Synopsis To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.
Drugs against flaviviruses such as dengue (DENV) and Zika (ZIKV) virus are urgently needed. We previously demonstrated that three fluoroquinolones, ciprofloxacin, enoxacin, and difloxacin, suppress replication of six flaviviruses. To investigate the barrier to resistance and mechanism(s) of action of these drugs, DENV-4 was passaged in triplicate in HEK-293 cells in the presence or absence of each drug. Resistance to ciprofloxacin was detected by the seventh passage and to difloxacin by the tenth, whereas resistance to enoxacin did not occur within ten passages. Two putative resistance-conferring mutations were detected in the envelope gene of ciprofloxacin and difloxacin-resistant DENV-4. In the absence of ciprofloxacin, ciprofloxacin-resistant viruses sustained a significantly higher viral titer than control viruses in HEK-293 and HuH-7 cells and resistant viruses were more stable than control viruses at 37 °C. These results suggest that the mechanism of action of ciprofloxacin and difloxacin involves interference with virus binding or entry.
The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species ( Xenopus laevis ) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter susceptibility to the lethal fungal pathogen Batrachochytrium dendrobatidis ( Bd ) in the adult life stage. While our treatments to reduce the microbiome during early development did not play a critical role in determining susceptibility to disease caused by Bd in X. laevis , they nevertheless indicate that developing a gnotobiotic amphibian model system may be highly useful for future immunological investigations. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’.
28Drugs against flaviviruses such as dengue (DENV) and Zika (ZIKV) virus are urgently 29 needed. We previously demonstrated that three fluoroquinolones, ciprofloxacin, enoxacin, and 30 difloxacin, suppress replication of six flaviviruses. To investigate the barrier to resistance and 31 mechanism(s) of action of these drugs, DENV-4 was passaged in triplicate in HEK-293 cells in 32 the presence or absence of each drug. Resistance to ciprofloxacin was detected by the seventh 33 passage and to difloxacin by the tenth, whereas resistance to enoxacin did not occur within ten 34 passages. Two putative resistance-conferring mutations were detected in the envelope gene of 35 ciprofloxacin and difloxacin-resistant DENV-4. In the absence of ciprofloxacin, ciprofloxacin-36 resistant viruses sustained a significantly higher viral titer than control viruses in HEK-293 and 37 HuH-7 cells and resistant viruses were more stable than control viruses at 37˚C. These results 38 suggest that the mechanism of action of ciprofloxacin and difloxacin involves interference with 39 virus binding or entry. 40 41 KEYWORDS 42Dengue virus, antiviral, fluoroquinolone, ciprofloxacin, enoxacin, difloxacin, evolution, 43 resistance, fitness, mechanism-of-action 44 45 Members of the genus Flavivirus, most notably DENV and ZIKV, pose a great and 47 growing threat to public health, but at present no approved antiviral therapies are available to 48 treat any flaviviral infection (1-3). Drug repurposing offers the fastest route to move anti-49 flaviviral drugs into the clinic (4), and to this end we have recently demonstrated that three FDA-50 approved fluoroquinolones, enoxacin, difloxacin, and ciprofloxacin, suppress replication of six 51 flaviviruses, including DENV and ZIKV, in cultured HEK-293 (human embryonic kidney) cells 52 (5). Additionally, we found that treatment with enoxacin suppressed replication of ZIKV in the 53 testes of interferon-deficient A129 mice in vivo, although it did not impact ZIKV titer in the 54 serum, brain, or liver in these mice (5). Further, Xu et al. (6) recently reported that enoxacin also 55 suppresses ZIKV replication in human neural progenitor cells (hNPCs) and brain organoids (6). 56Thus, fluoroquinolones offer a promising candidate for a repurposed therapy for flavivirus 57 infections. However, several key aspects of the fluoroquinolone-flavivirus interaction must be 58 elucidated before these drugs can move forward on the path to the clinic, particularly: (i) the 59 barrier to resistance to these drugs, (ii) patterns of cross-resistance among fluoroquinolones, (iii) 60 the fitness consequences of evolution of drug resistance, (iv) the mechanism(s)-of-action of 61 fluoroquinolones against flaviviruses. 62Resistance evolution has been a serious obstacle to successful treatment of other RNA 63 viruses, particularly HIV, HCV, and influenza (7-9). Moreover, cross-resistance to structurally 64 related drugs can occur and must be investigated as cross-resistance can render an entire class of 65 antivirals ineffective (...
To combat the loss of species due to emerging infectious diseases, scientists must incorporate ecological parameters, such as temperature and humidity, to understand how the environment affects host–pathogen interactions. The fungal disease chytridiomycosis is a compelling case study to investigate the role of both temperature and humidity on infectious disease, as both the fungal pathogen (Batrachochytrium dendrobatidis, Bd) and the host (amphibians) are heavily influenced by these abiotic factors. We performed two experiments to investigate the importance of relative humidity and temperature on frog immunity (production of antimicrobial skin secretions) and disease development in captive golden frogs (Atelopus zeteki) of Panama. We found that the quantity of skin secretions significantly decreased over time in frogs moved from low to medium and high relative humidity treatments. Following Bd exposure, frogs in high temperature (26–27 °C) and high relative humidity (80–90%) had lower pathogen loads and survived significantly longer than frogs kept in all other treatment conditions, including high temperature and low relative humidity. These results suggest that high relative humidity may be an important, although less understood, mediator of Bd infection and the survival of golden frogs. Because the environment can drastically alter disease dynamics, understanding how temperature and humidity influence chytridiomycosis outcomes in golden frogs may be essential for the success of the reintroduction of captive frogs.
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