Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 7;17(5):691.
doi: 10.3390/ijms17050691.

Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23

Patrick C Y Woo  1   2   3   4   5 , Susanna K P Lau  6   7   8   9   10 , Rachel Y Y Fan  11 , Candy C Y Lau  12 , Emily Y M Wong  13 , Sunitha Joseph  14 , Alan K L Tsang  15 , Renate Wernery  16 , Cyril C Y Yip  17 , Chi-Ching Tsang  18 , Ulrich Wernery  19 , Kwok-Yung Yuen  20   21   22   23   24
Affiliations

Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23

Patrick C Y Woo et al. Int J Mol Sci. .

Abstract

Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5'-UCUAAAC-3' as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 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 Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1.

Keywords: characterization; coronavirus; dromedary camel; isolation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(a) HRT-18G cells infected with dromedary camel coronavirus (DcCoV) UAE-HKU23 showing cytopathic effects with rounded, aggregated, fused, and granulated giant cells rapidly detaching from the monolayer at day 5 after incubation (arrows) (original magnification 40×); (b) Negative 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). Bar = 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 (original magnification 100× for both).
Figure 2
Figure 2
(a) Northern blot analysis for total RNA isolated from dromedary camel coronavirus (DcCoV) UAE-HKU23–infected HRT-18G cells. RNA species are indicated by arrows. NS2, non-structural NS2; HE, hemagglutinin; S, spike; NS5, non-structural NS5; E, envelope; M, membrane; N, nucleocapsid. Lane 1, 1 μg total RNA from uninfected cells; Lane 2, 1 μg total RNA from infected cells; (b) DcCoV UAE-HKU23 subgenomic mRNA (sg mRNA) leader-body junction and flanking sequences. The subgenomic mRNA sequences are shown in alignment with the leader and the genomic sequences. The start codon AUG in each subgenomic mRNA is depicted in bold. The putative transcription regulatory sequences (TRS) was underlined and base mismatch between the body TRS and the leader TRS or the corresponding genomic region was indicated by asterisk. The 43N and 115N in the parentheses indicate that 43 and 115 nucleotides at that region are not shown.
Figure 2
Figure 2
(a) Northern blot analysis for total RNA isolated from dromedary camel coronavirus (DcCoV) UAE-HKU23–infected HRT-18G cells. RNA species are indicated by arrows. NS2, non-structural NS2; HE, hemagglutinin; S, spike; NS5, non-structural NS5; E, envelope; M, membrane; N, nucleocapsid. Lane 1, 1 μg total RNA from uninfected cells; Lane 2, 1 μg total RNA from infected cells; (b) DcCoV UAE-HKU23 subgenomic mRNA (sg mRNA) leader-body junction and flanking sequences. The subgenomic mRNA sequences are shown in alignment with the leader and the genomic sequences. The start codon AUG in each subgenomic mRNA is depicted in bold. The putative transcription regulatory sequences (TRS) was underlined and base mismatch between the body TRS and the leader TRS or the corresponding genomic region was indicated by asterisk. The 43N and 115N in the parentheses indicate that 43 and 115 nucleotides at that region are not shown.
Figure 3
Figure 3
Comparison of neutralization antibody titer and immunofluorescence antibody titer of dromedary serum samples for dromedary camel coronavirus UAE-HKU23. Numbers of serum samples with that particular neutralization antibody and immunofluorescence antibody titers are indicated by points with different colors.
Figure 4
Figure 4
Western blotting analysis of dromedary camel coronavirus (DcCoV) UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV) N proteins expressed in E. coli. Lane 1: MERS-CoV N protein reacted with 1:16,000 dilution of serum from mouse immunized with MERS-CoV N protein; Lane 2: MERS-CoV N protein reacted with 1:16,000 dilution of serum from mouse immunized with DcCoV UAE-HKU23 N protein; Lane 3: DcCoV UAE-HKU23 N protein reacted with 1:16,000 dilution of serum from mouse immunized with MERS-CoV N protein, Lane 4: DcCoV UAE-HKU23 N protein reacted with 1:16,000 dilution of serum from mouse immunized with DcCoV UAE-HKU23 N protein.
Figure 5
Figure 5
Phylogenetic analyses of RNA-dependent RNA polymerase (RdRp), S, and N genes of dromedary camel coronavirus (DcCoV) UAE-HKU23. Included in the analysis were 2784, 4101, and 1347 nucleotide positions in RdRp, S and N, respectively. For RdRp, the scale bar indicates the estimated number of substitutions per 200 nucleotides. For S, the scale bars indicate the estimated number of substitutions per 50 nucleotides. For N, the scale bars indicate the estimated number of substitutions per 100 nucleotides. Bootstrap values were calculated from 1000 trees and those below 70% are not shown. The 14 strains of DcCoV UAE-HKU23 characterized in this and our previous studies are shown in bold. BCoV, bovine coronavirus; CRCoV, canine respiratory coronavirus; SDCoV, sambar deer coronavirus; WbCoV, waterbuck coronavirus; WtDCoV, white-tailed deer coronavirus; BRCoV, bovine respiratory coronavirus; GiCoV, giraffe coronavirus; SACoV, sable antelope coronavirus.
Figure 5
Figure 5
Phylogenetic analyses of RNA-dependent RNA polymerase (RdRp), S, and N genes of dromedary camel coronavirus (DcCoV) UAE-HKU23. Included in the analysis were 2784, 4101, and 1347 nucleotide positions in RdRp, S and N, respectively. For RdRp, the scale bar indicates the estimated number of substitutions per 200 nucleotides. For S, the scale bars indicate the estimated number of substitutions per 50 nucleotides. For N, the scale bars indicate the estimated number of substitutions per 100 nucleotides. Bootstrap values were calculated from 1000 trees and those below 70% are not shown. The 14 strains of DcCoV UAE-HKU23 characterized in this and our previous studies are shown in bold. BCoV, bovine coronavirus; CRCoV, canine respiratory coronavirus; SDCoV, sambar deer coronavirus; WbCoV, waterbuck coronavirus; WtDCoV, white-tailed deer coronavirus; BRCoV, bovine respiratory coronavirus; GiCoV, giraffe coronavirus; SACoV, sable antelope coronavirus.
Figure 6
Figure 6
(a) Scatter plot of the corresponding analysis (CA) using relative synonymous codon usage (RSCU) of the RdRp, S, and N genes of members of Betacoronavirus 1 and RbCoV HKU14. Different coronaviruses are indicated in different colored markers. The group of bovine coronavirus-like viruses is circled; (b) SimPlot analysis of complete RdRp, S, and N genes of DcCoV UAE-HKU23, alpaca CoV, BCoV and other wild ruminant CoVs. Each point plotted is the percent genetic distance within a sliding window of 200 nt wide, centered on the position plotted, with a step size of 20 nt. Each curve represents a comparison of the sequence data of DcCoV UAE-HKU23, BCoV, and other wild ruminant CoV strains to the reference sequence data of alpaca CoV. Alpaca CoV, alpaca coronavirus; BCoV, bovine coronavirus; BuCoV, Bubalus bubalis coronavirus; CRCoV, canine respiratory coronavirus; DcCoV, dromedary camel coronavirus, ECoV, equine coronavirus; GiCoV, giraffe coronavirus; HCoV-OC43, human coronavirus OC43; HTCoV, Himalayan tahr coronavirus; NyCoV, nyala coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; RbCoV, rabbit coronavirus; SACoV, sable antelope coronavirus; SDCoV, sambar deer coronavirus; SiCoV, sitatunga coronavirus; WbCoV, waterbuck coronavirus; WiCoV, wisent coronavirus; WtDCoV, white-tailed deer coronavirus.
Figure 6
Figure 6
(a) Scatter plot of the corresponding analysis (CA) using relative synonymous codon usage (RSCU) of the RdRp, S, and N genes of members of Betacoronavirus 1 and RbCoV HKU14. Different coronaviruses are indicated in different colored markers. The group of bovine coronavirus-like viruses is circled; (b) SimPlot analysis of complete RdRp, S, and N genes of DcCoV UAE-HKU23, alpaca CoV, BCoV and other wild ruminant CoVs. Each point plotted is the percent genetic distance within a sliding window of 200 nt wide, centered on the position plotted, with a step size of 20 nt. Each curve represents a comparison of the sequence data of DcCoV UAE-HKU23, BCoV, and other wild ruminant CoV strains to the reference sequence data of alpaca CoV. Alpaca CoV, alpaca coronavirus; BCoV, bovine coronavirus; BuCoV, Bubalus bubalis coronavirus; CRCoV, canine respiratory coronavirus; DcCoV, dromedary camel coronavirus, ECoV, equine coronavirus; GiCoV, giraffe coronavirus; HCoV-OC43, human coronavirus OC43; HTCoV, Himalayan tahr coronavirus; NyCoV, nyala coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; RbCoV, rabbit coronavirus; SACoV, sable antelope coronavirus; SDCoV, sambar deer coronavirus; SiCoV, sitatunga coronavirus; WbCoV, waterbuck coronavirus; WiCoV, wisent coronavirus; WtDCoV, white-tailed deer coronavirus.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Peck K.M., Burch C.L., Heise M.T., Baric R.S. Coronavirus host range expansion and Middle East Respiratory Syndrome coronavirus emergence: Biochemical mechanisms and evolutionary perspectives. Annu. Rev. Virol. 2015;2:95–117. doi: 10.1146/annurev-virology-100114-055029. - DOI - PubMed
    1. Lai M.M., Cavanagh D. The molecular biology of coronaviruses. Adv. Virus Res. 1997;48:1–100. - PMC - PubMed
    1. Ziebuhr J. Molecular biology of severe acute respiratory syndrome coronavirus. Curr. Opin. Microbiol. 2004;7:412–419. doi: 10.1016/j.mib.2004.06.007. - DOI - PMC - PubMed
    1. Brian D.A., Baric R.S. Coronavirus genome structure and replication. Curr. Top. Microbiol. Immunol. 2005;287:1–30. - PMC - PubMed
    1. De Groot R.J., Baker S.C., Baric R., Enjuanes L., Gorbalenya A.E., Holmes K.V., Perlman S., Poon L., Rottier P.J.M., Talbot P.J., et al. Family—Coronaviridae. In: Adams M.J., Carstens E.B., Lefkowitz E.J., editors. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, International Union of Microbiological Societies, Virology Division. Elsevier; San Diego, CA, USA: 2012. pp. 806–828.

LinkOut - more resources

-