A recombinant murine-like rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission

Abstract

Rotaviruses (RVs) are one of the main causes of severe gastroenteritis, diarrhea, and death in children and young animals. While suckling mice prove to be highly useful small animal models of RV infection and pathogenesis, direct visualization tools are lacking to track the temporal dynamics of RV replication and transmissibility in vivo. Here, we report the generation of the first recombinant murine-like RV that encodes a Nano-Luciferase reporter (NLuc) using a newly optimized RV reverse genetics system. The NLuc-expressing RV was replication-competent in cell culture and both infectious and virulent in neonatal mice in vivo. Strong luciferase signals were detected in the proximal and distal small intestines, colon, and mesenteric lymph nodes. We showed, via a noninvasive in vivo imaging system, that RV intestinal replication peaked at days 2 to 5 post infection. Moreover, we successfully tracked RV transmission to uninoculated littermates as early as 3 days post infection, 1 day prior to clinically apparent diarrhea and 3 days prior to detectable fecal RV shedding in the uninoculated littermates. We also observed significantly increased viral replication in Stat1 knockout mice that lack the host interferon signaling. Our results suggest that the NLuc murine-like RV represents a non-lethal powerful tool for the studies of tissue tropism and host and viral factors that regulate RV replication and spread, as well as provides a new tool to facilitate the testing of prophylactic and therapeutic interventions in the future.

Keywords: Nano-luciferase; in vivo imaging system; rotavirus; tissue tropism; transmission.

Figure 1

Generation and validation of a bioluminescent rD6/2-2g-NLuc. (A) A schematic diagram (not to scale) of a genetically engineered pT7 plasmid that encodes NLuc with nucleotide positions indicated. UTR, untranslated region; P2A, self-cleaving P2A peptide gene of porcine teschovirus-1. (B) BHK-T7 cells were transfected with pT7-NSP3 and increasing amounts of pT7-NSP3-NLuc for 48 hours, and cell lysates were analyzed by western blot. (C) BHK-T7 cells were transfected with pT7 or pT7-NSP3-NLuc plasmids for 48 hours. The luciferase activity was determined by Nano-Glo^® luciferase assay. Data are presented as the average of three experiments and error bars indicate standard error of the mean (SEM) (Student t test; *** P < 0.001). (D) MA104 cells were infected with rD6/2-2g and rD6/2-2g-NLuc viruses (MOI=0.1) for 24 hours, and cell lysates were analyzed by western blot. (E) dsRNA profiles. Viral RNA was extracted from sucrose cushion-concentrated virus, separated on a 10% polyacrylamide gel, and then stained with ethidium bromide. The dsRNA segment numbers are indicated and the position of the engineered segment 7 is marked with a yellow arrowhead. (F) Luciferase activity of rD6/2-2g and rD6/2-2g-NLuc. MA104 cells were infected with 10-fold serially diluted rD6/2-2g or rD6/2-2g-NLuc. Cells were harvested at 48 hpi and the luciferase activity was determined by Nano-Glo^® luciferase assay. Results are expressed as the mean luminescence of triplicates and error bars show the SEM (one-way ANOVA with Dunnett’s test; ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Figure 2

Growth kinetics of bioluminescent rD6/2-2g-NLuc in MA104 cells. (A) MA104 cells were infected with rD6/2-2g or rD6/2-2g-NLuc (MOI=0.01) in the presence of trypsin (0.5 μg/ml) and harvested at the indicated time points. The viral mRNA level was determined by RT-qPCR assay and normalized to that of GAPDH. Data are the average of three experiments, error bars indicate SEM (two-way ANOVA test; ns, not significant, * P < 0.05, ** P < 0.01). (B) Multi-step growth curves of rD6/2-2g-NLuc. MA104 cells were infected with rD6/2-2g or rD6/2-2g-NLuc (MOI=0.01) in the presence of trypsin (0.5 μg/ml) and harvested at the indicated time points. The viral titers were determined by an immunoperoxidase focus-forming assay. Data are the average of three experiments, error bars indicate SEM (two-way ANOVA test; ns, not significant, * P < 0.05, ** P < 0.01). (C) Plaque formation of rD6/2-2g-NLuc. Plaques were generated on MA104 monolayers and detected by crystal violet staining at 7 dpi. The diameter of at least 25 randomly selected plaques from 2 independent plaque assays was measured by a bright-field microscope. Error bars indicate SEM (Student t test; **** P < 0.0001). (D) Functional stability of luciferase activity in rD6/2-2g-NLuc after sequential passage. rD6/2-2g-NLuc was sequentially passaged in MA104 cells. The luciferase activity for passages 3-8 was determined by Nano-Glo^® luciferase assay as described. Results are expressed as the mean luminesce of duplicates. Error bars show SEM. Luminescence from NLuc substrate from MA104 cells infected with rD6/2-2g were plotted as a reference.

Figure 3

Bioluminescence of rD6/2-2g-NLuc in the intestines and the systemic sites in wild-type 129sv mice. (A) Five-day-old wild-type 129sv pups (n=5) were orally infected with 1.3 × 10⁶ FFUs of rD6/2-2g-NLuc and diarrhea was monitored till 5 days post infection. (B–G) Five-day-old wild-type 129sv pups were orally infected with 1.3 × 10⁶ FFUs of rD6/2-2g-NLuc, then euthanized at indicated days post infection. Bioluminescence from indicated tissue homogenates was determined by Nano-Glo^® luciferase assay. Luminescence from NLuc substrate of uninfected mice tissues were plotted as a reference.

Figure 4

Infectivity and pathogenicity of rD6/2-2g-NLuc in vivo. (A) Five-day-old 129sv mice (n=6) were orally inoculated with 3.5 × 10³ FFUs of rD6/2-2g-NLuc. The diarrhea rate was monitored from 1 to 12 days post infection. (B) Viral shedding in stool samples was detected by an FFU assay and normalized to the feces weight. (C) Representative images of rD6/2-2g-NLuc infected pups (1 to 12 days). The bioluminescent signal is expressed in photons per second per square centimeter per steradian (p/sec/cm²/sr). (D) Quantification of the luminescence in (C). The dashed line indicates the upper limit of detection.

Figure 5

Transmission rD6/2-2g-NLuc in vivo. (A) Five-day-old 129sv mice were co-housed with 6 infected (3.5 × 10³ FFUs of rD6/2-2g-NLuc) and 6 uninfected littermates in the same cage. The diarrhea rate was monitored from 1 to 12 days post infection. (B) Viral shedding in stool samples was detected by an FFU assay and normalized to the feces weight. (C) Representative images of naive pups (1 to 12 days). The bioluminescent signal is expressed in photons per second per square centimeter per steradian (p/sec/cm²/sr). (D) Quantification of the luminescence in (C). The dashed line indicates the upper limit of detection.

Figure 6

Characterization of rD6/2-2g-NLuc infection in Stat1 KO 129sv mice. (A) Five-day-old Stat1 KO 129sv mice (n=9) were orally inoculated with 3.5× 10³ FFUs of rD6/2-2g-NLuc. The diarrhea rate was monitored from 1 to 12 days post infection. (B) Viral shedding in stool samples was detected by an FFU assay and normalized by to feces weight. (C) Representative images of rD6/2-2g-NLuc infected Stat1 KO pups (1 to 12 days). The bioluminescent signal is expressed in photons per second per square centimeter per steradian (p/sec/cm²/sr). (D) Quantification of the luminescence in (C). The dashed line indicates the upper limit of detection. (E) Statistical analysis of area under the curve (AUC) comparing data in Figure 4D and (D). Error bars show the SEM (one-way ANOVA test; * P < 0.05, ** P < 0.01).