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Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples.

ABSTRACT: Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix(®) and RotaTeq(®) are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix(®) and RotaTeq(®) vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix(®) and RotaTeq(®) vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix(®) (NSP2, VP4) and RotaTeq(®) (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix(®) NSP2 and VP4 qRT-PCR assays exhibited 92-100% sensitivity, 99-100% specificity, 94-105% efficiency, and a limit of detection of 2-3 copies per reaction. RotaTeq(®) VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94-100% specificity, 91-102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix(®) and RotaTeq(®) vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE.

SUBMITTER: Gautam R

PROVIDER: S-EPMC4130254 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples.

Gautam Rashi R Esona Mathew D MD Mijatovic-Rustempasic Slavica S Ian Tam Ka K Gentsch Jon R JR Bowen Michael D MD

Human vaccines & immunotherapeutics 20131216 3

Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix(®) and RotaTeq(®) are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix(®) and RotaTeq(®) vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor ...[more]

PMID: 24342877

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Project description:Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8-100% sensitivity, 100% specificity, 86-89% efficiency and a limit of detection of 12-400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82-90% efficiency and limit of detection of 120-4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

Project description:Group A rotavirus (RVA) is the leading cause of severe childhood diarrhea globally, even with all effective interventions, particularly in developing countries. Among the diverse genotypes of RVA, G1P[8] is a common genotype that has continued to pervade around the world, including Pakistan. Two universally accepted rotavirus vaccines-Rotarix™ and RotaTeq™ contain the genotype G1P[8]. The current work was aimed at identifying differences between antigenic epitopes of Pakistan's G1P[8] strains and those of the two licensed vaccines. We sequenced 6 G1P[8] rotavirus strains previously reported in Rawalpindi, Islamabad, Pakistan in 2015 and 2016 for their outer capsid genes (VP7 and VP4). Phylogenetic analysis was then conducted in order to classify their specific lineages and to detect their association with strains isolated throughout world. Compared with the Rotarix™ and RotaTeq™ vaccine strains (G1-lineage II, P[8]-lineage III), our study G1-lineage I, P[8]-lineage IV strains showed 3 and 5 variations in the VP7 epitopes, respectively, and 13 and 11 variations in the VP4 epitopes, respectively. The G1 lineage II strains showed no single amino acid change compared to Rotarix™ (lineage II), but exhibited changes at 2 positions compared to RotaTeq™ (lineage III). So, this has been proposed that these G1 strains exist in our natural setting, or that they may have been introduced in Pakistan from other countries of the world. The distinct P[8]-lineage IV (OP354-like) strains showed twelve and thirteen amino acid variations, with Rotarix™ and RotaTeq™ (lineages II and III) strains, respectively. Such findings have shown that the VP4-P[8] component of the G1P[8] strains circulating in Pakistan differs considerably from that of the vaccine viruses compared to that of the VP7-G1. To monitor the long-term effects of vaccines on the emergence of G1P[8] strains with different lineages, routine and successful monitoring of these strains will be crucial.

Dataset Information

Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples.

Publications

Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples.

Similar Datasets

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