Francisco De Jesús Andino

Susceptibility of Xenopus laevis tadpoles to infection by the ranavirus Frog-Virus 3 correlates with a reduced and delayed innate immune response in comparison with adult frogs

Xenopus laevis adults mount effective immune responses to ranavirus Frog Virus 3 (FV3) infections and clear the pathogen within 2-3 weeks. In contrast, most tadpoles cannot clear FV3 and succumb to infections within a month. While larval susceptibility has been attributed to ineffective adaptive immunity, the contribution of innate immune components has not been addressed. Accordingly, we performed a comprehensive gene expression analysis on FV3-infected tadpoles and adults. In comparison to adults, leukocytes and tissues of infected tadpoles exhibited modest (10-100 time lower than adult) and delayed (3 day later than adult) increase in expression of inflammation-associated (TNF-α, IL-1β and IFN-γ) and antiviral (Mx1) genes. In contrast, these genes were readily and robustly upregulated in tadpoles upon bacterial stimulation. Furthermore, greater proportions of larval than adult PLs were infected by FV3. Our study suggests that tadpole susceptibility to FV3 infection is partially due to poor virus-elicited innate immune responses.

Immune Evasion Strategies of Ranaviruses and Innate Immune Responses to These Emerging Pathogens

Ranaviruses (RV, Iridoviridae) are large double-stranded DNA viruses that infect fish, amphibians and reptiles. For ecological and commercial reasons, considerable attention has been drawn to the increasing prevalence of ranaviral infections of wild populations and in aquacultural settings. Importantly, RVs appear to be capable of crossing species barriers of numerous poikilotherms, suggesting that these pathogens possess a broad host range and potent immune evasion mechanisms. Indeed, while some of the 95–100 predicted ranavirus genes encode putative evasion proteins (e.g., vIFα, vCARD), roughly two-thirds of them do not share significant sequence identity with known viral or eukaryotic genes. Accordingly, the investigation of ranaviral virulence and immune evasion strategies is promising for elucidating potential antiviral targets. In this regard, recombination-based technologies are being employed to knock out gene candidates in the best-characterized RV member, Frog Virus (FV3). Concurrently, by using animal infection models with extensively characterized immune systems, such as the African clawed frog, Xenopus laevis, it is becoming evident that components of innate immunity are at the forefront of virus-host interactions. For example, cells of the macrophage lineage represent important combatants of RV infections while themselves serving as targets for viral infection, maintenance and possibly dissemination. This review focuses on the recent advances in the understanding of the RV immune evasion strategies with emphasis on the roles of the innate immune system in ranaviral infections.

The Amphibian (Xenopus laevis) Type I Interferon Response to Frog Virus 3: New Insight into Ranavirus Pathogenicity

ABSTRACT The increasing prevalence of ranavirus (RV; Iridoviridae) infections of wild and commercially maintained aquatic species is raising considerable concerns. While Xenopus laevis is the leading model for studies of immunity to RV, amphibian antiviral interferon (IFN) responses remain largely uncharacterized. Accordingly, an X. laevis type I interferon was identified, the expression of the gene for this IFN was examined in RV (frog virus 3 [FV3])-infected tadpoles and adult frogs by quantitative PCR, and a recombinant form of this molecule (recombinant X. laevis interferon [rXlIFN]) was produced for the purpose of functional studies. This rXlIFN protected the kidney-derived A6 cell line and tadpoles against FV3 infection, decreasing the infectious viral burdens in both cases. Adult frogs are naturally resistant to FV3 and clear the infection within a few weeks, whereas tadpoles typically succumb to this virus. Hence, as predicted, virus-infected adult X. laevis frogs exhibited significantly more robust FV3-elicited IFN gene expression than tadpoles; nevertheless, they also tolerated substantially greater viral burdens following infection. Although tadpole stimulation with rXlIFN prior to FV3 challenge markedly impaired viral replication and viral burdens, it only transiently extended tadpole survival and did not prevent the eventual mortality of these animals. Furthermore, histological analysis revealed that despite rXlIFN treatment, infected tadpoles had considerable organ damage, including disrupted tissue architecture and extensive necrosis and apoptosis. Conjointly, these findings indicate a critical protective role for the amphibian type I IFN response during ranaviral infections and suggest that these viruses are more pathogenic to tadpole hosts than was previously believed, causing extensive and fatal damage to multiple organs, even at very low titers. IMPORTANCE Ranavirus infections are threatening wild and commercially maintained aquatic species. The amphibian Xenopus laevis is extensively utilized as an infection model for studying ranavirus-host immune interactions. However, little is known about amphibian antiviral immunity and, specifically, type I interferons (IFNs), which are central to the antiviral defenses of other vertebrates. Accordingly, we identified and characterized an X. laevis type I interferon in the context of infection with the ranavirus frog virus 3 (FV3). FV3-infected adult frogs displayed more robust IFN gene expression than tadpoles, possibly explaining why they typically clear FV3 infections, whereas tadpoles succumb to them. Pretreatment with a recombinant X. laevis IFN (rXlIFN) substantially reduced viral replication and infectious viral burdens in a frog kidney cell line and in tadpoles. Despite reducing FV3 loads and extending the mean survival time, rXlIFN treatments failed to prevent tadpole tissue damage and mortality. Thus, FV3 is more pathogenic than was previously believed, even at very low titers.

Waterborne infectivity of the Ranavirus frog virus 3 in Xenopus laevis

Ranaviruses like frog virus 3 (FV3) are responsible for emerging infectious diseases spreading worldwide to fish, amphibian and reptilian species. We have developed, in Xenopus laevis, an experimental model to investigate viral transmission. We show that FV3 released in water by immunocompromised infected adults can infect adult and larval stages of Xenopus within 3h of exposure. Time course of virus load and viral transcription in different tissues suggests that early waterborne FV3 infection through the digestive tract leads to dissemination in the kidney. Finally, a fraction of adult macrophages becomes infected following exposure to waterborne FV3 as visualized by fluorescence microscopy using macrophage- and FV3-specific antibodies. Little cytopathicity and apoptosis were detected in infected macrophages, which is consistent with our proposition that macrophages are permissive to FV3. These data highlight the efficiency of FV3 infectivity by the water route and the ability of FV3 to adapt to its hosts.

Prominent amphibian (Xenopus laevis) tadpole type III interferon response to the Frog Virus 3 ranavirus

ABSTRACT Ranaviruses (Iridoviridae) are posing an increasing threat to amphibian populations, with anuran tadpoles being particularly susceptible to these viral infections. Moreover, amphibians are the most basal phylogenetic class of vertebrates known to possess both type I and type III interferon (IFN)-mediated immunity. Moreover, little is known regarding the respective roles of the IFN mediators in amphibian antiviral defenses. Accordingly, we transcriptionally and functionally compared the amphibian Xenopus laevis type I (IFN) and III (IFN-λ) IFNs in the context of infections by the ranavirus frog virus 3 (FV3). X. laevis IFN and IFN-λ displayed distinct tissue expression profiles. In contrast to our previous findings that X. laevis tadpoles exhibit delayed and modest type I IFN responses to FV3 infections compared to the responses of adults, here we report that tadpoles mount timely and robust type III IFN gene responses. Recombinant forms of these cytokines (recombinant X. laevis IFN [rXlIFN] and rXlIFN-λ) elicited antiviral gene expression in the kidney-derived A6 cell line as well as in tadpole leukocytes and tissues. However, rXlIFN-λ was less effective than rXlIFN in preventing FV3 replication in A6 cells and tadpoles and inferior at promoting tadpole survival. Intriguingly, FV3 impaired A6 cell and tadpole kidney type III IFN receptor gene expression. Furthermore, in A6 cultures rXlIFN-λ conferred equal or greater protection than rXlIFN against recombinant viruses deficient for the putative immune evasion genes, the viral caspase activation and recruitment domain (vCARD) or a truncated vIF-2α gene. Thus, in contrast to previous assumptions, tadpoles possess intact antiviral defenses reliant on type III IFNs, which are overcome by FV3 pathogens. IMPORTANCE Anuran tadpoles, including those of Xenopus laevis, are particularly susceptible to infection by ranavirus such as FV3. We investigated the respective roles of X. laevis type I and type III interferons (IFN and IFN-λ, respectively) during FV3 infections. Notably, tadpoles mounted timely and more robust IFN-λ gene expression responses to FV3 than adults, contrasting with the poorer tadpole type I IFN responses. However, a recombinant X. laevis IFN-λ (rXlIFN-λ) conferred less protection to tadpoles and the A6 cell line than rXlIFN, which may be explained by the FV3 impairment of IFN-λ receptor gene expression. The importance of IFN-λ in tadpole anti-FV3 defenses is underlined by the critical involvement of two putative immune evasion genes in FV3 resistance to IFN- and IFN-λ-mediated responses. These findings challenge the view that tadpoles have defective antiviral immunity and suggest, rather, that their antiviral responses are predominated by IFN-λ responses, which are overcome by FV3.

Negative effects of low dose atrazine exposure on the development of effective immunity to FV3 in Xenopus laevis

The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxicology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses.

Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes

To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvβHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α-FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis.

Ranavirus Host Immunity and Immune Evasion

Ranaviruses (RV, Iridoviridae) are now known to infect fish, amphibians, and reptiles, raising considerable ecological and commercial concerns due to the escalating infection prevalence and the resulting die-offs of wild and aquacultural species. Notably, ranaviruses exhibit uncanny capacities to cross host species barriers of their poikilothermic hosts, likely owing to their potent immune evasion mechanisms. In turn, the species infected by these pathogens possess immune systems that are less well understood than those of mammals, and are often comprised of unique immune genes or multiple copy orthologs of the single hallmark mammalian immune factors. Thus, garnering greater insight into ranavirus infection strategies is largely contingent on gaining further insights into host immune barriers faced by these emerging infectious agents. Accordingly, here we coalesce the current state of understanding of the distinct facets of lower vertebrate immune responses to ranaviral infections and underline some of the evasion strategies by which these pathogens circumvent these host defenses.

Nonclassical MHC-Restricted Invariant Vα6 T Cells Are Critical for Efficient Early Innate Antiviral Immunity in the Amphibian Xenopus laevis

Nonclassical MHC class Ib–restricted invariant T (iT) cell subsets are attracting interest because of their potential to regulate immune responses against various pathogens. The biological relevance and evolutionary conservation of iT cells have recently been strengthened by the identification of iT cells (invariant Vα6 [iVα6]) restricted by the nonclassical MHC class Ib molecule XNC10 in the amphibian Xenopus laevis. These iVα6 T cells are functionally similar to mammalian CD1d-restricted invariant NKT cells. Using the amphibian pathogen frog virus 3 (FV3) in combination with XNC10 tetramers and RNA interference loss of function by transgenesis, we show that XNC10-restricted iVα6 T cells are critical for early antiviral immunity in adult X. laevis. Within hours following i.p. FV3 infection, iVα6 T cells were specifically recruited from the spleen into the peritoneum. XNC10 deficiency and concomitant lack of iVα6 T cells resulted in less effective antiviral and macrophage antimicrobial responses, which led to impaired viral clearance, increased viral dissemination, and more pronounced FV3-induced kidney damage. Together, these findings imply that X. laevis XNC10-restricted iVα6 T cells play important roles in the early anti-FV3 response and that, as has been suggested for mammalian invariant NKT cells, they may serve as immune regulators polarizing macrophage effector functions toward more effective antiviral states.

Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals.