Northern Blotting Total RNA was extracted from DcCoV UAE-HKU23-infected HRT-18G cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA)

Northern Blotting Total RNA was extracted from DcCoV UAE-HKU23-infected HRT-18G cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of [20,21,22]. As for F9995-0144 animal CoVs, we and others have described the discovery of SARS-CoV-like viruses in Chinese horseshoe bats in Hong Kong and other horseshoe bats in other provinces of China [23,24]. Recently, the discovery of SARS-CoV-like viruses in Chinese horseshoe bats in Yunnan has further highlighted the importance for hunting the animal origin of human infections [25]. In addition, we have also discovered 21 other animal CoVs, which include two novel lineages in and a novel genus F9995-0144 [26,27,28,29,30,31,32,33,34,35,36]. From our studies it was shown that bats are the gene source for and and birds are the gene source for and to fuel CoV evolution and dissemination [33]. In 2012, a novel CoV, named Middle East respiratory syndrome coronavirus (MERS-CoV), closely related to bat CoV HKU4 (Ty-BatCoV HKU4) and bat CoV HKU5 (Pi-BatCoV HKU5), has emerged as a cause of severe respiratory infections associated with high mortalities [37,38,39,40]. It has been shown that dromedaries in the Middle East possessed neutralizing antibodies against MERS-CoV [41,42]. Furthermore, MERS-CoV was also detected in the nasal swabs of dromedaries in Qatar, Saudi Arabia, Egypt, and the United Arab Emirates [43,44,45,46,47]. The recent emergence of MERS and the discovery of MERS-CoV in dromedaries have boosted interest in the search for other novel viruses in dromedaries [48,49]. In a recent molecular epidemiology study, we discovered a novel betacoronavirus, named dromedary camel CoV UAE-HKU23 (DcCoV UAE-HKU23), from fecal samples of dromedaries from Dubai [50]. In this study, we report the isolation of DcCoV UAE-HKU23 from the fecal sample of a dromedary and its characterization. 2. Results 2.1. Isolation of DcCoV UAE-HKU23 Of the seven cell lines inoculated with dromedary fecal samples positive for F9995-0144 DcCoV UAE-HKU23, viral replication was detected by RT-PCR in the supernatants of HRT-18G in two of the 14 dromedary fecal samples at day 7, with viral loads of 2.6 1010 and 9.7 106 copies/mL in HRT-18G cells in the presence of trypsin. Cytopathic effects (CPE), mainly in the form of rounded, fused and granulated huge cells rapidly detaching from your monolayer, were also observed in infected HRT-18G cells five days after inoculation (Number 1a), which showed viral nucleocapsid manifestation by immunofluorescence in 20% of cells. Electron microscopy of ultracentrifuged cell tradition extracts from infected HRT-18G cells showed the presence of CoV-like particles around 70C100 nm in diameter with standard club-shaped surface projections (Number 1b). Open in a separate window Number 1 (a) HRT-18G cells infected with dromedary camel coronavirus (DcCoV) UAE-HKU23 showing F9995-0144 cytopathic effects with rounded, aggregated, fused, and granulated huge cells rapidly detaching from your monolayer at day time 5 after incubation (arrows) (unique magnification 40); (b) Bad contrast electron microscopy of ultracentrifuged deposit of HRT-18G cell culture-grown DcCoV UAE-HKU23, showing typical club-shaped surface projections (arrow) of coronavirus particles, with rabbit coronavirus HKU14 as the control (bottom left corner). Pub = 50 nm; Indirect immunofluorescent antigen detection in (c) uninfected and (d) infected HRT-18G cells using serum from dromedary showing apple green fluorescence in (arrows) DcCoV UAE-HKU23 infected HRT-18G cells (unique magnification 100 for both). 2.2. Subgenomic mRNAs and Their Leader-Body Junction Sequences CoVs are characterized by a unique Rabbit Polyclonal to CaMK2-beta/gamma/delta mechanism of discontinuous transcription with the synthesis of a nested set of subgenomic mRNAs [51,52]. To assess.