Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences

Abstract

Bats may host emerging viruses, including coronaviruses (CoV). We conducted an evaluation of CoV in rhinolophid and vespertilionid bat species common in Europe. Rhinolophids carried severe acute respiratory syndrome (SARS)-related CoV at high frequencies and concentrations (26% of animals are positive; up to 2.4×10(8) copies per gram of feces), as well as two Alphacoronavirus clades, one novel and one related to the HKU2 clade. All three clades present in Miniopterus bats in China (HKU7, HKU8, and 1A related) were also present in European Miniopterus bats. An additional novel Alphacoronavirus clade (bat CoV [BtCoV]/BNM98-30) was detected in Nyctalus leisleri. A CoV grouping criterion was developed by comparing amino acid identities across an 816-bp fragment of the RNA-dependent RNA polymerases (RdRp) of all accepted mammalian CoV species (RdRp-based grouping units [RGU]). Criteria for defining separate RGU in mammalian CoV were a >4.8% amino acid distance for alphacoronaviruses and a >6.3% distance for betacoronaviruses. All the above-mentioned novel clades represented independent RGU. Strict associations between CoV RGU and host bat genera were confirmed for six independent RGU represented simultaneously in China and Europe. A SARS-related virus (BtCoV/BM48-31/Bulgaria/2008) from a Rhinolophus blasii (Rhi bla) bat was fully sequenced. It is predicted that proteins 3b and 6 were highly divergent from those proteins in all known SARS-related CoV. Open reading frame 8 (ORF8) was surprisingly absent. Surface expression of spike and staining with sera of SARS survivors suggested low antigenic overlap with SARS CoV. However, the receptor binding domain of SARS CoV showed higher similarity with that of BtCoV/BM48-31/Bulgaria/2008 than with that of any Chinese bat-borne CoV. Critical spike domains 472 and 487 were identical and similar, respectively. This study underlines the importance of assessments of the zoonotic potential of widely distributed bat-borne CoV.

FIG. 1.

Distribution of European rhinolophid bats. For each of the five rhinolophid bat species occurring in Europe, the area of distribution is depicted in separate colors (the underlying map of Europe was retrieved from http://visibleearth.nasa.gov). The map in the bottom left corner contains a white frame showing the position of the map in the bottom right corner, where the study region within Bulgaria is marked by a red outline. Plots are by A. Seebens and were adapted from reference , with permission. FYROM, former Yugoslavian Republic of Macedonia.

FIG. 2.

RdRp-based phylogeny including novel bat coronaviruses. (A) Bayesian phylogeny of CoV on an 816-bp gap-free alignment of a fragment of the RNA-dependent RNA-polymerase (RdRp) gene corresponding to nucleotides 14781 to 15596 in SARS CoV strain Frankfurt 1 (GenBank accession no. AY291315). Analysis was done with MrBayes v3.1 (53). For clarity of presentation, only posterior probability values above 0.65 are shown and values at crown positions were removed. Novel European bat coronaviruses from this study are shown in red type. Additional European and African bat coronaviruses described previously by our group are shown in blue type. Taxa are named according to the following pattern: identification code/strain or isolate/typical host/country/collection year. Min pus, Miniopterus pusillus; Min sch, Miniopterus schreibersii; Min tri, Miniopterus tristis; Min mag, Miniopterus magnater; Nyc lei, Nyctalus leisleri; Sco kuh, Scotophilus kuhlii; Sus scr, Sus scrofa; Myo ric, Myotis ricketti; Myo dau, Myotis daubentonii; Hom sap, Homo sapiens; Hip sp, Hipposideros sp.; Car per, Carollia perspicillata; Rhi sin, Rhinolophus sinicus; Rhi eur, Rhinolophus euryale; Can lup, Canis lupus familiaris; Fel sil, Felis silvestris; Rhi bla, Rhinolophus blasii; Rhi fer, Rhinolophus ferrumequinum; Rhi meh, Rhinolophus mehelyi; Rhi mac, Rhinolophus macrotis; Pag lar, Paguma larvata; Rhi pea, Rhinolophus pearsoni; Rou les, Rousettus leschenaulti; Pip abr, Pipistrellus abramus; Tyl pac, Tylonycteris pachypus; Mus mus, Mus musculus; Gir cam, Giraffa camelopardalis; Hip nig, Hippotragus niger; Bos pri, Bos primigenius; Gal gal, Gallus gallus; Mel gal, Meleagris gallopavo; Del leu, Delphinapterus leucas; Lon str, Lonchura striata; Pri ben, Prionailurus bengalensis; Tur hor, Turdus hortulorum; Pyc sin, Pycnonotus sinensis. The right-hand column shows a classification of clades into RdRp-based grouping units (RGU). Hosts and GenBank accession numbers of all viruses are listed in Tables S1 and S2 in the supplemental material. (B) Distance-based phylogeny (neighbor-joining algorithm in MEGA) of the HKU7- and HKU8-related RGU confirming the monophyly of European and Chinese representatives in both clades.

FIG. 3.

Coronavirus shedding and seasonality. (A) Numbers of genome RNA copies per gram of feces of bats are shown for Miniopterus alphacoronaviruses and Rhinolophus alpha- and betacoronaviruses. (B) Virus concentrations of a Rhinolophus SARS-like betacoronavirus and a Rhinolophus alphacoronavirus are shown separately for each sampling season. Boxes show medians (horizontal lines) and interquartile ranges (box lengths). Whiskers represent extensions of the 25th or 75th percentiles by 1.5 times the interquartile range. Datum points beyond the whisker range are considered outliers and extreme values and are marked as x's and inverted y's, respectively.

FIG. 4.

Distribution of pairwise divergence scores between coronaviruses. Jukes-Cantor corrected nucleotide sequence distances among members of the genus Alphacoronavirus (A) and Betacoronavirus (B); uncorrected amino acid percentage distances among alphacoronaviruses (C) and betacoronaviruses (D). The y axis indicates the number of pairwise sequence comparisons. Braces indicate pairwise distances within groups, within a genus, and between genera. Pairwise distances between coronaviruses were calculated with MEGA 1.0 (62). Seven betacoronaviruses from different groups were used for comparison with all alphacoronaviruses. Similarly, nine alphacoronaviruses were used for comparison with all betacoronaviruses. The distribution of pairwise distances was plotted with SYSTAT 11.

FIG. 5.

Comparison of the receptor-binding domains of SARS CoV spike proteins with homologous sequences in civet and bat CoV. SARS CoV spike protein residues 319 to 518 were aligned with homologous regions of closely related civet and bat CoV. The European SARS-related CoV BtCoV/BM48-31/Rhi bla/Bulgaria/2008 is highlighted in red. The alignment was done in MEGA and corrected manually, with preference for parsimonious nucleotide exchanges rather than gap extensions. (A) Sequences homologous to the receptor binding motif (RBM) of SARS CoV. Those amino acids interacting directly with the human ACE-2 molecule are identified by numbers. (B) Alignment of only those amino acids upstream and downstream of the SARS CoV RBM that vary across strains. The alignment in panel A corresponds to the column marked with X's. (C) Amino acid-based neighbor-joining phylogeny, using the p distance model in an alignment of sequences homologous to the SARS CoV RBD, aa 319 to 512.

FIG. 6.

Immunofluorescence staining of SARS CoV and SARS-related spike proteins. (A) The SARS CoV spike protein and SARS-related spike proteins from bats (strains BtCoV/Rp3/Rhi pea/China/2004 and BtCoV/BM48-31/Rhi bla/Bulgaria/2008) were expressed in BHK-21 cells. Cells were fixed and permeabilized, and immunofluorescence was performed by using a polyclonal rabbit serum raised against SARS CoV spike protein (CUHK-W1 strain) (top panels) or human patient sera (middle and bottom panels) (patient 1 from Germany [GER] and patient 2 from Hong Kong [HK]). After incubation with IgG-specific fluorescein isothiocyanate (FITC)-labeled secondary antibodies, fluorescence was visualized by fluorescence microscopy. (B) For detection of FLAG-tagged spike protein of BtCoV/BM48-31/Rhi bla/Bulgaria/2008, a monoclonal anti-FLAG antibody was used, followed by incubation with a secondary FITC-conjugated antibody.