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. 2014 Jun;88(12):7070-82.
doi: 10.1128/JVI.00631-14. Epub 2014 Apr 9.

Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China

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Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China

Biao He et al. J Virol. 2014 Jun.

Abstract

Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats in China, Europe, and Africa, most have a genetic organization significantly distinct from human/civet SARS CoVs in the receptor-binding domain (RBD), which mediates receptor binding and determines the host spectrum, resulting in their failure to cause human infections and making them unlikely progenitors of human/civet SARS CoVs. Here, a viral metagenomic analysis of 268 bat rectal swabs collected from four counties in Yunnan Province has identified hundreds of sequences relating to alpha- and betacoronaviruses. Phylogenetic analysis based on a conserved region of the RNA-dependent RNA polymerase gene revealed that alphacoronaviruses had diversities with some obvious differences from those reported previously. Full genomic analysis of a new SARS-like CoV from Baoshan (LYRa11) showed that it was 29,805 nucleotides (nt) in length with 13 open reading frames (ORFs), sharing 91% nucleotide identity with human/civet SARS CoVs and the most recently reported SARS-like CoV Rs3367, while sharing 89% with other bat SARS-like CoVs. Notably, it showed the highest sequence identity with the S gene of SARS CoVs and Rs3367, especially in the RBD region. Antigenic analysis showed that the S1 domain of LYRa11 could be efficiently recognized by SARS-convalescent human serum, indicating that LYRa11 is a novel virus antigenically close to SARS CoV. Recombination analyses indicate that LYRa11 is likely a recombinant descended from parental lineages that had evolved into a number of bat SARS-like CoVs.

Importance: Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been discovered in bats worldwide, there are significant different genic structures, particularly in the S1 domain, which are responsible for host tropism determination, between bat SARS-like CoVs and human SARS CoVs, indicating that most reported bat SARS-like CoVs are not the progenitors of human SARS CoV. We have identified diverse alphacoronaviruses and a close relative (LYRa11) to SARS CoV in bats collected in Yunnan, China. Further analysis showed that alpha- and betacoronaviruses have different circulation and transmission dynamics in bat populations. Notably, full genomic sequencing and antigenic study demonstrated that LYRa11 is phylogenetically and antigenically closely related to SARS CoV. Recombination analyses indicate that LYRa11 is a recombinant from certain bat SARS-like CoVs circulating in Yunnan Province.

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Figures

FIG 1
FIG 1
(A) Geo-distribution of bat CoVs in China (gray provinces); (B) locations of bat alphacoronaviruses (open circles) and SARS-like CoVs identified in our study (solid circle) and in Ge et al.'s study (open triangle) (29). α, alphacoronavirus; β, betacoronavirus; S, SARS-like CoV.
FIG 2
FIG 2
Taxonomic summary of viral reads with BLASTn (E < 10−5) results exhibited in MEGAN 4. The number of reads in each taxonomic level is shown after the level name.
FIG 3
FIG 3
Phylogenetic analysis of RdRp amplicons obtained in this study and representatives of species in genera Alphacoronavirus and Betacoronavirus based on the maximum likelihood method. All sequences were classified into two groups: group Alphacoronavirus comprising 17 clades, and group Betacoronavirus comprising 10 clades. Clades containing approved species are in italics; clades containing unapproved novel species are marked with an asterisk. All amplicons in this study are marked as filled triangles, with previously reported bat CoVs as open triangles. Middle letters identify the viral host: H, human; C, civet; B, bat; Bo, bovine; M, murine; Ca, canine; F, feline.
FIG 4
FIG 4
Characterization of S1 domains of SARS and SARS-like CoVs. (A) Phylogenetic analysis of entire S1 amino acid sequences based on the maximum likelihood method; (B) phylogenetic analysis of RBD amino acid sequences based on the maximum likelihood method; (C) sequence comparison of entire RBMs of SARS CoVs, LYRa11 (boxed), and other closely related bat SARS-like CoVs. The sequences of SARS-like CoVs in this study are marked as filled triangles, with other bat SARS-like CoVs as open triangles. Middle letters: H, human SARS CoV; C, civet SARS CoV; B, bat SARS-like CoV. Amino acid (aa) positions refer to SARS CoV Tor2 (AY274119). Critical residues that play key roles in receptor binding are indicated with asterisks.
FIG 5
FIG 5
(A) Expression of EGFP-S1 fusion proteins in BHK-21 cells; (B) Western blot of expressed EGFP-S1 fusion proteins using rabbit anti-EGFP antibody (left) and SARS-convalescent human serum (right). The molecular masses are given on the right. BJ, LY, Rp3, and E, respectively, represent EGFP-S1 proteins of SARS CoV BJ01, bat SARS-like CoVs LYRa11 and Rp3, and EGFP control.
FIG 6
FIG 6
Recombination analysis of LYRa11 and other SARS-like CoVs. Similarity plots (A) and bootscan analyses (B) were conducted with LYRa11 as the query and bat SARS-like CoVs, including Rs3367, Yunnan2011, and Rf1, as potential parental sequences. (C) A gene map of LYRa11 is used to position breakpoints. Four breakpoints at nt 20968, 23443, 24643, and 26143 in the LYRa11 genome were detected, generating three recombinant fragments, 1, 2, and 3. Phylogenetic trees were constructed based on the three fragments (D to F, corresponding to fragments 1 to 3) by the maximum likelihood method. LYRa11 (bold italic), Rs3367, Yunnan2011, and Rf1 used in SimPlot are shaded. Leading capitals: H, human SARS CoV; C, civet SARS CoV; B, bat SARS-like CoV.
FIG 7
FIG 7
CoV-like particle considered to be LYRa11.

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