Complete genome analysis of a SARS-like bat coronavirus identified in the Republic of Korea.

Complete genome analysis of a SARS-like bat coronavirus identified in the Republic of Korea.

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Abstract

Bats have been widely known as natural reservoir hosts of zoonotic diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) caused by coronaviruses (CoVs). In the present study, we investigated the whole genomic sequence of a SARS-like bat CoV (16BO133) and found it to be 29,075 nt in length with a 40.9% G+C content. Phylogenetic analysis using amino acid sequences of the ORF 1ab and the spike gene showed that the bat coronavirus strain 16BO133 was grouped with the Beta-CoV lineage B and was closely related to the JTMC15 strain isolated from Rhinolophus ferrumequinum in China. However, 16BO133 was distinctly located in the phylogenetic topology of the human SARS CoV strain (Tor2). Interestingly, 16BO133 showed complete elimination of ORF8 regions induced by a frame shift of the stop codon in ORF7b. The lowest amino acid identity of 16BO133 was identified at the spike region among various ORFs. The spike region of 16BO133 showed 84.7% and 75.2% amino acid identity with Rf1 (SARS-like bat CoV) and Tor2 (human SARS CoV), respectively. In addition, the S gene of 16BO133 was found to contain the amino acid substitution of two critical residues (N479S and T487 V) associated with human infection. In conclusion, we firstly carried out whole genome characterization of the SARS-like bat coronavirus discovered in the Republic of Korea; however, it presumably has no human infectivity. However, continuous surveillance and genomic characterization of coronaviruses from bats are necessary due to potential risks of human infection induced by genetic mutation.

Electronic supplementary material

The online version of this article (10.1007/s11262-019-01668-w) contains supplementary material, which is available to authorized users.

Keywords: Bat, SARS-like coronavirus, Zoonotic disease, Frame shift, Whole genome

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Introduction

Coronaviruses (CoVs) are enveloped viruses containing a single-stranded, positive-sense RNA genome of approximately 27–32 kb [1]. Currently, CoVs are grouped under four distinct genera: AlphacoronavirusBetacoronavirusGammacoronavirus, and Deltacoronavirus [2, 3].

Bat species have been recognized as major reservoirs of several emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) [4–6]. SARS is caused by a member of the Betacoronavirus genus and is the first global pandemic disease that has emerged in the Guangdong Province of China in 2002. SARS has spread to 25 countries across five continents, infecting 8096 people worldwide with a 9.5% (774/8096) fatality [7–9].

The four structural proteins (S, E, M, and N) are essential for viral entry and assembly. The S gene is the most important structural protein. The receptor-binding motif (RBM) within the receptor-binding domain (RBD) located in the S gene determines host tropism by binding angiotensin-converting enzyme 2 (ACE2) receptor [10, 11]. The RBD has two critical residues (N479 and T487) that play key roles in ACE2 receptor recognition and binding associated with human transmission [7, 12].

Novel coronaviruses are continuously being discovered in bat species around the world, especially in China [7, 13]. Due to relatively close geographic locations of bat species between China and the Republic of Korea, the surveillance of CoV prevalence and the analysis of their genetic information may be crucial for preventing a future outbreak [14]. However, there have been few investigations into SARS-related bat Beta-CoV prevalence [4]. In addition, whole genome analysis of SARS-related bat Beta-CoV has not yet been carried out in the Republic of Korea.

Together with the fact that bats are reservoirs of CoVs, genetic information about these CoVs may provide valuable information regarding the possible risk of these viruses infecting humans. In the present study, the complete genome sequence of SARS-related Beta-CoV (16BO133) isolated from Rhinolophus ferrumequinum was first characterized. The genome of 16BO133 was then compared with that of reference CoVs to demonstrate genetic diversity and a potential genetic feature associated with host tropism.

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Results and discussion

An oral swab was collected from bats living in their natural habitat in 2016. Bats were captured using a net for collection of oral swabs and were released immediately after sampling. Oral swab samples were kept in a viral transport medium at 4 °C. The oral swab sample was suspended in 1% antibiotic–antimycotic solution (Corning, USA) diluted in phosphate-buffered saline (PBS), and clarified by centrifugation at 3500×g for 10 min. RNA from the 200 μL sample was extracted with the QIAamp® Viral RNA mini kit (Qiagen, Germany) and eluted in 60 μL RNase-free water. cDNA was synthesized using a PrimeScript First Strand cDNA Synthesis kit (Takara, Japan) according to the manufacturer’s instructions. Bat-CoV screening was performed by a pancoronavirus PCR method based on primers as follows: (Corona forward, 5′-GGTTGGGACTATCCTAAGTGTGA-3′ and Corona reverse, 5′-CCATCATCAGATAG AATCATCATA-3′). The pancoronavirus primers were used to amplify and sequence a 440-bp segment of the highly conserved RNA-dependent RNA polymerase (RdRp) gene. Fifty-nine pairs of primers were synthesized by the Genotech corporation (Daejeon, Korea) and PCR was performed using an ABI 9800 GeneAmp system (Applied Biosystems, Foster City, CA, USA). The products were purified using a QIAquick gel extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. The purified PCR products were sequenced using the BigDye® Terminator Cycle Sequencing kit version 1.1 (Applied Biosystems, Foster City, CA, USA) and an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Whole genome sequences were submitted to GenBank (accession number KY938558). The nucleotide and amino acid sequences were aligned and compared to CoV sequences available from the GenBank database using ClustalW software implemented in BioEdit version 7.0.9.0. The phylogenetic trees were drawn using the neighboring joining method using the maximum composite likelihood model with MEGA 7 software. The bootstrap values were calculated with 1000 replicates.

The amino acid sequences of ORF 1ab and spike gene were analyzed for phylogenetic characterization. 16BO133 was grouped with the SARS-related Beta-CoV lineage B, not only due to sequence similarity with ORF 1ab but also with the spike gene (Fig. 1). The RF 1ab and spike amino acids were closely related to JTMC15. However, 16BO133 was distinctly located in the phylogenetic topology of the human SARS CoV strain (Tor2, Urbani, Frankfurt1, and ShanghaiQXC1)

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Jawahar Raina

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