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An in-vitro transcription assay for development of Rotavirus VP7.

ABSTRACT: Background and Objectives:Human rotavirus (RV) is responsible for most cases of acute gastroenteritis in infants, worldwide. Today, in vitro transcription (IVT) assay is widely used to develop efficient RNA for the biological experiments such as gene function analysis and reverse genetics. The aim of this study was to develop optimal full-length transcripts of the VP7 segment, using in vitro transcription assay. Materials and Methods:Special primers were designed in order to synthesize VP7 sequence of sense RNA in the process of IVT using T7 RNA polymerase. RT-PCR was performed using forward and reverse primers, containing T7 promoter sequence and BstUI restriction enzyme site, respectively. In order to synthesize ssRNA VP7, in accordance with the IVT technique, RV4-VP7 fragment was subcloned into PTZ57 R/T plasmid and digested by BstUI enzyme. Results:The sequencing of the VP7 gene showed 99% identity withVP7 gene of rotavirus RV4 strain (Sequence ID: M64666.1). The analysis of purity of DNA fragment and ssRNA VP7 segment revealed that OD ratio of A260/A280 and quantity of nucleic acids were (1.9, 0.036 ?g/?L) and (2.02, 0.98 ?g/?L), respectively. Conclusion:In the present study, a modified methodology of RNA synthetase was described by IVT assay, using T7RNA polymerase in order to transcribe the full-length transcripts of human VP7-RV4 strain. This method is applicable for reverse genetic approaches, especially for the production of reassortant RV vaccine.

SUBMITTER: Jalilian S

PROVIDER: S-EPMC5719513 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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An <i>in-vitro</i> transcription assay for development of Rotavirus VP7.

Jalilian Shahram S Teimoori Ali A Makvandi Manoochehr M Zandi Milad M

Iranian journal of microbiology 20170601 3

<h4>Background and objectives</h4>Human rotavirus (RV) is responsible for most cases of acute gastroenteritis in infants, worldwide. Today, <i>in vitro</i> transcription (IVT) assay is widely used to develop efficient RNA for the biological experiments such as gene function analysis and reverse genetics. The aim of this study was to develop optimal full-length transcripts of the VP7 segment, using <i>in vitro</i> transcription assay.<h4>Materials and methods</h4>Special primers were designed in ...[more]

PMID: 29225758

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Project description:Species A rotaviruses (RVs) are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years. Currently available RV vaccines were adapted from wild-type RV strains by serial passage of cultured cells or by reassortment between human and animal RV strains. These traditional methods require large-scale screening and genotyping to obtain vaccine candidates. Reverse genetics is a tractable, rapid, and reproducible approach to generating recombinant RV vaccine candidates carrying any VP4 and VP7 genes that provide selected antigenicity. Here, we developed a vaccine platform by generating recombinant RVs carrying VP4 (P[4] and P[8]), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P[2]) as a backbone. Neutralization assays using monoclonal antibodies and murine antisera revealed that recombinant VP4 and VP7 monoreassortant viruses exhibited altered antigenicity. However, replication of VP4 monoreassortant viruses was severely impaired. Generation of recombinant RVs harboring a chimeric VP4 protein for SA11 and human RV gene components revealed that the VP8* fragment was responsible for efficient infectivity of recombinant RVs. Although this system must be improved because the yield of vaccine viruses directly affects vaccine manufacturing costs, reverse genetics requires less time than traditional methods and enables rapid production of safe and effective vaccine candidates.IMPORTANCE Although vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral antigenicity, growth capacity, and pathogenicity. Here, we generated recombinant simian RVs (strain SA11) carrying heterologous VP4 and VP7 genes cloned from clinical isolates and showed that VP4- or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.

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An in-vitro transcription assay for development of Rotavirus VP7.

Publications

An <i>in-vitro</i> transcription assay for development of Rotavirus VP7.

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