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. 2017 Dec 19;14(1):239.
doi: 10.1186/s12985-017-0907-1.

Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy

Ana Moreno  1 , Davide Lelli  2 , Luca de Sabato  3   4 , Guendalina Zaccaria  3 , Arianna Boni  3 , Enrica Sozzi  2 , Alice Prosperi  2 , Antonio Lavazza  2 , Eleonora Cella  5 , Maria Rita Castrucci  3 , Massimo Ciccozzi  5 , Gabriele Vaccari  3
Affiliations

Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy

Ana Moreno et al. Virol J. .

Erratum in

Abstract

Background: Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii.

Methods: Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB).

Results: Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs.

Conclusions: This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity.

Keywords: Bats; Full genome sequencing; Italy; MERS-like Beta-CoV viruses; Phylogenetic and molecular analyses.

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Figures

Fig. 1
Fig. 1
Sequence identity between IT bat CoVs and other prototype clade c betacoronaviruses and MERS related strains. Similarity plots were generated using SSE version 1.2 using a sliding window of 600 and a step size of 100 nucleotides
Fig. 2
Fig. 2
Maximum phylogenetic tree based on alpha and beta CoV full genomes. Ultrafast bootstrap approximation approach was performed to compute the support of phylogenetic groups. Best-fit model according to BIC was GTR + G4. Bat sequences close related to MERS-CoVs are reported in colors: light blue for IT bat CoVs, pink for NeoCoV from South Africa, green for PREDICT from Uganda and red for SC2013 from China. Bat-CoV/H.savii/Italy/206645–40/2011 and Bat-CoV/P.khulii/Italy/206645–63/2011 sequences can be retrieved under accession numbers MG596802 and MG596803 respectively
Fig. 3
Fig. 3
Maximum phylogenetic tree based on deduced amino acid sequences of S1 protein of beta CoVs. Best-fit model according to BIC was WAG + F + I + G4. Clades 2a, 2b, 2c and 2d corresponding to the sequences of human CoVs OC43, SARS-like, MERS-like and HKU9 CoV respectively are identified
Fig. 4
Fig. 4
Comparison of secondary and tertiary structures of MERS-like CoV RBD. a Predicted 3D MMS of the core and external subdomains of HKU4 RBD and human DPP4 (right and left lateral view). The two deletions observed in IT Bat CoVs RBD are evidenced in green. b Structure –based sequence alignment . The secondary structure elements are defined based on an ESPript algorithm and are labeled as in a previous report on the MERS RBD structure [24]. Spiral lines indicate helices, while arrows represent β strands. The external subdomain is highlighted by enclosure in a blue box. The two deletions found in IT Bat CoVs and HKU5 RBD are marked with blue lines. The Arabic numerals 1–4 indicate cysteine residues that pair to form disulfide bonds
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