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.

Virulence and Pathogenicity of Chytrid Fungi Causing Amphibian Extinctions

Ancient enzootic associations between wildlife and their infections allow evolution to innovate mechanisms of pathogenicity that are counterbalanced by host responses. However, erosion of barriers to pathogen dispersal by globalization leads to the infection of hosts that have not evolved effective resistance and the emergence of highly virulent infections. Global amphibian declines driven by the rise of chytrid fungi and chytridiomycosis are emblematic of emerging infections. Here, we review how modern biological methods have been used to understand the adaptations and counteradaptations that these fungi and their amphibian hosts have evolved. We explore the interplay of biotic and abiotic factors that modify the virulence of these infections and dissect the complexity of this disease system. We highlight progress that has led to insights into how we might in the future lessen the impact of these emerging infections. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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.

Targeted Transcriptomics of Frog Virus 3 in Infected Frog Tissues Reveal Non-Coding Regulatory Elements and microRNAs in the Ranaviral Genome and Their Potential Interaction with Host Immune Response

BackgroundFrog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae. Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions.ResultsUsing a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis-regulatory elements (CREs) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3’-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs).ConclusionsUsing the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response.

Effects of Road De-Icing Salts on Two Amphibian Species, Rana Sylvatica and Rana Pipiens, and Their Trematode Parasites

With ongoing amphibian declines, it is essential to determine possible contributors such as diseases and environmental contaminants that may increase susceptibility. A potential contaminant is road salt (mainly NaCl), which leaches into aquatic environments. I examined whether road salts make larval amphibians (tadpoles) more susceptible to trematode parasite infection, and also how these affect free-living trematode infectious stages (cercariae). I exposed Rana sylvatica (wood frogs) and R. pipiens (northern leopard frogs) to control, medium (400 mg/L), and high salt (800 mg/L) treatments, and then to trematodes. High salt tended to reduce wood frog anti-parasite behaviour and resistance to infection but the opposite was seen for R. pipiens, although these tadpoles had elevated lymphocyte counts in high salinity. Trematodes were differentially affected by increased salinities. The results suggest that host-parasite-environment interactions are complex, with species differentially affected by contaminants, which may lead to community shifts in predominant hosts and parasite species.

Effects of Road De-Icing Salts on Two Amphibian Species, Rana Sylvatica and Rana Pipiens, and Their Trematode Parasites

With ongoing amphibian declines, it is essential to determine possible contributors such as diseases and environmental contaminants that may increase susceptibility. A potential contaminant is road salt (mainly NaCl), which leaches into aquatic environments. I examined whether road salts make larval amphibians (tadpoles) more susceptible to trematode parasite infection, and also how these affect free-living trematode infectious stages (cercariae). I exposed Rana sylvatica (wood frogs) and R. pipiens (northern leopard frogs) to control, medium (400 mg/L), and high salt (800 mg/L) treatments, and then to trematodes. High salt tended to reduce wood frog anti-parasite behaviour and resistance to infection but the opposite was seen for R. pipiens, although these tadpoles had elevated lymphocyte counts in high salinity. Trematodes were differentially affected by increased salinities. The results suggest that host-parasite-environment interactions are complex, with species differentially affected by contaminants, which may lead to community shifts in predominant hosts and parasite species.

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

Frog Virus 3 (FV3) is a large dsDNA virus that cause global infections in amphibians, fish and reptiles, and contribute to amphibian declines. FV3's genome contains near 100 putative open reading frames (ORFs). 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 genome-wide characterize ranaviral gene expression, 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, a 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 annotated 98 ORFs and profiled their differential expression in a tissue-, virus-, and temporal class-dependent manners. 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 instrumental reference. It also presents evidence implying that ranaviruses like FV3 have acquired previously unknown molecular mimics interfering with host IFN signaling during evolution.

Targeted Transcriptomics of Frog Virus 3 in Infected Frog Tissues Reveal Non-Coding Regulatory Elements and microRNAs in the Ranaviral Genome and Their Potential Interaction With Host Immune Response

Background: Frog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae . Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions. Results: Using a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis -regulatory elements ( CREs ) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3’-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs). Conclusions: Using the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially in the host IFN response.

Co-infecting pathogen lineages have additive effects on host bacterial communities

ABSTRACT Amphibian skin bacteria may confer protection against the fungus Batrachochytrium dendrobatidis (Bd), but responses of skin bacteria to different Bd lineages are poorly understood. The global panzootic lineage (Bd-GPL) has caused amphibian declines and extinctions globally. However, other lineages are enzootic (Bd-Asia-2/Brazil). Increased contact rates between Bd-GPL and enzootic lineages via globalization pose unknown consequences for host-microbiome-pathogen dynamics. We conducted a laboratory experiment and used 16S rRNA amplicon-sequencing to assess: (i) whether two lineages (Bd-Asia-2/Brazil and Bd-GPL) and their recombinant, in single and mixed infections, differentially affect amphibian skin bacteria; (ii) and the changes associated with the transition to laboratory conditions. We determined no clear differences in bacterial diversity among Bd treatments, despite differences in infection intensity. However, we observed an additive effect of mixed infections on bacterial alpha diversity and a potentially antagonistic interaction between Bd genotypes. Additionally, observed changes in community composition suggest a higher ability of Bd-GPL to alter skin bacteria. Lastly, we observed a drastic reduction in bacterial diversity and a change in community structure in laboratory conditions. We provide evidence for complex interactions between Bd genotypes and amphibian skin bacteria during coinfections, and expand on the implications of experimental conditions in ecological studies.