G and P genotyping of group ‘A’ rotavirus using RT-PCR and DNA probes

Siwatch, Deepti

G and P genotyping of group ‘A’ rotavirus using RT-PCR and DNA probes

Date

2005

Authors

Siwatch, Deepti

Publisher

LUVAS

Abstract

Group ‘A’ rotavirus is the major aetiologic agent associated with diarrheal diseases of human infants and young ones of many farm animals. It is the prototype virus of the genus Rotavirus in the family Reoviridae. Due to segmented nature of the RNA genome and wide host range, vast genetic and antigenic diversity exists amongst different isolates of rotavirus. Keeping this point in view, the present study was undertaken to characterize human group ‘A’ rotavirus circulating in Haryana. Of 110 human diarrheic stool samples collected, 75 (68.18%) were found positive for rotavirus by RNA-PAGE. Of the 75 RNA-PAGE positive samples, 58 (77.33%) were of long electropherotype and 17 (22.67%) were of short electropherotype. Fifty four of the seventy five RNA-PAGE rotavirus positive samples were subjected to characterization into G and P genotypes by RT-PCR. Fourty eight APPENDIX -viisamples (88.89%) were typed for various G types and 40 samples (74.07%) for various P types. G2 and G1 were the most prevalent G types followed by G4, G9 and G3. In case of P types, P4 was the most prevalent followed by P8, P6 and P10. There was high prevalence (24.07%) of mixed G and P genotypes in the strains circulating in this geographic region. G1P8 and G2P4 were found to be associated with 27.78% of rotavirus infection in the present study population. G2P4 being the most prevalent genotypic combination. Apart from the usual combinations, several unusual combinations such as G1P4, G1P6, G3P4, G4P4, G4P6, G9P4 and G9P6 were observed which constituted 22.22% of the locally prevalent rotavirus strains. Several unusual electropherotypes and genotype combinations were also seen. Nucleic acid hybridization assays using biotinlayted full length (1062 bp) type specific probes for G1, G2 and partial length (876 bp) DNA probe P4 type yielded reasonably good results for G and P genotyping of rotavirus positive field samples. Further characterization of the strains in terms of intratypic variation with in the prevalent G i.e. G1, G2 and P types i.e. P4 and P8 using XmnI, EcoRI, VspI, AluI , PstI and StyI restriction enzymes yielded results similar to the ones inferred from in silico analysis of the reference strains of the respective types. However, G2 strains remained undigested suggesting possible absence of restriction site for the enzymes used for the digestion of PCR product or possible mutation in a single base of restriction site which needs to be further validated using sequence studies.