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      Simultaneous detection of severe acute respiratory syndrome, Middle East respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR

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          Abstract

          Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50–100 copies/mL and 5–100 copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.

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          Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia

          The source of human infection with Middle East respiratory syndrome coronavirus remains unknown. Molecular investigation indicated that bats in Saudi Arabia are infected with several alphacoronaviruses and betacoronaviruses. Virus from 1 bat showed 100% nucleotide identity to virus from the human index case-patient. Bats might play a role in human infection.
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            Close Relative of Human Middle East Respiratory Syndrome Coronavirus in Bat, South Africa

            To the Editor: The severe acute respiratory syndrome (SARS) outbreak of 2002–03 and the subsequent implication of bats as reservoir hosts of the causative agent, a coronavirus (CoV), prompted numerous studies of bats and the viruses they harbor. A novel clade 2c betacoronavirus, termed Middle East respiratory syndrome (MERS)–CoV, was recently identified as the causative agent of a severe respiratory disease that is mainly affecting humans on the Arabian Peninsula ( 1 ). Extending on previous work ( 2 ), we described European Pipistrellus bat–derived CoVs that are closely related to MERS-CoV ( 3 ). We now report the identification of a South Africa bat derived CoV that has an even closer phylogenetic relationship with MERS-CoV. During 2011–2012, fecal pellets were collected from 62 bats representing 13 different species in the KwaZulu-Natal and Western Cape Provinces of South Africa and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA). Details about the bat sample are available in the Technical Appendix. RNA was extracted by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Screening for CoVs was done by nested reverse transcription PCR using broadly reactive oligonucleotide primers targeting a conserved region in the RNA-dependent RNA polymerase (RdRp) gene (online Technical Appendix). PCR results were positive for 5 (8%) of the 62 specimens. PCR amplicons for 4 positive specimens yielded alphacoronavirus sequences related to recently described bat alphacoronaviruses from South Africa ( 4 ). The other positive specimen, termed PML/2011, was from an adult female Neoromicia cf. zuluensis bat sampled in 2011; the specimen yielded a novel betacoronavirus (GenBank accession no. KC869678). Technical Appendix Figure 1 shows the distribution of this bat species. To obtain better phylogenetic resolution, we extended the 398-nt RdRp fragment generated by the screening PCR to 816 nt, as described ( 5 ). PML/2011 differed from MERS-CoV by only 1 aa exchange (0.3%) in the translated 816-nt RdRp gene fragment. Thus, PML/2011 was much more related to MERS-CoV than any other known virus. The amino acid sequence of the next closest known relatives of MERS-CoV, from European Pipistrellus bats ( 3 ), differed from MERS-CoV by 1.8%. The amino acid sequences of viruses from Nycteris bats in Ghana ( 3 ) and the 2c prototype bat CoVs, HKU4 and HKU5, from China ( 6 ) differed by 5.5%–7.7% from MERS-CoV. The smaller 152- to 396-nt RdRp fragments of 2c bat CoVs from a Hypsugo savii bat in Spain ( 7 ), bat guano in Thailand ( 8 ), and a Nyctinomops bat in Mexico ( 9 ) showed no or only partial overlap with the 816-nt fragment generated in this study; thus, a direct comparison could not be done. However, in their respective RdRp fragments, these CoVs yielded amino acid sequence distances of 3.5%–8.0% and were thus probably more distant from MERS-CoV than the virus described here. A Bayesian phylogenetic analysis of the 816-nt RdRp sequence confirmed the close relationship between PML/2011 and MERS-CoV (Figure). Their phylogenetic relatedness was as close as that of SARS-CoV and the most closely related bat coronavirus known, Rs672 from a Rhinolophus sinicus bat (Figure). Like PML/2011 and MERS-CoV, Rs672 and SARS-CoV showed only 1 aa exchange in the translated 816-nt RdRp fragment. To confirm this relatedness, we amplified and sequenced a short 269-nt sequence encompassing the 3′-terminus of the spike gene for PML/2011 (oligonucleotide primers available upon request from the authors). A partial spike gene–based phylogeny using this sequence yielded the same topology as that using the partial RdRp sequence (Technical Appendix Figure 2). Again, PML/2011 was most closely related to MERS-CoV, showing only a 10.9% aa sequence distance in this gene, which encodes the glycoprotein responsible for CoV attachment and cellular entry. This distance was less than the 13.3% aa sequence distance between MERS-CoV and the European Pipistrellus CoVs ( 3 ) and less than the 20.5%–27.3% aa sequence distance between MERS-CoV and HKU5 and between MERS-CoV and HKU4 ( 6 ) in the same sequence fragment. Figure Partial RNA-dependent RNA polymerase (RdRp) gene phylogeny, including the novel betacoronavirus from a Neoromicia zuluensis bat in South Africa (GenBank accession no. KC869678 for both partial RdRp and spike gene sequences). The Bayesian phylogeny was done on a translated 816-nt RdRp gene sequence fragment, as described ( 5 ). MrBayes V3.1 (http://mrbayes.sourceforge.net/) was used with a WAG substitution model assumption over 2,000,000 generations sampled every 100 steps, resulting in 20,000 trees, of which 25% were discarded as burn-in. A whale gammacoronavirus was used as an outgroup. The novel N. zuluensis bat virus is highlighted in gray. Values at deep nodes represent statistical support from posterior probabilities. Only values >0.9 are shown. Coronavirus clades are depicted to the right of taxa. Scale bar represents genetic distance. MERS-CoV, Middle East respiratory syndrome coronavirus; SARS, severe acute respiratory syndrome; Bt-CoV, bat coronavirus; HCoV, human coronavirus, MHV, mouse hepatitis virus; FCoV, feline coronavirus; TGEV, transmissible gastroenteritis coronavirus. Our results further support the hypothesis that, like human CoV-229E and SARS-CoV, ancestors of MERS-CoV might exist in Old World insectivorous bats belonging to the family Vespertilionidae, to which the genera Neoromicia and Pipistrellus belong ( 3 ). Knowledge of the close relatedness of PML/2011 and MERS-CoV, which contrasts with the more distant relatedness of CoVs in bats from the Americas and Asia, enables speculations of an African origin for bat reservoir hosts of MERS-CoV ancestors. This hypothesis is limited by a global sampling bias, the small sample size, and the single clade 2c betacoronavirus detection in this study. Still, a putative transfer of MERS-CoV ancestors from Africa to the Arabian Peninsula would parallel the transfer of other viruses (e.g., the exportation of Rift Valley fever virus from East Africa, which led to a severe outbreak in Saudi Arabia in 2000) ( 10 ). Studies of Vespertilionidae bats and potential intermediate hosts (e.g., carnivores and ungulates, such as camels) are urgently needed to elucidate the emergence of MERS-CoV. Such studies should focus on the Arabian Peninsula and Africa. Technical Appendix Description of bat sampling, screened bat species, distribution of Neoromicia zuluensis bats, and spike gene phylogeny of the 2c betacoronavirus clade.
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              Structural characterization of the fusion-active complex of severe acute respiratory syndrome (SARS) coronavirus.

              The causative agent of a recent outbreak of an atypical pneumonia, known as severe acute respiratory syndrome (SARS), has been identified as a coronavirus (CoV) not belonging to any of the previously identified groups. Fusion of coronaviruses with the host cell is mediated by the envelope spike protein. Two regions within the spike protein of SARS-CoV have been identified, showing a high degree of sequence conservation with the other CoV, which are characterized by the presence of heptad repeats (HR1 and HR2). By using synthetic and recombinant peptides corresponding to the HR1 and HR2 regions, we were able to characterize the fusion-active complex formed by this novel CoV by CD, native PAGE, proteolysis protection analysis, and size-exclusion chromatography. HR1 and HR2 of SARS-CoV associate into an antiparallel six-helix bundle, with structural features typical of the other known class I fusion proteins. We have also mapped the specific boundaries of the region, within the longer HR1 domain, making contact with the shorter HR2 domain. Notably, the inner HR1 coiled coil is a stable alpha-helical domain even in the absence of interaction with the HR2 region. Inhibitors binding to HR regions of fusion proteins have been shown to be efficacious against many viruses, notably HIV. Our results may help in the design of anti-SARS therapeutics.
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                Author and article information

                Contributors
                dgjeong@kribb.re.kr
                khk1329@kribb.re.kr
                Journal
                Arch Virol
                Arch. Virol
                Archives of Virology
                Springer Vienna (Vienna )
                0304-8608
                1432-8798
                20 February 2017
                2017
                : 162
                : 6
                : 1617-1623
                Affiliations
                [1 ]ISNI 0000 0004 0636 3099, GRID grid.249967.7, Infectious Disease Research Center, , Korea Research Institute of Bioscience and Biotechnology, ; Daejeon, Republic of Korea
                [2 ]ISNI 0000 0000 9611 0917, GRID grid.254229.a, College of Veterinary Medicine, , Chungbuk National University, ; Cheongju, 28644 Republic of Korea
                [3 ]ISNI 0000 0004 1791 8264, GRID grid.412786.e, Bio-Analytical Science Division, , Korea University of Science and Technology (UST), ; Daejeon, Republic of Korea
                [4 ]ISNI 0000 0001 0840 2678, GRID grid.222754.4, Department of Pharmacy, College of Pharmacy, , Korea University, ; Sejong, Republic of Korea
                Article
                3281
                10.1007/s00705-017-3281-9
                7086956
                28220326
                © Springer-Verlag Wien 2017

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100003715, Korea Research Institute of Bioscience and Biotechnology;
                Award ID: KGM4691511
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003621, Ministry of Science, ICT and Future Planning;
                Award ID: H-GUARD 2013M3A6B2078954
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003625, Ministry of Health and Welfare;
                Award ID: HI15C3036
                Award Recipient :
                Categories
                Original Article
                Custom metadata
                © Springer-Verlag Wien 2017

                Microbiology & Virology

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