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. 2015 Aug;51(2):298-304.
doi: 10.1016/j.dci.2015.01.006. Epub 2015 Jan 26.

A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance

K A Gabor  1 , J R Charette  2 , M J Pietraszewski  2 , D J Wingfield  2 , J S Shim  2 , P J Millard  1 , C H Kim  3
Affiliations

A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance

K A Gabor et al. Dev Comp Immunol. 2015 Aug.

Abstract

Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus.

Keywords: Antiviral; Danio rerio; Mda5; Rhabdovirus; Type I IFN; Zebrafish.

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Figures

Figure 1
Figure 1. Over-expression of mda5 Protects Against Virus Infection
ZFL cells were nucleofected with empty vector or mda5 or left as a no transfection control (NTC). 24 h post transfection (hpt), cells were exposed to the indicated amounts of SHRV and incubated for an additional 72 h. Supernatants were removed and viral burden was measured by TCID50 assay (B) (*, p<0.05). The remaining cells were fixed and stained with crystal violet (A).
Figure 2
Figure 2. Development of Tg(actb:mda5,myl7:EGFP) DN-mda5 zebrafish
Anesthetized three day post fertilization DN-mda5 F2 zebrafish were screened for the presence of the transgene EGFP marker and imaged with an Olympus IX-81 inverted epifluorescence microscope equipped with a Hamamatsu ORCA-ER charge-coupled-device (CCD) camera and IPLab software (BD Biosciences, Rockville, MD). The myl7 promoter driven EGFP expression is restricted to the heart as seen at 4× (A) and 20× (B). (C) A schematic of the bicistronic expression vector with flanking Tol2 transposable elements. Black arrows indicate direction of transcription.
Figure 3
Figure 3. Rescue of DN-mda5 demonstrates critical role for mda5 in IFNϕ1 response both in vitro and in vivo
A) ZFL cells were transfected with empty vector (frm) or the expression vector containing full length mda5 or the dominant negative ΔCARD mda5 (250ng) along with reporter construct pGL3-IFN (250ng) and the internal control pRL-CMV (6.25ng). Twenty four hpt cells were exposed to SHRV at MOI=0.1. After an additional 24 h cells were lysed and reporter activity was measured. Brackets indicate significant difference between uninfected and infected treatments for vector and mda5 groups. Individual * over each treatment indicates a significant difference between mda5 and vector and DN-mda5. A two-tailed heteroscedastic T-test was performed to calculate signifiance (*, p<0.05). B) DN-mda5 zebrafish were injected at the 1-cell stage with mda5 RNA or pCS2+ empty vector. Wild-type AB lines were kept as controls. Fish were infected with 1×106 TCID50/ml SHRV and collected for RNA isolation at 48 and 72 hpi. RNA was purified and IFNϕ1 was subsequently measured by qPCR (*, p<0.05).
Figure 4
Figure 4. mda5 is critical for immune response to SHRV in vivo
A) Control, DN-mda5, and rescued DN-mda5 zebrafish embryos were treated as uninfected (UI) or infected by static immersion 48 hpf with 1×106 TCID50/ml SHRV and monitored daily for mortality. Results are representative of three separate experiments. Statistical analysis (Wilcoxon test) of the Kaplan-Meier curve was performed (**, p<0.01; ***, p<0.001) where ** is the comparison between DN-mda5 SHRV versus DN-mda5 rescue SHRV while *** is the comparison between control SHRV and DN-mda5 SHRV. B) Control, DN-mda5, and rescued DN-mda5 zebrafish embryos were infected 48 hpf 1×106 TCID50/ml virus and harvested for viral burden analysis. The graph indicates that at 24, 48 ,and 72 h post infection there is an increased viral burden in the DN-mda5 embryos, while the control and rescued DN-mda5 embryos had comparable viral burden. Figure is representative of three experiments; error bars are standard error of the mean (*, p<0.05; **, p<0.01).
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