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
<p>(<b>a</b>) 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>b</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 <b>left</b> corner). Bar = 50 nm; Indirect immunofluorescent antigen detection in (<b>c</b>) uninfected and (<b>d</b>) 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).</p> "> Figure 2
<p>(<b>a</b>) 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>b</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.</p> "> Figure 2 Cont.
<p>(<b>a</b>) 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>b</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.</p> "> Figure 3
<p>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.</p> "> Figure 4
<p>Western blotting analysis of dromedary camel coronavirus (DcCoV) UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV) N proteins expressed in <span class="html-italic">E. coli</span>. 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.</p> "> Figure 5
<p>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.</p> "> Figure 5 Cont.
<p>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.</p> "> Figure 6
<p>(<b>a</b>) Scatter plot of the corresponding analysis (CA) using relative synonymous codon usage (RSCU) of the RdRp, S, and N genes of members of <span class="html-italic">Betacoronavirus 1</span> and RbCoV HKU14. Different coronaviruses are indicated in different colored markers. The group of bovine coronavirus-like viruses is circled; (<b>b</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, <span class="html-italic">Bubalus bubalis</span> 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.</p> "> Figure 6 Cont.
<p>(<b>a</b>) Scatter plot of the corresponding analysis (CA) using relative synonymous codon usage (RSCU) of the RdRp, S, and N genes of members of <span class="html-italic">Betacoronavirus 1</span> and RbCoV HKU14. Different coronaviruses are indicated in different colored markers. The group of bovine coronavirus-like viruses is circled; (<b>b</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, <span class="html-italic">Bubalus bubalis</span> 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.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Isolation of DcCoV UAE-HKU23
2.2. Subgenomic mRNAs and Their Leader-Body Junction Sequences
2.3. Immunofluorescence Antibody Tests and Neutralization Antibody Assays
2.4. Cross-Antigenicity between DcCoV UAE-HKU23 and MERS-CoV
2.5. Complete RdRp, S and N Gene Sequence Analysis
2.6. Codon Usage Analysis and Genetic Distance of RdRp, S and N Genes
3. Discussion
4. Materials and Methods
4.1. Virus Culture
4.2. Real-Time Quantitative RT-PCR
4.3. Electron Microscopy
4.4. Northern Blotting
4.5. Determination of Leader-Body Junction Sequence
4.6. Immunofluorescence Antibody Tests
4.7. Neutralization Antibody Assays
4.8. Cloning and Purification of (His)6-Tagged Recombinant DcCoV UAE-HKU23 and MERS-CoV Nucleocapsid Proteins from Escherichia coli
4.9. Animal Immunization Using N Proteins of DcCoV UAE-HKU23 and MERS-CoV
4.10. Western Blot Analysis
4.11. Animal Immunization Using Heat-Inactivated DcCoV UAE-HKU23
4.12. Sequencing of Complete RdRp, S, and N Genes of DcCoV UAE-HKU23 Strains
4.13. Phylogenetic Analysis
4.14. Codon Usage Analysis and Genetic Distance of RdRp, S, and N Genes
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Antibody Titer | Number (%) of Samples |
---|---|
Immunofluorescence Antibody Test | |
<20 | 1 (1.7) |
80 | 21 (35.6) |
320 | 23 (39.0) |
1280 | 13 (22.0) |
5120 | 1 (1.7) |
Neutralization Antibody Test | |
10 | 2 (3.4) |
20 | 4 (6.8) |
40 | 6 (10.2) |
80 | 18 (30.5) |
160 | 11 (18.6) |
320 | 9 (15.3) |
640 | 5 (8.5) |
1280 | 2 (3.4) |
2560 | 2 (3.4) |
Primer | Sequence (5′-3′) | Use |
---|---|---|
LPW25800 | GATTGTGAGCGATTTGCGTGC | Forward primer for all sg mRNAs PCR |
LPW18463 | GTAAACCTTTATAATTTAACACA | Reverse primer for mRNA (NS2) PCR |
LPW25801 | AATCGGTAAAGTGAAAACTCC | Reverse primer for mRNA (HE) PCR |
LPW25802 | CCACAATGTACTCAACAATAAAG | Reverse primer for mRNA (S) PCR |
LPW25803 | TAGCGAATGCTGTAAAACCAG | Reverse primer for mRNA (NS5) PCR |
LPW25804 | CTCATAAAACTGCCTACCTCT | Reverse primer for mRNA (E) PCR |
LPW18468 | CCAAGATACACATTATTCAACG | Reverse primer for mRNA (M) PCR |
LPW18469 | GAGTAATTCCAGAGAACCAAGA | Reverse primer for mRNA (N) PCR |
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Woo, P.C.Y.; Lau, S.K.P.; Fan, R.Y.Y.; Lau, C.C.Y.; Wong, E.Y.M.; Joseph, S.; Tsang, A.K.L.; Wernery, R.; Yip, C.C.Y.; Tsang, C.-C.; et al. 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. Int. J. Mol. Sci. 2016, 17, 691. https://doi.org/10.3390/ijms17050691
Woo PCY, Lau SKP, Fan RYY, Lau CCY, Wong EYM, Joseph S, Tsang AKL, Wernery R, Yip CCY, Tsang C-C, et al. 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. International Journal of Molecular Sciences. 2016; 17(5):691. https://doi.org/10.3390/ijms17050691
Chicago/Turabian StyleWoo, Patrick C. Y., Susanna K. P. Lau, Rachel Y. Y. Fan, Candy C. Y. Lau, Emily Y. M. Wong, Sunitha Joseph, Alan K. L. Tsang, Renate Wernery, Cyril C. Y. Yip, Chi-Ching Tsang, and et al. 2016. "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" International Journal of Molecular Sciences 17, no. 5: 691. https://doi.org/10.3390/ijms17050691