Background: Coronaviruses (CoVs) are the largest enveloped single-strand RNA viruses and belong to the Coronaviridae family in the Nidovirales order and are divided into four genera named Alphacoronaviruses, Betacoronaviruses (divided into the four clades A to D), Deltacoronaviruses and Gammacoronaviruses, based on the phylogenetic distance of highly conserved domains. Until now six human coronaviruses have been identified and HCoV-OC43 is the most common human coronavirus and has high genetically diversity. Five genotypes of HCoV-OC43 (A to E) have been identified and genotype D has been dominant from 2004 to 2012. Until now only 90 complete genome sequences of HCoV-OC43 were available in GenBank. In this study, we investigate of the presence of different genotypes among HCoVs strains and comparison their potential similarity.
Methods: Patients hospitalized with acute respiratory tract infections were included in the study. All nasopharyngeal swabs were sent to the laboratory of the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, for the routine detection of respiratory viruses, including respiratory syncytial virus (RSV), human rhinoviruses (hRV), human metapneumovirus (hMPV), human coronaviruses (HCoVs), human bocavirus (HBoV), adenoviruses (AdV), parainfluenza virus (PIV) and influenza viruses A and B (Flu A-B) by real-time RT-PCR. HCoVs positive samples with high viral load (low Ct value) and those negative for all other respiratory viruses were include into further testing by amplifying a 440-bp-long fragment of the highly conserved polymerase gene.
Results: From December 2013 to February 2016, a total 16686 nasopharyngeal swabs from patients with acute respiratory tract infections were enrolled in the study. From these 976 (5.8%) were positive for HCoVs and 523 (58.6%) were negative for RSV, hRV, hMPV, HCoVs, HBoV, AdV, PIV, Flu A and FluB by real-time RT-PCR. From 523 HCoVs positive sample 129 were further tested for all HCoVs species, including 47 HCoV-HKU1, 44 HCoV-OC43, 24 HCoV-NL63, 11 HCoV-229E, 1 HCoV-HKU1/HCoV-229E and 1 HCoV-NL63/HCoV-229E. Only HCoVs positive samples (HCoV-HKU1 and HCoV-OC43) with high viral load (Ct-value less than 30) were include into further testing. To characterize the overall diversity of coronavirus sequences, 65 sequences have been included in phylogenetic analysis; 31 sequences of HCoV-OC43 and 34 sequences of HCoV-HKU1.
Conclusions: Among four circulating HCoVs, HCoV-HKU1 and HCoV-OC43 seem to show the highest prevalence and incidence in hospitalized patients. The phylogenetic analysis shows that Slovenian human coronavirus strains from this study belong to the four clusters, two grouping HCoV-OC43 and two HCoV-HKU1. The present study draws genetically diversity of human coronaviruses in Slovenian hospitalized patients.