| Nucleic acid probes were developed for the detection and differentiation of porcine rotaviruses with different serotype specificities. Initial studies were conducted with nucleic acid probes prepared from full- and partial-length gene 4 (coding for VP4) and gene 9 (coding for VP7) cDNA of the Gottfried (G serotype 4, P serotype 6), and OSU (G serotype 5, P serotype 7) porcine rotaviruses. Partial-length gene 4 and 9 cDNA were prepared by polymerase chain reaction (PCR) amplification using specific primers, and encompassed areas of major sequence variability (nucleotides 51 to 392, 212 to 612, and 211 to 607 for Gottfried and OSU genes 9, OSU gene 4, and Gottfried gene 4, respectively) among rotaviruses of different serotypes. The full-length gene 9 probes were specific and hybridized with the respective Gottfried or OSU porcine rotaviruses, and serologically defined human and animal rotaviruses of the same G serotype by dot hybridization. The Gottfried gene 9 probe hybridized with human rotaviruses ST3 and VA70, and porcine rotaviruses Gottfried, SB-1A, SB2, SB3, and SB5. The OSU gene 9 probe hybridized with the equine H1 rotavirus, and porcine rotaviruses OSU and EE. The PCR-derived gene 9 probes were equally sensitive, however, the hybridization specificities of the Gottfried probe was restricted to porcine rotaviruses. The full-length gene 4 probes were in general 4 probes were specific for gene 4 by Northern blot hybridization. Analysis of 52 rotavirus-positive field samples collected in the U.S. and Canada revealed a high percentage (67.3%) of rotaviruses that did not hybridize with the probes. Analysis of these samples with PCR-derived gene 9 probes prepared from human rotaviruses Wa (G!), DS-1 (G2), and P (G3) did not detect rotaviruses with homologous gene specificities. Hybridization analyses with a PCR-derived gene 9 probe from the G11 porcine rotavirus YM detected 11 rotaviruses with similar genetic homologies. |
