Implementation of a multiplex PCR amplfication system combined with next-generation genome sequencing to decipher the circulation of Human coronavirus 229E lineages in Southern France

Abstract

Coronaviruses are known to evolve rapidly and be prone to new virus emergence. Human coronavirus-229E is one of the seven coronaviruses currently known to cause respiratory symptoms in humans. Genomic data are very scarce for this virus. Here, we implemented an in-house multiplex PCR strategy to amplify HCoV-229E genomes from nasopharyngeal samples diagnosed as positive for this virus RNA in Southeastern France. Then, next-generation sequencing was performed using Nanopore or Illumina technologies on Gridion or Novaseq 6000 instruments, respectively. HCoV-229E genomes were assembled and analyzed using MAFFT, MEGA, Itol, Nexstrain and Nextclade softwares. Thirty-one PCR primer pairs were designed to amplify overlapping fragments of the HCoV-229E genome. They allowed obtaining 123 genomes, which were classified in an emerging HCoV-229E lineage first reported in China, with two sublineages being delineated. Relatively to genome NC_002645.1 dating back to 1962, regarding nucleotide mutations, 1,167 substitutions, 72 insertions and 34 deletions were detected in viral genomes obatined here, while regarding amino acid mutations, 415 susbstitutions, 39 deletions and 14 amino acid insertions were detected. The genes with the greatest diversity were S (that encodes the spike protein) then Nsp3. Signature mutations were identified for the two sublineages. In summary, we almost doubled the set of HCoV-229E genomes available worldwide and provided the first genomes from France. Further studies are needed to strengthen the knowledge about the phylogenomics and evolutionary dynamics of this neglected respiratory virus, and about their specificities, which may also purvey clues to contribute improving knowledge for all other human coronaviruses.