Detection and Genotyping of Human Group A Rotaviruses by Oligonucleotide Microarray Hybridization

Abstract

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotypespecific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.Rotaviruses are the most important etiological agents of severe diarrhea in infants and young children in both developed and developing countries (15). The rotavirus outer capsid proteins VP4 and VP7 represent neutralization antigens that are the basis for the classification of rotaviruses by P and G serotypes, respectively. VP4-and VP7-specific antibodies provide serotype-specific protection against rotavirus diarrhea (14). Accurate and rapid serological identification is important for the diagnosis of rotavirus infections, host range determination, and global epidemiological surveillance. It is also an important part of molecular epidemiology and can be used for tracing lines of viral transmission, monitoring molecular evolution, identifying new strains, and determining genotype distribution in clinical trials of experimental vaccines. The current classification of rotaviruses is based on neutralization assays with polyclonal or monoclonal antibodies (13). At least 14 G serotypes (VP7 protein) and 13 (not including subtypes) P serotypes (VP4 protein) have been identified by this approach (7,15).Nucleotide sequence analysis of the VP7 and VP4 genes allows rotaviruses to be classified into a number of distinct genotypes. The G genotypes have been shown to consi...

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