Detection of field isolates of human and animal group C rotavirus by reverse transcription-polymerase chain reaction and digoxigenin-labeled oligonucleotide probes.
Rotaviruses (RV) are important etiological agents of acute gastroenteritis in infants and young children, as well as the young of a variety of animals worldwide. These viruses belong to Reoviridae family and contain a genome of 11 segments of double-stranded RNA (dsRNA). Two major proteins, VP4 and VP7, encoded by genome segments 4 and 7, 8 or 9, respectively, evoke a neutralizing antibody response and form the basis for the current classification of group (gp) A rotavirus into P (VP4) and G (VP7) serotypes. Although much recent progress has been made on the molecular biology of gp C RV, routine methods to detect and discriminate human, porcine, and bovine strains are not available widely. In this study, a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and digoxigenin-labeled (dig) oligonucleotide probes using chemiluminescence has been developed to detect and discriminate VP7 genes from culture-adapted and field isolates of human, porcine and bovine gp C RV. The multiplex RT-PCR and dig-probes were specific for the VP7 genes of human, porcine and bovine gp C RV and allowed detection and characterization of single and mixed infections of porcine gp C RV with porcine gp A or gp B rotaviruses. Detection rates for gp C RV were more than 50% when compared with polyacrylamide gel electrophoresis. These new diagnostic assays may help determine the epidemiological importance of these viruses in human and animal infections.
Alfieri
A A
AA
Laboratory of Animal Virology, Londrina State University (UEL), PR, Brazil.
Leite
J P
JP
Alfieri
A F
AF
Jiang
B
B
Glass
R I
RI
Gentsch
J R
JR
D000818:Animals; D000956:Antigens, Viral; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002417:Cattle; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D004076:Digoxigenin; D005759:Gastroenteritis / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D005814:Genes, Viral; D006801:Humans; D007223:Infant; D017720:Molecular Epidemiology; D015336:Molecular Probe Techniques; D015345:Oligonucleotide Probes; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D013552:Swine; D014773:Virology / Q000379:methods*
D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D015345:Oligonucleotide Probes; C050779:VP7 protein, Rotavirus; D004076:Digoxigenin