Shared Common Ancestry of Rodent Alphacoronaviruses Sampled Globally

Abstract

The recent discovery of novel alphacoronaviruses (alpha-CoVs) in European and Asian rodents revealed that rodent coronaviruses (CoVs) sampled worldwide formed a discrete phylogenetic group within this genus. To determine the evolutionary history of rodent CoVs in more detail, particularly the relative frequencies of virus-host co-divergence and cross-species transmission, we recovered longer fragments of CoV genomes from previously discovered European rodent alpha-CoVs using a combination of PCR and high-throughput sequencing. Accordingly, the full genome sequence was retrieved from the UK rat coronavirus, along with partial genome sequences from the UK field vole and Poland-resident bank vole CoVs, and a short conserved ORF1b fragment from the French rabbit CoV. Genome and phylogenetic analysis showed that despite their diverse geographic origins, all rodent alpha-CoVs formed a single monophyletic group and shared similar features, such as the same gene constellations, a recombinant beta-CoV spike gene, and similar core transcriptional regulatory sequences (TRS). These data suggest that all rodent alpha CoVs sampled so far originate from a single common ancestor, and that there has likely been a long-term association between alpha CoVs and rodents. Despite this likely antiquity, the phylogenetic pattern of the alpha-CoVs was also suggestive of relatively frequent host-jumping among the different rodent species.

Keywords: alphacoronavirus; ancestry; coronavirus; evolution; recombination; rodents.

Figure 1

Genome organisation of rodent alphacoronaviruses. Schematic representation of the UKRn3 genome organisation along with those of other rodent alphacoronaviruses (α-CoVs): Lucheng Rn CoV (LRNV), AcCoV-JC34, RtRl-CoV, RtMruf-CoV, and the related Bat-HKU2 and Swine acute diarrhoea syndrome coronaviruses (CoV) (SADS-CoV). Its genome organisation was similar to those of members of the genus Alphacoronavirus, with the characteristic 5′- ORF1ab - spike (S) - envelope (E) – membrane (M) - nucleocapsid (N) - 3′ gene order. The red colour represents genes that belong to the genus Alphacoronavirus, whereas light blue denotes genes that belong to the genus Betacoronavirus, indicative of recombination. The Human 229E genome represents a typical alpha-CoV genome organisation.

Figure 2

Phylogenetic relationships of the rodent alphacoronaviruses based on the ORF1b gene. Maximum likelihood phylogenetic analysis of coronavirus partial 576 nt ORF1b gene sequences, revealing that the rodent alphaviruses formed a single clade with the novel rabbit L232 CoV within the genus alphacoronavirus. The novel coronavirus sequence obtained from a French rabbit L232 CoV was analysed alongside reference sequences representing three different coronavirus genera (Alphacoronavirus, Betacoronavirus, and Gammacoronavirus as an outgroup). Reference sequences are indicated by their GenBank accession numbers. Branch lengths are drawn to a scale of nucleotide substitutions per site. Numbers above individual branches indicate the percentage that that branch was found in 1000 bootstrap replicates; only values >80% are shown. Alpha-CoVs are highlighted in brown, betacoronaviruses in blue, gammacoronaviruses in green, and the rodent coronaviruses in light red.

Figure 3

Phylogenetic relationships of the rodent alphacoronaviruses based on the N gene. Maximum likelihood phylogenetic analysis of coronavirus partial N gene sequences corresponding to positions 10–1173 of the Lucheng Rn CoV (KF294380). Novel coronavirus sequences obtained from PL-resident Myodes glareolus (PLMg1) and the UK Microtus agrestis (UKMa1) and the UK Rattus norvegicus (UKRn3) were analysed alongside reference sequences representing three different coronavirus genera (Alphacoronavirus, Betacoronavirus, and Gammacoronavirus as an outgroup). Reference sequences are indicated by their GenBank accession numbers. Branch lengths are drawn to a scale of nucleotide substitutions per site. Numbers above individual branches indicate the percentage that that branch was found in 1000 bootstrap replicates; only values >80% are shown. Alpha-CoVs are highlighted in brown, betacoronaviruses in blue, gammacoronaviruses in green, and the rodent coronaviruses in light red.

Figure 4

Phylogenetic relationships of the rodent alphacoronaviruses based on the S gene. Maximum likelihood phylogenetic analysis of coronavirus partial S gene sequences corresponding to positions 1831–3228 of the Lucheng Rn CoV (KF294380), indicating that the rodent alpha-CoVs formed a distinct clade within the genus Betacoronavirus. The novel coronavirus sequence obtained from a UK-resident Microtus agrestis was analysed alongside reference sequences representing three different coronavirus genera (Alphacoronavirus, Betacoronavirus, and Gammacoronavirus as an outgroup). Reference sequences are indicated by their GenBank accession numbers. Branch lengths are drawn to a scale of nucleotide substitutions per site. Numbers above individual branches indicate the percentage that the branch was found in 1000 bootstrap replicates; only values >80% are shown. Alpha-CoVs are highlighted in brown, betacoronaviruses in blue, gammacoronaviruses in green, and the rodent coronaviruses in light red.

Figure 5

(A) Tanglegram of rodent cytochrome b and CoV ORF1b evolution. Maximum likelihood phylogenetic analysis of full-length rodent cytochrome b nucleotide sequences compared to rodent alpha-CoV ORF1b phylogeny. Cytochrome b sequences were obtained from GenBank for the rodent species Microtus, Myodes, Rattus, and Apodemus, and the rabbit Oryctolagus cuniculus, which was used as an outgroup. Branch lengths are drawn to scale: the bar indicates 0.1 nucleotide substitutions per site. Numbers above individual branches indicate the percentage that that branch was found in 1000 bootstrap replicates; only percentages >80 are shown. Blue indicates the Myodes spp., cyan the Microtus spp., purple the Apodemus spp., red Rattus spp., and black indicates Oryctolagus cuniculus. (B) Reconciliation of the sampled rodent alpha-CoV phylogeny with that of their mammalian hosts, utilizing the co-phylogenetic method implemented in the Jane package [16]. This figure shows the maximum possible co-divergence, duplication, and host switching events.