Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing

Alquezar-Planas, David E.; Mourier, Tobias; Bruhn, Christian A. W.; Hansen, Anders J.; Vitcetz, Sarah Nathalie; Mørk, Søren; Gorodkin, Jan; Nielsen, Hanne Abel; Guo, Yan; Sethuraman, Anand; Paxinos, Ellen E.; Shan, Tongling; Delwart, Eric L.; Nielsen, Lars P.

Microbial screening -Immunological and PCR based assays All secretions were initially tested for respiratory syncytial virus (RSV) using immunological tests including Immunofluorescence or membrane ELISA. As only 36% of the samples collected in the RSV season tested positive, we additionally tested them for a range of viral and bacterial pathogens (see methods). The full results are available upon request. Briefly, the extensive PCR testing revealed that ~46% of the secretions tested positive for RSV, ~20% for various picornaviruses and smaller proportions of a range of other respiratory pathogens (Supplementary Table S1 ). In 75% of secretions a pathogen was found, where in 20% of the samples more than one pathogen was identified. A summary of detected viral and bacterial pathogens using PCRbased-assays in children with one or multiple infections is listed in Supplementary Table S2 . With the exception HPIV4, all viruses identified using deep sequencing in this study had been previously screened using hybridization-based methods (Supplementary Table S1 ). Despite the increased sensitivity of PCR based assays, these results emphasize the limitations of PCR based identification of microbial pathogens in the public health setting. The largest proportion of humanvirus-sequences identified (61%) was taxonomically classified as HPIV4, with most sequences predominating from one high titer sample. The second largest portion of human-virus-reads derived from human metapneumovirus (hMPV) (19%), which also predominately originated from one specimen. All identified coronavirus sequences were identified as the OC43 strain, accounting for 7% of the sequence reads. Finally, small subsets of reads to four known respiratory viruses were identified in some samples, including influenza A (n=1), human parainfluenza virus 3 (n=1), adenovirus (n=2) and human rhinovirus (n=3), two of which mapped to the recently proposed type C species 9 . Efforts in characterizing the HRV-C genomes are currently ongoing.