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Low-pathogenic avian influenza virus (LPAIV) remains the most common subtype of type-A influenza virus that causes moderate to severe infection in poultry with significant zoonotic and pandemic potential. Due to high mutability, increasing drug resistance, and limited vaccine availability, the conventional means to prevent intra- or interspecies transmission of AIV is highly challenging. As an alternative to control AIV infections, cytokine-based approaches to augment antiviral host defense have gained significant attention. However, the selective application of cytokines is critical since unregulated expression of cytokines, particularly proinflammatory ones, can cause substantial tissue damage during acute phases of immune responses. Moreover, depending on the type of cytokine and its impact on intestinal microbiota, outcomes of cytokine-gut microflora interaction can have a critical effect on overall host defense against AIV infections. Our recent study demonstrated some prominent roles of chicken IL-17A (ChIL-17A) in regulating antiviral host responses against AIV infection, however, in an in vitro model. For more detailed insights into ChIL-17A function, in the present study, we investigated whether ChIL-17A-meditated elevated antiviral host responses can translate into effective immune protection against AIV infection in an in vivo system. Moreover, considering the role of gut health in fostering innate or local host responses, we further studied the contributory relationships between gut microbiota and host immunity against AIV infection in chickens. For this, we employed a recombinant lactic acid-producing bacterial (LAB) vector, Lactococcus lactis, expressing ChIL-17A and analyzed the in vivo functionality in chickens against an LPAIV (A/H9N2) infection. Our study delineates that mucosal delivery of rL. lactis expressing ChIL-17A triggers proinflammatory signaling cascades and can drive a positive shift in phylum Firmicutes, along with a marked decline in phylum Actinobacteriota and Proteobacteria, favoring effective antiviral host responses against AIV infection in chickens. We propose that ChIL-17A-mediated selective expansion of beneficial gut microbiota might form a healthy microbial community that augments the effective immune protection against AIV infections in chickens.

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Interferons (IFNs) activate JAK-STAT pathways to induce downstream effector genes for host defense against invaded pathogens and tumors. Here both type I (ß) and II (γ) IFNs are shown that can activate the transcription factor IRF3 in parallel with STAT1. IRF3-deficiency impairs transcription of a subset of downstream effector genes induced by IFN-ß and IFN-γ. Mechanistically, IFN-induced activation of IRF3 is dependent on the cGAS-STING-TBK1 axis. Both IFN-ß and IFN-γ cause mitochondrial DNA release into the cytosol. In addition, IFNs induce JAK1-mediated tyrosine phosphorylation of cGAS at Y214/Y215, which is essential for its DNA binding activity and signaling. Furthermore, deficiency of cGAS, STING, or IRF3 impairs IFN-ß- or IFN-γ-mediated antiviral and antitumor activities. The findings reveal a novel IRF3 activation pathway parallel with the canonical STAT1/2 activation pathways triggered by IFNs and provide an explanation for the pleiotropic roles of the cGAS-STING-IRF3 axis in host defense.

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To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45+ cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T+ cells and 2 ± 0.9 % of CD45+ cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45+ leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRß, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45+ leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates.

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Human rhinovirus 16 (HRV16) may induce inflammatory and antiviral responses in the human lung vascular endothelium (ECs) and impair its barrier functions after infection. However, ECs may regain barrier and metabolic functions. Mechanisms of limitation of HRV16 infection in the lung vascular endothelium are unknown. Human lung vascular endothelium (HMVEC-L) was infected with HRV16. IFN-ß, OAS-1, and PKR expression was assessed by real-time PCR, flow cytometry, and confocal microscope. To prove the significance of IFN-ß in the limitation of HRV16 replication, HMVEC-Ls were preincubated with anti-IFN-ß Abs. To prove the involvement of OAS-1 and PKR in the IFN-dependent limitation of HRV16 replication, HMVEC-Ls were transfected with respective siRNA. HRV16 stimulated IFN-ß production and activated intracellular mechanisms of antiviral immunity based on OAS-1 and PKR activation. Blocking of IFN-ß contributed to the inhibition of intracellular mechanisms of antiviral immunity (OAS-1, PKR) and boosted replication of HRV16. Effective OAS-1 silencing by siRNA caused the increase of HRV16 copy numbers after HRV16 infection. siRNA upregulated the other genes related to the antiviral response. The infected lung vascular endothelium may limit the HRV16 infection. This limitation may be associated with the induction of IFN-ß-dependent intracellular mechanisms based on OAS-1 and PKR activity.

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Viral infections pose significant threats to human health, causing various diseases with varying severity. The intricate interactions between viruses and host cells determine the outcome of infection, including viral replication, immune responses, and disease progression. Cholesterol 25-hydroxylase (CH25H) is an enzyme that catalyzes the conversion of cholesterol to 25-hydroxycholesterol (25HC), a potent antiviral molecule. In recent years, increasing evidence has highlighted the critical involvement of CH25H in modulating immune responses and influencing viral infections. Notably, the review discusses the implications of CH25H in viral pathogenesis and the development of therapeutic strategies. It examines the interplay between CH25H and viral immune evasion mechanisms, highlighting the potential of viral antagonism of CH25H to enhance viral replication and pathogenesis. Furthermore, it explores the therapeutic potential of targeting CH25H or modulating its downstream signaling pathways as a strategy to control viral infections and enhance antiviral immune responses. This comprehensive review demonstrates the crucial role of CH25H in viral infections, shedding light on its mechanisms of action in viral entry, replication, and immune modulation. Understanding the complex interplay between CH25H and viral infections may pave the way for novel therapeutic approaches and the development of antiviral strategies aimed at exploiting the antiviral properties of CH25H and enhancing host immune responses against viral pathogens. In the current review, we tried to provide an overview of the antiviral activity and importance of CH25H in viral pathogenesis.

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BACKGROUND: IFN-ε is essential in combating viral infections, particularly in epithelial cells and protected mucosal tissues. Its protective effects have been demonstrated against HSV2, Zika virus, HIV and SARS-COV2. However, the specific expression and role of IFN-ε in skin keratinocytes and HPV infection are still not fully understood and require further investigation. OBJECTIVE: In this study, we aimed to investigate the functions and expression mechanism of IFN-ε in keratinocytes during HPV infection and the progression of condyloma acuminata. METHODS: Keratinocytes isolated from biopsied CA warts and normal skins samples were analyzed by MeRIP-seq analysis. IFN-ε and WTAP in CA warts and normal skins were analyzed by immunostaining and qPCR. RESULTS: In this study, we identified IFN-ɛ was markedly upregulated in CA warts and HPV-infected keratinocytes. IFN-ɛ expression also showed negatively correlation with the size of CA warts (R=-0.4646, P = 0.009). IFN-ɛ suppressed the susceptibility of HPV infection directly. m6A analysis reveals WTAP is a key m6A writer promoting the m6A modification of IFNE mRNA. CONCLUSION: Our research suggests that IFN-ɛ is an important Type I IFN cytokine involved in the development of genital warts. Furthermore, we found that HPV infection affects the m6A modifications of IFNE through a mechanism dependent on WTAP. This study provides insights into the innate immune response of the host to HPV infection and may contribute to the development of future strategies for regulating innate immunity to treat genital warts.

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Background: Toll-like receptor (TLR) agonists have been investigated due to their potential dual effects as latency reverting agents and immune modulatory compounds in people living with HIV (PLWH). Here, we investigated whether co-stimulation of TLR7/8 agonists with RIG-I-like receptor (RLR) agonists enhances antiviral immunity. Methods: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (DCs) were incubated with TLR and RLR-agonists for 24 h and innate and adaptive immune responses were determined (maturation markers, cytokines in supernatant, ISG expression). Results: Both TLR7 and TLR8 agonists induced pro-inflammatory cytokines in DCs as well as PBMCs. TLR8 agonists were more potent in inducing cytokine responses and had a stronger effect on DC-induced immunity. Notably, while all compounds induced IL-12p70, co-stimulation with TLR8 agonists and RLR agonist polyI: C induced significantly higher levels of IL-12p70 in PBMCs. Moreover, crosstalk between TLR8 and RLR agonists induced a strong type I Interferon (IFN) response as different antiviral IFN-stimulated genes were upregulated by the combination compared to the agonists alone. Conclusion: Our data strongly suggest that TLR crosstalk with RLRs leads to strong antiviral immunity as shown by induction of IL-12 and type I IFN responses in contrast to TLRs alone. Thus, co-stimulation of TLRs and RLRs might be a powerful strategy to induce reactivation of latent reservoir as well as antiviral immunity that eliminates the reactivated cells.

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Dengue virus (DENV) is the most important human virus transmitted by mosquitos. Dengue pathogenesis is characterized by a large induction of proinflammatory cytokines. This cytokine induction varies among the four DENV serotypes (DENV1 to 4) and poses a challenge for live DENV vaccine design. Here, we identify a viral mechanism to limit NF-κB activation and cytokine secretion by the DENV protein NS5. Using proteomics, we found that NS5 binds and degrades the host protein ERC1 to antagonize NF-κB activation, limit proinflammatory cytokine secretion, and reduce cell migration. We found that ERC1 degradation involves unique properties of the methyltransferase domain of NS5 that are not conserved among the four DENV serotypes. By obtaining chimeric DENV2 and DENV4 viruses, we map the residues in NS5 for ERC1 degradation, and generate recombinant DENVs exchanging serotype properties by single amino acid substitutions. This work uncovers a function of the viral protein NS5 to limit cytokine production, critical to dengue pathogenesis. Importantly, the information provided about the serotype-specific mechanism for counteracting the antiviral response can be applied to improve live attenuated vaccines.

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Recent studies have begun to reveal the complex and multifunctional roles of N6-methyladenosine (m6A) modifications and their associated writer, reader, and eraser proteins in infection by diverse RNA and DNA viruses. However, little is known about their regulation and functions during infection by several viruses, including poxviruses. Here, we show that members of the YTH Domain Family (YTHDF), in particular YTHDF2, are downregulated as the prototypical poxvirus, vaccinia virus (VacV) enters later stages of replication in a variety of natural target cell types, but not in commonly used transformed cell lines wherein the control of YTHDF2 expression appears to be dysregulated. YTHDF proteins also decreased at late stages of infection by herpes simplex virus 1 (HSV-1) but not human cytomegalovirus, suggesting that YTHDF2 is downregulated in response to infections that induce host shutoff. In line with this idea, YTHDF2 was potently downregulated upon infection with a VacV mutant expressing catalytically inactive forms of the decapping enzymes, D9 and D10, which fails to degrade dsRNA and induces a protein kinase R response that itself inhibits protein synthesis. Overexpression and RNAi-mediated depletion approaches further demonstrate that YTHDF2 does not directly affect VacV replication. Instead, experimental downregulation of YTHDF2 or the related family member, YTHDF1, induces a potent increase in interferon-stimulated gene expression and establishes an antiviral state that suppresses infection by either VacV or HSV-1. Combined, our data suggest that YTHDF2 is destabilized in response to infection-induced host shutoff and serves to augment host antiviral responses. IMPORTANCE There is increasing recognition of the importance of N6-methyladenosine (m6A) modifications to both viral and host mRNAs and the complex roles this modification plays in determining the fate of infection by diverse RNA and DNA viruses. However, in many instances, the functional contributions and importance of specific m6A writer, reader, and eraser proteins remains unknown. Here, we show that natural target cells but not transformed cell lines downregulate the YTH Domain Family (YTHDF) of m6A reader proteins, in particular YTHDF2, in response to shutoff of protein synthesis upon infection with the large DNA viruses, vaccinia virus (VacV), or herpes simplex virus type 1. We further reveal that YTHDF2 downregulation also occurs as part of the host protein kinase R response to a VacV shutoff mutant and that this downregulation of YTHDF family members functions to enhance interferon-stimulated gene expression to create an antiviral state.

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Several studies over the last decade have identified intimate links between cellular metabolism and macrophage function. Metabolism has been shown to both drive and regulate macrophage function by producing bioenergetic and biosynthetic precursors as well as metabolites (and other bioactive molecules) that regulate gene expression and signal transduction. Many studies have focused on lipopolysaccharide-induced reprogramming, assuming that it is representative of most inflammatory responses. However, emerging evidence suggests that diverse pathogen-associated molecular patterns (PAMPs) are associated with unique metabolic profiles, which may drive pathogen specific immune responses. Further, these metabolic pathways and processes may act as a rheostat to regulate the magnitude of an inflammatory response based on the biochemical features of the local microenvironment. In this review, we will discuss recent work examining the relationship between cellular metabolism and macrophage responses to viral PAMPs and describe how these processes differ from lipopolysaccharide-associated responses. We will also discuss how an improved understanding of the specificity of these processes may offer new insights to fine-tune macrophage function during viral infections or when using viral PAMPs as therapeutics.

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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative virus of pandemic acute respiratory disease called coronavirus disease 2019 (COVID-19). Most of the infected individuals have asymptomatic or mild symptoms, but some patients show severe and critical systemic inflammation including tissue damage and multi-organ failures. Immune responses to the pathogen determine clinical course. In general, the activation of innate immune responses is mediated by host pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) as well as host damage-associated molecular patterns (DAMPs), which results in the activation of the downstream gene induction programs of types I and III interferons (IFNs) and proinflammatory cytokines for inducing antiviral activity. However, the excessive activation of these responses may lead to deleterious inflammation. Here, we review the recent advances in our understanding of innate immune responses to SARS-CoV-2 infection, particularly in terms of innate recognition and the subsequent inflammation underlying COVID-19 immunopathology.

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Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease, while respiratory infections can elicit exacerbations in COPD patients to mediate increased mortality. Administration of Tanshinones (TS) derivatives has been demonstrated to protect against cigarette smoking (CS) and lipopolysaccharide (LPS)-induced COPD progression. However, the underlying molecular mechanisms and the roles of TS in mitigating the severity of viral-mediated exacerbations of COPD have not been elucidated. Here, we found that TS treatments significantly attenuated lung function decline, inflammatory responses and oxidative stress in CS and LPS-induced COPD mice. Subsequent RNA-seq analysis revealed significantly upregulated Hemopexin expression and enriched interferons (IFNs) signaling pathways in lung tissues of COPD mice upon TS treatments. Moreover, TS administration demonstrated Hemopexin-dependent beneficial roles in BEAS-2B lung cells and RAW264.7 macrophages, which was associated with the suppression of oxidative stress and ERK, NF-κB, and NLRP3 inflammasome signaling pathways-mediated inflammation. Furthermore, TS promoted IFN signaling and rescued impaired antiviral responses in CS and LPS-exposed lung cells that were infected by influenza virus. Notably, hemopexin over-expression in lung cells and macrophages recapitulated the pharmacological activities of TS. Taken together, these results indicate that TS administration is a promising and potential therapeutic strategy for treating COPD and preventing COPD exacerbations.

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Most alphaviruses are transmitted by mosquito vectors and infect a wide range of vertebrate hosts, with a few exceptions. Eilat virus (EILV) in this genus is characterized by a host range restricted to mosquitoes. Its chimeric viruses have been developed as safe and effective vaccine candidates and diagnostic tools. Here, we investigated the interactions between these insect-specific viruses (ISVs) and mosquito cells, unveiling their potential roles in determining vector competence and arbovirus transmission. By RNA sequencing, we found that these ISVs profoundly modified host cell gene expression profiles. Two EILV-based chimeras, consisting of EILV's nonstructural genes and the structural genes of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), namely, EILV/CHIKV (E/C) and EILV/VEEV (E/V), induced more intensive transcriptome regulation than parental EILV and activated different antiviral mechanisms in host cells. We demonstrated that E/C robustly promoted antimicrobial peptide production and E/V strongly upregulated the RNA interference pathway components. This also highlighted the intrinsic divergences between CHIKV and VEEV, representatives of the Old World and New World alphaviruses. In contrast, EILV triggered a limited antiviral response. We further showed that initial chimera infections efficiently inhibited subsequent pathogenic alphavirus replication, especially in the case of E/V infection, which almost prevented VEEV and Sindbis virus (SINV) superinfections. Altogether our study provided valuable information on developing ISVs as biological control agents. IMPORTANCE Mosquito-borne alphaviruses can cause emerging and reemerging infectious diseases, posing a considerable threat to human and animal health worldwide. However, no specific antivirals or commercial vaccines are currently available. Therefore, it is vital to develop biological control measures to contain virus transmission. Insect-specific EILV and its chimeras are supposed to induce superinfection exclusion owing to the close phylogenetical relationship with pathogenic alphaviruses. These viruses might also, like bacterial symbionts, modulate mosquito hosts' vector competence for arboviruses. However, little is known about the responses of mosquitoes or mosquito cells to ISV infections. Here, we found that EILV barely elicited antiviral defenses in host cells, while its chimeras, namely, E/C and E/V, potentiated the responses via different mechanisms. Furthermore, we showed that initial chimera infections could largely inhibit subsequent pathogenic alphavirus infections. Taken together, our study proposed insect-specific chimeras as a promising candidate for developing biological control measures against pathogenic alphaviruses.

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Promyelocytic leukemia protein (PML) bodies are implicated in one of the key pathways in the establishment of antiviral status in response to interferon (IFN), yet the molecular mechanisms bridging the cross talk remain elusive. Herein, we report that a major constitutive component of the PML body, Sp100A, is ubiquitously located in the cytosol of various cell types and is an immediate responder to multiple extracellular stimuli, including virus infection, IFN, epidermal growth factor (EGF), glial cell-derived nerve factor (GDNF), etc., signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. IFN-ß induces phosphorylation of Sp100A on Ser188, which fortifies the binding of Sp100A to pyruvate kinase 2 (PKM2) and facilitates its nuclear importation through the extracellular signal-regulated kinase 1/2 (ERK1/2)-PKM2-PIN1-importin axes. Blocking PI3K pathway signaling or interference with the ERK1/2-PKM2-PIN1-importin axes independently hampers nuclear translocation of Sp100A in response to IFN, reflecting a dual-regulation mechanism governing this event. In the nucleus, Sp100A is enriched in the promoter regions of essential antiviral interferon-stimulated genes (ISGs), such as those coding for IFI16, OAS2, and RIG-I, and activates their transcription. Importantly, nuclear importation of Sp100A, but not accumulation of a mutant Sp100A that failed to respond to IFN, during infection potently enhanced transcription of these antiviral ISGs and restricted virus propagation. These findings depict a novel IFN response mechanism by PML bodies in the cytosol and shed light on the complex sensing-regulatory network of PML bodies. IMPORTANCE PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection. It shows that PML body constitutive component Sp100A was phosphorylated on Ser188 by IFN signaling through the PI3K pathway in the cytosol, cotranslocated into the nucleus with PKM2, enriched on the promoter regions of essential antiviral ISGs such as those coding for IFI16, RIG-I, OAS2, etc., and mediating their transcriptional activation.

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Zika virus (ZIKV) exhibits distinct selectivity for infection of various cells and tissues, but how host cellular factors modulate varying permissivity remains largely unknown. Previous studies showed that the neuroblastoma cell line SK-N-AS (expressing low levels of cellular protein CD24) was highly restricted for ZIKV infection, and that this restriction was relieved by ectopic expression of CD24. We tested the hypothesis that CD24 expression allowed ZIKV replication by suppression of the antiviral response. SK-N-AS cells expressing an empty vector (termed CD24-low cells) showed elevated basal levels of phosphorylated STAT1, IRF-1, IKKE, and NFκB. In response to exogenously added type I interferon (IFN-I), CD24-low cells had higher-level induction of antiviral genes and activity against two IFN-I-sensitive viruses (VSV and PIV5-P/V) compared to SK-N-AS cells with ectopic CD24 expression (termed CD24-high cells). Media-transfer experiments showed that the inherent antiviral state of CD24-low cells was not dependent on a secreted factor such as IFN-I. Transcriptomics analysis revealed that CD24 expression decreased expression of genes involved in intracellular antiviral pathways, including IFN-I, NFκB, and Ras. Our findings that CD24 expression in neuroblastoma cells represses intracellular antiviral pathways support the proposal that CD24 may represent a novel biomarker in cancer cells for susceptibility to oncolytic viruses.

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Calcium, as a second intracellular messenger, participate in various physiological and biochemical processes, including cell growth and proliferation, energy metabolism, information transfer, cell death, and immune response. Ca2+ channels or pumps in plasma and organelle membranes and Ca2+-related proteins maintain Ca2+ homeostasis by regulating Ca2+ inflow, outflow and buffering to avoid any adverse effects caused by Ca2+ overload or depletion. Thus, Ca2+ signaling also provides a target for virus invasion, replication, proliferation and release. After hijacking the host cell, viruses exploit Ca2+ signaling to regulate apoptosis and resist host immunity to establish persistent infection. In this review, we discuss cellular Ca2+ signaling and channels, interaction of calcium-associated proteins with viruses, and host cell fate, as well as the role of Ca2+ in cell death and antiviral response during viral infection.

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Plasmacytoid dendritic cells (pDCs) are specialized cells of the immune system that are thought to be the main cellular source of type I interferon alpha (IFNα) in response to viral infections. IFNs are powerful antivirals, whereas defects in their function or induction lead to impaired resistance to virus infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. IFN production needs to be controlled, because sustained IFN production can also have detrimental effects on disease outcome. As such, pDCs are likely important for acute antiviral protection against SARS-CoV-2 infection but could potentially also contribute to chronic IFN levels. Here, we provide a historical overview of pDC biology and summarize existing literature addressing their involvement and importance during viral infections of the airways. Furthermore, we outline recent reports focused on the potential role of pDCs during SARS-CoV-2 infection, as well as the potential for this cellular subset to impact COVID-19 disease outcome.

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The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

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In invertebrate cells, RNA interference (RNAi) acts as a powerful immune defense that stimulates viral gene knockdown thereby preventing infection. With this pathway, virally produced long dsRNA (dsRNA) is cleaved into short interfering RNA (siRNA) by Dicer and loaded into the RNA-induced silencing complex (RISC) which can then destroy/disrupt complementary viral mRNA sequences. Comparatively, in mammalian cells it is believed that the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. In these cells, dsRNA acts as a potent inducer of the IFN system, which is dependent on dsRNA length, but not sequence, to stimulate an antiviral state. Although the cellular machinery for RNAi is intact and functioning in mammalian cells, its role to trigger an antiviral response using long dsRNA (dsRNAi) remains controversial. Here we show that dsRNAi is not only functional but has a significant antiviral effect in IFN competent mammalian cells. We found that pre-soaking mammalian cells with concentrations of sequence specific dsRNA too low to induce IFN production could significantly inhibit vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and the human coronaviruses (CoV) HCoV-229E and SARS-CoV-2 replication. This phenomenon was shown to be dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, knockout cell lines revealed that functional Dicer was required for viral inhibition, revealing that the RNAi pathway was indeed responsible. These results provide the first evidence that soaking with gene-specific long dsRNA can generate viral knockdown in mammalian cells. We believe that this novel discovery provides an explanation as to why the mammalian lineage retained its RNAi machinery and why vertebrate viruses have evolved methods to suppress RNAi. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform, both antiviral and gene targeting in nature.

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