Publications by authors named "Kazuhiko Katayama"

139 Publications

Direct derivation of human alveolospheres for SARS-CoV-2 infection modeling and drug screening.

Cell Rep 2021 06 19;35(10):109218. Epub 2021 May 19.

Department of Organoid Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan; Coronavirus Task Force, Keio University School of Medicine, Tokyo 160-8582, Japan. Electronic address:

Although the main cellular target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be alveolar cells, the absence of their tractable culture system precludes the development of a clinically relevant SARS-CoV-2 infection model. Here, we establish an efficient human alveolosphere culture method and sphere-based drug testing platform for SARS-CoV-2. Alveolospheres exhibit indolent growth in a Wnt- and R-spondin-dependent manner. Gene expression, immunofluorescence, and electron microscopy analyses reveal the presence of alveolar cells in alveolospheres. Alveolospheres express ACE2 and allow SARS-CoV-2 to propagate nearly 100,000-fold in 3 days of infection. Whereas lopinavir and nelfinavir, protease inhibitors used for the treatment of human immunodeficiency virus (HIV) infection, have a modest anti-viral effect on SARS-CoV-2, remdesivir, a nucleotide prodrug, shows an anti-viral effect at the concentration comparable with the circulating drug level. These results demonstrate the validity of the alveolosphere culture system for the development of therapeutic agents to combat SARS-CoV-2.
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http://dx.doi.org/10.1016/j.celrep.2021.109218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133488PMC
June 2021

Molecular and clinical characterization of the equine-like G3 rotavirus that caused the first outbreak in Japan, 2016.

J Gen Virol 2021 Mar 15;102(3). Epub 2021 Feb 15.

Department of Pediatrics, Sapporo Medical University School of Medicine, Sapporo, Japan.

Since 2013, equine-like G3 rotavirus (eG3) strains have been detected throughout the world, including in Japan, and the strains were found to be dominant in some countries. In 2016, the first eG3 outbreak in Japan occurred in Tomakomai, Hokkaido prefecture, and the strains became dominant in other Hokkaido areas the following year. There were no significant differences in the clinical characteristics of eG3 and non-eG3 rotavirus infections. The eG3 strains detected in Hokkaido across 2 years from 2016 to 2017 had DS-1-like constellations (i.e. G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2), and the genes were highly conserved (97.5-100 %). One strain, designated as To16-12 was selected as the representative strain for these strains, and all 11 genes of this strain (To16-12) exhibited the closest identity to one foreign eG3 strain (STM050) seen in Indonesia in 2015 and two eG3 strains (IS1090 and MI1125) in another Japanese prefecture in 2016, suggesting that this strain might be introduced into Japan from Indonesia. Sequence analyses of VP7 genes from animal and human G3 strains found worldwide did not identify any with close identity (>92 %) to eG3 strains, including equine RV Erv105. Analysis of another ten genes indicated that the eG3 strain had low similarity to G2P[4] strains, which are considered traditional DS-1-like strains, but high similarity to DS-1-like G1P[8] strains, which first appeared in Asia in 2012. These data suggest that eG3 strains were recently generated in Asia as mono-reassortant strain between DS-1-like G1P[8] strains and unspecified animal G3 strains. Our results indicate that rotavirus surveillance in the postvaccine era requires whole-genome analyses.
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http://dx.doi.org/10.1099/jgv.0.001548DOI Listing
March 2021

Impact of the Heterogeneity in Free Chlorine, UV, and Ozone Susceptibilities Among Coxsackievirus B5 on the Prediction of the Overall Inactivation Efficiency.

Environ Sci Technol 2021 03 15;55(5):3156-3164. Epub 2021 Feb 15.

Department of Urban Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan.

The disinfection susceptibilities of viruses vary even among variants, yet the inactivation efficiency of a certain virus genotype, species, or genus was determined based on the susceptibility of its laboratory strain. The objectives were to evaluate the variability in susceptibilities to free chlorine, UV, and ozone among 13 variants of coxsackievirus B5 (CVB5) and develop the model allowing for predicting the overall inactivation of heterogeneous CVB5. Our results showed that the susceptibilities differed by up to 3.4-fold, 1.3-fold, and 1.8-fold in free chlorine, UV, and ozone, respectively. CVB5 in genogroup B exhibited significantly lower susceptibility to free chlorine and ozone than genogroup A, where the laboratory strain, Faulkner, belongs. The capsid protein in genogroup B contained a lower number of sulfur-containing amino acids, readily reactive to oxidants. We reformulated the Chick-Watson model by incorporating the probability distributions of inactivation rate constants to capture the heterogeneity. This expanded Chick-Watson model indicated that up to 4.2-fold larger free chlorine CT is required to achieve 6-log inactivation of CVB5 than the prediction by the Faulkner strain. Therefore, it is recommended to incorporate the variation in disinfection susceptibilities for predicting the overall inactivation of a certain type of viruses.
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http://dx.doi.org/10.1021/acs.est.0c07796DOI Listing
March 2021

A Longitudinal Study on Enteric Virus Contamination in Bivalves along the Coast of Ibaraki Prefecture, Japan.

J Food Prot 2021 Jun;84(6):946-952

Gunma Paz University Graduate School of Health Science, Gunma, Japan.

Abstract: During the 2014 to 2018 seasons, we conducted a longitudinal study involving enteric virus surveillance in bivalves, including natural oysters and clams harvested in Ibaraki Prefecture, Japan. Some norovirus (NoV) contaminations were detected in natural oysters, whereas no enteric virus was found in clams. NoVs detected in oysters were of the genotypes GII.4 and GII.6, both of which are closely related genetically to the NoV strains prevalent in humans. We found low level of enteric virus contamination in bivalves collected along the coast of Ibaraki Prefecture. The possibility of food poisoning caused by these viruses appears low, and few cases of infectious disease have been observed in the surrounding area. The harvest timing was more related to contamination quantity than the harvest area in many enteric viruses. Our results highlight that contamination of bivalves by enteric viruses may depend upon the prevalence of human diarrhea and illness.

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http://dx.doi.org/10.4315/JFP-20-353DOI Listing
June 2021

Molecular epidemiology and genetic diversity of norovirus infection in children hospitalized with acute gastroenteritis in East Java, Indonesia in 2015-2019.

Infect Genet Evol 2021 Mar 2;88:104703. Epub 2021 Jan 2.

Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan. Electronic address:

Noroviruses are recognized as a leading cause of outbreaks and sporadic cases of acute gastroenteritis (AGE) among individuals of all ages worldwide, especially in children <5 years old. We investigated the epidemiology of noroviruses among hospitalized children at two hospitals in East Java, Indonesia. Stool samples were collected from 966 children with AGE during September 2015-July 2019. All samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for the amplification of both the RNA-dependent RNA polymerase (RdRp) and the capsid genes of noroviruses. The genotypes were determined by phylogenetic analyses. In 2015-2019, noroviruses were detected in 12.3% (119/966) of the samples. Children <2 years old showed a significantly higher prevalence than those ≥2 years old (P = 0.01). NoV infections were observed throughout the year, with the highest prevalence in December. Based on our genetic analyses of RdRp, GII.[P31] (43.7%, 31/71) was the most prevalent RdRp genotype, followed by GII.[P16] (36.6%, 26/71). GII.[P31] was a dominant genotype in 2016 and 2018, whereas GII.[P16] was a dominant genotype in 2015 and 2017. Among the capsid genotypes, the most predominant norovirus genotype from 2015 to 2018 was GII.4 Sydney_2012 (33.6%, 40/119). The most prevalent genotype in each year was GII.13 in 2015, GII.4 Sydney_2012 in 2016 and 2018, and GII.3 in 2017. Based on the genetic analyses of RdRp and capsid sequences, the strains were clustered into 13 RdRp/capsid genotypes; 12 of them were discordant, e.g., GII.4 Sydney[P31], GII.3[P16], and GII.13[P16]. The predominant genotype in each year was GII.13[P16] in 2015, GII.4 Sydney[P31] in 2016, GII.3[P16] in 2017, and GII.4 Sydney[P31] in 2018. Our results demonstrate high detection rates and genetic diversity of norovirus GII genotypes in pediatric AGE samples from Indonesia. These findings strengthen the importance of the continuous molecular surveillance of emerging norovirus strains.
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http://dx.doi.org/10.1016/j.meegid.2020.104703DOI Listing
March 2021

Dynamic rotation of the protruding domain enhances the infectivity of norovirus.

PLoS Pathog 2020 07 2;16(7):e1008619. Epub 2020 Jul 2.

National Institute for Physiological Sciences, Okazaki, Japan.

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the resting conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two similar P domain conformations were also found simultaneously in the human norovirus GII.3 capsid, although the mechanism of the conformational change is not yet clear. These results provide new insights into the mechanisms of non-enveloped norovirus transmission that invades host cells, replicates, and sometimes escapes the hosts immune system, through dramatic environmental changes in the gastrointestinal tract.
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http://dx.doi.org/10.1371/journal.ppat.1008619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331980PMC
July 2020

Molecular pharmacology of ciclesonide against SARS-CoV-2.

J Allergy Clin Immunol 2020 08 13;146(2):330-331. Epub 2020 Jun 13.

Department of Microbiology, Yokohama City University School of Medicine, Kanagawa, Japan.

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http://dx.doi.org/10.1016/j.jaci.2020.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293530PMC
August 2020

Molecular evolution of the capsid () region in human norovirus genogroup II genotype 3.

Heliyon 2020 May 3;6(5):e03835. Epub 2020 May 3.

Department of Health Science, Gunma Paz University Graduate School of Health Sciences, 1-7-1 Tonyamachi, Takasaki-shi, Gunma 370-0006, Japan.

Norovirus GII.3 has been suggested to be a prevalent genotype in patients with acute gastroenteritis. However, the genetic properties of the region encoding the major GII.3 antigen remain unclear. Here, we performed molecular evolutionary analyses of the GII.3 region detected in various countries. We performed time-scaled phylogenetic analyses, selective pressure analyses, phylogenetic distance analyses, and conformational epitope analyses. The time-scaled phylogenetic tree showed that an ancestor of the GII.3 region diverged from the common ancestors of the GII.6, GII.11, GII.18, and GII.19 approximately 70 years ago with relatively low divergence. The evolutionary rate of the GII.3 region was rapid (4.82 × 10 substitutions/site/year). Furthermore, one positive site and many negative selection sites were observed in the capsid protein. These results suggest that the GII.3 region rapidly evolved with antigenic variations.
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http://dx.doi.org/10.1016/j.heliyon.2020.e03835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205756PMC
May 2020

Identification of novel norovirus polymerase genotypes from pediatric fecal samples collected between the year 1997 and 2000 in Japan.

Infect Genet Evol 2020 08 4;82:104313. Epub 2020 Apr 4.

Laboratory of Viral Infection I, Department of Infection Control and Immunology, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan.

We analyzed 46 pediatric fecal samples collected between the years 1997 and 2000 to retrospectively evaluate the norovirus strains circulating during that era and to identify possible re-emergence patterns. From the tested fecal samples, we detected GII.1, GII.3, GII.4 (95/96-US) and GII.6 strains. Most importantly, two novel polymerase genotypes (GI.PNA4 and GII.PNA7) were detected during the study. Two possible recombinant strains (GII.6[P7] and GII.3[P29]) were identified and SimPlot analysis confirmed that GII.6[P7] is a recombinant strain. The study emphasizes the importance of retrospective evaluation of human fecal samples in obtaining a better understanding of norovirus circulation, re-emergence and evolution.
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http://dx.doi.org/10.1016/j.meegid.2020.104313DOI Listing
August 2020

Molecular Evolution of the Protease Region in Norovirus Genogroup II.

Front Microbiol 2019 14;10:2991. Epub 2020 Jan 14.

Graduate School of Health Sciences, Gunma Paz University, Takasaki, Japan.

Noroviruses are a major cause of viral epidemic gastroenteritis in humans worldwide. The protease (Pro) encoded in open reading frame 1 (ORF1) is an essential enzyme for proteolysis of the viral polyprotein. Although there are some reports regarding the evolutionary analysis of norovirus GII-encoding genes, there are few reports focused on the region. We analyzed the molecular evolution of the region of norovirus GII using bioinformatics approaches. A time-scaled phylogenetic tree of the region constructed using a Bayesian Markov chain Monte Carlo method indicated that the common ancestor of GII diverged from GIV around 1680 CE [95% highest posterior density (HPD), 1607-1749]. The GII region emerged around 1752 CE (95%HPD, 1707-1794), forming three further lineages. The evolutionary rate of GII region was estimated at more than 10 substitutions/site/year. The distribution of the phylogenetic distances of each genotype differed, and showed genetic diversity. Mapping of the negative selection and substitution sites of the Pro structure showed that the substitution sites in the Pro protein were mostly produced under neutral selection in positions structurally adjacent to the active sites for proteolysis, whereas negative selection was observed in residues distant from the active sites. The phylodynamics of GII.P4, GII.P7, GII.P16, GII.P21, and GII.P31 indicated that their effective population sizes increased during the period from 2005 to 2016 and the increase in population size was almost consistent with the collection year of these genotypes. These results suggest that the region of the norovirus GII evolved rapidly, but under no positive selection, with a high genetic divergence, similar to that of the RNA-dependent RNA polymerase () region and the region of noroviruses.
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http://dx.doi.org/10.3389/fmicb.2019.02991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971112PMC
January 2020

Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids.

Proc Natl Acad Sci U S A 2020 01 2;117(3):1700-1710. Epub 2020 Jan 2.

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030;

Human noroviruses (HuNoVs) cause sporadic and epidemic outbreaks of gastroenteritis in all age groups worldwide. We previously reported that stem cell-derived human intestinal enteroid (HIE) cultures support replication of multiple HuNoV strains and that some strains (e.g., GII.3) replicate only in the presence of bile. Heat- and trypsin-treatment of bile did not reduce GII.3 replication, indicating a nonproteinaceous component in bile functions as an active factor. Here we show that bile acids (BAs) are critical for GII.3 replication and replication correlates with BA hydrophobicity. Using the highly effective BA, glycochenodeoxycholic acid (GCDCA), we show BAs act during the early stage of infection, BA-dependent replication in HIEs is not mediated by detergent effects or classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling but involves another G protein-coupled receptor, sphingosine-1-phosphate receptor 2, and BA treatment of HIEs increases particle uptake. We also demonstrate that GCDCA induces multiple cellular responses that promote GII.3 replication in HIEs, including enhancement of 1) endosomal uptake, 2) endosomal acidification and subsequent activity of endosomal/lysosomal enzyme acid sphingomyelinase (ASM), and 3) ceramide levels on the apical membrane. Inhibitors of endosomal acidification or ASM reduce GII.3 infection and exogenous addition of ceramide alone permits infection. Furthermore, inhibition of lysosomal exocytosis of ASM, which is required for ceramide production at the apical surface, decreases GII.3 infection. Together, our results support a model where GII.3 exploits rapid BA-mediated cellular endolysosomal dynamic changes and cellular ceramide to enter and replicate in jejunal HIEs.
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http://dx.doi.org/10.1073/pnas.1910138117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983410PMC
January 2020

Immune-Focusing Properties of Virus-like Particles Improve Protective IgA Responses.

J Immunol 2019 12 8;203(12):3282-3292. Epub 2019 Nov 8.

Department of Immunology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan;

Virus-like particles (VLPs) provide a well-established vaccine platform; however, the immunogenic properties acquired by VLP structure remain poorly understood. In this study, we showed that systemic vaccination with norovirus VLP recalls human IgA responses at higher magnitudes than IgG responses under a humanized mouse model that was established by introducing human PBMCs in severely immunodeficient mice. The recall responses elicited by VLP vaccines depended on VLP structure and the disruption of VLP attenuated recall responses, with a more profound reduction being observed in IgA responses. The IgA-focusing property was also conserved in a murine norovirus-primed model under which murine IgA responses were recalled in a manner dependent on VLP structure. Importantly, the VLP-driven IgA response preferentially targeted virus-neutralizing epitopes located in the receptor-binding domain. Consequently, VLP-driven IgA responses were qualitatively superior to IgG responses in terms of the virus-neutralizing activity in vitro. Furthermore, the IgA in mucosa obtained remarkable protective function toward orally administrated virus in vivo. Thus, our results indicate the immune-focusing properties of the VLP vaccine that improve the quality/quantity of mucosal IgA responses, a finding with important implications for developing mucosal vaccines.
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http://dx.doi.org/10.4049/jimmunol.1900481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900486PMC
December 2019

Evolutionary Analysis of the and RNA-Dependent RNA Polymerase Regions of Human Norovirus GII.P17-GII.17 in 2013-2017.

Front Microbiol 2019 27;10:2189. Epub 2019 Sep 27.

Graduate School of Health Sciences, Gunma Paz University, Takasaki, Japan.

Human norovirus (HuNoV) GII.P17-GII.17 (Kawasaki2014 variant) reportedly emerged in 2014 and caused gastroenteritis outbreaks worldwide. To clarify the evolution of both and RNA-dependent RNA polymerase () regions of GII.P17-GII.17, we analyzed both global and novel Japanese strains detected during 2013-2017. Time-scaled phylogenetic trees revealed that the ancestral GII.17 region diverged around 1949, while the ancestral GII.P17 region diverged around 2010. The evolutionary rates of the and regions were estimated at ~2.7 × 10 and ~2.3 × 10 substitutions/site/year, respectively. The phylogenetic distances of the region exhibited no overlaps between intra-cluster and inter-cluster peaks in the GII.17 strains, whereas those of the region exhibited a unimodal distribution in the GII.P17 strains. Conformational epitope positions in the VP1 protein of the GII.P17-GII.17 strains were similar, although some substitutions, insertions and deletions had occurred. Strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the VP1 protein. Moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the RdRp protein. These results suggest that the GII.P17-GII.17 virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past 10 years.
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http://dx.doi.org/10.3389/fmicb.2019.02189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777354PMC
September 2019

ICTV Virus Taxonomy Profile: .

J Gen Virol 2019 11;100(11):1469-1470

School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, Australia.

The family includes viruses with single-stranded, positive-sense RNA genomes of 7.4-8.3 kb. The most clinically important representatives are human noroviruses, which are a leading cause of acute gastroenteritis in humans. Virions are non-enveloped with icosahedral symmetry. Members of seven genera infect mammals (, , , , , and ), members of two genera infect birds ( and ), and members of two genera infect fish ( and ). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family , which is available at ictv.global/report/caliciviridae.
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http://dx.doi.org/10.1099/jgv.0.001332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011698PMC
November 2019

Updated classification of norovirus genogroups and genotypes.

J Gen Virol 2019 10;100(10):1393-1406

Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

Noroviruses are genetically diverse RNA viruses associated with acute gastroenteritis in mammalian hosts. Phylogenetically, they can be segregated into different genogroups as well as P (polymerase)-groups and further into genotypes and P-types based on amino acid diversity of the complete VP1 gene and nucleotide diversity of the RNA-dependent RNA polymerase (RdRp) region of ORF1, respectively. In recent years, several new noroviruses have been reported that warrant an update of the existing classification scheme. Using previously described 2× standard deviation (sd) criteria to group sequences into separate clusters, we expanded the number of genogroups to 10 (GI-GX) and the number of genotypes to 48 (9 GI, 27 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI and 1 genotype each for GVII, GVIII, GIX [formerly GII.15] and GX). Viruses for which currently only one sequence is available in public databases were classified into tentative new genogroups (GNA1 and GNA2) and genotypes (GII.NA1, GII.NA2 and GIV.NA1) with their definitive assignment awaiting additional related sequences. Based on nucleotide diversity in the RdRp region, noroviruses can be divided into 60 P-types (14 GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII and 1 GX), 2 tentative P-groups and 14 tentative P-types. Future classification and nomenclature updates will be based on complete genome sequences and will be coordinated and disseminated by the international norovirus classification-working group.
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http://dx.doi.org/10.1099/jgv.0.001318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011714PMC
October 2019

Whole genome characterization and evolutionary analysis of OP354-like P[8] Rotavirus A strains isolated from Ghanaian children with diarrhoea.

PLoS One 2019 14;14(6):e0218348. Epub 2019 Jun 14.

Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Ghana.

In 2010, the rare OP354-like P[8]b rotavirus subtype was detected in children less than 2 years old in Ghana. In this follow-up study, to provide insight into the evolutionary history of the genome of Ghanaian P[8]b strains RVA/Human-wt/GHA/GHDC949/2010/G9P[8] and RVA/Human-wt/GHA/GHM0094/2010/G9P[8] detected in an infant and a 7-month old child hospitalised for acute gastroenteritis, we sequenced the complete genome using both Sanger sequencing and Illumina MiSeq technology followed by phylogenetic analysis of the near-full length sequences. Both strains possessed the Wa-like/genotype 1 constellation G9P[8]b-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequence comparison and phylogenetic inference showed that both strains were identical at the lineage level throughout the 11 genome segments. Their VP7 sequences belonged to the major sub-lineage of the G9-lineage III whereas their VP4 sequences belonged to P[8]b cluster I. The VP7 and VP4 genes of the study strains were closely related to a Senegalese G9P[8]b strain detected in 2009. In the remaining nine genome segments, both strains consistently clustered together with Wa-like RVA strains possessing either P[8]a or P[8]b mostly of African RVA origin. The introduction of a P[8]b subtype VP4 gene into the stable Wa-like strain backbone may result in strains that might propagate easily in the human population, with a potential to become an important public health concern, especially because it is not certain if the monovalent rotavirus vaccine (Rotarix) used in Ghana will be efficacious against such strains. Our analysis of the full genomes of GHM0094 and GHDC949 adds to knowledge of the genetic make-up and evolutionary dynamics of P[8]b rotavirus strains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218348PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570025PMC
February 2020

Variation of human norovirus GII genotypes detected in Ibaraki, Japan, during 2012-2018.

Gut Pathog 2019 24;11:26. Epub 2019 May 24.

Gunma Paz University Graduate School of Health Science, Gunma, 370-0006 Japan.

Background: Human norovirus (HuNoV) is the major cause of viral acute gastroenteritis for all age groups in various countries. HuNoV GII in particular accounted for the majority of norovirus outbreaks, among which GII.4 caused repeated outbreaks for a long time. Besides GII.4, other norovirus genotypes, GII.2, GII.6, and GII.17, have also been prevalent in various contexts in recent years, but few detailed epidemiological studies of them have been performed and are poorly understood. We thus conducted an epidemiological analysis of HuNoV GII in Ibaraki Prefecture, Japan, by performing surveillance in the six seasons from September 2012 to August 2018.

Results: HuNoV GI occurred almost sporadically for all genotypes; however, each genotype of GII exhibited its typical epidemiological characteristics. Although the number of outbreaks of GII.4 decreased season by season, it reemerged in 2017/2018 season. The timing of the epidemic peak in terms of number of cases for GII.17 differed from that for the other genotypes. The patients age with GII.2 and GII.6 were younger and outbreak of GII.17 occurred frequently as food poisoning. Namely, the primarily infected outbreak group differed for each genotype of HuNoV GII. Moreover, the viral load of patients differed according to the genotype.

Conclusions: Various HuNoV genotypes including GII.2, GII.4, GII.6, and GII.17 were shown to be associated with various types of outbreak sites (at childcare and educational facilities, involving cases of food poisoning, and at elderly nursing homes) in this study. These genotypes emerged in recent years, and their prevalence patterns differed from each other. Moreover, differences in outbreak sites and viral load of patients among the genotypes were identified.
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http://dx.doi.org/10.1186/s13099-019-0303-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533662PMC
May 2019

Molecular Epidemiology and Clinical Features of Rotavirus Infection Among Pediatric Patients in East Java, Indonesia During 2015-2018: Dynamic Changes in Rotavirus Genotypes From Equine-Like G3 to Typical Human G1/G3.

Front Microbiol 2019 3;10:940. Epub 2019 May 3.

Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan.

Group A rotavirus (RVA) is the most important cause of severe gastroenteritis among children worldwide, and effective RVA vaccines have been introduced in many countries. Here we performed a molecular epidemiological analysis of RVA infection among pediatric patients in East Java, Indonesia, during 2015-2018. A total of 432 stool samples were collected from hospitalized pediatric patients with acute gastroenteritis. None of the patients in this cohort had been immunized with an RVA vaccine. The overall prevalence of RVA infection was 31.7% (137/432), and RVA infection was significantly more prevalent in the 6- to 11-month age group than in the other age groups ( < 0.05). Multiplex reverse transcription-PCR (RT-PCR) revealed that the most common G-P combination was equine-like G3P[8] (70.8%), followed by equine-like G3P[6] (12.4%), human G1P[8] (8.8%), G3P[6] (1.5%), and G1P[6] (0.7%). Interestingly, the equine-like strains were exclusively detected until May 2017, but in July 2017 they were completely replaced by a typical human genotype (G1 and G3), suggesting that the dynamic changes in RVA genotypes from equine-like G3 to typical human G1/G3 in Indonesia can occur even in the country with low RVA vaccine coverage rate. The mechanism of the dynamic changes in RVA genotypes needs to be explored. Infants and children with RVA-associated gastroenteritis presented more frequently with some dehydration, vomiting, and watery diarrhea, indicating a greater severity of RVA infection compared to those with non-RVA gastroenteritis. In conclusion, a dynamic change was found in the RVA genotype from equine-like G3 to a typical human genotype. Since severe cases of RVA infection were prevalent, especially in children aged 6 to 11 months or more generally in those less than 2 years old, RVA vaccination should be included in Indonesia's national immunization program.
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http://dx.doi.org/10.3389/fmicb.2019.00940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510320PMC
May 2019

Improvement of Rotavirus Genotyping Method by Using the Semi-Nested Multiplex-PCR With New Primer Set.

Front Microbiol 2019 29;10:647. Epub 2019 Mar 29.

Laboratory of Viral Infection I, Department of Infection Control and Immunology, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan.

Rotavirus A (RVA) is a major cause of gastroenteritis in infants and young children. After vaccine introduction, RVA surveillance has become more important for monitoring changes in genotype distribution, and the semi-nested multiplex-PCR is a popular method for RVA genotyping. In particular, the VP7 primer set reported by Gouvea and colleagues in 1990 is still widely used worldwide as the recommended WHO primer set in regional and national reference RVA surveillance laboratories. However, this primer set yielded some mistakes with recent epidemic strains. The newly emerged equine-like G3 strains were mistyped as G1, G8 strains were mistyped as G3, the G9 lineage 3 strains showed very weak band, and the G9 lineage 6 strains showed a G9-specific band and a non-specific band. Gouvea's standard protocol has become relatively unreliable for identifying genotypes correctly. To overcome this limitation, we redesigned the primer set to include recent epidemic strains. Our new primer set enabled us to correctly identify the VP7 genotypes of representative epidemic strains by agarose gel electrophoresis (G1, G2, human typical G3, equine-like G3, G4, G8, G9, and G12). We believe that the multiplex-PCR method with our new primer set is a useful and valuable tool for surveillance of RVA epidemics.
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http://dx.doi.org/10.3389/fmicb.2019.00647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449864PMC
March 2019

Predicting Directions of Changes in Genotype Proportions Between Norovirus Seasons in Japan.

Front Microbiol 2019 5;10:116. Epub 2019 Feb 5.

Kitasato Institute for Life Sciences, Kitasato University, Minato, Japan.

The norovirus forecasting system (NOROCAST) has been developed for predicting directions of changes in genotype proportions between human norovirus (HuNoV) seasons in Japan through modeling herd immunity to structural protein 1 (VP1). Here 404 nearly complete genomic sequences of HuNoV were analyzed to examine whether the performance of NOROCAST could be improved by modeling herd immunity to VP2 and non-structural proteins (NS) in addition to VP1. It was found that the applicability of NOROCAST may be extended by compensating for unavailable sequence data and observed genotype proportions of 0 in each season. Incorporation of herd immunity to VP2 and NS did not appear to improve the performance of NOROCAST, suggesting that VP1 may be a suitable target of vaccines.
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http://dx.doi.org/10.3389/fmicb.2019.00116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370659PMC
February 2019

Study of Complete Genome Sequences of Rotavirus A Epidemics and Evolution in Japan in 2012-2014.

Front Microbiol 2019 31;10:38. Epub 2019 Jan 31.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

A comprehensive molecular epidemiological study using next-generation sequencing technology was conducted on 333 rotavirus A (RVA)-positive specimens collected from six sentinel hospitals across Japan over three consecutive seasons (2012-2014). The majority of the RVA isolates were grouped into five genotype constellations: Wa-like G1P[8], DS-1-like G1P[8], G2P[4], G3P[8] and G9P[8]. Phylogenetic analysis showed that the distribution of strains varied by geographical locations and epidemic seasons. The VP7 genes of different G types were estimated to evolve at 7.26 × 10-1.04 × 10 nucleotide substitutions per site per year. The Bayesian time-scaled tree of VP7 showed that the time to the most recent common ancestor of epidemic strains within a region was 1-3 years, whereas that of the epidemic strains across the country was 2-6 years. This study provided, for the first time, the timeframe during which an epidemic strain spread locally and within the country and baseline information needed to predict how rapidly RVAs spread.
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http://dx.doi.org/10.3389/fmicb.2019.00038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365416PMC
January 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

Molecular Evolutionary Analyses of the RNA-Dependent RNA Polymerase Region in Norovirus Genogroup II.

Front Microbiol 2018 18;9:3070. Epub 2018 Dec 18.

Graduate School of Health Sciences, Gunma Paz University, Takasaki, Japan.

Noroviruses are the leading cause of viral gastroenteritis in humans across the world. RNA-dependent RNA polymerase (RdRp) plays a critical role in the replication of the viral genome. Although there have been some reports on a limited number of genotypes with respect to the norovirus evolution of the region, no comprehensive molecular evolution examination of the norovirus GII genotype has yet been undertaken. Therefore, we conducted an evolutionary analysis of the 25 genotypes of the norovirus GII region (full-length), collected globally using different bioinformatics technologies. The time-scaled phylogenetic tree, generated using the Bayesian Markov Chain Monte Carlo (MCMC) method, indicated that the common ancestor of GII diverged from GIV around 1443 CE [95% highest posterior density (HPD), 1336-1542]. The GII region emerged around 1731 CE (95% HPD, 1703-1757), forming three lineages. The evolutionary rate of the region of the norovirus GII strains was estimated at over 10 substitutions/site/year. The evolutionary rates were significantly distinct in each genotype. The composition of the phylogenetic distances differed among the strains for each genotype. Furthermore, we mapped the negative selection sites on the RdRp protein and many of these were predicted in the GII.P4 RdRp proteins. The phylodynamics of GII.P4, GII.P12, GII.P16, and GII.Pe showed that their effective population sizes increased during the period from 2003 to 2014. Our results cumulatively suggest that the region of the norovirus GII rapidly and uniquely evolved with a high divergence similar to that of the norovirus gene.
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http://dx.doi.org/10.3389/fmicb.2018.03070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305289PMC
December 2018

Free-Chlorine Disinfection as a Selection Pressure on Norovirus.

Appl Environ Microbiol 2018 07 18;84(13). Epub 2018 Jun 18.

Department of Civil and Environmental Engineering, Tohoku University, Sendai, Miyagi, Japan

Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure. This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions.
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http://dx.doi.org/10.1128/AEM.00244-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007107PMC
July 2018

Foodborne Outbreaks Caused by Human Norovirus GII.P17-GII.17-Contaminated Nori, Japan, 2017.

Emerg Infect Dis 2018 05;24(5):920-923

Seven foodborne norovirus outbreaks attributable to the GII.P17-GII.17 strain were reported across Japan in 2017, causing illness in a total of 2,094 persons. Nori (dried shredded seaweed) was implicated in all outbreaks and tested positive for norovirus. Our data highlight the stability of norovirus in dehydrated food products.
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http://dx.doi.org/10.3201/eid2405.171733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938760PMC
May 2018

Equine-like G3 rotavirus strains as predominant strains among children in Indonesia in 2015-2016.

Infect Genet Evol 2018 07 31;61:224-228. Epub 2018 Mar 31.

Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan. Electronic address:

Rotavirus A (RVA) is a major cause of acute gastroenteritis in humans and animals worldwide. As a result of the segmented nature of the rotavirus genome, genetic reassortment commonly occurs. This study aims to clarify the genetic characteristics of RVAs circulating in Indonesia. From June 2015 through August 2016, stool samples were collected from 134 children aged <5 years (71 male and 63 female) with acute gastroenteritis who were inpatients at a private hospital in Surabaya, Indonesia. All stool samples were screened for RVA antigen using immunochromatography. Forty-two samples (31.3%, 42/134) were RVA antigen-positive. All RVA positive samples tested showed the unusual combinations of G3P[8] (n = 36) and G3P[6] (n = 3) with a short RNA pattern by G/P typing and polyacrylamide gel electrophoresis (PAGE). Whole genome analysis by next-generation sequencing (NGS) was performed for 11 strains to determine the RVA genotypes. Eleven rotavirus strains were found to carry a DS-like genetic backbone; nine strains showed a G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genome constellation, which was recently reported in Australia, Hungary, Spain and Brazil; as well, two strains showed a G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genome constellation. The phylogenetic tree based on the VP7 gene showed that all 11 strains were classified as equine-like G3, which is genetically distinct and different in origin from typical human G3 strains. The phylogenetic tree based on the NSP4 gene showed that six strains were classified as bovine-like strain and the remaining five were classified as human strain. In conclusion, we identified the strains which are intergenogroup reassortants containing an equine-like G3 VP7, a P[8])/P[6] VP4, with a DS-1-like genetic backbone. These findings suggest that equine-like G3P[8] and P[6] RVA strains have been circulating in the Indonesian population for at least 1 year and probably longer, indicating a diversity of RVAs in this area.
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http://dx.doi.org/10.1016/j.meegid.2018.03.027DOI Listing
July 2018

Evaluation of the use of various rat strains for immunogenic potency tests of Sabin-derived inactivated polio vaccines.

Biologicals 2018 Mar 21;52:12-17. Epub 2018 Feb 21.

Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan.

Slc:Wistar rats have been the only strain used in Japan for purpose of evaluating a national reference vaccine for the Sabin-derived inactivated polio vaccine (sIPV) and the immunogenicity of sIPV-containing products. However, following the discovery that the Slc:Wistar strain was genetically related to the Fischer 344 strain, other "real" Wistar strains, such as Crlj:WI, that are available worldwide were tested in terms of their usefulness in evaluating the immunogenicity of the past and current lots of a national reference vaccine. The response of the Crlj:WI rats against the serotype 1 of sIPV was comparable to that of the Slc:Wistar rats, while the Crlj:WI rats exhibited a higher level of response against the serotypes 2 and 3. The immunogenic potency units of a national reference vaccine determined using the Slc:Wistar rats were reproduced on tests using the Crlj:WI rats. These results indicate that a titer of the neutralizing antibody obtained in response to a given dose of sIPV cannot be directly compared between these two rat strains, but that, more importantly, the potency units are almost equivalent for the two rat strains.
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http://dx.doi.org/10.1016/j.biologicals.2018.02.001DOI Listing
March 2018

Genetic Analysis of Human Norovirus Strains in Japan in 2016-2017.

Front Microbiol 2018 18;9. Epub 2018 Jan 18.

Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Musashimurayama, Japan.

In the 2016/2017 winter season in Japan, HuNoV GII.P16-GII.2 strains (2016 strains) emerged and caused large outbreaks of acute gastroenteritis. To better understand the outbreaks, we examined the molecular evolution of the gene and region in 2016 strains from patients by studying their time-scale evolutionary phylogeny, positive/negative selection, conformational epitopes, and phylodynamics. The time-scale phylogeny suggested that the common ancestors of the 2016 strains gene and region diverged in 2006 and 1999, respectively, and that the 2016 strain was the progeny of a pre-2016 GII.2. The evolutionary rates of the gene and region were around 10 substitutions/site/year. Amino acid substitutions (position 341) in an epitope in the P2 domain of 2016 strains were not found in pre-2016 GII.2 strains. Bayesian skyline plot analyses showed that the effective population size of the gene in GII.2 strains was almost constant for those 50 years, although the number of patients with NoV GII.2 increased in 2016. The 2016 strain may be involved in future outbreaks in Japan and elsewhere.
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http://dx.doi.org/10.3389/fmicb.2018.00001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778136PMC
January 2018