2 ml tube

For adult MNoV infections, 6–9-week-old mice were orally inoculated with 1e6 plaque-forming units (PFU) of CR6 in a volume of 25μl. For neonatal and juvenile infections, postnatal day (P)6 and P15 mice, respectively, were gavaged with 10⁶ PFU of CR6 in a volume of 50μl using a 22ga plastic feeding tube. Virus stocks contained a range of 10^7.4-10^7.7 genome copies in 10⁶ PFU. Stool samples were collected by gently palpating the abdomen to encourage defecation. Given the challenge of collecting stool from young mice, stool was collected as possible, and the number of samples collected was generally representative of a much greater number of mice. Tissues were collected from mice at time of euthanasia or shortly after natural death when tissues were still intact. Stool and tissues were harvested into 2-ml tubes (Sarstedt, Germany) with 1-mm-diameter zirconia/silica beads (Biospec, Bartlesville, OK). Samples were frozen and stored at −80C until RNA extraction or plaque assay. For controls for treatment groups, infected groups include mice treated with PBS or PBS containing 2% Tween-80.

MNoV stocks for MNoV^CR6 (CR6) and MNoV^CW3 (CW3) were generated from molecular clones as previously described^{31 (link),32 (link)}. Briefly, plasmids encoding the MNoV genome were transfected into 293T cells to generate infectious virus, which was subsequently passaged on BV2 cells. After two passages, BV2 cultures were frozen and thawed to liberate virions. Then, cultures were cleared of cellular debris and concentrated by ultracentrifugation. Titers of virus stocks were determined by plaque assay on BV2 cells as described^{32 (link)}. For MNoV infections, conventional specific-pathogen free and germ-free mice were inoculated with a dose of 10⁶ pfu of the indicated viral strain, except as otherwise mentioned. Germ-free mice were infected with a dose of 10⁷ pfu of the indicated viral strain. All mice were infected at 6 to 8 weeks of age by the oral route in a volume of 25 μl. Mouse rotavirus (RV) strain EC (RV^EC) was provided by Mary Estes (Baylor College of Medicine)^{33 (link)}. Mice received 10⁵ 50% shedding doses (SD50) of RV in 100μl, preceded by 100μl 1.33% (w/v) sodium bicarbonate (Sigma-Aldrich) by oral gavage. For all experiments, stool and tissues were harvested from mice into 2-ml tubes (Sarstedt, Germany) with 1-mm diameter zirconia/silica beads (Biospec, Bartlesville, OK). Tissues were frozen on dry ice and either processed on the same day or stored at −80°C.

At specific days after virus infection, serum was obtained by intracardiac heart puncture and followed by extensive perfusion. Tissues were harvested into 2 mL tubes (Sarstedt, Germany) with 1-mm diameter zirconia/silica beads (Biospec), flash frozen in a bath of ethanol and dry ice, weighted and stored at −80°C. Tissues were homogenized using a bead-beater apparatus and levels of virus were determined by plaque assay on BHK21 or Vero cells, or by quantitative reverse transcription-PCR using WNV- or ZIKV-specific primers and probes as described previously (Brien et al., 2013 ; Govero et al., 2016 (link)).