Use of alkaline northern blot hybridization for the identification of genetic relatedness of the fourth gene of rotaviruses

Abstract

A recently developed alkaline northern blot hybridization assay (Li, J. K. K., Parker, B. & Kowalik, T., Analytical Biochemistry 163, 210-18, 1987) was used to assess the genetic relatedness of the fourth gene of human rotavirus strains recovered from children with diarrhea and from asymptomatic neonates. Cenomic double stranded (ds) RNAs of the rotavirus strains were separated by polyacrylamide gel electrophoresis and were blotted to nylon membranes (Gene Screen Plus or Zeta Probe membranes). The blotted RNAs were then probed with 32P-labelled single-stranded (ss) RNA probes prepared by in vitro transcription from single-shelled particles of the different strains. When analysed under a condition of high stringency (52 °C, 2.5 × SSC, 50% formamide) that allowed up to 21% of nucleotide mismatch, a high degree of the fourth gene homology was observed among strains recovered from asymptomatic neonates (asymptomatic rotaviruses) or among strains recovered from infants and children with diarrhea (symptomatic rotaviruses), while the homology of the fourth gene between the asymptomatic and symptomatic strains was considerably lower. It is of particular interest that the fourth gene of the AU-1 and AU228 strains recovered from children with diarrhea failed to hybridize to the corresponding gene of either asymptomatic or symptomatic rotavirus strains but showed a high degree of homology with the fourth gene of a feline rotavirus recovered from an apparently healthy cat. These data indicate that a new group of the fourth gene is present among symptomatic rotaviruses and that the fourth gene of this group is genetically related to the corresponding gene of a feline rotavirus.