Development of a replication competent murine norovirus reporter system

Fig 1. Development and Characterization of MNoV-HiBiT Viruses. A) (Left) Cartoon illustration of the MNoV genome organization with a focus on the NS4-NS5 cleavage site and the introduction of the HiBiT tag (red). Scissors indicate the MNoV protease cleavage site. (Right) Cartoon illustration of the utility of the MNoV-HiBiT virus and the complementation with LgBiT supplied in lysis buffer to form a functional nanoluciferase. B) Representative western blot of BV2 cells either uninfected or infected with MNoVCW3-HiBiT at a multiplicity of infection (MOI) of 1.0 for 12 hours and probed for indicated antibodies. Predicted molecular weight of MNoV nonstructural proteins are indicated. C) BV2 cells were challenged with MNoVCW3 or MNoVCW3-HiBiT at an MOI of 0.05. Viral production was enumerated using plaque assays (PFU; plaque forming units) at the indicated time points. D-E) BV2 or BV2ΔCD300lf cells were challenged with MNoVCW3 or MNoVCW3-HiBiT at a multiplicity of infection (MOI) of 5.0. At the indicated time points, infection was quantified using Nano-Glo HiBiT Lytic Detection System (RLU; relative luminescent units). F) HeLa or HeLa-CD300lf cells were challenged with MNoVCW3 or…

Fig 2. Stability of the HiBiT reporter during repeated passaging. A) Cartoon schematic of the passaging strategy. First, plasmids containing the MNoV-HiBiT molecular clone are transfected into HEK293T cells and subsequently passaged onto BV2 cells. Passaged stocks are titered and tested for luminescence capacity via low MOI infection of BV2 cells. B) Two independent passaging experiments with MNoVCW3-HiBiT (Left) and MNoVCR6-HiBiT (Right) with luminescence values being normalized to the initial p1 stock. RLU quantified using Nano-Glo HiBiT Lytic Detection System. All data are shown as mean ± S.D. from three or four independent experiments and analyzed by one way ANOVA with Tukey’s multiple comparison test. Statistical significance is annotated as follows: ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 3. Utility of the MNoV-HiBiT system for in vivo applications and antiviral discovery. A-B) STAT1-/- mice were inoculated with 2.5 x 105 PFU of MNoVCW3-HiBiT and euthanized 3 days post-infection. (A)Tissue titers for mesenteric lymph nodes (MLN), spleen, liver, colon, and ileum were analyzed by qPCR for MNoV genome copies and normalized to actin. (B) Relative Luminescence Units (RLU) from indicated tissue homogenates quantified using Nano-Glo HiBiT Lytic Detection System. Raw RLU values from uninfected matched tissues were used to background subtract from infected samples. Dotted lines represent limit of detection of both assays. C) Correlation of MNoV genome copies (y-axis) and RLU (x-axis) for indicated tissues. R2 and P values for linear regression analyses for each tissue are included in the legend. D-F) Dose response curves of BV2 cells treated with indicated doses of 2CMC (D) GO289 (E), or HPA-12 (F) infected with MNoVCW3-HiBiT (left axis) or uninfected and measured for cell viability using CellTiterGlo (right axis). Inhibitory Concentration 50 (IC50) and Cytotoxic Concentration 50 (CC50) are listed in bottom left corner. All data are shown as mean ± S.D. from three independent…