Detection and Differentiation of Bovine Group A Rotavirus Serotypes using Polymerase Chain Reaction-Generated Probes to the VP7 Gene

Abstract

Abstract. Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Cracker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture-propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.

Molecular and Serologic Characterization of a Group a Bovine Rotavirus with a Short Genome Pattern

“…The G and P types of 2 other short genome pattern BRV strains, ID and OK, have been previously characterized. 20,21 Both strains have a G6, P5 specificity, similar to the IND BRV strain.…”

“…2,3,7-13, 16,18,21,26,32,41 Rotaviruses are also typed using similar procedures on the basis of VP4 specificity, and currently there are at least 4 P types of BRV (P1, P5, P11, and P12). 18,20,33 Upon analysis by PAGE, the 11 dsRNA segments of rotaviruses produce characteristic patterns referred to as genome electropherotypes.…”

“…29 The sources and serotypes (P and G) of the reference rotaviruses used in this study have been described previously. 20,21,24 In addition to the BRV strains described previously, 20,21 strain 2292B was obtained from Califomia.…”

“…Partial length VP7 cDNA (nucleotides 51-392) and VP4 cDNA (nucleotides 211-686), encompassing areas of major sequence diversity, were prepared by PCR amplification using specific primers. 20,21 The heat-denatured dsRNA was diluted 1:4 (5% DMSO) in a reaction mixture containing 10 mM Tris (pH 8. The tubes were placed in a thermocycler h and incubated at 42 C to generate cDNA copies of rotavirus dsRNA.…”

Detection of Serotype G6 Rotaviruses in Bovine Field Samples Using a Nonradioactive PCR-Derived cDNA Probe

“…Recently, characterization of rotavirus serotypes has been performed using serotype-specific monoclonal antibody (MAb)-based ELISA 1,6,11 and lately using tests based on the detection of the divergence of the VP7 gene sequence among rotaviruses of different serotypes such as nucleic acid hybridization, polymerase chain reaction (PCR) and gene sequencing. 3,4,[7][8][9][10][11][12] Nucleic acid probes obtained by PCR amplification of hyperviariable regions of VP7 genes have been used successfully for the G typing of human and animal rotaviruses.…”