Objectives HCoV-HKU1 diversity and evolution was scarcely studied. We performed next-generation sequencing (NGS) and analysis of HCoV-HKU1 genomes over five years. Methods NGS used Illumina technology on NovaSeq 6000 following whole genome PCR amplification by a in-house set of primers designed using Gemi and PrimalScheme. Genome assembly and analyses used CLC Genomics, Mafft, BioEdit, Nextstrain, Nextclade, MEGA and iTol bioinformatic tools. Spike molecular modelling and dynamics simulations used Molegro Molecular Viewer/Hyperchem. Results Twenty-eight PCR systems allowed obtaining 158 HCoV-HKU1 genomes including 69 and 89 of genotype A and B, respectively. Both genotypes co-circulated during the study period but one predominated each year. 1,683 amino acid substitutions including 80 in ≥10 genomes were detected in genotype A relatively to a 2004 reference. H512R in spike, first detected in 2009 and reported as involved in antibody neutralization, was found in all genotype A, almost always with V387I and K478N, and was predicted here to significantly improve cellular TMPRSS2 protein binding. 1,802 amino acid substitutions including 64 in ≥10 genomes were detected in genotype B relatively to a 2005 reference. Conclusion This study substantially expands the global set of HCoV-HKU1 genomes. Genomics with protein structural analyses contributed to our understanding of HCoV-HKU1 evolution.

The number of SARS-CoV-2 recombinants identified during the pandemic has increased since the era of Omicron variants, but XBB.1.5 (or Omicron 23A) is the first lineage comprised of hybrid genomes to predominate at the country and global scales. Very interestingly, the XBB.1.5 recombinant, like the Marseille-4B subvariant (B.1.160/20A.EU2) and the pandemic variant B.1.1.7 (20I/Alpha) previously, has its ORF8 gene inactivated by a stop codon. XBB.1.5 was generated through two successive main events: a recombination between SARS-CoV-2 of lineages BA.2.10.1.1 (BJ.1) and BA.2 75.3.1.1.1 (BM.1.1.1) that generated the XBB (22F) lineage; then ORF8 gene inactivation by a stop codon. We further identified that a stop codon was present at 89 (74%) codons of the ORF8 gene in ≥1 of 15,222,404 genomes available in GISAID, and at 15 codons (12%) in ≥1,000 genomes. Thus, it is very likely that stop codons in ORF8 gene contributed on at least 3 occasions and independently during the SARS-CoV-2 pandemic to the evolutionary success of a lineage that became transiently predominant, most recently XBB.1.5. Such association of gene loss with evolutionary success, which suits the recently described Mistigri rule, is an important biological phenomenon very unknown in virology while largely described in cellular organisms.