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      Identification of Diverse Bat Alphacoronaviruses and Betacoronaviruses in China Provides New Insights Into the Evolution and Origin of Coronavirus-Related Diseases

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          Abstract

          Outbreaks of severe acute respiratory syndrome (SARS) in 2002, Middle East respiratory syndrome in 2012 and fatal swine acute diarrhea syndrome in 2017 caused serious infectious diseases in humans and in livestock, resulting in serious public health threats and huge economic losses. All such coronaviruses (CoVs) were confirmed to originate from bats. To continuously monitor the epidemic-related CoVs in bats, virome analysis was used to classify CoVs from 831 bats of 15 species in Yunnan, Guangxi, and Sichuan Provinces between August 2016 and May 2017. We identified 11 CoV strains from 22 individual samples of four bat species. Identification of four alpha-CoVs from Scotophilus kuhlii in Guangxi, which was closely related to a previously reported bat CoV and porcine epidemic diarrhea virus (PEDV), revealed a bat-swine lineage under the genus Alphacoronavirus. A recombinant CoV showed that the PEDV probably originated from the CoV of S. kuhlii. Another alpha-CoV, α-YN2018, from Rhinolophus affinis in Yunnan, suggested that this alpha-CoV lineage had multiple host origins, and α-YN2018 had recombined with CoVs of other bat species over time. We identified five SARS-related CoVs (SARSr-CoVs) in Rhinolophus bats from Sichuan and Yunnan and confirmed that angiotensin-converting enzyme 2 usable SARSr-CoVs were continuously circulating in Rhinolophus spp. in Yunnan. The other beta-CoV, strain β-GX2018, found in Cynopterus sphinx of Guangxi, represented an independently evolved lineage different from known CoVs of Rousettus and Eonycteris bats. The identification of diverse CoVs here provides new genetic data for understanding the distribution and source of pathogenic CoVs in China.

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          Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats.

          Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and analysis of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were observed between human and civet SARS-CoV genomes. In addition, the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals.
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            Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus.

            Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination.
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              Coronavirus Genomics and Bioinformatics Analysis

              The drastic increase in the number of coronaviruses discovered and coronavirus genomes being sequenced have given us an unprecedented opportunity to perform genomics and bioinformatics analysis on this family of viruses. Coronaviruses possess the largest genomes (26.4 to 31.7 kb) among all known RNA viruses, with G + C contents varying from 32% to 43%. Variable numbers of small ORFs are present between the various conserved genes (ORF1ab, spike, envelope, membrane and nucleocapsid) and downstream to nucleocapsid gene in different coronavirus lineages. Phylogenetically, three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with Betacoronavirus consisting of subgroups A, B, C and D, exist. A fourth genus, Deltacoronavirus, which includes bulbul coronavirus HKU11, thrush coronavirus HKU12 and munia coronavirus HKU13, is emerging. Molecular clock analysis using various gene loci revealed that the time of most recent common ancestor of human/civet SARS related coronavirus to be 1999–2002, with estimated substitution rate of 4×10−4 to 2×10−2 substitutions per site per year. Recombination in coronaviruses was most notable between different strains of murine hepatitis virus (MHV), between different strains of infectious bronchitis virus, between MHV and bovine coronavirus, between feline coronavirus (FCoV) type I and canine coronavirus generating FCoV type II, and between the three genotypes of human coronavirus HKU1 (HCoV-HKU1). Codon usage bias in coronaviruses were observed, with HCoV-HKU1 showing the most extreme bias, and cytosine deamination and selection of CpG suppressed clones are the two major independent biological forces that shape such codon usage bias in coronaviruses.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                14 August 2019
                2019
                : 10
                : 1900
                Affiliations
                [1] 1NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing, China
                [2] 2Key Laboratory of Zoonosis of Liaoning Province, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University , Shenyang, China
                [3] 3EcoHealth Alliance , New York, NY, United States
                Author notes

                Edited by: Ashley C. Banyard, Animal & Plant Health Agency, United Kingdom

                Reviewed by: Frank van der Meer, University of Calgary, Canada; Nicholas Johnson, Animal & Plant Health Agency, United Kingdom

                *Correspondence: Zhiqiang Wu, wuzq2009@ 123456ipbcams.ac.cn

                These authors have contributed equally to this work

                This article was submitted to Virology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2019.01900
                6702311
                31474969
                30959df7-78fb-4b61-8a4b-129292d6d044
                Copyright © 2019 Han, Du, Su, Zhang, Zhu, Zhang, Wu and Jin.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 16 February 2019
                : 31 July 2019
                Page count
                Figures: 5, Tables: 3, Equations: 0, References: 49, Pages: 12, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                bats,coronaviruses,porcine epidemic diarrhea virus,severe acute respiratory syndrome coronavirus,ecological and genetic diversity

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