HRV genotyping
To determine HRV genotypes, the VP4/2 region was amplified using previously described primers (33) with PrimeScriptâ„¢ One Step RT-PCR Kit (v.2, Takara, Japan). PCR-amplified samples were then sequenced using the Nanopore MinION with the Rapid Barcoding Kit 96 (Oxford Nanopore Technologies, United Kingdom).
The genotype of each sample was determined through phylogenetic analysis based on the rhinovirus VP2/4 region. HRV VP4/2 sequences with collection dates after 2010 were downloaded from NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed 20-09-2022). After the removal of duplicates, VP4/2 sequences were subsampled to include one random representative from each year, country, and genotype. Sequences were aligned using MAFFT (v7.487) (34), and maximum likelihood (ML) trees were constructed using IQ-TREE (v.2.0.3) based on the best-fit nucleotide substitution model (35).