守口 匡子

The group A rotavirus (RVA) genome comprising 11 double-stranded RNAs encodes six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6). Among these 12 rotaviral proteins, NSP6 has been less studied as to its function. We previously prepared a recombinant NSP6-deficient RVA derived from simian strain SA11-L2 by reverse genetics, and found that the NSP6-deficient virus grew well in cell culture, although its growth was less abundant than that of the parental SA11-L2 strain. In this study, we examined the potency of a recombinant RVA incapable of NSP6 expression to cause diarrhoea in suckling mice. The suckling mice infected with the NSP6-deficient virus apparently experienced diarrhoea, although the symptom was milder and the duration of diarrhoea was shorter than in the mice infected with the authentic SA11-L2 strain. Thus, together with the results obtained for cultured cells in the previous study, it can be concluded that NSP6 is not necessarily required for replication and pathogenicity in vitro and in vivo.

A highly sensitive biosensor to detect norovirus in environment is desired to prevent the spread of infection. In this study, we investigated a design of surface plasmon resonance (SPR)-assisted fluoroimmunosensor to increase its sensitivity and performed detection of norovirus virus-like particles (VLPs). A quantum dot fluorescent dye was employed because of its large Stokes shift. The sensor design was optimized for the CdSe-ZnS-based quantum dots. The optimal design was applied to a simple SPR-assisted fluoroimmunosensor that uses a sensor chip equipped with a V-shaped trench. Excitation efficiency of the quantum dots, degree of electric field enhancement by SPR, and intensity of auto fluorescence of a substrate of the sensor chip were theoretically and experimentally evaluated to maximize the signal-to-noise ratio. As the result, an excitation wavelength of 390 nm was selected to excite SPR on an Al film of the sensor chip. The sandwich assay of norovirus VLPs was performed using the designed sensor. Minimum detectable concentration of 0.01 ng/mL, which corresponds to 100 virus-like particles included in the detection region of the V-trench, was demonstrated. (C) 2016 The Authors. Published by Elsevier B.V.

Human rotavirus (RV) infection is a leading cause of dehydrating diarrhea in infants and young children worldwide. Since therapeutic approaches to RV gastroenteritis are limited to alleviation of dehydration with oral rehydration solutions, more direct approaches to palliate symptoms of RV gastroenteritis are required. Treatments with probiotics have been increasingly recognized as alternative safe and low cost treatments for moderate infectious diarrhea. In this study, Bifidobacterium bifidum G9-1 (BBG9-1), which has been used as an intestinal drug for several decades, was shown to have a remarkable protective effect against RV gastroenteritis in a suckling mice model. As well as prophylactic oral administration of BBG9-1 from 2 days before RV infection, therapeutic oral administration of BBG9-1 from 1 day after RV infection significantly alleviated RV-induced diarrhea. Therapeutic administration of BBG9-1 reduced various types of damage in the small intestine, such as epithelial vacuolization and villous shortening, and significantly diminished the infectious RV titer in mixtures of cecal contents and feces. It was also shown that therapeutic administration of BBG9-1 significantly increased the number of acidic mucin-positive goblet cells and the gene expression of mucosal protective factors including MUC2, MUC3, MUC4, TG931 and TFF3 in the small intestine. This led to alleviation of low gut permeability shown as decreased gene expression levels of occludin, claudin-1 and villin-1 after RV infection. Furthermore, in the small intestine, therapeutic administration of BBG9-1 significantly palliated the decreased gene expression of SGLT-1, which plays an important role in water absorption. In the large intestine, administered BBG9-1 was shown to replicate to assimilate undigested nutrients, resulting in normalization of the abnormally high osmotic pressure. These results suggested that water malabsorption caused by RV infection was alleviated in mice administered BBG9-1. Thus, the present study showed that oral administration of BBG9-1 palliated diarrhea partly through protection against RV-induced lesions by inducing mucosal protective factors. Oral administration of BBG9-1 is thought to be an efficient method for management of an RV epidemic for both prophylactic and therapeutic purposes.

The rotavirus-induced diarrhea of human and animal neonates is a major public health concern worldwide. Until recently, no effective therapy is available to specifically inactivate the rotavirion particles within the gut. Passive immunotherapy by oral administration of chicken egg yolk antibody (IgY) has emerged of late as a fresh alternative strategy to control infectious diseases of the alimentary tract and has been applied in the treatment of diarrhea due to rotavirus infection. The purpose of this concise review is to evaluate evidence on the properties and performance of anti-rotavirus immunoglobulin Y (IgY) for prevention and treatment of rotavirus diarrhea in human and animal neonates. A survey of relevant anti-rotavirus IgY basic studies and clinical trials among neonatal animals (since 1994-2015) and humans (since 1982-2015) have been reviewed and briefly summarized. Our analysis of a number of rotavirus investigations involving animal and human clinical trials revealed that anti-rotavirus IgY significantly reduced the severity of clinical manifestation of diarrhea among IgY-treated subjects relative to a corresponding control or placebo group. The accumulated information as a whole depicts oral IgY to be a safe and efficacious option for treatment of rotavirus diarrhea in neonates. There is however a clear need for more randomized, placebo controlled and double-blind trials with bigger sample size to further solidify and confirm claims of efficacy and safety in controlling diarrhea caused by rotavirus infection especially among human infants with health issues such as low birth weights or compromised immunity in whom it is most needed.

Human rotavirus samples from 54 children with acute gastroenteritis in Myanmar in 2011 were subjected to reverse transcription-PCR to determine their G and P types. On G typing, G2 (24/54; 44.4%) was found to be the most prevalent, followed by G12 (17/54; 31.5%) and 01 (1/54; 1.9%). Mixed cases with G2 and G12 were found in 12 of the 54 (22.2%) samples. On P typing, P[4] was found to be the most predominant (29/54; 53.7%), followed by P[8] (17/54; 31.5%) and P[6] (4/54; 7.4%). Mixed cases with P[4] and P[8] were detected in 4 of 54 (7.4%) samples. Thus, occurrence of G2 and unusual G12 in high proportions was characteristic of human rotaviruses in Myanmar in this study setting.

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/ Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/ Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/ Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.

In order to identify the repertoire of antibodies generated on natural infection of norovirus (NoV) in humans, and to characterize the human monoclonal antibodies against NoV, three phage-displayed antibody libraries originating from healthy person(s) were screened using purified virus-like particles (VLPs) of strain Narita 104 (r104, genogroup II, genotype 4) or strain Chiba 407 (rCV, genogroup I, genotype 4) as antigens. On screening with r104, 62 clones were isolated. Among these antibodies, two clones, 12A11 and 12B10, showed intra-genogroup cross-reactivity to genotypes 1, 3-7, 12, and 14, and genotypes 1, 4, 6, and 7 of genogroup II, respectively. In addition, antibodies belonging to the same group were isolated from two different libraries. On screening with rCV, five clones were isolated, two of which were cross-reactive. One, CV-2F5, reacted to genotypes 1-4, and 8 of genogroup I, and the other, CV-1A5, showed inter-genogroup cross-reactivity to all the VLPs employed in this study. The blocking activities of the monoclonal antibodies against the interaction of homotypic VLPs (VLPs used in the panning procedure) with histo-blood group antigens were also assessed as an alternative to neutralization assay. Although the blocking activity of 12A11 was partially limited 12B10 prevented the binding of r104 to histo-blood group antigens that had been reported to bind r104. The blocking activity of CV-2F5 against the attachment of rCV to suitable histo-blood group antigens was weak, but the blocking activity of CV-1A5 was well recognized. Thus, 12B10 and CV-1A5 were suggested to be cross-reactive monoclonal antibodies with neutralizing activity. J. Med. Virol. 86:558-567, 2014. (c) 2013 Wiley Periodicals, Inc.

This study aims to evaluate the effect of hyperimmune immunoglobulin Y (IgY) against human rotavirus (HRV) among pediatric patients receiving standard supportive treatment for rotavirus-associated diarrhea mostly with an enteric non-cholera co-pathogen in a hospital setting. Two natural HRV reassortant clinical strains ATCC VR 2273 and ATCC VR 2274 were used as mixed immunizing antigens in poultry hens to generate anti-HRV IgY (Rotamix IgY). The Rotamix IgY was used in laboratory and clinical studies against control or placebo IgY. The control or placebo IgY was prepared using tissue culture medium from mock-infected MA104 cell line as antigen for poultry immunization. In vitro, Rotamix IgY exhibited multi-serotypic cross neutralization activities along with synergistic effects against major global serotypes G1. G2, G3, G4 and other human or animal rotavirus strains when compared with mono-specific IgY. Suckling mice (ICR strain) pre-treated orally once with Rotamix IgY and then challenged with rotavirus 3 h later showed a significant dose-dependent reduction in frequency (p < 0.05) and duration (p < 0.05) of diarrhea compared to placebo IgY-treated mice. Out of 114 children aged between 3 and 14 months and with diarrhea upon admission in a Myanmar hospital, 54 dehydrated and rotavirus-positive children were randomized into Rotamix IgY group and placebo IgY group. Of these, only 52 children had complete data with n = 26 children per study group. Ninety-two percent of patients in each of these groups were positive for co-infecting enteric non-cholera pathogen and all patients received standard supportive therapy for diarrhea. The patients were monitored for volume and duration of oral rehydration fluid (ORF) and intravenous fluid (IVF) intake, daily stool frequency and overall duration of diarrhea, and frequency and duration of rotavirus shedding. Compared to placebo IgY group, the Rotamix IgY group had statistically significant reduction in mean ORF intake (p = 0.004), mean duration of intravenous fluid administration (p = 0.03), mean duration of diarrhea from day of admission (p < 0.01) and mean duration of rotavirus clearance from stool from day of admission (p = 0.05). Overall, our novel approach using oral Rotamix IgY for rotavirus-infected children mostly with non-cholera enteric pathogen co-infection appears to be a promising, safe and effective adjunct to management of acute diarrhea in pediatric patients. (C) 2012 Elsevier Ltd. All rights reserved.

The infectivity of rotavirus (RV) is dependent on an activation process triggered by the proteolytic cleavage of its spike protein VP4. This activation cleavage is performed by exogenous trypsin in the lumen of the intestines in vivo. Here, we report the generation and characterization of a recombinant RV expressing cDNA-derived VP4 with a modified cleavage site (arginine at position 247) recognized by endogenous furin as well as exogenous trypsin. Unexpectedly, the mutant virus (KU//rVP4-R247Furin) was incapable of plaque formation without an exogenous protease, although the mutant VP4s on virions were efficiently cleaved by endogenous furin. Furthermore, KU//rVP4-R247Furin showed impaired infectivity in MA104 and CV-1 cells even in the presence of trypsin compared with the parental virus carrying authentic VP4 (KU//rVP4). Although the total titre of KU//rVP4-R247Furin was comparable to that of KU//rVP4, the extracellular titre of KU//rVP4-R247Furin was markedly lower than its cell-associated titre in comparison with that of KU//rVP4. In contrast, the two viruses showed similar growth in a furin-defective LoVo cell line. These results suggest that intracellular cleavage of VP4 by furin may be disadvantageous for RV infectivity, possibly due to an inefficient virus release process.

Purpose To confirm the presence of the susceptible gene for the thymoma development in the region that was assumed by the previous linkage study by Oyabu et al. (J Natl Cancer Inst 91:279-282, 1999), we tried to establish a congenic strain of rats. Methods Backcrossings between the BUF/Mna strain as a donor strain and the ACI/NMna strain as an inbred partner were repeated for 12 generations, examining whether rats had the thymoma development region, and then homozygous rats were yielded by mating among the heterozygotes. To detect the phenotypic expression, heterozygous ACI.BUF/Mna-Tsr1/+ (ACI-Tsr1/+) rats were generated by crossing female ACI.BUF/Mna-Tsr1/Tsr1 (ACI-Tsr1/Tsr1) rats with male ACI/NMna rats and were maintained for 24 months. Results These ACI-Tsr1/+ rats produced thymoma in 71%, showing a dominant trait. The thymomas were of the lymphocyte predominant type, as those developed in rats of the original BUF/Mna strain. Conclusions Thus, a new rat congenic strain, ACI-Tsr1/Tsr1, was established, revealing that thymoma develops in the dominant trait in ACI-Tsr1/+ rats.

The most intensively studied rotavirus strains initially attach to cells when the "heads" of their protruding spikes bind cell surface sialic acid. Rotavirus strains that cause disease in humans do not bind this ligand. The structure of the sialic acid binding head (the VP8* core) from the simian rotavirus strain RRV has been reported, and neutralization epitopes have been mapped onto its surface. We report here a 1.6-angstrom resolution crystal structure of the equivalent domain from the sialic acid-independent rotavirus strain DS-1, which causes gastroenteritis in humans. Although the RRV and DS-1 VP8* cores differ functionally, they share the same galectin-like fold. Differences between the RRV and DS-1 VP8* cores in the region that corresponds to the RRV sialic acid binding site make it unlikely that DS-1 VP8* binds an alternative carbohydrate ligand in this location. In the crystals, a surface cleft on each DS-1 VP8* core binds N-terminal residues from a neighboring molecule. This cleft may function as a ligand binding site during rotavirus replication. We also report an escape mutant analysis, which allows the mapping of heterotypic neutralizing epitopes recognized by human monoclonal antibodies onto the surface of the VP8* core. The distribution of escape mutations on the DS-1 VP8* core indicates that neutralizing antibodies that recognize VP8* of human rotavirus strains may bind a conformation of the spike that differs from those observed to date.

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 mug/ml.