Swabs from participants with confirmed infection were further assessed in a quantitative RT-PCR assay targeted at the M gene as described previously.27 The target sequence was cloned and quantified using pico green to prepare a standard curve for quantitation. Standard curves were run in duplicate. Samples were generally tested once but RT-PCR was repeated to validate fluctuations. Results were expressed as cDNA equivalent copies of viral RNA. The limit of detection was 5 RNA copies/reaction. De novo whole genome sequencing was performed on combined nose and throat swabs with Ct values below 33. All 8 virus gene

segments were amplified in two RT-PCR reactions by using primers that target the conserved termini: (5′-GCCGGAGCTCTGCAGATATCAGCRAAAGCAGG-3′) or learn more (5′-GCCGGAGCTCTGCAGATATCAGCGAAAGCAGG-3′) HSP inhibitor cancer with (5′-CAGGAAACAGCTATGACAGTAGAAACAAGG-3′).28 454 sequencing adaptors and molecular identifier tags were ligated to combined PCR products using the SPRIworks Fragment Library System II

for Roche GS FLX* DNA Sequencer. Emulsion PCR, bead recovery and enrichment were performed manually according to the manufacturer’s protocol followed by sequencing on a Roche GS FLX+. Analysis was limited to the envelope gene sequences in the current study. Sequences will be made available in Genbank. Sera were tested in haemagglutination inhibition (HI) and microneutralization (MN) assay as previously described.26 A reference antigen supplied by WHO (A/California/7/2009(H1N1)-like) was used with turkey erythrocytes. Titres were read as the reciprocal of the highest serum dilution causing complete inhibition of agglutination. If there was no inhibition of HI at the highest serum concentration (1:10 dilution) the titre was designated as 5. Influenza infection was defined as a positive RT-PCR, regardless of the presence of symptoms. Household members with RT-PCR confirmed infection but no increase in mouth temperature

and none of the symptoms listed earlier were defined as asymptomatic infection. Serology was not routinely performed on acute sera so was not considered in the definition of secondary infection. Nevertheless, else seroconversion was reported if there was a 4-fold or greater rise in HI or MN titre between pre- and post-pandemic sera. Household secondary infection risk (SIR) was calculated as the number of household contacts infected 1–8 days after symptom onset in the index case divided by the number of household contacts, similar to other studies.6, 7, 13, 15 and 17 Serial interval was defined as the number of days between symptom onset in the index case and the first secondary case. Other secondary household cases were only included in the serial interval calculation if their symptoms started on the same day as the first secondary case. Children were defined as those up to 15 years of age. Oseltamivir treatment was considered to be timely if commenced within 2 days of symptom onset.