title: Microarray assay for detection and discrimination of Orthopoxvirus species.
abstract: A microarray method was developed for simultaneous detection and identification of six species of Orthopoxvirus (OPV) including Variola, Monkeypox, Cowpox, Camelpox, Vaccinia, and Ectromelia viruses. The method allowed us to discriminate OPV species from varicella-zoster virus (VZV), Herpes Simplex 1 virus (HSV-1), and Herpes Simplex 2 virus (HSV-2) that cause infections with clinical manifestations similar to OPV infections. The nucleotide sequences of the C23L/B29R and the B19R genes identified for 86 and 72 different OPV strains, respectively, were used to design species-specific microarray oligonucleotide probes (oligoprobes). The microarray also contained several oligoprobes selected from the ORF31, US4, and US5 genes of VZV, HSV-1, and HSV-2, respectively. The samples (from HSVs or OPVs) of ssDNAs for analyses were prepared by using asymmetric PCR followed by chemical labeling of ssDNA with Cy3 dye. DNA from 52 samples of various OPV species, two isolates of VZV, two of HSV-1, and three of HSV-2 were tested using the developed microarray assay; all tested viruses were accurately identified. To ensure the robustness of the microarray assay, three additional unrelated variola virus strains with unknown sequences of the C23L/B29R and the B19R genes were tested. In each instance the microarray unambiguously identified them as Variola virus species. The results obtained in this study demonstrated that this new microarray method is a valuable tool for the rapid and accurate detection and differentiation of these important viral pathogens.
authors.affiliation: State Research Center of Virology and Biotechnology Vector, Novosibirsk Region, Kol'tsovo, Russia.
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Ryabinin
authors.forename: Leonid A
authors.initials: LA
authors.lastname: Shundrin
authors.forename: Elena B
authors.initials: EB
authors.lastname: Kostina
authors.forename: Majid
authors.initials: M
authors.lastname: Laassri
authors.forename: Vladimir
authors.initials: V
authors.lastname: Chizhikov
authors.forename: Sergei N
authors.initials: SN
authors.lastname: Shchelkunov
authors.forename: Konstantin
authors.initials: K
authors.lastname: Chumakov
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Sinyakov
mesh_terms: D002232:Carbocyanines; D004277:DNA, Single-Stranded / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D018155:Orthopoxvirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D011213:Poxviridae Infections / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013194:Staining and Labeling
chemical_list: D002232:Carbocyanines; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D015345:Oligonucleotide Probes; C089187:cyanine dye 3