Determination of Human Rotavirus VP6 Genogroups I and II by Reverse Transcription-PCR

Abstract

Based on nucleotide sequence and phylogenetic analysis of the partial VP6 genes, group A rotaviruses can be mainly differentiated into two genogroups. In this study, a method employing reverse transcription-PCR (RT-PCR) and degenerate primers was established to assign the VP6 genogroup. VP6 genogroup I and genogroup II could be determined according to the sizes of the amplicons: 380 and 780 bp, respectively. The VP6 genogroup of human reference strains of G1 to G4 and G9 types and RotaTeq vaccine strains could be properly assigned by RT-PCR. Eighty rotavirus-positive fecal samples were subjected to enzyme-linked immunosorbent assay (ELISA), RT-PCR, and sequencing of the partial VP6 gene for subgroup and genogroup determination. The results correlated well among these three methods, except for seven samples whose subgroups could not be determined by ELISA. VP6 genogroups of another 150 rotavirus strains recovered between 1981 and 2005 were determined by RT-PCR and sequencing, and the same results were obtained by these two methods. Furthermore, an additional 524 rotavirus-positive fecal samples were tested by RT-PCR, and the VP6 genogroups could be easily determined. The RT-PCR assay developed here provided a reliable and convenient method for assigning the VP6 genogroups of human rotaviruses with a wide range of genetic variation.

Rotavirus genotype distribution during the pre-vaccine period in Bolivia: 2007–2008

“…19 The extracted RNA was used for RT-PCR as described to ascertain G, P, and VP6 genotypes. 20–22 Amplicons were analyzed on a 1.5% Tris–borate–EDTA (TBE) agarose gel and viewed under ultraviolet illumination after ethidium bromide staining.…”

Rotavirus infection and the current status of rotavirus vaccines

“…78,79 The fifth reassortant virus expresses the attachment protein (P1A [8]) from the human rotavirus parent strain and the G6 outer capsid protein from the bovine rotavirus parent strain. 72,96 RotaTeq is administered in three oral doses at 1-to 2-month intervals, beginning at 6e12 weeks of age. 96 Table 2 outlines the major phase III trials on human rotavirus efficacy in various settings.…”

Multiple‐gene characterization of rotavirus strains: Evidence of genetic linkage among the VP7‐, VP4‐, VP6‐, and NSP4‐encoding genes

“…The cDNA of the rotavirus VP6-encoding gene was synthesized by RT-PCR using the primers 6BEG.303 and VP6R [Lin et al, 2008]. Primers NSP41a and NSP41b were used for the NSP4-encoding gene [Kudo et al, 2001].…”

“…The amplified products were subjected to a nested-PCR reaction with genotype-specific primers that were described earlier for both genes. A set of primers that allow identification of the main human I genotypes (I1 and I2, former genogroups SG II and SG I, respectively) was used for VP6 A [Lin et al, 2008]. The PCR conditions were as follows: 3 min at 948C, followed by 10 cycles of PCR, each consisting of 1 min at 948C, 40 sec at 468C, and 1 min at 728C, then 10 cycles of 1 min at 948C, 40 sec at 488C, and 1 min at 728C, and finally 15 cycles of 1 min at 948C, 40 sec at 508C, and 1 min at 728C.…”