Intraspecific variation of the hedgehog arteriviruses, which may constitute a new genus in the subfamily Heroarterivirinae of the family Arteriviridae

Abstract

We recently discovered a novel member of the family Arteriviridae, hedgehog arterivirus 1 (HhAV-1), in the brains of hedgehogs with fatal encephalitis. In this study, we classified this virus and investigated its intrahost genomic diversity using next-generation sequencing. We sequenced HhAV-1 genomes from specimens from seven hedgehogs (two males and five females) with signs of encephalitis that were collected in Buckinghamshire, Gloucestershire, and Cambridgeshire, England, and had died or been euthanised between 2013 and 2024. Analysis of the intrahost populations of these seven HhAV-1 isolates and a previously described isolate revealed the presence of single-nucleotide polymorphisms (SNPs), which were most frequent in open reading frames 5, 6, and 7, encoding glycoprotein 5, the membrane protein, and the nucleocapsid protein. Pairwise comparisons of the eight HhAV-1 variants showed that the nucleotide sequence identity values in their combined complete coding sequences ranged from 76.2% to 100%. The eight HhAV-1 variants also shared at least 82.8% amino acid sequence identity in five domains that are involved in replication and are used for the classification of nidoviruses: 3CLpro, NiRAN, RdRp, ZBD, and HEL1. In a replicase-based phylogenetic tree of members of the family Arteriviridae, the HhAV-1 variants formed a sister cluster to African pouched rat arterivirus. A DEmARC-based pairwise distance analysis indicated that these viruses may comprise a new species, for which we propose the name "Xiarterivirus erinaceid", in a new genus in the subfamily Heroarterivirinae.

Fig. 1

Amino acid sequence divergence of the proteins of HhAV-1 isolates. Shown is a box-and-whisker plot of the pairwise sequence identity of the proteins of seven HhAV-1 isolates (Supplementary Table S1). Isolate 0045 was excluded from this analysis because its protein sequences were found to be identical to those of isolate 0047. The entire and 25-to-75% distance ranges and median distance are depicted for nsp1-12 and the structural proteins. For this analysis, overlapping parts of structural proteins were used only for the most distal protein in a pair, e.g., for GP5 in the GP4 and GP5 pair.

Fig. 2

Phylogenetic analysis of arteriviruses. For virus selection, alignment, and tree generation using MEGA X software, see Materials and methods. The tree with the highest log likelihood (−32651.18) is shown. The percentage of trees (bootstrap values) in which two or more viruses clustered together is shown next to the respective branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap values less than 50% have been omitted. Arterivirus subfamilies are shown at the right. Each virus on the tree is represented by its GenBank accession number and name. The designation of subfamilies is according to Brinton et al. [3]. The viruses analysed in this study are shown in bold.

Fig. 3

Hierarchical classification of arteriviruses according to DEmARC. Presented is the distribution of pairwise distances using a box-and-whiskers diagram for clusters at four levels that correspond to taxonomy ranks of the family Arteriviridae, from species to subgenus to genus to subfamily (from lowest to highest). Each level of a classification is coloured differently and shown from left to right. The number of virus strains/isolates/variants used in the analysis for each virus species is shown in parentheses after the abbreviation of the prototype virus for the respective species. PRRSV-1 and PRRSV-2, porcine reproductive and respiratory syndrome virus 1 and 2, respectively; RtEiAV, RtEi arterivirus; RtMrufAV, RtMruf arterivirus; RtClonAV, RtClon arterivirus; RtMcAV, RtMc arterivirus; LDV, lactate dehydrogenase-elevating virus; RtClanAV, RtClan arterivirus; KRCV, Kibale red colobus virus; SHFV, simian haemorrhagic fever virus; FSVV, Free State vervet virus; SHEV, simian haemorrhagic encephalitis virus; ZMbV, Zambian malbrouck virus; MYBV, Mikumi yellow baboon virus; KKCBV, Kafue kinda chacma baboon virus; KRTGV, Kibale red-tailed guenon virus; PBJV, Pebjah virus; DeBMAV, DeBrazza’s monkey arterivirus; EAV, equine arteritis virus; APRAV, African pouched rat arterivirus; WPDV, wobbly possum disease virus; OSV-1, Olivier's shrew virus 1. For other details of the plot design, see Lauber & Gorbalenya [18].