A gastrointestinal rotavirus infection mouse model for immune modulation studies. Methods
Viruses
To obtain a large quantity of virulent epizootic-diarrhea infant-mouse virus (EDIM), 78 neonatal mice were inoculated at the age of 4 days with 5 μl EDIM 7.8*107 focus forming units/ml, a kind gift from Dr. Richard Ward, Cincinnati Children’s Hospital Medical Center, USA. Stool samples were collected and pooled from day 5 until day 13 and EDIM was extracted with genetron (1,1,2-trichloro-1,2,2,-trifluoroethane, Sigma-Aldrich, Zwijndrecht, The Netherlands). The stool preparation of EDIM contained 400 μg rotavirus/ml as determined in the IDEIA ELISA (Dako diagnostics, Glostrup, Denmark) verified against a rotavirus stock with known concentration of rotavirus. To determine whether EDIM caused diarrheal disease when orally administered to neonatal mice, 7 mice were inoculated with 5 μl EDIM (400 μg rotavirus/ml) at the age of 7 days. At day 2 p.i., 70% of the mice were suffering from diarrhea, rising to 100% at day 5 and declining again from day 6.
The rhesus rotavirus (RRV) strain used in this study, also provided by Dr. Richard Ward, was grown in African green monkey kidney MA104 cells (ECACC, Salisbury, UK) and concentrated by ultracentrifugation. The titer was determined using a titration assay in MA104 cells resulting in a 50% cell culture infective dose (CCID50) of 1 × 107.7. Virus stocks were UV-inactivated overnight at short wave UV 254 nm (UV cabinet CM-10; Alltech, Breda, The Netherlands) and used for measuring delayed-type hypersensitivity (DTH) responses and T cell stimulation. Inactivation of rotavirus was confirmed using the titration assay in MA104 cells. For DTH responses, the EDIM and RRV stocks were diluted 100× in PBS. For use in T cell stimulation experiments, caesium chloride gradient purified EDIM double layered particles (provided by Dr. Richard Ward) were diluted to 1 μg/ml and RRV was diluted to a CCID50 of 5 × 107.
Simian rotavirus, SA-11, (ATCC, Middlesex, UK) was grown in MA104 cells and concentrated by ultracentrifugation. SA-11 contained 14 μg rotavirus/ml as determined in the IDEIA ELISA, verified against a rotavirus stock with known concentration of rotavirus. For the determination of rotavirus-specific antibodies in serum a concentration of 500 ng/ml SA-11 was used.
Mice
Pregnant female BALB/c mice were housed individually in sterile micro-isolation cages under standard housing conditions at the animal facility of the Dutch Vaccine Institute with a 12 h dark and light cycle. Animal care and use were performed in accordance with the guidelines of the Dutch Committee of Animal Experiments. Weaning of suckling pups occurred at 21 days of age. Alternatively, pups immunized and challenged prior to weaning remained with their dams.