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.
more »
« less
Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Use of Expanded Repertoires of Type I and Type III Interferon Cytokines
While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney (principal FV3 target) tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults.
more »
« less
- Award ID(s):
- 1749427
- PAR ID:
- 10094306
- Date Published:
- Journal Name:
- Viruses
- Volume:
- 10
- Issue:
- 7
- ISSN:
- 1999-4915
- Page Range / eLocation ID:
- 372
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Besides the central role of classical Major Histocompatibility Complex (MHC) class Ia-restricted conventional Cluster of Differentiation 8 (CD8) T cells in antiviral host immune response, the amphibian Xenopus laevis critically rely on MHC class I-like (mhc1b10.1.L or XNC10)-restricted innate-like (i)T cells (iVα6 T cells) to control infection by the ranavirus Frog virus 3 (FV3). To complement and extend our previous reverse genetic studies showing that iVα6 T cells are required for tadpole survival, as well as for timely and effective adult viral clearance, we examined the conditions and kinetics of iVα6 T cell response against FV3. Using a FV3 knock-out (KO) growth-defective mutant, we found that upregulation of the XNC10 restricting class I-like gene and the rapid recruitment of iVα6 T cells depend on detectable viral replication and productive FV3 infection. In addition, by in vivo depletion with XNC10 tetramers, we demonstrated the direct antiviral effector function of iVα6 T cells. Notably, the transitory iV6 T cell defect delayed innate interferon and cytokine gene response, resulting in long-lasting negative inability to control FV3 infection. These findings suggest that in Xenopus and likely other amphibians, an immune surveillance system based on the early activation of iT cells by non-polymorphic MHC class-I like molecules is important for efficient antiviral immune response.more » « less
-
The amphibian declines are compounded by emerging pathogens that often preferentially target distinct amphibian developmental stages. While amphibian immune responses remain relatively unexplored, macrophage (Mφ)-lineage cells are believed to be important to both amphibian host defenses and to their pathogen infection strategies. As such, a greater understanding of tadpole and adult amphibian Mφ functionality is warranted. Mφ biology is interdependent of interleukin-34 (IL-34) and colony-stimulating factor-1 (CSF-1) cytokines and we previously showed that CSF-1- and IL-34-derived Mφs of the Xenopus laevis frog are morphologically, transcriptionally, and functionally distinct. Presently, we directly compared the cytology and transcriptomes of X. laevis tadpole and frog CSF-1- and IL-34-Mφs. Our results indicate that tadpole and frog CSF-1-Mφs possess greater non-specific esterase activity, typically associated with Mφ-lineage cells. By contrast, both tadpole and frog IL-34-Mφs have greater specific esterase activity, which is typically attributed to granulocyte-lineage cells. Our comparisons of tadpole CSF-1-Mφ transcriptomes with those of tadpole IL-34-Mφs indicate that the two tadpole populations possess significantly different transcriptional profiles of immune and non-immune genes. The frog CSF-1-Mφ gene expression profiles are likewise significantly disparate from those of frog IL-34-Mφs. Compared to their respective tadpole Mφ subtypes, frog CSF-1- and IL-34-Mφs exhibited greater expression of genes associated with antigen presentation. Conversely, compared to their frog Mφ counterparts, tadpole CSF-1- and IL-34-Mφs possessed greater levels of select Fc-like receptor genes. Presumably, these cytological and transcriptional differences manifest in distinct biological roles for these respective tadpole and frog Mφ subtypes.more » « less
-
null (Ed.)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 with the host IFN response.more » « less
-
more » « less
Global amphibian declines are largely driven by deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). In the time since these disease outbreaks were first discovered, much has been learned about the roles of amphibian skin-produced antimicrobial components and skin microbiomes in controlling Bd. Yet almost nothing is known about the roles of skin-resident immune cells in anti-Bd defenses. Notably, mammalian mast cells reside within and serve as key immune sentinels in barrier tissues like the skin. Thus, they are critical to immune recognition of pathogens and to orchestrating the ensuing immune responses. Accordingly, we investigated the roles of Xenopus laevis frog mast cells during Bd infections. Our findings indicate that enrichment of X. laevis skin mast cells confers significant anti-Bd protection and ameliorates the inflammation-associated skin damage caused by Bd infection. Moreover, enriching X. laevis mast cells promotes greater mucin content within cutaneous mucus glands and protects frogs from Bd-mediated changes to their skin microbiomes. Together, this work underlines the importance of amphibian skin-resident immune cells in anti-Bd defenses and introduces a novel approach for investigating amphibian host-chytrid pathogen interactions.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/v10070372
