Risk factors and patterns of household clusters of respiratory viruses in rural Nepal

Scott, E. M.; Magaret, A.; Kuypers, J.; Tielsch, J. M.; Katz, J.; Khatry, S. K.; Stewart, L.; Shrestha, L.; LeClerq, S. C.; Englund, J. A.; Chu, H. Y.

Samples were tested by a real-time reverse transcriptase polymerase chain reaction (PCR) for 12 respiratory viruses, including RSV, MPV, influenza viruses A and B, parainfluenza virus 1-4 (PIV 1-4), adenovirus (AdV), human coronavirus (CoV), HRV and bocavirus [19] [20] [21] . Influenza transmission in household was the primary aim of the trial substudy and is being analysed separately. Bocavirus was not included because of its prolonged shedding patterns. Sequencing was performed for HRV-and RSV-positive samples from household illness clusters utilizing samples with PCR cycle threshold values <33 and 30 for HRV and RSV, respectively, based on previous difficulty sequencing low viral load samples [22, 23] . Briefly, nucleic acid was extracted, and cDNA was synthesised. A hemi-nested PCR protocol was used targeting the 5 ′ untranslated region and the second hypervariable region of the attachment (G) glycoprotein coding region for HRV and RSV, respectively [22, 23] . Sequences were aligned using MAFFT v7.309, and maximum likelihood phylogenetic trees using the HKY85 model with 100 bootstrap replicates were inferred using PhyML 3.1 within Geneious [24, 25] . Sequences were considered to be the same virus type with ⩾98% identity. Sequences >200 base pairs (bp) were submitted to GenBank under accession numbers MH266546 to MH266612.