Animal husbandry and monitoring of animal health
NOD mice and SOCS1-tg mice on a NOD background (both from in-house breeding) were housed in specific pathogen-free conditions at Karolinska Institutet, Stockholm, Sweden. Ethics approval was granted for all experiments by the local ethics committee and were conducted in accordance with the NIH Principles of Laboratory Animal Care and national laws in Sweden. Extended health monitoring of mice was performed. Additional information is provided in the ESM Methods: Animals, together with details of primers used for SOCS1-tg mouse genotyping (ESM Table 1).
Virus and vaccine production
A CVB1 field isolate (CVB1-V200; ), was propagated in Vero cells (ECACC no. 84113001; mycoplasma negative), purified and then formalin inactivated for 3 days at 37°C to produce CVB1 vaccine. See ESM Methods: Vaccine production and Hankaniemi et al  for more details. Mice were infected with CVB1-10796 (propagated in HeLa cells, originally obtained from R Glas, Karolinska Institutet, Stockholm, Sweden, mycoplasma negative).
Male and female age-matched NOD and SOCS1-tg mice (4–7 weeks old) were randomly assigned to groups and vaccinated on days 0, 14 and 28 with non-adjuvanted vaccine containing 1.8 μg protein (n = 8 and n = 7, respectively) or mock-vaccinated with vaccine buffer (M199-0.1% Tween80 (vol./vol.), 150 μl, n = 6 for NOD and for SOCS1-tg mice) by interscapular injection [6, 7]. Serum was collected before vaccinations and infection (day 35) by tail bleeding (experimental timeline displayed in Fig. 1a). The study was not blinded to the experimenter.
Mice were challenged with 106 plaque forming units (PFU) CVB1-10796 (diluted in serum-free RPMI to a final volume of 200 μl, administered by intraperitoneal injection, with the dose carefully optimised, data not shown) on day 35. Blood samples were collected on day 3 post infection (p.i.; 1:1 ratio with 12 mmol EDTA). NOD mice were killed on day 3 p.i. and pancreases saved for virus quantification and histological analysis. SOCS1-tg mice were monitored until diabetes development or day 21 p.i. and pancreases saved for histological analysis.
Monitoring of blood glucose and diabetes development
Blood glucose was measured in blood obtained from the tail-vein with a Bayer Contour XT blood glucose meter (Bayer, Basel, Switzerland) and a glucose concentration >18 mmol/l, or two consecutive measurements between 13 and 18 mmol/l were used to define diabetes and diabetic mice were killed.
Neutralising antibody detection
Neutralising antibody titres were determined by standard virus plaque reduction assay in green monkey kidney (GMK) cells (National Institute for Health and Welfare, Finland; mycoplasma negative) . Plaque number reduction ≥80% compared with untreated virus suspension was considered positive. The detection limit of the assay was a fourfold dilution (1:4), and positivity cut-off for serum samples was set to ≥1:16.
Pancreases were homogenised and lytic virus quantified in either blood or pancreas by standard plaque assay in GMK cells. Viral titres are expressed as PFU/g of tissue or ml of blood. See ESM Methods: Virus titration and tissue homogenisation for more details.
Enterovirus specific real-time PCR was performed using RNA extracted from blood samples; see ESM Methods: PCR analysis and Honkanen et al  for further details. Primers are shown in ESM Table 2.
Histological analysis and immunohistochemistry
Histological analysis and immunohistochemistry of pancreas samples with viral capsid protein 1 (VP1), insulin and glucagon were carried out as in Flodström et al and Larsson et al ([4, 6]; ESM Methods: Immunohistochemistry and antibodies).
Statistical analyses were executed using Prism 5 software (GraphPad, La Jolla, CA, USA). PCR and VP1 immunohistochemistry data were analysed by χ
2 tests. Plaque assay virus titrations were analysed by Mann–Whitney U test. Neutralising antibody data was analysed by one-way ANOVA. Diabetes incidence was analysed by log rank Mantel–Cox test. Data are expressed as the mean ± SD. A p value ≤0.05 was regarded statistically significant.