title: Development and validation of DNA microarray for genotyping group A rotavirus VP4 (P[4], P[6], P[8], P[9], and P[14]) and VP7 (G1 to G6, G8 to G10, and G12) genes.
abstract: Previously, we reported the development of a microarray-based method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) (V. Chizhikov et al., J. Clin. Microbiol. 40:2398-2407, 2002). The expanded version of the rotavirus microarray assay presented herein is capable of identifying (i) five clinically relevant human rotavirus VP4 genotypes (P[4], P[6], P[8], P[9], and P[14]) and (ii) five additional human rotavirus VP7 genotypes (G5, G6, G8, G10, and G12) on one chip. Initially, a total of 80 cell culture-adapted human and animal reference rotavirus strains of known P (P[1] to P[12], P[14], P[16], and P[20]) and G (G1-6, G8 to G12, and G14) genotypes isolated in various parts of the world were employed to evaluate the new microarray assay. All rotavirus strains bearing P[4], P[6], P[8], P[9], or P[14] and/or G1 to G6, G8 to G10, or G12 specificity were identified correctly. In addition, cross-reactivity to viruses of genotype G11, G13, or G14 or P[1] to P[3], P[5], P[7], P[10] to P[12], P[16], or P[20] was not observed. Next, we analyzed a total of 128 rotavirus-positive human stool samples collected in three countries (Brazil, Ghana, and the United States) by this assay and validated its usefulness. The results of this study showed that the assay was sensitive and specific and capable of unambiguously discriminating mixed rotavirus infections from nonspecific cross-reactivity; the inability to discriminate mixed infections from nonspecific cross-reactivity is one of the inherent shortcomings of traditional multiplex reverse transcription-PCR genotyping. Moreover, because the hybridization patterns exhibited by rotavirus strains of different genotypes can vary, this method may be ideal for analyzing the genetic polymorphisms of the VP7 or VP4 genes of rotaviruses.
authors.affiliation: Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-8026, USA.
authors.forename: Shinjiro
authors.initials: S
authors.lastname: Honma
authors.forename: Vladimir
authors.initials: V
authors.lastname: Chizhikov
authors.forename: Norma
authors.initials: N
authors.lastname: Santos
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Tatsumi
authors.forename: Maria do Carmo S T
authors.initials: Mdo C
authors.lastname: Timenetsky
authors.forename: Alexandre C
authors.initials: AC
authors.lastname: Linhares
authors.forename: Joana D'Arc P
authors.initials: JD
authors.lastname: Mascarenhas
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ushijima
authors.forename: George E
authors.initials: GE
authors.lastname: Armah
authors.forename: Jon R
authors.initials: JR
authors.lastname: Gentsch
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Hoshino
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D005243:Feces / Q000821:virology; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011110:Polymorphism, Genetic; D012401:Rotavirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000821:virology*; D012680:Sensitivity and Specificity
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus