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The Binding, Infection, and Promoted Growth of Batrachochytrium dendrobatidis by the Ranavirus FV3.
De Jesús Andino F
,
Davydenko A
,
Webb RJ
,
Robert J
.
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Increasing reports suggest the occurrence of co-infection between Ranaviruses such as Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) in various amphibian species. However, the potential direct interaction of these two pathogens has not been examined to date. In this study, we investigated whether FV3 can interact with Bd in vitro using qPCR, conventional microscopy, and immunofluorescent microscopy. Our results reveal the unexpected ability of FV3 to bind, promote aggregation, productively infect, and significantly increase Bd growth in vitro. To extend these results in vivo, we assessed the impact of FV3 on Xenopus tropicalis frogs previously infected with Bd. Consistent with in vitro results, FV3 exposure to previously Bd-infected X. tropicalis significantly increased Bd loads and decreased the host's survival.
Figure 1. Detection of FV3 binding to Bd using fluorescence microscopy. FV3 was added to Bd culture at 1 MOI in 1 mL volume and incubated for 24 h. Detection of FV3 was performed by staining with the anti-MCP mAb BG11 followed by FITC-conjugates goat anti-mouse Ab. Nuclei were stained with Hoechst. Negative control, Mock FV3-exposed Bd culture. Stained samples were visualized under a EVOS FL Digital Inverted Fluorescence Microscope.
Figure 2. Detection of FV3 binding to Bd using qPCR. FV3 was incubated with Bd at 1 MOI in 1 mL volume for 1 and 24 h. Samples were then centrifuged at 700× g and both pellets and supernatants were subjected to qPCR with FV3 specific primers (vDNA pol). * Statistical differences (p < 0.05).
Figure 3. Increased Bd aggregation in the presence of FV3. Zoospores were incubated without (A) or with FV3 at 1 MOI (B) and 10 MOI (C). Zoospore aggregation was photographed at 1 h post-FV3 addition. Arrows: aggregated Bd.
Figure 4. Effect of FV3 on Bd growth. Bd zoospores were co-cultured with FV3 at 1 and 10 MOI for 24 h and 48 h. Growth was determined by ATP consumption (A,B) and by determining the total Bd cell surface area per field of view (C). Negative control Bd was heat-inactivated (B). For all panels three replicates per condition were used. Statistical differences: ** (p < 0.01), * (p < 0.05).
Figure 5. FV3 infection of Bd detected using fluorescence microscopy. FV3 was added to Bd culture at 1 MOI in 1 mL volume and incubated for 24 h. Detection of FV3 was performed by staining with the anti-53R rabbit polyclonal Ab followed by FITC-conjugates goat anti-rabbit Ab (green). Nuclei were stained with Hoechst (blue). (A,C) Negative control, Mock FV3-exposed Bd culture displaying no FITC signal. (B) FV3-exposed Bd zoospores displaying FITC signal indicating active infection. (C,D,E) Higher magnification of mock- (C) and FV3-exposed Bd zoosporangia (D,E) with FITC signal. Arrow: Chytrid rhizoid with strong FITC signal.
Figure 6. FV3 infection of Bd detected using RT-PCR. FV3 was incubated with Bd at 1 MOI in 1 mL volume for 24 h. RT-PCR on DNAse-treated RNA was performed using a primer specific for early (vDNA pol) and late (MCP) FV3 genes (A). To control for viral genomic contamination, the same amount of RNA was assayed in absence of reverse transcriptase (B). As an additional control, the same assay was performed with heat-killed Bd (C).
Figure 7. Detection of productive Bd infection by FV3. FV3 was incubated with Bd at 1 MOI in 1 mL volume for 1, 3, and 6 days. Viral replication was determined by qPCR ((A); genome copy number) and the production of infectious particles by plaque assay (B). * Statistical differences (p < 0.05).
Figure 8. Effect of exposure to FV3 on host resistance to Bd infection in X. tropicalis. Adult X. tropicalis were infected with 1 × 106 Bd (zoospores) for 15 days before being exposed to 1 × 106 PFU FV3 (or shame-exposed). (A) Bd load was monitored by skin swabs at 15 days before FV3 infection and at 1, 3, and 6 days post-FV3 infection. (B) Survival of animals (N = 3) infected with Bd only or exposed after 15 days to FV3. (C) Bd loads of animals that succumbed from the infection. * Statistical differences (p < 0.05).
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