Identification of OP354-like human rotavirus strains with subtype P[8]b in Ghanaian children with diarrhoea.

Background: Rotaviruses with the P[8] genotype have been associated with majority of infections. Recent improvements in molecular diagnostics have delineated the P[8] genotype into P[8]a and P[8]b subtypes. P[8]a is the previously known P[8] genotype which is common whilst P[8]b subtype also known as OP354-like strain is genetically distinct, rarely detected and reported from a few countries. In a previous study, the P-types could not be determined for 80 RVA-positive samples by conventional RT-PCR genotyping methods with the recommended pool of P-genotype specific primers used in the WHO Regional Rotavirus Reference Laboratory in Ghana. The present study employed sequence-dependent cDNA amplification method to genotype previously non-typeable P-types.

Methods: Viral RNAs were extracted and rotavirus VP4 genes amplified by one step RT-PCR using gene specific primers. PCR amplicons were purified, sequenced and sequences aligned with cognate gene sequences available in GenBank using the ClustalW algorithm. Phylogenetic analysis was performed using the Neighbour-Joining method in MEGA v6.06 software. Phylogenetic tree was statistically supported by bootstrapping with 1000 replicates, and distances calculated using the Kimura-2 parameter model.

Results: Of the 80 RVA-positive samples, 57 were successfully sequenced and characterized. Forty-eight of these were identified as P[8] strains of which 5 were characterized as the rare P[8]b subtype. Phylogenetic analysis of the VP8* fragment of the VP4 genes of these P[8]b strains revealed a close relationship with prototype OP354-like P[8]b strain and P[8]b strains of Russian and South African P[8]b origin.

Conclusion: The study highlights the importance of regularly updating the primers employed for molecular typing of rotaviruses.