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. 2015 Mar;89(6):3076-92.
doi: 10.1128/JVI.02420-14. Epub 2014 Dec 31.

Discovery of a novel coronavirus, China Rattus coronavirus HKU24, from Norway rats supports the murine origin of Betacoronavirus 1 and has implications for the ancestor of Betacoronavirus lineage A

Susanna K P Lau  1 , Patrick C Y Woo  1 , Kenneth S M Li  2 , Alan K L Tsang  2 , Rachel Y Y Fan  2 , Hayes K H Luk  2 , Jian-Piao Cai  2 , Kwok-Hung Chan  2 , Bo-Jian Zheng  1 , Ming Wang  3 , Kwok-Yung Yuen  4
Affiliations

Discovery of a novel coronavirus, China Rattus coronavirus HKU24, from Norway rats supports the murine origin of Betacoronavirus 1 and has implications for the ancestor of Betacoronavirus lineage A

Susanna K P Lau et al. J Virol. 2015 Mar.

Abstract

We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (βCoVs) in conserved replicase domains support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble those of both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins but closer to murine coronavirus by G+C content, the presence of a single nonstructural protein (NS4), and an absent transcription regulatory sequence for the envelope (E) protein. Its N-terminal domain (NTD) demonstrated higher sequence identity to the bovine coronavirus (BCoV) NTD than to the mouse hepatitis virus (MHV) NTD, with 3 of 4 critical sugar-binding residues in BCoV and 2 of 14 contact residues at the MHV NTD/murine CEACAM1a interface being conserved. Molecular clock analysis dated the time of the most recent common ancestor of ChRCoV HKU24, Betacoronavirus 1, and rabbit coronavirus HKU14 to about the year 1400. Cross-reactivities between other lineage A and B βCoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide were demonstrated. Using the spike polypeptide-based Western blot assay, we showed that only Norway rats and two oriental house rats from Guangzhou, China, were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, possessed antibodies only against N protein and not against the spike polypeptide, suggesting infection by βCoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1, and rodents are likely an important reservoir for ancestors of lineage A βCoVs.

Importance: While bats and birds are hosts for ancestors of most coronaviruses (CoVs), lineage A βCoVs have never been found in these animals and the origin of Betacoronavirus lineage A remains obscure. We discovered a novel lineage A βCoV, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China with a high seroprevalence. The unique genome features and phylogenetic analysis supported the suggestion that ChRCoV HKU24 represents a novel CoV species, occupying a deep branch at the root of members of Betacoronavirus 1 and being distinct from murine coronavirus. Nevertheless, ChRCoV HKU24 possessed genome characteristics that resemble those of both Betacoronavirus 1 and murine coronavirus. Our data suggest that ChRCoV HKU24 represents the murine origin of Betacoronavirus 1, with interspecies transmission from rodents to other mammals having occurred centuries ago, before the emergence of human coronavirus (HCoV) OC43 in the late 1800s. Rodents are likely an important reservoir for ancestors of lineage A βCoVs.

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Figures

FIG 1
FIG 1
Comparison of genome organizations of ChRCoV HKU24, MHV, HCoV OC43, and HCoV HKU1. Papain-like proteases 1 and 2 (PL1pro and PL2pro, respectively) are represented by orange boxes. The residues at the cleavage site are indicated above or below the boundary of each nonstructural protein. The unique cleavage site in ChRCoV HKU24 is in bold.
FIG 2
FIG 2
Predicted model of ChRCoV HKU24 spike protein and NTD using the SWISS-MODEL tool. (A) Predicted domain structure of ChRCoV HKU24 spike protein. NTD, N-terminal domain; RBD, receptor-binding domain; HR, heptad repeat; TM, transmembrane anchor. The signal peptide corresponds to residues 1 to 15 and is cleaved during molecular maturation. (B) Sequence alignment of the ChRCoV HKU24 NTD with the BCoV, HCoV OC43, and MHV NTDs, performed using the PROMALS3D program. The three strains of ChRCoV HKU24 characterized in this study are in bold. β strands are shown as yellow arrows, and the alpha helix is shown as a red wavy line. Loops 10 and 11 are boxed. The 14 contact residues at the MHV NTD/mCEACAM1a interface are highlighted in blue, the four BCoV critical sugar-binding residues are highlighted in brown, and the BCoV noncritical sugar-binding residues are highlighted in yellow. The location of the residue substitution that might decrease the sugar-binding affinity of BCoV NTD is marked by an inverted triangle. Asterisks, positions that have fully conserved residues; colons, positions that have strongly conserved residues; periods, positions that have weakly conserved residues. (C) Predicted structure of the ChRCoV HKU24 NTD constructed through homology modeling from BCoV NTD (4h14) and close-up view of the pocket above the β-sandwich core. The global model quality estimation score of 0.83 and QMEAN4 Z-score of −1.82 indicate reliable overall model quality.
FIG 3
FIG 3
Phylogenetic analyses of the RdRp, S, N, and HE proteins of ChRCoV HKU24. The trees were constructed by the maximum likelihood method using the WAG+I+G substitution model and bootstrap values calculated from 100 trees. Bootstrap values below 70% are not shown. Nine hundred twenty-eight, 1,358, 443, and 425 aa positions in RdRp, S, N, and HE, respectively, were included in the analyses. The scale bar represents 0.3 substitution per site. The three strains of ChRCoV HKU24 characterized in this study are in bold. Definitions of the abbreviations are provided in footnote a of Table 2.
FIG 4
FIG 4
Estimation of tMRCA of the ChRCoV HKU24 strains, BCoV/HCoV OC43, and ChRCoV HKU24/members of Betacoronavirus 1/RbCoV HKU14 on the basis of the complete sequences of the RdRp and HE genes. The mean estimated dates (above the branch) and Bayesian posterior probabilities (below the branch) are labeled and are represented by gray squares. The taxa are labeled with their sampling dates.
FIG 5
FIG 5
Western blot analysis for antibodies against purified His6-tagged recombinant ChRCoV HKU24 N protein (∼50-kDa) (A) and spike polypeptide (∼50-kDa) (B) in rodent serum samples and serum samples from other animals or humans infected by different βCoVs, including HCoV OC43 (Betacoronavirus lineage A), RbCoV HKU14 (Betacoronavirus lineage A), and SARS-CoV (Betacoronavirus lineage B). Lanes: 1, negative control; 2, oriental house rat serum sample negative for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 3, Norway rat serum sample negative for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 4, oriental house rat serum sample positive for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 5, Norway rat serum sample positive for antibody against the ChRCoV HKU24 N protein and spike polypeptide; 6 and 7, serum samples from rabbits infected by RbCoV HKU14; 8 and 9, serum samples from patients with HCoV OC43 infection; 10 and 11, serum samples from patients with SARS-CoV infection; 12, positive control (anti-His antibody).
FIG 6
FIG 6
Evolution of CoVs from their ancestors in bat, bird, and rodent hosts to virus species that infect other animals. Dashed arrows, possible routes of transmission from bats or birds to rodents before establishment of Betacoronavirus lineage A.
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