Phylogenetic and bioinformatic analysis of rat hepatitis E virus : investigations for host adaptation factors of an emerging pathogen

Abstract

Rocahepevirus ratti genotype 1 (HEV-C1), colloquially known as rat HEV, is a hepatitis E virus (HEV) that circulates within rodents and is phylogenetically distant from the HEV species that classically infects humans (Paslahepevirus balayani genotypes 1–4, HEV-1–4, HEV-A1–4). Nonetheless, human cases of rat hepatitis E infection are being reported worldwide and no longer considered a sporadic, local phenomenon. Proper curation of sequenced rat HEV is required such that analyses on the phylogeny can be conducted to extricate any host adaptation factors of this novel zoonotic pathogen. The first part of this study thus concerns the intragenotypic characterisation of the HEV-C1 phylogeny. Sequences were first procured from GenBank and local screening in patients and rodents. Maximum likelihood patristic distance of both the genomic nucleotide (nt) and amino acid (aa) sequences was found to produce better cut-offs on the pairwise distance histogram than the conventionally used p distance. Following previous nomenclature, two subgenotypic levels, the clade (cut-off at 1.0227 with nt sequences and 0.2087 with aa sequences) and the subtype (nt: 0.5586, aa: 0.1153), were defined, and with this definition two clades and six subtypes were identified. Two human-derived recombinants were identified, each from a different clade. The second part contrasts the bioinformatic behaviour of HEV-C1. The central hypothesis was that human-derived HEV-C1 should demonstrate more codon usage bias (CUB) towards humans and/or positive selection than nonhuman- derived HEV-C1. Results were however conflicting: while many CUB indices in this study, including the guanine–cytosine bias, degree of segregation on the plot of the first two principal axes of the correspondence analysis of relative synonymous codon usage, and estimated number of codons, consistently showed that the by-host difference was not statistically significant and that the by-clade difference was; occasionally, there were evidence of human CUB in the overall HEV-C1 dataset with the codon adaptation index (p value of Homo sapiens versus Mus, Rattus spp. and Suncus murinus = 6.51–6.89E-18), selection relaxation in ORF2 (p = 5.48E-4) and intensification in ORF3 (p = 0.47) in the overall humanderived HEV-C1 dataset, and episodic positive selection in some human-derived rat HEV-C1 strains (pt 5 (GenBank accession number: MN450853.1) in ORF1 (p = 1.14E-06), KRS in ORF1 (p = 0.008), and HEV 17/1683 (MK050105.1) in ORF3 (p = 0.019)). Using a strict clock model, the time to most recent common ancestor (tMRCA) of clade I, the clade generally found to have more human CUB, was 393.933 years (95% confidence interval: 245.308–584.728 years), earlier than that of clade II (367.820 years, 95% CI: 224.955–537.235 years). An evolving host-adapting event specific to the Hong Kong HEV-C1 supercluster in clade II is unlikely. Rather, the phylogenetically earlier clade I is reporting more human cases recently, also contains a human-infecting recombinant, and is shown to be consistently more biased to human codon usage. The adaptation of HEV-C1 to humans therefore appears to be a pan-clade process, and more screening efforts tailored to HEV-C1 should be conducted to uncover more sequences for subgenotypic classification and analysis.