Department of Health and Human Services
Centers for Disease Control and Prevention

Emerging Infectious Diseases

ISSN: 1080-6059

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Article Contents

Volume 12, Number 11–November 2006

Letter

Chikungunya Fever, Hong Kong

Nelson Lee,* Chun K. Wong,* Wai Y. Lam,* Ann Wong,† Wilina Lim,† Christopher W.K. Lam,* Clive S. Cockram,* Joseph J.Y. Sung,* Paul K.S. Chan,* and Julian W. Tang*
*The Chinese University of Hong Kong Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, People's Republic of China; and †Department of Health, Kowloon, Hong Kong Special Administrative Region, People's Republic of China

Appendix

Chikungunya Cytokine Profile Determination (Prince of Wales Hospital)

Serum was prepared from clotted blood by centrifugation (2,000 ×g for 10 min). T helper (Th)1/Th2 cytokines, inflammatory cytokines, and chemokines in serum were measured by cytometric bead array (CBA) by using a 4-color FACSCalibur flow cytometer (Becton Dickinson [BD], San Jose, CA, USA) located in a biosafety level-2 laboratory (1,2). In CBA, 5or 6 bead populations with distinct fluorescence intensities had been coated with capturing antibodies specific for different cytokines or chemokines. These bead populations could be resolved in the fluorescence channels of the flow cytometer. After the beads had been incubated with 50 μL of serum, different cytokines or chemokines in the sample were captured by their corresponding beads. The cytokine/chemokine captured beads were then mixed with phycoerythrin-conjugated detection antibodies to form sandwich complexes. Following incubation, washing, and acquisition of fluorescence data, the results were generated in graphic format by the BD CBA software.

The following concentrations were measured by using the IFN-γ CBA flex set, human inflammatory cytokine and chemokine CBA kits (BD PharMingen, San Diego, CA, USA): Th1/2 cytokines (interferon-γ [IFN-γ] and interleukin 10 [IL-10]), inflammatory cytokines (IL-1β, IL-6, tumor necrosis factor-α, and IL-12p70), and chemokines (IL-8/CXCL8, regulated upon activation normal T cell-expressed and secreted [RANTES/CCL5], monocyte chemoattractant protein-1 [MCP-1/CCL2], IFN-γ-inducible protein-10 [IP-10/CXCL10], and monokine induced by IFN-γ [MIG/CXCL9]. The assay sensitivities of these cytokines and chemokines were 7.1, 3.3, 7.2, 2.5, 3.7, 1.9, 0.2, 1.0, 2.7, 2.8, and 2.5 ng/L, respectively. The coefficients of variation for all cytokine and chemokine assays were <10%. Their respective normal ranges have been derived from measurement of concentrations in >100 healthy subjects (1).

In-house Chikungunya Immunofluorescent Slide Serologic IgG/IgM assays (Public Health Laboratory Service)

For measurement of the chikungunya-specific antibody response, an in-house serologic assay was developed. Chikungunya virus obtained from the patient's viremic sera was grown in Vero E6 cells, which were then fixed onto glass slides. Dilutions of patient sera, starting at 1:10, were made up, and 20 μL of each dilution was added to the glass slide and incubated for 45 min at 37°C. They were then washed in phosphate-buffered saline (PBS) for 10 min and blotted dry. Next, 20 μL of a fluorescein-labeled antihuman immunoglobulin M (IgM and polyvalent, from Biosource International, Camarillo, CA, USA) was added to each sample on the slide and incubated for a further 45 min at 37°C. After this, the slides were washed again with PBS for 10 min, blotted dry, mounted with phosphate-buffered glycerol and a coverslip, and then viewed under a UV microscope. The reported immunofluorescent titer was the highest serum dilution showing a positive reaction (apple-green fluorescent cytoplasmic granules).

Chikungunya RT-PCR and Sequencing (Prince of Wales Hospital)

Genomic viral RNA was extracted from 140 μL of clinical plasma using the QIAamp ViralRNA MiniKit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturer's instructions, and resuspended in 60 μL of RNase-free water. reverse transcription (RT)-PCR was carried out with SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocols. In brief, a 50-μL reaction mix, containing 0.5 μM of each forward and reverse primers, and 10 μL of extracted RNA template were used for the RT-PCR. Two previously published primer sets were selected to amplify the nonstructural protein 1 (NSP1) and envelope glycoprotein 1 (E1) genomic regions of the chikungunya viral genome. Primer set 1: (forward: CHIK/NSP1-S 5´-TAGAGCAGGAAATTGATCCC-3´ and reverse: CHIK/NSP1-C 5´- CTTTAATCGCCTGGTGGTAT-3´) (3) was used to amplify the NSP1 region. Primer set 2: (forward: OP16 5´-AGCTGTAAGGTCTTCACCGG-3´ and reverse: OP17 5´- GTATTTTGTTACTATTCAGG-3´) (4) was used to amplify the E1 region. The RT reaction (57°C for 30 min) was followed by 94°C for 2 min and 40 cycles of PCR (94°C for 40 sec, 55°C for 40 sec, and 68°C for 1 min 30 sec, for each cycle) and a final extension at 68°C for 10 min.

Appendix Figure 1

Appendix Figure 1. The maximum-likelihood phylogenetic tree for chikungunya envelope glyprotein 1 (E1) sequences (199 bp) constructed by using PAUP...

Appendix Figure 2

Appendix Figure 2. The maximum likelihood phylogenetic tree for chikungunya nonstructural protein 1 (NSP1) sequences (314 bp) constructed using PAUP...

The NSP1 (354 bp) and E1 (1.2 kb) PCR products were purified from 1.5% agarose gels by using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Sequencing reactions were undertaken with the BigDye Terminator version 3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA). The RT-PCR primer sets were also used for the sequencing reactions. Sequencing was carried out with an automatic sequence analyzer (ABI 3130 Genetic Analyzer, Applied Biosystems) following the manufacturer's protocol. Nucleotide sequences were aligned by using the ClustalX 1.83 software (5). The NSP1 and E1 sequences have been submitted to the National Center for Biotechnology's GenBank under accession nos. DQ489787 (E1) and DQ489788 (NSP1) (available from http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar).

For phylogenetic analysis, existing NSP1 and E1 sequences were downloaded from GenBank and aligned with ClustalX version 1.83 (5) and edited with Bioedit version 7.0.4.1 (available from http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Many of the NSP1 and E1 sequences downloaded from GenBank were quite short, and we decided to include as many sequences as possible in the final trees, to show the diverse origin of the sequences. Therefore, after editing, the lengths of the sequences used in the final tree construction were 314 bp and 199 bp for NSP1 and E1, respectively. Phylogenetic tree construction was performed with PAUP* version 4.0b10 (6) by using a maximum likelihood (ML) approach, under an optimum model of evolution as determined by MODELTEST version 3.7 (7). Optimal trees were searched for by using a tree-bisection-reconnection heuristic search strategy. Bootstrapping was performed within PAUP*. The tree was initially displayed in Treeview version 1.6.6. (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html), then opened in NJPlot (http://pbil.univ-lyon1.fr/software/njplot.html), and bootstrap values added to the significant (bootstrap values >70) branches by using Microsoft Word. Pairwise comparison of the NSP1 and E1 sequences obtained at PWH by using BioEdit were identical (i.e., 100% similarity) to the corresponding sequences in the full-length chikungunya genome sequence (LR2006_OPY1_Reunion_2006) deposited in GenBank (accession no. DQ443544) from the same outbreak in La Reunion.

Appendix Figure 1 shows the phylogenetic tree for chikungunya E1 sequences (199 bp) constructed by using PAUP* under a Tamura-Nei 1993 model with invariable sites (i.e., TrN+I), as selected by MODELTEST (version 3.7) under the Akaike information criteria (AIC). The bracket shows that the PWH patient E1 sequence (DQ489787) clusters most closely with the outbreak La Reunion sequence (LR2006_OPY1_Reunion_2006_E1, DQ443544). Only bootstrap values >70 are shown and considered significant. Appendix Figure 2 shows the phylogenetic tree for chikungunya NSP1 sequences (314 bp), constructed using PAUP* under a transitional model with equal base frequencies with a γ-distributed rate of substitution (i.e., TIM+G), as selected by MODELTEST (version 3.7) under the AIC. The bracket shows that the PWH patient NSP1 sequence (GenBank accession no. DQ489788) clusters most closely with the La Reunion chikungunya outbreak sequence (LR2006_OPY1_Reunion_2006_NSP1, DQ443544).

Appendix References

Wong CK, Lam CW, Wu AK, Ip WK, Lee NL, Chan IH, et al. Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol. 2004;136:95–103.
Tang NL, Chan PK, Wong CK, To KF, Wu AK, Sung YM, et al. Early enhanced expression of interferon-inducible protein-10 (CXCL-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome. Clin Chem. 2005;51:2333–40.
Pastorino B, Muyembe-Tamfum JJ, Bessaud M, Tock F, Tolou H, Durand JP, et al. Epidemic resurgence of Chikungunya virus in democratic Republic of the Congo: identification of a new central African strain. J Med Virol. 2004;74:277–82.
Hasebe F, Parquet MC, Pandey BD, Mathenge EG, Morita K, Balasubramaniam V, et al. Combined detection and genotyping of Chikungunya virus by a specific reverse transcription-polymerase chain reaction. J Med Virol. 2002;67:370–4.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25:4876–82.
Swofford DL. PAUP*. Phylogenetic analysis using parsimony (*andother methods). Version 4. Sunderland (MA): Sinauer Associates;2001. Available from http://paup.csit.fsu.edu/win.html
Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998;14:817–8. Available from http://darwin.uvigo.es/software/modeltest.html

Appendix Figures

Appendix Figure 1. The maximum-likelihood phylogenetic tree for chikungunya envelope glyprotein 1 (E1) sequences (199 bp) constructed by using PAUP...
Appendix Figure 2. The maximum likelihood phylogenetic tree for chikungunya nonstructural protein 1 (NSP1) sequences (314 bp) constructed using PAUP...