Phylogenetic analysis of enteroviruses from non-human primates reveals two new species within the genus Enterovirus and inter-species recombination

To date, 15 species have been described within the genus Enterovirus. Previous studies suggested the existence of another species comprising strains isolated from the stool specimens of non-human primates (NHPs) in Central Africa. Moreover, numerous full-length or partial genomic sequences of NHP enteroviruses (EVs) can be found in GenBank without being properly classified. To our knowledge, no comprehensive synthesis of NHP EV data exists, leaving genetic relationships between strains across independent studies unclear. To address these gaps, we sequenced the complete genome of four NHP EVs from our stool collection and conducted an extensive search of NHP EV sequences in GenBank to perform a comprehensive phylogenetic analysis. Our analyses revealed two new species tentatively named Enterovirus mbel and Enterovirus noa, which contain at least 6 and 2 virus types, respectively. We also identified new virus types within the known species EV-J. Phylogenetic analyses strongly suggest interspecies recombination events between NHP EVs in the non-structural region of the genome, challenging the long-held view that recombination is confined within narrowly defined subsets of EVs belonging to the same species. We also performed the first comprehensive comparative analysis of full length human and NHP EV genomes, focusing on GC content, dinucleotide frequencies and codon-usage bias. GC content emerged as the most robust host-associated marker: all NHP-associated virus types within species E. alphacoxsackie and E. betacoxsackie displayed GC % below 47 %, whereas human-derived virus types exhibited GC % above 47 %. Dinucleotide frequency, Effective Number of Codons (ENC) and Relative Synonymous Codon Usage highlighted distinct codon-bias clusters that mirror the phylogenetic relationships between EVs but only partially correlate with their respective hosts of origin. This work enhances our understanding of EVs circulating in NHPs and paves the way for future research aiming at understanding the mechanisms underlying host-adaptation among EVs.