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Application of Restriction Fragment Length Polymorphism Analysis of VP7-Encoding Genes: Fine Comparison of Irish and Global Rotavirus Isolates

Molecular Diagnostics Unit, Cork Institute of Technology, Bishopstown,¹ Department of Medical Microbiology, Cork University Hospital, Wilton, Cork, Ireland,² Viral Gastroenteritis Section, MS G-04, Centers for Disease Control and Prevention, Atlanta, Georgia 30333³

Received 3 August 2001/ Returned for modification 27 September 2001/ Accepted 18 November 2001

A restriction fragment length polymorphism (RFLP) detection assay was developed to examine the genetic relationship(s) among VP7-encoding genes from 100 Irish rotavirus isolates and 30 randomly selected global rotavirus isolates (from the current databases). RFLP analysis of the VP7 gene segments was performed independently with three enzymes (RsaI, AluI, and EcoRV) in separate reactions by direct digestion of the DNA product amplified by reverse transcriptase (RT)-mediated PCR (RT-PCR) or by using computational methods. Thirty-six RFLP patterns were identified for all 130 strains, and of these, only nine patterns were associated with the Irish isolates. A correlation between the G type of the Irish isolates and certain single or combined enzyme profiles was apparent. These data suggested that the Irish wild-type rotavirus population was homogeneous and could be distinguished by RFLP analysis from global isolates of the same serotype(s). The deduced amino acid sequences of the VP7 RT-PCR products from six Irish isolates known to be of the G serotype revealed significant amino acid substitutions within major antigenic regions. In addition, these data identified the existence of at least two genetic lineages within serotype G1 strains which were distinguishable by RFLP analysis.

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