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      Isolation and characterization of a highly pathogenic strain of Porcine enteric alphacoronavirus causing watery diarrhoea and high mortality in newborn piglets

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          Abstract

          Porcine enteric alphacoronavirus ( PEAV) was first discovered in China in February 2017, and the origin and virulence of this novel porcine coronavirus were not fully characterized. Here, we isolated a strain of PEAV, named GDS04 that is identified by immunofluorescence and typical crown‐shaped particles observed with electron microscopy. Genomic analysis reveals that PEAV GDS04 shares a close relationship with SADS‐CoV and Se ACoV. Furthermore, newborn piglets orally challenged with PEAV GDS04 developed typical clinical symptoms as watery diarrhoea in neonatal piglets. Viral RNA was detected in faeces and various tissues of the infected piglets. Moreover, macroscopic and microscopic lesions in whole intestinal tract were observed, and viral antigen could be detected in the small intestines by immunohistochemical staining and electron microscopy. Importantly, the mortality rate of inoculated‐newborn piglets was 100% and half of the cohabiting piglets died. Collectively, we demonstrate that PEAV is highly pathogenic in newborn piglets.

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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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            A new coronavirus-like particle associated with diarrhea in swine

            Summary Coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on 4 swine breeding farms. Diarrhea was reproduced in experimental pigs with one of the isolates, designated CV777, which was found to be distinct from the 2 known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus.
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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
                caoych@mail.sysu.edu.cn
                Journal
                Transbound Emerg Dis
                Transbound Emerg Dis
                10.1111/(ISSN)1865-1682
                TBED
                Transboundary and Emerging Diseases
                John Wiley and Sons Inc. (Hoboken )
                1865-1674
                1865-1682
                10 September 2018
                January 2019
                : 66
                : 1 ( doiID: 10.1111/tbed.2019.66.issue-1 )
                : 119-130
                Affiliations
                [ 1 ] State Key Laboratory of Biocontrol School of Life Science Sun Yat‐sen University Guangzhou China
                [ 2 ] Guangdong Wen's Group Academy Guangdong Wen’s Foodstuffs Group Co., Ltd Xinxing China
                Author notes
                [*] [* ] Correspondence

                Yongchang Cao, School of Life Science, Higher Education Mega Center, Sun Yat‐sen University, Guangzhou 510006, China.

                Email: caoych@ 123456mail.sysu.edu.cn

                Author information
                http://orcid.org/0000-0002-5697-5734
                http://orcid.org/0000-0002-0311-5474
                Article
                TBED12992
                10.1111/tbed.12992
                7168553
                30103259
                43d2c96f-6471-4cd0-b2c7-80d741079427
                © 2018 Blackwell Verlag GmbH

                This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

                History
                : 09 April 2018
                : 03 August 2018
                : 06 August 2018
                Page count
                Figures: 8, Tables: 0, Pages: 12, Words: 7700
                Funding
                Funded by: National Key Research and Development Program
                Award ID: 2016YFD0500101
                Funded by: National Natural Science Foundation , open-funder-registry 10.13039/501100001809;
                Award ID: 31741118
                Funded by: Guangdong Natural Science Foundation , open-funder-registry 10.13039/501100003453;
                Award ID: 2018B030314003
                Categories
                Original Article
                Original Articles
                Custom metadata
                2.0
                January 2019
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.0 mode:remove_FC converted:15.04.2020

                Infectious disease & Microbiology
                newborn piglets,pathogenicity,porcine enteric alphacoronavirus (peav)

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