Developmental exposure to chemicals associated with unconventional oil and gas extraction alters immune homeostasis and viral immunity of the amphibian Xenopus.

Although aquatic vertebrates and humans are increasingly exposed to water pollutants associated with unconventional oil and gas extraction (UOG), the long-term effects of these pollutants on immunity remains unclear. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable and sensitive model for evaluating the effects of waterborne pollutants. X. laevis tadpoles were exposed to a mixture of equimass amount of UOG chemicals with endocrine disrupting activity (0.1 and 1.0 µg/L) for 3 weeks, and then long-term effects on immune function at steady state and following viral (FV3) infection was assessed after metamorphosis. Notably, developmental exposure to the mixture of UOG chemicals at the tadpole stage affected metamorphic development and fitness by significantly decreasing body mass after metamorphosis completion. Furthermore, developmental exposure to UOGs resulted in perturbation of immune homeostasis in adult frogs, as indicated by significantly decreased number of splenic innate leukocytes, B and T lymphocytes; and a weakened antiviral immune response leading to increased viral load during infection by the ranavirus FV3. These findings suggest that mixture of UOG-associated waterborne endocrine disruptors at low but environmentally-relevant levels have the potential to induce long-lasting alterations of immune function and antiviral immunity in aquatic vertebrates and ultimately human populations.

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 ß-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-, ΔvßHSD- 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 vß-HSD (52L) genes contribute to viral pathogenesis.

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.