Nucleotide variation in the VP7 gene affects PCR genotyping of G9 rotaviruses identified in Italy

Abstract

A modified (aFT9m) and a degenerate (aFT9d) version of the rotavirus G9-specific primer (aFT9) allowed strains that were previously untypable, because of point mutations accumulating at the primer binding site, to be G typed by reverse transcription-polymerase chain reaction. The strains were collected during 2001-2002 in Italy in hospitals of the Apulia region, from children affected by severe rotavirus-associated enteritis. Using a wide selection of G9 rotaviruses detected worldwide, sequencing of the G9 untypable strains, sequence comparison, and phylogenetic analysis showed that the Italian strains have strong genetic similarity (< or =99.4%) to G9 rotaviruses identified recently in many parts of the world and different from the old G9 strains identified during the 1980s (less than 90%). Genetic variation of G9 rotaviruses explains the constraints encountered in the typing assays and presumably accounts, together with genetic reassortment events, for the emergence on a global scale of the G9 serotype.

Sequencing and Phylogenetic Analysis of Human Genotype P[6] Rotavirus Strains Detected in Hungary Provides Evidence for Genetic Heterogeneity within the P[6] VP4 Gene

“…However, substantial differences were observed in the VP8‫ء‬ sequences of two additional P [6] strains, namely, AU19, a unique human G1 rotavirus displaying a supershort RNA electropherotype that was isolated from a child affected with severe diarrhea and dehydration in Japan (26), and the porcine G4 strain, Gottfried, that was originally isolated from the intestinal content of a suckling pig with diarrhea (3). These findings indicate that strains AU19 and Gottfried may be considered prototypes of two additional P [6] lineages (26) and also suggest that the P[6] VP4 gene has undergone genetic and antigenic drift, similar to what has been described for the P[8] VP4 gene and for the G1, G3, G4, G6, and G9 VP7 genes (4,12,16,17,22,24,25,34,36). Interestingly, all three lineages of genotype P [6] represent serologically distinct subtypes, designated P2A [6] (M37-like) and P2C [6] (AU19-like) for the human strains and P2B [6] (Gottfried-like) for the porcine strain (20,26).…”

Predominance of Rotavirus Genotype G9 during the 1999, 2000, and 2002 Seasons among Hospitalized Children in the City of Salvador, Bahia, Brazil: Implications for Future Vaccine Strategies

“…Previous studies have reported either a failure to genotype or the mistyping of contemporary G9 rotavirus strains, due to the accumulation of point mutations at the primer binding site (18,27,37) or the occurrence of antigenic variability (4). In this study, we used two different sets of primer pools (H2 pool and C pool), each of which contained a distinct G9 type-specific primer (aFT9 in the H2 pool and 9T-9B in the C pool), in order to identify correctly the VP7 specificity of the isolates, as previously suggested (37).…”

Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

“…A previous study, for example, demonstrated the mistyping of rotavirus G8 strains as being G3 by typing PCR due to the cross-reactivity of the G3-specific primer with G8 strains (1). Moreover, the mistyping of G9 strains has been reported, particularly when the H-1 pool is used, due to a cross-reactivity of the G4-specific primer with G9 strains (52,69). The use of three different probes per genotype in the PCR-ELISA minimized the chances of mistyping those variant strains.…”

“…Consequently, the methodologies used for rotavirus typing need to be monitored closely and updated accordingly to improve the capability in detecting and typing such strains. Indeed, a number of studies published lately have reported problems in genotyping certain human rotavirus field strains using the currently available methodologies such as typing PCR or monoclonal antibody-based ELISA (1,13,20,21,31,39,42,52,58,60,65,69,79).…”

“…Typing PCR for the detection and identification of human and animal rotavirus VP4 and VP7 genotypes that was developed in the early 1990s (15,24,27,28,29,33,38,76) has become the method of choice for strain genotyping, which can now be regarded as the "gold standard" (20). However, this technique also suffers from inherent shortcomings: point mutation(s) on the VP4 or VP7 gene at the primer-binding site may lead to failure in the typing process, and cross-priming of primers between different genotypes may produce false-positive reactivity (1,20,21,31,39,42,51,52,58,60,65,69). Sensitive and reliable diagnostic techniques that do not bear such disadvantages (11,17,36,49) are needed not only for accurate rotavirus strain surveillance but also for effective rotavirus vaccine development and evaluation (for example, analyses of homotypic versus heterotypic protection).…”