Monitoring Shedding of Five Genotypes of RotaTeq Vaccine Viruses by Genotype-Specific Real-Time Reverse Transcription-PCR Assays

Abstract

RotaTeq (RV5) is a widely used live attenuated pentavalent rotavirus (RV) vaccine. Although fecal shedding of RV vaccine strains persists for long time periods, it is unclear how each vaccine strain replicates in intestinal tissue and is excreted in stool. To examine this issue, we established RV5 genotype-specific real-time reverse transcription-PCR (RT-PCR) assays. Five real-time RT-PCR assays were designed for the VP7 gene in genotypes G1, G2, G3, G4, and G6. All assays exhibited excellent linearity, and the detection limit was 1 infectious unit (IU)/reaction for G2, G4, and G6 and 10 IUs/reaction for G1 and G3. No cross-reactivity was observed among G genotypes. The inter- and intra-assay coefficients of variation were less than 3%. The assays were used to examine 129 stool samples collected from eight infants who received RV5. In cases 1 and 2, who received three rounds of vaccination, RV shedding decreased gradually with the number of vaccinations. G1 and G6 shedding appeared to be predominant in comparison to shedding of the other genotypes. Patterns of fecal shedding of the five genotypes of vaccine viruses differed between the eight vaccine recipients. RV5 genotype-specific real-time RT-PCR assays will be useful to study the molecular biology of RV5 replication in infants and experimental animals.

Persistent systemic rotavirus vaccine infection in a child with X‐linked severe combined immunodeficiency

“…Our recent study based on the RV5 G genotype‐specific real‐time RT‐PCR analysis suggested that patterns of fecal shedding of the five genotypes of vaccine viruses varied in vaccine recipients . Meanwhile, although RV5 contains five different genotypes, only G1 was detected in this patient.…”

“…Viral double‐stranded RNA (dsRNAs) were extracted from the stool suspensions and 140 μL sera using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. RV5‐specific real‐time reverse transcription polymerase chain reactions (RT‐PCRs) were developed by our institute . PrimerQuest (Integrated DNA Technologies, Coralville, IA) was used to design primers and probes to differentiate between the VP7 genes (G1, G2, G3, G4, and G6) based on the reference sequences of the vaccine strains.…”

“…Our recent study based on the RV5 G genotype-specific real-time RT-PCR analysis suggested that patterns of fecal shedding of the five genotypes of vaccine viruses varied in vaccine recipients. 15 Meanwhile, although RV5 contains five different genotypes, only G1 was detected in this patient. Next-generation sequence analysis revealed that persistent RV5 genotype G1 was produced by at least reassortment between two of the vaccine strains VP7 segment from G1P [5] and VP4 segment from G6P [8] (Table 1).…”

“…RV5-specific real-time reverse transcription polymerase chain reactions (RT-PCRs) were developed by our institute. 15 PrimerQuest (Integrated DNA Technologies, Coralville, IA) was used to design primers and probes to differentiate between the VP7 genes (G1, G2, G3, G4, and G6) based on the reference sequences of the vaccine strains. ZEN Double-Quenched Probes (Integrated DNA Technologies) were used to enhance assay sensitivity.…”

Genetic Characterizations and Molecular Evolution of VP7 Gene in Human Group A Rotavirus G1

“…The two licensed vaccines both include the G1 genotype, and G1 still remains a largely dominant G genotype worldwide [ 12 , 13 , 14 ]. It was also reported that G1 was the predominant shedding vaccine-related viral particle in vaccines [ 15 , 16 ], which may have an effect on the evolution of wild-type G1 circulating in populations. Thus, research on the long-term evolutionary characterizations of RVA G1 from a global perspective is desirable and is currently inadequate.…”