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.


CONCLUSIONS
The study highlights the importance of regularly updating the primers employed for molecular typing of rotaviruses.
Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana. sdamanka@noguchi.ug.edu.gh.
Susan
S
Damanka
Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana.
Francis E
FE
Dennis
Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana.
Chantal
C
Agbemabiese
Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana.
Belinda
B
Lartey
Department of Microbiology, University of Ghana Medical School, College of Health Sciences, Accra, Ghana.
Theophilus
T
Adiku
Department of Epidemiology and Disease Control, School of Public Health, College of Health Sciences, University of Ghana, Legon, Accra, Ghana.
Kofi
K
Nyarko
Department of Child Health, Korle Bu Teaching Hospital, College of Health Sciences, University of Ghana, Accra, Ghana.
Christabel C
CC
Enweronu-Laryea
Department of Microbiology, University of Ghana Medical School, College of Health Sciences, Accra, Ghana.
Kwamena W
KW
Sagoe
Department of Immunology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana.
Michael
M
Ofori
Department of Child Health, Korle Bu Teaching Hospital, College of Health Sciences, University of Ghana, Accra, Ghana.
Onike
O
Rodrigues
Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana.
George E
GE
Armah
D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D003967:Diarrhea / Q000821:virology*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D005869:Ghana; D006801:Humans; D007223:Infant; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000821:virology*; D017422:Sequence Analysis, DNA
D017931:DNA Primers
Ghana; Nucleotide sequence; P[8]b subtype; Phylogenetic analysis; Rotavirus