Identification of G8 rotavirus strains determined as G12 by rotavirus genotyping PCR: updating the current genotyping methods.
BACKGROUND
Rotaviruses are classified into G- and P-types, which are determined by the reactivity with antibodies to the outer viral proteins, VP7 and VP4, respectively, or sequence variation in the genes encoding these proteins. There are presently a number of different rotavirus strains co-circulating within the UK, with the common human strains G1P[8], G2P[4] and G9P[8] being the most prevalent. As part of strain surveillance for the European Rotavirus Network (EuroRotaNet) a cluster (n=29) of G8 strains was detected in the UK between February and May 2009.


OBJECTIVE
G8 strains were initially mistyped as G12 through multiplex RT-PCR, therefore further investigation was performed to ascertain the reasons behind this mistyping.


METHODS
The genes encoding the VP7 of these G8 strains were sequenced and aligned with the existing G8- and G12-specific oligonucleotide primers.


RESULTS
Multiple alignment of sequences derived from these strains and the G8- and G12-specific oligonucleotide primers revealed a series of point mutations which resulted in mismatches at the 3' end of the G8-specific primer binding site that prevented amplification with the G8-specific primer, whilst a close homology with the G12-specific primer allowed mis-priming. Both the G8 and G12 primers were redesigned and their ability to correctly identify G8 and G12 strains was evaluated and confirmed.


CONCLUSIONS
These findings highlight the importance of monitoring the specificity and sensitivity of the genotyping methods in order to detect changes in the genotype distribution and changes associated with genetic drift of common or uncommon genotypes.
Health Protection Agency, Centre for Infections, 61 Colindale Avenue, London, NW9 5HT, United Kingdom.
Farah
F
Aladin
Sameena
S
Nawaz
Miren
M
Iturriza-Gómara
Jim
J
Gray
D000956:Antigens, Viral / Q000235:genetics; D020137:Base Pair Mismatch; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D003951:Diagnostic Errors; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D017354:Point Mutation; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000175:diagnosis / Q000821:virology*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D006113:United Kingdom
D000956:Antigens, Viral; D036022:Capsid Proteins; D017931:DNA Primers; D012367:RNA, Viral; C050779:VP7 protein, Rotavirus