Abstract
BackgroundRotaviruses 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.MethodsViral 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.ResultsOf 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.ConclusionThe study highlights the importance of regularly updating the primers employed for molecular typing of rotaviruses.
Highlights
Rotaviruses with the P[8] genotype have been associated with majority of infections
RVA-positive samples (n = 80) whose VP4 genes could not be characterised using the pool of genotype specific primers recommended for use in the WHO Regional Reference Laboratory in Ghana were included in the study
Sequence mismatches were detected in the primer binding region of the target gene of the 43 Ghanaian P[8]a rotavirus strains when aligned with the available WHO P[8]a primers (1 T-1, 1 T-1Wa and 1 T1-VN) which might have resulted in genotyping failure
Summary
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. 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. Rotaviruses with the P[8] genotype have been associated with majority of these infections, accounting for approximately 74 % of global prevalence [2]. In previous WHO sponsored rotavirus surveillance studies in selected African countries, [8] the genotypes of 80 RVA-positive samples with first round RT-PCR amplification products could not be determined with the recommended primers presently available within the WHO Regional Reference Laboratory in Ghana. This study sought to determine genotypes of previously non-typeable rotavirus VP4 genes using sequence-dependent cDNA amplification methods
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