Improvement of Rotavirus Genotyping Method by Using the Semi-Nested Multiplex-PCR With New Primer Set.

Yoshiki Fujii,Yen Hai Doan,Ikuo Shoji,Maria Inge Lusida,Takako Utsumi,Kazuhiko Katayama,Rury Mega Wahyuni

doi:10.3389/fmicb.2019.00647

Abstract

Rotavirus A (RVA) is a major cause of gastroenteritis in infants and young children. After vaccine introduction, RVA surveillance has become more important for monitoring changes in genotype distribution, and the semi-nested multiplex-PCR is a popular method for RVA genotyping. In particular, the VP7 primer set reported by Gouvea and colleagues in 1990 is still widely used worldwide as the recommended WHO primer set in regional and national reference RVA surveillance laboratories. However, this primer set yielded some mistakes with recent epidemic strains. The newly emerged equine-like G3 strains were mistyped as G1, G8 strains were mistyped as G3, the G9 lineage 3 strains showed very weak band, and the G9 lineage 6 strains showed a G9-specific band and a non-specific band. Gouvea’s standard protocol has become relatively unreliable for identifying genotypes correctly. To overcome this limitation, we redesigned the primer set to include recent epidemic strains. Our new primer set enabled us to correctly identify the VP7 genotypes of representative epidemic strains by agarose gel electrophoresis (G1, G2, human typical G3, equine-like G3, G4, G8, G9, and G12). We believe that the multiplex-PCR method with our new primer set is a useful and valuable tool for surveillance of RVA epidemics.

Highlights

Rotavirus A (RVA), a member of the Reoviridae family, is a major cause of gastroenteritis in infants and young children worldwide
Ten representative RVA strains circulating in Japan (Supplementary Table S1, except for G12 strain) were selected for evaluating the Gouvea primer set
Equine-like G3 strain showed an incorrect band near the size of G1

Summary

Introduction

Rotavirus A (RVA), a member of the Reoviridae family, is a major cause of gastroenteritis in infants and young children worldwide. In 2016, RVA caused 128,500 deaths in children under 5 years of age globally (Troeger et al, 2018). RVA imposes a huge burden even on developed countries including Japan (Nakagomi et al, 2013). Two live attenuated RVA vaccines, Rotarix (GlaxoSmithKline, Biologicals, Belgium) and RotaTeq (Merck & Co., Inc., United States), were introduced in Japan in November 2011 and July 2012, respectively. The vaccines are very effective (Lambert et al, 2009; Leshem et al, 2014; Karafillakis et al, 2015), but the selective pressure of vaccines may induce an epidemic strain shift

Methods

Results

Conclusion