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      Epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of HCoV-OC43 during 2010-2015 in Guangzhou

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          Abstract

          Human coronavirus (HCoV) is one of the most common causes of respiratory tract infection throughout the world. To investigate the epidemiological and genetic variation of HCoV in Guangzhou, south China, we collected totally 13048 throat and nasal swab specimens from adults and children with fever and acute upper respiratory infection symptoms in Gunazhou, south China between July 2010 and June 2015, and the epidemiological features of HCoV and its species were studied. Specimens were screened for HCoV by real-time RT-PCR, and 7 other common respiratory viruses were tested simultaneously by PCR or real-time PCR. HCoV was detected in 294 cases (2.25%) of the 13048 samples, with most of them inpatients (251 cases, 85.4% of HCoV positive cases) and young children not in nursery (53.06%, 156 out of 294 HCoV positive cases). Four HCoVs, as OC43, 229E, NL63 and HKU1 were detected prevalent during 2010–2015 in Guangzhou, and among the HCoV positive cases, 60.20% were OC43, 16.67% were 229E, 14.97% were NL63 and 7.82% were HKU1. The month distribution showed that totally HCoV was prevalent in winter, but differences existed in different species. The 5 year distribution of HCoV showed a peak-valley distribution trend, with the detection rate higher in 2011 and 2013 whereas lower in 2010, 2012 and 2014. The age distribution revealed that children (especially those <3 years old) and old people (>50 years) were both high risk groups to be infected by HCoV. Of the 294 HCoV positive patients, 34.69% (101 cases) were co-infected by other common respiratory viruses, and influenza virus was the most common co-infecting virus (30/101, 29.70%). Fifteen HCoV-OC43 positive samples of 2013–2014 were selected for S gene sequencing and phylogenetic analysis, and the results showed that the 15 strains could be divided into 2 clusters in the phylogenetic tree, 12 strains of which formed a separate cluster that was closer to genotype G found in Malaysia. It was revealed for the first time that genotype B and genotype G of HCoV-OC43 co-circulated and the newly defined genotype G was epidemic as a dominant genotype during 2013–2014 in Guanzhou, south 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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            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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              Rapid and quantitative detection of human adenovirus DNA by real-time PCR.

              Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was
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                Author and article information

                Contributors
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Data curation
                Role: Data curation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Writing – review & editing
                Role: Funding acquisitionRole: Supervision
                Role: Funding acquisitionRole: Project administrationRole: Supervision
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 January 2018
                2018
                : 13
                : 1
                : e0191789
                Affiliations
                [1 ] Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong Province, China
                [2 ] Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-Sen University, Guangzhou, Guangdong Province, China
                [3 ] Clinical Laboratory and Institute of Medical Genetics, Women and Children's Healthcare Hospital of Zhuhai City, Zhuhai, Guangdong Province, China
                [4 ] Sun Yat-sen University—University of Hong Kong Joint Laboratory of Infectious Disease Surveillance, Sun Yat-sen University, Guangzhou, Guangdong Province, China
                [5 ] Medical ICU, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China
                [6 ] Department of Microbiology, University of Hong Kong, Hong Kong SAR, China
                Deutsches Primatenzentrum GmbH - Leibniz-Institut fur Primatenforschung, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-1506-8801
                Article
                PONE-D-17-40448
                10.1371/journal.pone.0191789
                5788356
                29377913
                e649de7c-77c4-4ea5-9590-96af95fd359a
                © 2018 Zhang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 15 November 2017
                : 11 January 2018
                Page count
                Figures: 6, Tables: 5, Pages: 20
                Funding
                This research was supported by National Major Projects of Major infectious Disease Control and Prevention, the Ministry of Science and Technology of the People’s Republic of China (grant number 2009ZX10004-213, 2012ZX10004213-001 and 2017ZX10103011). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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