two ranavirus isolates were recovered during a wildlife disease investigation in California in 1996. Preliminary testing at the time of sample collection indicated that the two isolates were identical. Phylogenetic analysis of the full genomes of these two isolates revealed that they are a single strain of frog virus 3.

Sustained Ranavirus outbreak causes mass-mortality and morbidity in imperiled amphibians.

A persistent two-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and signs in N. perstriatus continued for five weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a Frog Virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

Comparative Genomics Analysis between frog Virus 3-like Ranavirus from the First Canadian Reptile Mortality Event and Similar Viruses from Amphibians

Ranaviruses are emerging pathogens that threaten the biodiversity of wild and captive cold-blooded vertebrates. Reports of ranavirus-induced mortality events are increasing and ranavirus disease is reportable to the World Organization for Animal Health. Previous studies have suggested interclass transmission of ranaviruses and Frog virus 3 (FV3)-like viruses are of particular interest. This study presents the whole-genome assembly of a 106 kb FV3-like genome obtained from the liver tissue of a reptile (wild Chelydra serpentina, common snapping turtle) that died of ranavirus disease in Canada. The FV3-like ON turtle/2018 strain shares the highest genome-wide nucleotide identity (99.71%) with the wild-type FV3 virus detected in the USA from a Northern leopard frog and an FV3-like strain identified from a wood frog in 2017 in Alberta, Canada. The novel genome contains all 26 Iridoviridae core genes, 11 FV3-like genes, and 9 unique truncations, three of which are core Iridoviridae ORFs. Additionally, the two most closely related FV3-like strains from amphibians, were compared to a non-FV3-like amphibian infecting and a fish infecting ranavirus species that showed similar codon usage patterns. G/C-ending codons were the preferred codons for all five strains. Investigation of putative recombination events identified four potential recombination events in the FV3-like ON turtle/2018 genome consistent with this FV3-like reptile infecting strain originating from an amphibian infecting FV3-like ranavirus. Altogether, this study provides insights into the genome structure and the differences in the novel FV3-like genome compared to other ranavirus genomes.

Discovery of frog virus 3 microRNAs and their roles in evasion of host antiviral responses

Frog virus 3 (FV3, genus Ranavirus) causes devastating disease in amphibian populations and is capable of subverting host immune responses. Evidence suggests that virus-encoded microRNAs (v-miRNAs) are implicated in host immunoevasion tactics. Thus, we sought to discover FV3-encoded v-miRNAs and to uncover their putative roles in immunoevasion. Small RNA libraries were generated from FV3-infected Xela DS2, a Xenopus laevis dorsal skin epithelial-like cell line, at 24- and 72-hours post-infection (hpi). We discovered 43 FV3 v-miRNAs and identified that 15 are upregulated at 24 hpi, while 18 are upregulated at 72 hpi. Target prediction analyses revealed that FV3 v-miRNAs target host genes involved in key antiviral signaling pathways, while gene ontology analyses suggest that FV3 v-miRNAs may broadly impact host cell function. This is the first study to experimentally detect mature v-miRNAs produced by FV3. Our findings highlight the possibility that ranaviral v-miRNAs facilitate immunoevasion of frog antiviral responses.

Molecular Confirmation of Ranavirus Infection in Amphibians From Chad, Africa

Ranaviruses are DNA viruses (Family Iridoviridae; Subfamily Alphairidovirinae) and ranaviral disease is considered an emerging infectious disease of ectothermic vertebrates. Ranavirus infection can have varying pathological effects on infected amphibians, reptiles, and fish, most notably causing significant mortality events and population declines. Despite having a broad global range with reports from six continents, only a single incidental finding in Xenopus longipes from mainland Africa (Cameroon) is known and lacks molecular confirmation. Thus, there is a considerable knowledge gap concerning ranaviruses in Africa. We opportunistically obtained tissue samples from 160 amphibians representing five genera (Hoplobatrachus, Hylarana, Ptychadena, Pyxicephalus, and Xenopus) and two turtles (Pelomedusa sp.) from Chad, Africa. Samples were tested for ranavirus infection using a conventional PCR assay targeting the major capsid protein (MCP). A total of 25/160 (16%) frogs tested positive including 15/87 (17%) Hoplobatrachus occipitalis, 10/58 (17%) Ptychadena spp., 0/3 Pyxicephalus spp., 0/9 Xenopus spp., and 0/3 Hylarana spp. One of two turtles tested positive. Partial MCP gene sequences indicated all samples were >98% similar to several frog virus 3 (FV3)-like sequences. Additional gene targets (DNA polymerase [DNApol], ribonucleotide reductase alpha [RNR- α], ribonucleotide reductase beta subunit [RNR- β]) were sequenced to provide further detailed classification of the virus. Sequences of individual gene targets indicate that the ranavirus detected in frogs in Chad is most similar to tiger frog virus (TFV), a FV3-like virus previously isolated from diseased amphibians cultured in China and Thailand. Full genome sequencing of one sample indicates that the Chad frog virus (CFV) is a well-supported sister group to the TFVs previously determined from Asia. This work represents the first molecular confirmation of ranaviruses from Africa and is a first step in comparing ranavirus phylogeography on a local and global scale.

Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Antiviral Responses to Intestinal Frog Virus 3 Infections

The global amphibian declines are compounded by ranavirus infections such as Frog Virus 3 (FV3), and amphibian tadpoles more frequently succumb to these pathogens than adult animals. Amphibian gastrointestinal tracts represent a major route of ranavirus entry, and viral pathogenesis often leads to hemorrhaging and necrosis within this tissue. Alas, the differences between tadpole and adult amphibian immune responses to intestinal ranavirus infections remain poorly defined. As interferon (IFN) cytokine responses represent a cornerstone of vertebrate antiviral immunity, it is pertinent that the tadpoles and adults of the anuran Xenopus laevis frog mount disparate IFN responses to FV3 infections. Presently, we compared the tadpole and adult X. laevis responses to intestinal FV3 infections. Our results indicate that FV3-challenged tadpoles mount more robust intestinal type I and III IFN responses than adult frogs. These tadpole antiviral responses appear to be mediated by myeloid cells, which are recruited into tadpole intestines in response to FV3 infections. Conversely, myeloid cells bearing similar cytology already reside within the intestines of healthy (uninfected) adult frogs, possibly accounting for some of the anti-FV3 resistance of these animals. Further insight into the differences between tadpole and adult frog responses to ranaviral infections is critical to understanding the facets of susceptibility and resistance to these pathogens.

Virus-Targeted Transcriptomic Analyses Implicate Ranaviral Interaction with Host Interferon Response in Frog Virus 3-Infected Frog Tissues

Ranaviruses (Iridoviridae), including Frog Virus 3 (FV3), are large dsDNA viruses that cause devastating infections globally in amphibians, fish, and reptiles, and contribute to catastrophic amphibian declines. FV3’s large genome (~105 kb) contains at least 98 putative open reading frames (ORFs) as annotated in its reference genome. Previous studies have classified these coding genes into temporal classes as immediate early, delayed early, and late viral transcripts based on their sequential expression during FV3 infection. To establish a high-throughput characterization of ranaviral gene expression at the genome scale, we performed a whole transcriptomic analysis (RNA-Seq) using total RNA samples containing both viral and cellular transcripts from FV3-infected Xenopus laevis adult tissues using two FV3 strains, a wild type (FV3-WT) and an ORF64R-deleted recombinant (FV3-∆64R). In samples from the infected intestine, liver, spleen, lung, and especially kidney, an FV3-targeted transcriptomic analysis mapped reads spanning the full-genome coverage at ~10× depth on both positive and negative strands. By contrast, reads were only mapped to partial genomic regions in samples from the infected thymus, skin, and muscle. Extensive analyses validated the expression of almost all of the 98 annotated ORFs and profiled their differential expression in a tissue-, virus-, and temporal class-dependent manner. Further studies identified several putative ORFs that encode hypothetical proteins containing viral mimicking conserved domains found in host interferon (IFN) regulatory factors (IRFs) and IFN receptors. This study provides the first comprehensive genome-wide viral transcriptome profiling during infection and across multiple amphibian host tissues that will serve as an instrumental reference. Our findings imply that Ranaviruses like FV3 have acquired previously unknown molecular mimics, interfering with host IFN signaling during evolution.

Epigenetic regulation of frog innate immunity: Discovery of frog microRNAs associated with antiviral responses and ranavirus infection using a Xenopus laevis skin epithelial-like cell line

Epigenetic regulators such as microRNAs are emerging as conserved regulators of innate antiviral immunity in vertebrates, yet their roles in amphibian antiviral responses remain uncharacterized. We profiled changes in microRNA expressions in the Xenopus laevis skin epithelial–like cell line Xela DS2 in response to poly(I:C) – an analogue of double-stranded viral RNA and inducer of type I interferons – or frog virus 3 (FV3), an immunoevasive virus associated with amphibian mortality events. We sequenced small RNA libraries generated from untreated, poly(I:C)–treated, and FV3–infected cells. We detected 136 known X. laevis microRNAs and discovered 133 novel X. laevis microRNAs. Sixty–five microRNAs were differentially expressed in response to poly(I:C), many of which were predicted to target regulators of antiviral pathways such as cGAS–STING, RIG–I/MDA–5, TLR signaling, and type I interferon signaling, as well as products of these pathways (NF–κB–induced and interferon-stimulated genes). In contrast, only 49 microRNAs were altered by FV3 infection, fewer of which were predicted to interact with antiviral pathways. Interestingly, poly(I:C) treatment or FV3 infection downregulated transcripts encoding factors of the host microRNA biogenesis pathway. Our study is the first to suggest that host microRNAs regulate innate antiviral immunity in frogs, and sheds light on microRNA–mediated mechanisms of immunoevasion by FV3.