Enterovirus seroneutralization assays
Neutralizing antibodies were measured in serum or plasma against 41 EV serotypes listed in Table 2 as previously described23. Viruses were isolated and cultivated, and seroneutralization assays were performed using the A549, Vero, RD (all bought from American Type Culture Collection) and GMK (from the National Institute for Health and Welfare, Finland) cell lines. Most of the viruses were analyzed using a standard plaque neutralization assay24, whereas viruses that did not form clear plaques were analyzed using a microneutralization assay (CAV5, CAV6, CAV10, CAV16, EV71, EV78 and EV94). The neutralization assay was considered positive when the serum dilution 1:4 reduced the number of plaques by more than 75% (plaque assay) or inhibited the ability of the virus to kill cells (microneutralization assay). The serum/plasma samples analyzed were taken between 1 and 5 years of age. The sample volume was adequate for the analysis of neutralizing antibodies against all 41 serotypes in 183 children. In 83 children the volume was adequate for the analysis of 39-40 serotypes and in nine children for 4-21 serotypes.