Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice

Abstract

Small-animal models and reverse genetics systems are powerful tools for investigating the molecular mechanisms underlying viral replication, virulence, and interaction with the host immune response in vivo. Rotavirus (RV) causes acute gastroenteritis in many young animals and infants worldwide. Murine RV replicates efficiently in the intestines of inoculated suckling pups, causing diarrhea, and spreads efficiently to uninoculated littermates. Because RVs derived from human and other non-mouse animal species do not replicate efficiently in mice, murine RVs are uniquely useful in probing the viral and host determinants of efficient replication and pathogenesis in a species-matched mouse model. Previously, we established an optimized reverse genetics protocol for RV and successfully generated a murine-like RV rD6/2-2g strain that replicates well in both cultured cell lines and in the intestines of inoculated pups. However, rD6/2-2g possesses three out of eleven gene segments derived from simian RV strains, and these three heterologous segments may attenuate viral pathogenicity in vivo. Here, we rescued the first recombinant RV with all 11 gene segments of murine RV origin. Using this virus as a genetic background, we generated a panel of recombinant murine RVs with either N-terminal VP8* or C-terminal VP5* regions chimerized between a cell-culture-adapted murine ETD strain and a non-tissue-culture-adapted murine EW strain and compared the diarrhea rate and fecal RV shedding in pups. The recombinant viruses with VP5* domains derived from the murine EW strain showed slightly more fecal shedding than those with VP5* domains from the ETD strain. The newly characterized full-genome murine RV will be a useful tool for dissecting virus-host interactions and for studying the mechanism of pathogenesis in neonatal mice.

Keywords: reverse genetics; rotavirus; small-animal model.

Figure 1

Schematic presentation of the murine RV VP4 gene. (A) Schematic presentation of RV gene segment 4. The 5′ and 3′ UTRs are shown as black boxes. VP8* and the body and foot regions of the VP5* domain in the VP4 gene are shown in light blue boxes. The numbers above the box indicate the amino acid positions. (B) Schematic presentation of the murine RV ETD_822 and EW strains and the VP4 chimeric viruses generated in this study. The five amino acids that differ between the ETD_822 and EW strains are highlighted in red inside the blue boxes. The number above the box indicates the amino acid positions.

Figure 2

Percentage of diarrhea caused by the wild-type murine RV and recombinant murine RVs. Five-day-old 129sv pups were inoculated with (A) 1 × 10³ DD₅₀ of EW, or 1 × 10³ FFU of (B) rD6/2, (C) rEW/ETD-VP4, (D) rEW/ETD-VP4-EW-VP8*, (E) rEW/ETD-VP4-ETD-VP5*-body, or (F) rEW/ETD-VP4-ETD-VP5*-foot. The infected mice were monitored for diarrheal stool for 12 days by gentle abdominal pressure.

Figure 3

Fecal RV shedding by wild-type murine RV and recombinant murine RVs. Five-day-old 129sv pups were inoculated with the same doses and viruses as in Figure 2. (A) 1 × 10³ DD₅₀ of EW, or 1 × 10³ FFU of (B) rD6/2, (C) rEW/ETD-VP4, (D) rEW/ETD-VP4-EW-VP8*, (E) rEW/ETD-VP4-ETD-VP5*-body, or (F) rEW/ETD-VP4-ETD-VP5*-foot. The amount of RV in the stool samples was determined by ELISA. Each dot shows data from one pup and the line shows the average score. The dotted lines indicate the score of the limit of detection determined from the stool of uninfected pups.