Viral RNA Extraction and Sequencing

Viral RNA was extracted using MagNA pure TNA high throughput kit (Roche). NPS samples were screened for respiratory viruses using multiplex real-time reverse-transcription PCR (rRT-PCR) [32 (link)]. A sample was determined positive if rRT-PCR cycle threshold (Ct) value was <40.0 [33 (link)]. Ct value was assumed to be related to viral load. VP4/VP2 sequencing (approximately 450 bp) was used for species and type assignment. Primers amplifying approximately 549 bp of the VP4/VP2 region (F1: 5′-CCGGCCCCTGAATGYGGCTAA-3′, F2: 5′-ACCRACTACTTTGGGTGTCCGTG-3′; R1: 5′-TCWGG HARYTTCCAMCACCANCC-3′, R2: 5′- ACATRTTYTSN CCAAANAYDCCCAT-3′) were used in a nested 25-μL reaction [34 (link)]. PCR products were purified (QIAquick PCR purification Kit, Qiagen) and sequenced on an ABI 3130xl instrument (Applied Biosystems).