Publications by authors named "Satoshi Komoto"

81 Publications

Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes.

J Gen Virol 2021 Apr;102(4)

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.
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http://dx.doi.org/10.1099/jgv.0.001587DOI Listing
April 2021

Fibroblast activation protein targeted near infrared photoimmunotherapy (NIR PIT) overcomes therapeutic resistance in human esophageal cancer.

Sci Rep 2021 Jan 18;11(1):1693. Epub 2021 Jan 18.

Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8558, Japan.

Cancer-associated fibroblasts (CAFs) have an important role in the tumor microenvironment. CAFs have the multifunctionality which strongly support cancer progression and the acquisition of therapeutic resistance by cancer cells. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment that uses a highly selective monoclonal antibody (mAb)-photosensitizer conjugate. We developed fibroblast activation protein (FAP)-targeted NIR-PIT, in which IR700 was conjugated to a FAP-specific antibody to target CAFs (CAFs-targeted NIR-PIT: CAFs-PIT). Thus, we hypothesized that the control of CAFs could overcome the resistance to conventional chemotherapy in esophageal cancer (EC). In this study, we evaluated whether EC cell acquisition of stronger malignant characteristics and refractoriness to chemoradiotherapy are mediated by CAFs. Next, we assessed whether the resistance could be rescued by eliminating CAF stimulation by CAFs-PIT in vitro and in vivo. Cancer cells acquired chemoradiotherapy resistance via CAF stimulation in vitro and 5-fluorouracil (FU) resistance in CAF-coinoculated tumor models in vivo. CAF stimulation promoted the migration/invasion of cancer cells and a stem-like phenotype in vitro, which were rescued by elimination of CAF stimulation. CAFs-PIT had a highly selective effect on CAFs in vitro. Finally, CAF elimination by CAFs-PIT in vivo demonstrated that the combination of 5-FU and NIR-PIT succeeded in producing 70.9% tumor reduction, while 5-FU alone achieved only 13.3% reduction, suggesting the recovery of 5-FU sensitivity in CAF-rich tumors. In conclusion, CAFs-PIT could overcome therapeutic resistance via CAF elimination. The combined use of novel targeted CAFs-PIT with conventional anticancer treatments can be expected to provide a more effective and sensible treatment strategy.
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http://dx.doi.org/10.1038/s41598-021-81465-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814141PMC
January 2021

Genomic characterization of a novel G3P[10] rotavirus strain from a diarrheic child in Thailand: Evidence for bat-to-human zoonotic transmission.

Infect Genet Evol 2021 Jan 5;87:104667. Epub 2020 Dec 5.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.

An unusual rotavirus strain with the G3P[10] genotype (RVA/Human-wt/THA/MS2015-1-0001/2015/G3P[10]) was identified in a stool sample from a hospitalized child aged 11 months with severe gastroenteritis in Thailand. In the current study, we sequenced and characterized the full genome of strain MS2015-1-0001. On full-genomic analysis, strain MS2015-1-0001 exhibited the following genotype configuration: G3-P[10]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is identical or closely related to those of bat and bat-like rotavirus strains (MYAS33-like). Furthermore, phylogenetic analysis revealed that all 11 genes of strain MS2015-1-0001 appeared to be of bat origin. Our findings provide evidence for bat-to-human interspecies transmission of rotaviruses and important insights into dynamic interactions between human and bat rotavirus strains.
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http://dx.doi.org/10.1016/j.meegid.2020.104667DOI Listing
January 2021

Genomic analysis of group A rotavirus G12P[8] including a new Japanese strain revealed evidence for intergenotypic recombination in VP7 and VP4 genes.

Infect Genet Evol 2021 Jan 3;87:104656. Epub 2020 Dec 3.

Division of Microbiology, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan. Electronic address:

Group A rotavirus is a leading cause of severe acute gastroenteritis worldwide. In this study, the first complete coding sequences of 11 RNA segments of human group A rotavirus G12P[8] in Japan were determined by an unbiased viral metagenomics. Its genomic constellation (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes) was identified as G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. When performing the genetic analysis, we discovered an intergenotypic recombination event in the pig group A rotavirus G12P[8] strain BUW-14-A008. The novel recombination was found between two different genotypes G12 and G3 in the VP7 gene, and P[8] and P[13] in the VP4 gene.
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http://dx.doi.org/10.1016/j.meegid.2020.104656DOI Listing
January 2021

Reduction of severe acute respiratory syndrome coronavirus-2 infectivity by admissible concentration of ozone gas and water.

Microbiol Immunol 2021 Jan 14;65(1):10-16. Epub 2020 Dec 14.

Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Because complete elimination of SARS-CoV-2 appears difficult, decreasing the risk of transmission is important. Treatment with 0.1 and 0.05 ppm ozone gas for 10 and 20 hr, respectively, decreased SARS-CoV-2 infectivity by about 95%. The magnitude of the effect was dependent on humidity. Treatment with 1 and 2 mg/L ozone water for 10 s reduced SARS-CoV-2 infectivity by about 2 and 3 logs, respectively. Our results suggest that low-dose ozone, in the form of gas and water, is effective against SARS-CoV-2.
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http://dx.doi.org/10.1111/1348-0421.12861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753712PMC
January 2021

Unusual mono-reassortant of a Wa-like G1P[8] species A rotavirus containing a DS-1-like (genotype 2) NSP4 gene.

Virus Genes 2020 Oct 22;56(5):638-641. Epub 2020 Jul 22.

Division of Microbiology, Nihon University School of Medicine, Itabashi-ku, Tokyo, Japan.

Species A rotaviruses are a major cause of acute gastroenteritis in infants and young children worldwide. Reassortment is a common phenomenon due to the segmented nature of the rotavirus genome. The complete coding sequences of a species A rotavirus strain isolated from the feces of a child with acute gastroenteritis in Japan in 2018 were determined using an unbiased viral metagenomics approach. The genetic analysis revealed that the rotavirus strain had an unusual genomic constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), suggesting reassortment of a genotype 1 with a genotype 2 rotavirus, from which the NSP4-encoding gene was acquired.
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http://dx.doi.org/10.1007/s11262-020-01780-2DOI Listing
October 2020

Generation of recombinant rotaviruses from just 11 cDNAs encoding a viral genome.

Virus Res 2020 09 24;286:198075. Epub 2020 Jun 24.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.

Reverse genetics technology allows one to engineer replication-competent viruses from cloned cDNAs at will. Since the establishment of the initial reverse genetics system for species A rotaviruses (RVAs) requiring a helper virus in 2006, attempts have been successfully made to improve this technology. Efficient generation of replication-competent RVAs is now possible from just 11 T7-driven plasmids encoding an RVA genome when the quantity ratio of the two rescue T7-driven plasmids for the NSP2 and NSP5 segments is increased by 3-fold in relation to that of the other nine plasmids (11 plasmid-only system). Further, it is now possible to generate recombinant RVAs even with severely less efficient infectivity by using the 11 plasmid-only system, which has not been possible with the existing approaches. More importantly, the 11 plasmid-only system does not need any helper expression plasmid, and thus this simplest and robust system has a clear advantage over the existing systems in terms of safety. This 11 plasmid-only system should contribute to the development of safe next-generation vaccines and vaccine vectors.
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http://dx.doi.org/10.1016/j.virusres.2020.198075DOI Listing
September 2020

Molecular characterization of rotaviruses obtained from patients with rotavirus-associated encephalitis/encephalopathy.

Microbiol Immunol 2020 Aug 4;64(8):541-555. Epub 2020 Aug 4.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

Group A rotavirus (RVA) rarely causes severe complications such as encephalitis/encephalopathy. However, the pathophysiology of this specific complication remains unclear. Next-generation sequence analysis was used to compare the entire genome sequences of RVAs detected in patients with encephalitis/encephalopathy and gastroenteritis. This study enrolled eight patients with RVA encephalitis/encephalopathy and 10 with RVA gastroenteritis who were treated between February 2013 and July 2014. Viral RNAs were extracted from patients' stool, and whole-genome sequencing analysis was carried out to identify the specific gene mutations in RVA obtained from patients with severe neurological complications. Among the eight encephalitis/encephalopathy cases, six strains were DS-1-like G1P[8] and the remaining two were Wa-like G1P[8] (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). Meanwhile, eight of the 10 viruses detected in rotavirus gastroenteritis patients were DS-1-like G1P[8], and the remaining two were Wa-like G1P[8]. These strains were further characterized by conducting phylogenetic analysis. No specific clustering was demonstrated in RVAs detected from encephalitis/encephalopathy patients. Although the DS-1-like G1P[8] strain was predominant in both groups, no specific molecular characteristics were detected in RVAs from patients with severe central nervous system complications.
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http://dx.doi.org/10.1111/1348-0421.12827DOI Listing
August 2020

Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics.

J Gen Virol 2020 08 3;101(8):806-815. Epub 2020 Jun 3.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.

Reassortment is an important mechanism in the evolution of group A rotaviruses (RVAs), yielding viruses with novel genetic and phenotypic traits. The classical methods for generating RVA reassortants with the desired genetic combinations are laborious and time-consuming because of the screening and selection processes required to isolate a desired reassortant. Taking advantage of a recently developed RVA reverse genetics system based on just 11 cloned cDNAs encoding the RVA genome (11 plasmid-only system), we prepared a panel of simian SA11-L2 virus-based single-gene reassortants, each containing 1 segment derived from human KU virus of the G1P[8] genotype. It was shown that there was no gene-specific restriction of the reassortment potential. In addition to these 11 single-gene reassortants, a triple-gene reassortant with KU-derived core-encoding VP1-3 gene segments with the SA11-L2 genetic background, which make up a virion composed of the KU-based core, and SA11-L2-based intermediate and outer layers, could also be prepared with the 11 plasmid-only system. Finally, for possible clinical application of this system, we generated a series of VP7 reassortants representing all the major human RVA G genotypes (G1-4, G9 and G12) efficiently. The preparation of each of these single-gene reassortants was achieved within just 2 weeks. Our results demonstrate that the 11 plasmid-only system allows the rapid and reliable generation of RVA single-gene reassortants, which will be useful for basic research and clinical applications.
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http://dx.doi.org/10.1099/jgv.0.001443DOI Listing
August 2020

Full genome characterization of novel DS-1-like G9P[8] rotavirus strains that have emerged in Thailand.

PLoS One 2020 22;15(4):e0231099. Epub 2020 Apr 22.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231099PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176146PMC
July 2020

Reverse genetics system for human rotaviruses.

Microbiol Immunol 2020 Jun 2;64(6):401-406. Epub 2020 May 2.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.
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http://dx.doi.org/10.1111/1348-0421.12795DOI Listing
June 2020

High prevalence of equine-like G3P[8] rotavirus in children and adults with acute gastroenteritis in Thailand.

J Med Virol 2020 02 19;92(2):174-186. Epub 2019 Sep 19.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

Group A rotavirus (RVA) is a major cause of acute gastroenteritis in infants and young children worldwide. This study aims to clarify the distribution of G/P types and genetic characteristics of RVAs circulating in Thailand. Between January 2014 and September 2016, 1867 stool specimens were collected from children and adults with acute gastroenteritis in six provinces in Thailand. RVAs were detected in 514/1867 (27.5%) stool specimens. G1P[8] (44.7%) was the most predominant genotype, followed by G3P[8] (33.7%), G2P[4] (11.5%), G8P[8] (7.0%), and G9P[8] (1.3%). Unusual G3P[9] (0.8%), G3P[10] (0.4%), G4P[6] (0.4%), and G10P[14] (0.2%) were also detected at low frequencies. The predominant genotype, G1P[8] (64.4%), in 2014 decreased to 6.1% in 2016. In contrast, the frequency of G3P[8] markedly increased from 5.5% in 2014 to 65.3% in 2015 and 89.8% in 2016. On polyacrylamide gel electrophoresis, most (135/140; 96.4%) of the G3P[8] strains exhibited a short RNA profile. Successful determination of the nucleotide sequences of the VP7 genes of 98 G3P[8] strains with a short RNA profile showed that they are all equine-like G3P[8] strains. On phylogenetic analysis of genome segments of two representative Thai equine-like G3P[8] strains, it was noteworthy that they possessed distinct NSP4 genes, one bovine-like and the other human-like. Thus, we found that characteristic equine-like G3P[8] strains with a short RNA electropherotype are becoming highly prevalent in children and adults in Thailand.
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http://dx.doi.org/10.1002/jmv.25591DOI Listing
February 2020

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

Cell Microbiol 2019 12 26;21(12):e13101. Epub 2019 Aug 26.

Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata, India.

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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http://dx.doi.org/10.1111/cmi.13101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162324PMC
December 2019

Characterization of an Unusual DS-1-Like G8P[8] Rotavirus Strain from Japan in 2017: Evolution of Emerging DS-1-Like G8P[8] Strains through Reassortment.

Jpn J Infect Dis 2019 Jul 28;72(4):256-260. Epub 2019 Feb 28.

Department of Virology and Parasitology, Fujita Health University School of Medicine.

The emergence of unusual DS-1-like intergenogroup reassortant rotaviruses with a bovine-like G8 genotype (DS-1-like G8P[8] strains) has been reported in several Asian countries. During the rotavirus surveillance program in Japan in 2017, a DS-1-like G8P[8] strain (RVA/Human-wt/JPN/SO1162/2017/G8P[8]) was identified in 43 rotavirus-positive stool samples. Strain SO1162 was shown to have a unique genotype constellation, including genes from both genogroup 1 and 2: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that the VP1 gene of strain SO1162 appeared to have originated from DS-1-like G1P[8] strains from Thailand and Vietnam, while the remaining 10 genes were closely related to those of previously reported DS-1-like G8P[8] strains. Thus, SO1162 was suggested to be a reassortant strain that acquired the VP1 gene from Southeast Asian DS-1-like G1P[8] strains on the genetic background of co-circulating DS-1-like G8P[8] strains. Our findings provide important insights into the evolutionary dynamics of emerging DS-1-like G8P[8] strains.
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http://dx.doi.org/10.7883/yoken.JJID.2018.484DOI Listing
July 2019

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

J Virol 2019 04 3;93(8). Epub 2019 Apr 3.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

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. 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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http://dx.doi.org/10.1128/JVI.02207-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450123PMC
April 2019

A Novel Combination Cancer Therapy with Iron Chelator Targeting Cancer Stem Cells via Suppressing Stemness.

Cancers (Basel) 2019 Feb 3;11(2). Epub 2019 Feb 3.

Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan (Y.K.).

Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as , , , , and , and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy.
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http://dx.doi.org/10.3390/cancers11020177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406536PMC
February 2019

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

J Med Virol 2019 06 6;91(6):1008-1013. Epub 2019 Feb 6.

Department of Pediatrics, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan.

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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http://dx.doi.org/10.1002/jmv.25410DOI Listing
June 2019

[Reverse genetics of rotaviruses: Generation of recombinant human rotaviruses from just 11 cDNAs encoding the rotavirus genome].

Uirusu 2019 ;69(1):1-12

Department of Virology and Parasitology, Fujita Health University School of Medicine.

An entirely plasmid-based reverse genetics system for animal rotavirus was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 T7 plasmids for its 11 genes under the condition of increasing the ratio (3- or 5-fold) of the cDNA plasmids for NSP2 and NSP5 genes (11-plasmid system). Utilizing this highly efficient system, we engineered the first infectious recombinant rotaviruses harboring fluorescent (EGFP and mCherry) protein genes. In addition to these recombinant animal viruses, the first infectious recombinant human rotavirus (strain KU (G1P[8])) was also generated with the 11-plasmid system with some modifications. The availability of recombinant human rotaviruses will provide a genetic platform for a better understanding of the replication, pathogenicity, and other biological characteristics of this medically important virus and enable the rational development of next-generation human rotavirus vaccines.
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http://dx.doi.org/10.2222/jsv.69.1DOI Listing
January 2019

Genomic characterization of uncommon human G3P[6] rotavirus strains that have emerged in Kenya after rotavirus vaccine introduction, and pre-vaccine human G8P[4] rotavirus strains.

Infect Genet Evol 2019 03 10;68:231-248. Epub 2018 Dec 10.

Kenya Research Station, Institute of Tropical Medicine (NEKKEN), Kenya Medical Research Institute (KEMRI)/Nagasaki University, Nairobi 19993-00202, Kenya.

A monovalent rotavirus vaccine (RV1) was introduced to the national immunization program in Kenya in July 2014. There was increased detection of uncommon G3P[6] strains that coincided temporally with the timing of this vaccine introduction. Here, we sequenced and characterized the full genomes of two post-vaccine G3P[6] strains, RVA/Human-wt/KEN/KDH1951/2014/G3P[6] and RVA/Human-wt/KEN/KDH1968/2014/G3P[6], as representatives of these uncommon strains. On full-genomic analysis, both strains exhibited a DS-1-like genotype constellation: G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that all 11 genes of strains KDH1951 and KDH1968 were very closely related to those of human G3P[6] strains isolated in Uganda in 2012-2013, indicating the derivation of these G3P[6] strains from a common ancestor. Because the uncommon G3P[6] strains that emerged in Kenya are fully heterotypic as to the introduced vaccine strain regarding the genotype constellation, vaccine effectiveness against these G3P[6] strains needs to be closely monitored.
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http://dx.doi.org/10.1016/j.meegid.2018.12.004DOI Listing
March 2019

Cancer-associated fibroblasts (CAFs) promote the lymph node metastasis of esophageal squamous cell carcinoma.

Int J Cancer 2019 02 3;144(4):828-840. Epub 2018 Dec 3.

Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

Lymph node metastasis is a pathognomonic feature of spreading tumors, and overcoming metastasis is a challenge in attaining more favorable clinical outcomes. Esophageal cancer is an aggressive tumor for which lymph node metastasis is a strong poor prognostic factor, and the tumor microenvironment (TME), and cancer-associated fibroblasts (CAFs) in particular, has been implicated in esophageal cancer progression. CAFs play a central role in the TME and have been reported to provide suitable conditions for the progression of esophageal cancer, similar to their role in other malignancies. However, little is known concerning the relevance of CAFs to the lymph node metastasis of esophageal cancer. Here, we used clinical samples of esophageal cancer to reveal that CAFs promote lymph node metastasis and subsequently verified the intercellular relationships in vitro and in vivo using an orthotopic metastatic mouse model. In the analysis of clinical samples, FAP CAFs were strongly associated with lymph node metastasis rather than with other prognostic factors. Furthermore, CAFs affected the ability of esophageal cancer cells to acquire metastatic phenotypes in vitro; this finding was confirmed by data from an in vivo orthotopic metastatic mouse model showing that the number of lymph node metastases increased upon injection of cocultured cancer cells and CAFs. In summary, we verified in vitro and in vivo that the accumulation of CAFs enhances the lymph node metastasis of ESCC. Our data suggest that CAF targeted therapy can reduce lymph node metastasis and improve the prognosis of patients with esophageal cancer in the future.
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http://dx.doi.org/10.1002/ijc.31953DOI Listing
February 2019

Cancer-Associated Fibroblasts Affect Intratumoral CD8 and FoxP3 T Cells Via IL6 in the Tumor Microenvironment.

Clin Cancer Res 2018 10 19;24(19):4820-4833. Epub 2018 Jun 19.

Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a central role in tumor progression. We investigated whether CAFs can regulate tumor-infiltrating lymphocytes (TILs) and their role in tumor immunosuppression. A total of 140 cases of esophageal cancer were analyzed for CAFs and CD8 or forkhead box protein 3 (FoxP3) TILs by IHC. We analyzed cytokines using murine or human fibroblasts and cancer cells. Murine-derived fibroblasts and cancer cells were also inoculated into BALB/c or BALB/c- mice and the tumors treated with recombinant IL6 or anti-IL6 antibody. CD8 TILs and CAFs were negatively correlated in intratumoral tissues ( < 0.001), whereas FoxP3 TILs were positively correlated ( < 0.001) in esophageal cancers. Cocultured Colon26 cancer cells and fibroblasts resulted in accelerated tumor growth in BALB/c mice, along with decreased CD8 and increased FoxP3 TILs, compared with cancer cells alone. , IL6 was highly secreted in both murine and human cancer cell/fibroblast cocultures. IL6 significantly increased Colon26 tumor growth in immune-competent BALB/c ( < 0.001) with fewer CD8 TILs than untreated tumors ( < 0.001), whereas no difference in BALB/c- mice. In contrast, FoxP3 TILs increased in IL6-treated tumors ( < 0.001). IL6 antibody blockade of tumors cocultured with fibroblasts resulted not only in regression of tumor growth but also in the accumulation of CD8 TILs in intratumoral tissues. CAFs regulate immunosuppressive TIL populations in the TME via IL6. IL6 blockade, or targeting CAFs, may improve preexisting tumor immunity and enhance the efficacy of conventional immunotherapies. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0205DOI Listing
October 2018

Characterization of a G10P[14] rotavirus strain from a diarrheic child in Thailand: Evidence for bovine-to-human zoonotic transmission.

Infect Genet Evol 2018 09 15;63:43-57. Epub 2018 May 15.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.

An unusual rotavirus strain, DB2015-066 with the G10P[14] genotype (RVA/Human-wt/THA/DB2015-066/2015/G10P[14]), was detected in a stool sample from a child hospitalized with acute gastroenteritis in Thailand. Here, we sequenced and characterized the full-genome of the strain DB2015-066. On whole genomic analysis, strain DB2015-066 was shown to have a unique genotype constellation: G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The backbone genes of this strain (I2-R2-C2-M2-A3-N2-T6-E2-H3) are commonly found in rotavirus strains from artiodactyls such as cattle. Furthermore, phylogenetic analysis indicated that each of the 11 genes of strain DB2015-066 could be of artiodactyl (likely bovine) origin. Thus, strain DB2015-066 appeared to be derived from through zoonotic transmission of a bovine rotavirus strain. Of note, the VP7 gene of strain DB2015-066 was located in G10 lineage-6 together with ones of bovine and bovine-like rotavirus strains, away from the clusters comprising other G10P[14] strains in G10 lineage-2/4/5/9, suggesting the occurrence of independent bovine-to-human interspecies transmission events. Our observations provide important insights into the origins of rare G10P[14] strains, and into dynamic interactions between artiodactyl and human rotavirus strains.
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http://dx.doi.org/10.1016/j.meegid.2018.05.009DOI Listing
September 2018

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

J Virol 2018 07 13;92(13). Epub 2018 Jun 13.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors. Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.
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http://dx.doi.org/10.1128/JVI.00588-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002737PMC
July 2018

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

J Clin Microbiol 2018 06 25;56(6). Epub 2018 May 25.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

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.
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http://dx.doi.org/10.1128/JCM.00035-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971533PMC
June 2018

Impact of rotavirus vaccination on rotavirus hospitalisation rates among a resource-limited rural population in Mbita, Western Kenya.

Trop Med Int Health 2018 04 8;23(4):425-432. Epub 2018 Mar 8.

Institute of Tropical Medicine, Kenya Research Station, KEMRI/Nagasaki University, Nairobi, Kenya.

Objectives: A two-dose oral monovalent rotavirus vaccine (RV1) was introduced into the Kenyan National Immunization Program in July 2014. We assessed trends in hospitalisation for rotavirus-specific acute gastroenteritis (AGE) and strain distribution among children <5 years in a rural, resource-limited setting in Kenya before and after the nationwide implementation of the vaccine.

Methods: Data on rotavirus AGE and strain distribution were derived from a 5-year hospital-based surveillance. We compared rotavirus-related hospitalisations and strain distribution in the 2-year post-vaccine period with the 3-year pre-vaccine baseline. Vaccine administrative data from the Unit of Vaccines and Immunization Services (UVIS) for Mbita sub-county were used to estimate rotavirus immunisation coverage in the study area.

Results: We observed a 48% (95% CI: 27-64%) overall decline in rotavirus-related hospitalisations among children aged <5 years in the post-vaccine period. Coverage with the last dose of rotavirus vaccine increased from 51% in year 1% to 72% in year 2 of the vaccine implementation. Concurrently, reductions in rotavirus hospitalisations increased from 40% in the first year to 53% in the second year of vaccine use. The reductions were most pronounced among the vaccine-eligible group, with the proportion of cases in this age group dropping to 14% in post-vaccine years from a high of 51% in the pre-vaccine period. A diversity of rotavirus strains circulated before the introduction of the vaccine with G1P[8] being the most dominant strain. G2P[4] replaced G1P[8] as the dominant strain after the vaccine was introduced.

Conclusions: Rotavirus vaccination has resulted in a notable decline in hospital admissions for rotavirus infections in a rural resource-limited population in Kenya. This provides early evidence for continued use of rotavirus vaccines in routine childhood immunisations in Kenya. Our data also underscore the need for expanding coverage on second dose so as to maximise the impact of the vaccine.
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http://dx.doi.org/10.1111/tmi.13040DOI Listing
April 2018

[Radical Thoracoscopic Esophagectomy for Elderly Patients with Advanced Esophageal Cancer].

Gan To Kagaku Ryoho 2017 Nov;44(12):1784-1786

Dept. of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences.

We report a case of an elderly patient with advanced esophageal cancer who underwent multidisciplinary treatment. An 86-year-old male consulted our hospital with complaints of pharynx discomfort and difficulty in swallowing. He was preoperatively diagnosed with esophageal cancer, T3N2M0, Stage III . We performed 2 courses of cisplatin plus 5-FU therapy as neoadjuvant chemotherapy. The primary tumor and metastatic lymph nodes reduced in size, and thoracoscopic esophagectomy in the prone position was performed. Pathological findings were esophageal cancer, pT3-Ad, INF b, ly2, v1, IM0, pPM0, pDM0, pRM1, pN3, pStage III . As the radical margin was positive, chemoradiotherapy was performed. We continued postoperative chemotherapy for approximately 1 year, and the patient has survived without relapse for 4 years from esophagectomy. Even in patients over 80 years old, long-term prognosis can be expected by performing radical surgery and chemoradiotherapy.
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November 2017

[Successful Multimodality Treatment Including Three-Stage Operation for Esophageal Cancer with Esophagorespiratory Fistula - A Case Report].

Gan To Kagaku Ryoho 2017 Nov;44(12):1053-1055

Dept. of Gastroenterological Surgery, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences.

The esophagorespiratory fistula(ERF)is a fatal complication ofesophageal cancer, because ofadvanced oncological status and poor conditions due to pneumonia and/or malnutrition.We report here a case of patient who was successfully treated for esophageal cancer with ERF with multimodality therapy including three-stage operation. A 65-year-old woman ofesophageal cancer received preoperative chemotherapy, and developed EFR before operation. Prolonged conservative therapies for ERF let her general condition get worse. Therefore, the patient underwent esophagostomy and gastrostomy to recover her condition. She received chemo-radiotherapy followed by esophagectomy. And she was performed the reconstruction next month. She is still alive without recurrence at 20 months after resection. In previous reports, a total of 6 cases have been performed esophagectomy for esophageal cancer with ERF in Japan. Only one case was reported that had survived longer than 12 months. This multimodality therapy can be one ofthe best strategies for the patients ofesophageal cancer with ERF, even ifthey have poor condition.
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November 2017

Characterization of unusual DS-1-like G3P[8] rotavirus strains in children with diarrhea in Japan.

J Med Virol 2018 05 5;90(5):890-898. Epub 2018 Feb 5.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

The emergence and rapid spread of novel DS-1-like intergenogroup reassortant rotaviruses having the equine-like G3 genotype (DS-1-like G3P[8] strains) have been recently reported from several countries. During rotavirus surveillance in Japan in 2015-2016, three DS-1-like G3P[8] strains were identified from children with severe diarrhea. In the present study, we sequenced and characterized the full genomes of these three strains. On full-genomic analysis, all three strains showed a unique genotype constellation including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that each of the 11 genes of the three strains was closely related to that of Japanese DS-1-like G1P[8] and/or Japanese equine-like G3P[4] human strains. Thus, the three study strains were suggested to be reassortants that acquired the G3-VP7 gene from equine G3 rotaviruses on the genetic background of DS-1-like G1P[8] strains. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G3P[8] strains.
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http://dx.doi.org/10.1002/jmv.25016DOI Listing
May 2018

Rotavirus Vaccination Can Be Performed Without Viral Dissemination in the Neonatal Intensive Care Unit.

J Infect Dis 2018 01;217(4):589-596

Department of Pediatrics, School of Medicine, Fujita Health University Toyoake, Konan, Japan.

Background: This study was conducted to assess the transmissibility of rotavirus vaccine strains after rotavirus vaccination in a neonatal intensive care unit (NICU).

Methods: Pentavalent (RV5) or monovalent (RV1) rotavirus vaccine was administered to infants admitted to the NICU. Nineteen vaccinated infants and 49 unvaccinated infants whose beds were located in close proximity to the vaccinated infants were enrolled in this study. Dissemination and fecal shedding of vaccine viruses within the NICU were examined using real-time reverse transcription-polymerase chain reaction.

Results: Shedding of the vaccine strain was detected in all 19 vaccinated infants. RV5 virus shedding started 1 day after the first vaccination and persisted for 8 days after the first vaccination, and viral shedding terminated by day 5 after administration of the second RV5 dose. The kinetics of RV1 virus shedding differed among vaccinated infants. The duration of RV1 virus shedding was longer after the first vaccination than after the second vaccination. In contrast to the vaccinated infants, no vaccine virus genomes were detected in any of the stool samples collected from the 49 unvaccinated infants.

Conclusions: This study is direct evidence of no transmission of rotavirus vaccine strains between vaccinated infants and unvaccinated infants in close proximity within a NICU.
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http://dx.doi.org/10.1093/infdis/jix590DOI Listing
January 2018

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

J Virol 2017 11 13;91(21). Epub 2017 Oct 13.

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.
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http://dx.doi.org/10.1128/JVI.00695-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640853PMC
November 2017