RESTRICTION ENDONUCLEASES WHOSE SITES ARE PREDICTABLE FROM THE AMINO-ACID-SEQUENCE OFFER AN IMPROVED STRATEGY FOR TYPING BOVINE ROTAVIRUSES

Variation in the third base of a codon hampers genotypic characterizat ion, particularly of RNA viruses. Some restriction endonucleases, howe ver, have a recognition site with a variable base at the third positio n and will always cleave when a certain amino acid pair occurs (such a s glycine-proline for Sau961 and glutamic or aspartic acid followed by serine usually for Hinfl). We developed a restriction fragment length polymorphism (RFLP) procedure based on these enzymes for P-typing bov ine group A rotaviruses (BRV). Employing this procedure 20 BRV local s trains, isolated in tissue culture as well as the original faecal samp le, could be typed in one of three patterns. More variability was obse rved when restriction endonucleases were employed whose cleavage sites cannot be predicted from the amino acid sequence (Taql and Tsp5091). These RFLP results agreed with the PCR-VP4 typing assay, neutralizatio n tests, and nucleotide sequence analysis. RFLP with Sau961 and Hinfl provided quick and objective P-typing of strains and could detect mult iple genotypes in the same sample. (C) 1997 Academic Press Limited.