4 Determination of VP4 Genotype. The strategy for PCR typing of gene 4 is identical, in principle, to that used for VP7.64 First strand synthesis (reverse transcription) takes advantage of highly conserved areas of VP4 utilizing primers Con2 and Con3 (Box 3), which are complimentary to the 3' ends of both viral RNA strands within gene segment 4. 1 to 5 µL of viral RNA is added to 0.5 ml microcentrifuge tubes containing 3.5 µl of dimethylsulfoxide (Sigma) in a final volume of 8.5 µl, and the samples are mixed and denatured at 97°C for 5 min. The samples are then cooled on ice for 5 min and collected by brief centrifugation. 13.5 µL of H20, 16 µL of deoxynucleoside triphosphates (1.25 mM each dATP, dGTP, dCTP, and dTTP), 5 µL 1OX buffer (100 mM Trishydrochloride [pH 8.3], 500 mM KCl) (Perkin-Elmer Cetus), 3.5 µL of 25 mM MgCl2, 2 µl of primer (containing 25 µM of each primer, Con 2 and Con 3), and 9 U reverse transcriptase is then added to each denatured dsRNA sample tube (to give a final reaction volume of 49 µ1) and incubated at 42oC for 60 minutes.