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 μ g/μ L) and (2. 02, 0. 98 μ g/μ 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.

Introduction: Vaccination is the most effective measure to prevent Rotavirus infection in children under five years of age. The most important targets of neutralizing and protective antibodies against this virus are VP4 and VP7 proteins of Rotavirus. Today, the recombinant protein produced in yeast plays an important role in producing highly effective and cost-effective vaccines. Methods: The effect of different linkers, including flexible and rigid, were evaluated on the stability and immunogenicity of the protein via in silico assays. A suitable linker was selected and expressed in Pichia pastoris yeast. Prediction and validation were carried out using bioinformatics tools, including Expasy's ProtPara, Phyre2 online server, I-TASSER server, ElliPro. Moreover, an appropriate linker was selected for cloning into pPICZα and expression in P. pastoris. Results: The results showed that as a flexible linker, (GGGGS)3 was the best structure to provide stability for VP4-VP7 target fusion protein. The produced recombinant protein was stable after the expression. Conclusion: These in silico results and expression data on P. pastoris suggested that VP4– (GGGGS)3-VP7 construct can potentially serve as a potent immunogenic candidate for recombinant Rotavirus vaccines.

Background and Aims: In humans the group A rotaviruses are associated with endemic diarrhea in children under the age of 5, leading to approximately 800, 000 deaths every year. Introduction of rotavirus vaccines into childhood immunization programs can contribute to substantial reduction in mortality from rotavirus gastroenteritis in developing countries and virtually eliminating hospitalizations due to rotavirus gastroenteritis, a heavy burden in developed countries. A reassortant Rhesus-human rotavirus vaccine, Rotashield was manufactured and marketed in 1998. Nine months later a small number of cases of intussusceptions were identified, leading to the withdrawal of vaccine.Important questions remain for these oral live vaccines related with safety andside effects as well as production costs, thus development of second generation of nonreplicating rotavirus vaccine should be considered as an alternative to live vaccines. VP7 is major outer capsid glycoprotein; can induce production of neutralizing antibodies. Regarding these concepts we cloned VP7 gene of SA11 rotavirus in pGEM vector for future expression.Methods: With large scale expression and purification of VP7 protein, it will be convenient to study VP7 antigenic, immunogenic, biologic functions and its potential for vaccine development.Results: BSC-1 cells were grown as a monolayer. Simian rotavirus, SA11 was cultured. Oligonucleotide forward and reverse primers, specific for gene segment 9 which encodes VP7, were synthesized, according to dsRNA gene segment 9 of simian rotavirus SA11 sequence data at NCBI GeneBank. Rotavirus ds-RNA was extracted and used as template for reverse transcription to synthesize cDNA from both viral strands.cDNA product of RT-PCR was cloned into pGEM@-5Zf (-) vector and the results showed that rotavirus genome segment 9 was cloned into pGEM@-5Zf (-) vector and confirmed by restriction enzyme and sequencing.Conclusion: Sequencing result was analyzed by BLAST software showing 100% homology with 91SA11 rotavirus genome segment in NCBI GeneBank.a

In humans the group A rotaviruses are associated with endemic diarrhea in children under the age of 5, leading to approximately 800,000 deaths every year. Introduction of rotavirus vaccines into childhood immunization programs can contribute to substantial reduction in mortality from rotavirus gastroenteritis in developing countries and virtually eliminating hospitalizations due to rotavirus gastroenteritis, a heavy burden in developed countries. A reassortant Rhesus-human rotavirus vaccine, Rotashield was manufactured and marketed in 1998. Nine months later a small number of cases of intussusceptions were identified, leading to the withdrawal of vaccine. Important questions remain for these oral live vaccines related with safety and side effects as well as production costs, thus development of second generation of nonreplicating rotavirus vaccine should be considered as an alternative to live vaccines. VP7 is major outer capsid glycoprotein; can induce production of neutralizing antibodies. Regarding these concepts we cloned VP7 gene of SA11 rotavirus in pGEM vector for future expression. With large scale expression and purification of VP7 protein, it will be convenient to study VP7 antigenic, immunogenic, biologic functions and its potential for vaccine development.BSC-1 cells were grown as a monolayer. Simian rotavirus, SA11 was cultured. Oligonucleotide forward and reverse primers, specific for gene segment 9 which encodes VP7, were synthesized, according to dsRNA gene segment 9 of simian rotavirus SA 11 sequence data at NCBI GeneBank. Rotavirus ds-RNA was extracted and used as template for reverse transcription to synthesize cDNA from both viral strands. cDNA product of RT-PCR was cloned into pGEMÒ-5Zf(-) vector and the results showed that rotavirus genome segment 9 was cloned into pGEMÒ-5Zf(-) vector and confirmed by restriction enzyme and sequencing. Sequencing result was analyzed by BLAST software showing 100% homology with 9th SA11 rotavirus genome segment in NCBI GeneBank.

Acute gastroenteritis caused by Rotavirus remains the leading cause of child mortality worldwide. Rotavirus genotype G9 circulates in humans throughout the world. Antibodies against the outer glycoproteins VP7 and VP4 Rotavirus capsid are the main neutralization antibodies in the vaccine assessment. This study aimed to select an epitope to evoke T and B cells' response, as a favorable candidate for vaccine development using in Silico evaluation. In the present study, Rotavirus genotypes were determined in 100 stools specimens collected from children with acute diarrhea. The results showed predominant G genotype, G9 (38. 5%) followed by G2 (22. 9%), G1 (16. 5%), G12 (11. 4%), G4 (6. 4%), and G3 (4. 3%). The G9 was dominant in this study and other regions of Iran; thus, this study was conducted to select an epitope from Rotavirus genotype G9 as a promising epitope candidate for future vaccine development. For this reason, several works including a complete sequence of VP7 G9, phylogenetic analysis, Prediction of Protein Structure, Physicochemical Properties of Protein and Epitope prediction were carried out. The outcomes of this study revealed that the complete sequence of VP7 (G9) was comprised of 1062 nt with 326 amino acids (accession number MH824633). The selected epitope contained amino acid sequence of STLCLYYPTEASTQIGDTEWKN with the best score for T and B cells response. Based on data of computational biology, the selected epitope can optimistically have considered as an epitope candidate for Rotavirus vaccine development.