'Vivaldi': an amplicon-based whole-genome sequencing method for the four seasonal human coronaviruses, 229E, NL63, OC43 and HKU1, alongside SARS-CoV-2.

Prior to the emergence of SARS-CoV-2 in 2019, alphacoronaviruses 229E and NL63 and betacoronaviruses OC43 and HKU1 were already established endemic 'common cold' viral infections. Despite their collective contribution towards global respiratory morbidity and mortality and potential to inform the future trajectory of SARS-CoV-2 endemicity, they are infrequently sequenced. We therefore developed a 1,200 bp amplicon whole-genome sequencing scheme targeting all four seasonal coronaviruses and SARS-CoV-2. The 'Vivaldi' method was applied retrospectively and prospectively using Oxford Nanopore Technology to ~400 seasonal coronavirus infections diagnosed in Nottingham, UK, from February 2016 to July 2023. We demonstrate that the amplicon multiplex strategy can be applied agnostically to determine the complete genomes of five different species from two coronaviral genera. Three hundred and four unique seasonal coronavirus genomes of greater than 95% coverage were achieved: 64 for 229E, 85 for NL63, 128 for OC43 and 27 for HKU1. They collectively indicated a dynamic seasonal coronavirus genomic landscape, with co-circulation of multiple variants emerging and declining over the UK winter respiratory infection season, with further geographical distinction when compared to a global dataset. Prolonged infection with concomitant intra-host evolution was also observed for both alpha- (NL63) and betacoronaviruses (OC43). This data represents the largest single cohort of seasonal coronavirus genomes to date and also a novel amplicon scheme for their future global surveillance suitable for widespread and easy adoption in the post-SARS-CoV-2 era of viral genomics.