Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia

Abstract

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5-100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.

Fig. 1

Locations of sampling shown on a map of Slovenia. Each number (1–27) represents one location where bat samples were collected. Coronavirus RT-PCR-positive samples were found at the following locations: Spodnji Log in commune Kočevje (14), Livold in commune Kočevje (15), Tinje in commune Slovenska Bistrica (17), Knežja Njiva in commune Loška dolina (22) and Mali Otok in commune Postojna (23). Other sampling locations are Vnanje Gorice (1), Verd (2), Goče (3), Krašnja (4), Volovščica (5), Trebnje (6), Dolnji Suhor (7), Kobiljača (8), Završe (9), Kobilje (10), Mali Obrh (11), Močnik (12), Račna (13), Škocjan (16), Puščava (18), Dobrova (19), Selca (20), Snežnik (21), Velike Lašče (24), Iška vas (25), Cerkno (26) and Rižana (27)

Fig. 2

Phylogenetic tree based on nucleotide sequences of a partial pol gene sequence with representative isolates from bats, other mammals and birds (Table 2). Analyses were conducted using the PHYLIP program with partial sequences (405 nt) of each isolate taken from GenBank (corresponding to nt 15,234–15,638 of the genome of bat strain Rp3/2004 (DQ071615), using the neighbor-joining algorithm and a bootstrap test of phylogeny. Boostrap values were obtained from 1,000 datasets, and only nodes with bootstrap values higher than 70% are presented. The new group of 14 coronaviruses from this study was found to belong to Group 2 (bootstrap value 100%)

Fig. 3

EM micrograph of a coronavirus particle detected in a bat stool suspension