A single influenza B virus isolated from a participant during 2008, and propagated in MDCK cells was used to assess serum for both the first and second seasons. The virus had EPZ015666 purchase a titer of 320 with B/Wisconsin/1/2010 (Yamagata) reference antisera and of <10 with B/Brisbane/60/2008

(Victoria) antisera. A reference antigen supplied by WHO (A/California/7/2009(H1N1)-like) was used to assess season 3/pandemic plasma. The HI titer was read as the reciprocal of the highest serum dilution causing complete inhibition of agglutination, partial agglutination was not scored as inhibition of agglutination. If there was no inhibition of HI at the highest serum concentration (1:10 dilution) the titer was designated as 5. Only one sample had a titer >1280 and this was not

adjusted. Influenza infection’ was defined as either the detection of influenza RNA in a swab sample by RT-PCR or a four fold or greater rise in HI titer, with a second titer of at least 40. Participants were excluded from analysis of each season if they were not present for ILI surveillance during the periods of LGK 974 confirmed influenza transmission or if paired-plasma were not collected. Additionally, participants were excluded from the analysis of effect of infection in one season on infection in subsequent season if they had not been available or fully assessed for infection in both seasons. The risk of an infection was modeled as depending on

the (log2-transformed) pre-season titer using a marginal logistic regression model, which takes into account potential household clustering. Unadjusted titer effects and titer effects adjusted for age (modeled as a natural cubic spline with 3 degrees of freedom and knots at 10 and 20 years) were calculated. We also tested for potential non-linear effects of the log2-titer on outcome by additionally including a quadratic term into the model and for titer–age interactions. The risk of infection was also modeled as depending on infection in the preceding season with each strain that did not induce HI antibodies (i.e. prior heterologous infections). As above, marginal logistic regression was used to account for potential household clustering and results adjusted for effects of age and pre-season HI titer. Statistical analyses Methane monooxygenase were performed with the statistical software R version 2.15.0 (R foundation for Statistical Computing, Vienna, Austria) and the companion R package geepack version 1.1-6. A detailed description of the cohort and of the infections and illnesses detected has been presented previously.21 In brief, 940 individuals were studied for three consecutive influenza seasons, from December 2007 through April 2010, resulting in 1793 person-seasons of influenza surveillance. The age of participants ranged from <1 to 90 years and none had ever received influenza vaccination.