Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides

Abstract

Variola virus (VARV), causing smallpox, is a potential biological weapon. Methods to detect VARV rapidly and to differentiate it from other viruses causing similar clinical syndromes are needed urgently. We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples.

Fig. 1

Amplification of C23L/B29R genes of different OPV strains with specific primers bioB29r-U1and shortB29r-L1 and amplification of the region in ORF 62 of chickenpox virus with primers 1099r and 769f. (A) 1 Kb DNA-marker (lane 1), CPXV strains RP-9 (lane 2), MPXV strain CDC# 77-666 (lane 3), VACV strains Wyeth (lane 4); Chambon St-Yves Menard (lane5), EP-267 (lane 6); EM-63 (lane 7), EP-7 (lane 8), EP-5 (lane 9), EP-2 (lane 10), Elstree 3399 (lane 11), 1Kb DNA-marker (lane 12). (B) 1 Kb DNA-marker (lane 1), VARV strain Kuw-5 (lane 2), VACV strains LIVP clone 4 (lane 3), CPXV strains Turk-74 (lane 4), EP-4 (lane 5) CVI-78 (lane 6), Copenhagen (lane 7); EP-3 (lane 8), EP-8 (lane 9), EP-6 (lane 10), EP-1 (lane 11), 1 Kb DNA-marker (lane 12). (C) 1 Kb DNA-marker (lane 1), VARV strains Congo-2 (lane 2), CPXV strain Brighton (lane 3), Ind-3A (lane 4), VZV OKA (lane 5), VZV wild type (lane 6), 1 Kb DNA-marker (lane 7).

Fig. 2

Dependence of fluorescence intensity on concentrations of labeled DNA amplicon and oligoprobe. (A) Dependence of fluorescence intensity on oligoprobe concentrations, (B) dependence of fluorescence intensity on fluorescent DNA concentrations.

Fig. 3

Comparison of the intensity of hybridization with fluorescent DNA amplicon generated by different methods from OPV C23L/B29R gene (A) Image of microarray profile (B) Quantification of fluorescence by quantarray Software. Fluorescent DNA amplicons used in this study were; Dm, dsDNA generated with multiplex PCR; DL, dsDNA PCR Product; Bm, ssDNA separated from dsDNA multiplex PCR product on streptavidin beads; BL, ssDNA separated from dsDNA PCR product on streptavidin beads; Am, product of multiplex asymmetric PCR; Al, product of asymmetric PCR.

Fig. 4

Patterns of detection and discrimination between the OPV species pathogenic for humans and their differentiation from HHV 3 (VZV) (A) composition microarray for OPV species identification. (B) Patterns of discriminated species; B1 VARV strain Congo-2, B2; VARV strain Ind-3A, B3; VARV strain Kuw-5, B4; VACV strain Elstree 3399, B5; VACV strain Copenhagen, B6; VACV strain CVI-78, B7; CPXV-A strain CPV Turk-74, B8; CPXV-A strain CPV Brighton, B9; CPXV-B strain EP-1, B10; CPXV-B strain EP-2, B11; CPXV-B strain EP-7, B12; MPXV strain CDC# 77-666, B13; VZV strain Oka. B14; Pattern of the mixture hybridization of VARV strain Ind-3A and VZV strain Oka. (C) Patterns of unclassified OPVs detected in blinded samples; C1; CmPV CP-5 (Camelpox), C2; CmPV CP-1 (Camelpox), C3; CmPV 1269/95 (Camelpox), C4; CmPV 1231 (Camelpox), C5; ECT. 33221 (Ectomelia), C6; ECT.4908 (Ectomelia), C7; ECT. MP-1 (Ectomelia), C8; ECT. MP-2 (Ectomelia).

Fig. 4

Patterns of detection and discrimination between the OPV species pathogenic for humans and their differentiation from HHV 3 (VZV) (A) composition microarray for OPV species identification. (B) Patterns of discriminated species; B1 VARV strain Congo-2, B2; VARV strain Ind-3A, B3; VARV strain Kuw-5, B4; VACV strain Elstree 3399, B5; VACV strain Copenhagen, B6; VACV strain CVI-78, B7; CPXV-A strain CPV Turk-74, B8; CPXV-A strain CPV Brighton, B9; CPXV-B strain EP-1, B10; CPXV-B strain EP-2, B11; CPXV-B strain EP-7, B12; MPXV strain CDC# 77-666, B13; VZV strain Oka. B14; Pattern of the mixture hybridization of VARV strain Ind-3A and VZV strain Oka. (C) Patterns of unclassified OPVs detected in blinded samples; C1; CmPV CP-5 (Camelpox), C2; CmPV CP-1 (Camelpox), C3; CmPV 1269/95 (Camelpox), C4; CmPV 1231 (Camelpox), C5; ECT. 33221 (Ectomelia), C6; ECT.4908 (Ectomelia), C7; ECT. MP-1 (Ectomelia), C8; ECT. MP-2 (Ectomelia).