Generation of Recombinant Rotaviruses Expressing Fluorescent Proteins by Using an Optimized Reverse Genetics System

Komoto, Satoshi; Fukuda, Seisuke; Ide, Tomihiko; Ito, Naoto; Sugiyama, Makoto; Yoshikawa, Tetsushi; Murata, Takayuki; Taniguchi, Koki

doi:10.1128/jvi.00588-18

Cited by 78 publications

(145 citation statements)

References 39 publications

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“…To determine whether this hypothesis is correct or not, it is necessary to evaluate the superiority of G1 type VP7 and P [8] type VP4 in RVA infection by using a reverse genetics system of RVA in future studies. [19][20][21] Gene rearrangements have been detected in chronic RVA infection in immunodeficient children, 22,23 and in vitro during passages at a high multiplicity of infection in cell culture. 24,25 Since PAGE analysis suggested that the gene rearrangements occurred in several genes including NSP5 gene in RVAs collected from the patient (Figure 2), sequence analysis was carried out to confirm the gene rearrangements.…”

Section: Discussionmentioning

confidence: 99%

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

Yoshikawa

Ihira²,

Higashimoto

et al. 2019

Journal of Medical Virology

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Objective The main aims of the present study were to elucidate the systemic group A rotavirus (RVA) infection and to clarify the genetic changes of persistent virus in the X‐linked severe combined immunodeficiency (SCID) patient. Methods RotaTeq vaccine (RV5) genotype‐specific real‐time reverse transcription polymerase chain reaction was used to monitor viral RNA load in serially collected serum and stool samples. Next‐generation sequence analysis was used to determine the genotype of the virus by sequencing 11 gene segments. Polyacrylamide gel electrophoresis (PAGE) analysis was used to identify rearrangement of viral genes. The gene rearrangement was examined in NSP5 gene by using Sanger sequence. Results A 7‐month‐old boy demonstrated chronic diarrhea following the third administration of RV5 and failure to thrive. He was diagnosed with X‐linked SCID and successfully underwent cord blood transplantation. High copy numbers of RV5 genotype G1 RNA were detected in serially collected stool and serum samples and the kinetics of viral RNA loads were correlated with the degree of clinical disease. Next‐generation sequence analysis revealed genetic reassortment at least between the strains WI79‐9/G1P7[5] and WI79‐4/G6P1A[8] in the VP7 gene and the VP4 gene among the vaccine‐derived rotavirus strains. In addition, PAGE analysis suggested genetic rearrangements in several genes, and it was confirmed in the NSP5 gene by sequence analysis. Conclusions The kinetics of RVA RNA load in serum and stool samples was consistent with the clinical course of the patient. Among five genotypes of RV5 vaccine, G1 genotype replicated well in this patient. Reassortment and rearrangements were demonstrated in persistently infected G1 genotype of RV5.

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Section: Discussionmentioning

confidence: 99%

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

Yoshikawa

Ihira²,

Higashimoto

et al. 2019

Journal of Medical Virology

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“…All antibodies were used at manufacturer's recommended dilution. Rabbit polyclonal antibodies against RV‐SA11 structural proteins VP1 and VP4 (Komoto et al, ) and nonstructural proteins NSP1 (Komoto et al, ), NSP3, NSP4, and NSP5 (Komoto et al, ) were raised according to standard protocols at the Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan.…”

Section: Methodsmentioning

confidence: 99%

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2

Mukhopadhyay

Chanda

Patra

et al. 2019

Cellular Microbiology

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RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter‐defensive measures by producing virulence determinants called viral‐suppressors‐of‐RNAi (VSRs). Interestingly, in spite of dominance of interferon‐based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double‐stranded RNA genome. Intriguingly, a time point‐dependent bimodal regulation of RNAi was observed in RV‐infected cells, where short interfering RNA (siRNA)‐based RNAi was rendered non‐functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA‐mediated RNAi, via ubiquitin–proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.

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“…Alternatively, a versatile entirely plasmidbased reverse genetics system was developed recently (14). Entirely plasmid-based reverse genetics systems have been developed only for a single animal RVA strain, simian SA11-L2, which grows to high titers in cell cultures (14)(15)(16). Three strategies for entirely plasmid-based reverse genetics have been developed for this animal virus.…”

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Generation of Infectious Recombinant Human Rotaviruses from Just 11 Cloned cDNAs Encoding the Rotavirus Genome

Komoto

Fukuda

Kugita

et al. 2019

J Virol

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The generation of recombinant group A rotaviruses (RVAs) entirely from cloned cDNAs has been described only for a single animal RVA strain, simian SA11-L2. We recently developed an optimized RVA reverse genetics system based on only RVA cDNAs (11-plasmid system), in which the concentration of cDNA plasmids containing the NSP2 and NSP5 genes is 3- or 5-fold increased in relation to that of the other plasmids. Based on this approach, we generated a recombinant human RVA (HuRVA)-based monoreassortant virus containing the VP4 gene of the simian SA11-L2 virus using the 11-plasmid system. In addition to this monoreassortant virus, authentic HuRVA (strain KU) was also generated with the 11-plasmid system with some modifications. Our results demonstrate that the 11-plasmid system involving just RVA cDNAs can be used for the generation of recombinant HuRVA and recombinant HuRVA-based reassortant viruses. IMPORTANCE Human group A rotavirus (HuRVA) is a leading pathogen causing severe diarrhea in young children worldwide. In this paper, we describe the generation of recombinant HuRVA (strain KU) from only 11 cloned cDNAs encoding the HuRVA genome by reverse genetics. The growth properties of the recombinant HuRVA were similar to those of the parental RVA, providing a powerful tool for better understanding of HuRVA replication and pathogenesis. Furthermore, the ability to manipulate the genome of HuRVAs “to order” will be useful for next-generation vaccine production for this medically important virus and for the engineering of clinical vectors expressing any foreign genes.

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Generation of Recombinant Rotaviruses Expressing Fluorescent Proteins by Using an Optimized Reverse Genetics System

Cited by 78 publications

References 39 publications

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

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

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2

Generation of Infectious Recombinant Human Rotaviruses from Just 11 Cloned cDNAs Encoding the Rotavirus Genome

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