Modification of rotavirus multiplex RT-PCR for the detection of G12 strains based on characterization of emerging G12 rotavirus strains from South India

Abstract

Rotaviruses are the major etiological agents of diarrhea in children less than 5 years of age. The commonest G types in humans are G1-4 and G9. G12 is a rare human rotavirus (HRV) strain first reported in the Philippines. In this study, 13 G12 strains obtained from a community-based cohort and a hospital-based surveillance system in 2005 were characterized by phylogenetic analysis of partial nucleotide sequences of VP7, VP6, and NSP4 genes. Sequence and phylogenetic analysis of VP7 gene sequences showed that these southern Indian strains had the greatest homology with G12 strains recently reported from eastern India (97-99% identity both at the nucleotide level and deduced amino acid level) and less homology with the prototype G12 strain, L26 (89-90% identity at the nucleotide level and 90-94% at the deduced amino acid level). Phylogenetic analysis of the VP6 and the NSP4 genes revealed that the Vellore G12 strains belonged to VP6 subgroup II and NSP4 genotype B. The P types associated with these strains were P[6] and P[8]. A G12 type-specific primer was designed for inclusion in an established VP7 G-typing multiplex RT PCR, and tested against a panel of known G types and untyped samples and was found to detect G12 strains in the multiplex-PCR. Close homology of the South Indian G12 strains to those from Kolkata suggests that G12 HRV strains are emerging in India. Methods for characterization of rotaviruses in epidemiological studies need to be updated frequently, particularly in developing countries.

Fig. 1

Human rotavirus gene 9. Illustration of VP7 specific primer positions and type-specific amplicon sizes after incorporation of the new G12 primer in the VP7 multiplex PCR. The size of the expected product of amplification from first round amplification is 881 bp. The second round amplification products for gene 9 typing PCR are 754 bp (G8), 682 bp (G3), 618 bp (G1), 521 bp (G2), 452 bp (G4), 387 bp (G12), 266 bp (G10), and 179 bp (G9).

Fig. 2

Phylogenetic tree constructed using the maximum parsimony method and sequences of the VP7 encoding gene of the human G12 rotavirus strains form Vellore and of other strains, available in GenBank or the European rotavirus genotyping database. The name or reference number of the strains is indicated in the first column, G and P types are indicated in the second and third columns, respectively, and the geographical origin of the strains is indicated in the last two columns.

Fig. 3

Dendogram constructed by using NSP4 nucleotide sequences of the G12 rotavirus strains using the maximum parsimony method. Sequences representative of the different existing NSP4 genotypes were included for comparison. The name or reference number of the strains is indicated in the first column, G and P types are indicated in the second and third columns, respectively, the NSP4 genotype is in the third column, and the species from which the strain was obtained is indicated in the last column.

Fig. 4

Dendogram constructed with the maximum parsimony method using rotavirus VP6 partial sequences derived from G12 strains and other representative strains from GenBank and the European rotavirus genotyping database. The name or reference number of the strains is indicated in the first column, G and P types are indicated in the second and third columns, respectively, the subgroup is in the fourth column, and the geographical origin of the strains is indicated in the last two columns.

Fig. 5

Agarose gel electrophoresis of PCR products from the second round of VP7 specific multiplex RT-PCR. A panel of rotavirus strains of other genotypes, as well as the G12 strains, were identified after incorporation of the new G12 primer in the multiplex PCR.