Screening of natural extracts with anti-norovirus effects and analysis of this mechanism in grape seed extract.

Norovirus (NoV）is a major causative virus of viral gastroenteritis and requires a general disinfection method because it is resistant to common disinfectants such as ethanol and chlorhexidine. This study aimed to find natural extracts as candidates for versatile disinfectant ingredients. The antiviral effect of natural extracts against NoV can be evaluated using the feline calicivirus (FCV）-inactivation test and NoV virus-like particle (NoV-VLP）-binding inhibition test. In this study, screening of natural extracts with anti- NoV effects was performed using these two methods. Of the 63 natural extracts examined, 14 were found to have high FCV-inactivation and NoV-VLP-binding inhibitory effects. In addition, we evaluated the NoV-VLPbinding inhibitory effect of grape seed extract（GSE）containing proanthocyanidins under multiple concentration conditions and treatment times and determined that the binding inhibitory effect of GSE was concentration- and time-dependent. Electron microscopy showed that GSE-treated NoV-VLPs aggregated, distorted, and swelled, suggesting that GSE directly interacts with NoV particles. The results suggest that some natural extracts containing GSE can be used as components of disinfectants against NoV.

Improved detection methods by genetic and immunological techniques for botulinum C/D and D/C mosaic neurotoxins.

Clostridium botulinum type C and D strains produce serotype-specific or mosaic botulinum neurotoxin (BoNT). Botulinum C/D and D/C mosaic neurotoxins (BoNT/CD and /DC) are related to avian and bovine botulism, respectively. The two mosaic BoNTs cannot be differentiated from authentic type C and D BoNTs by the conventional serotyping method. In this study, we attempted to establish novel methods for the specific detection of BoNT/CD or/DC. Comparison with nontoxic component genes in type C and D strains revealed that the nucleotide sequence of the ha70 gene is well conserved among either serotype-specific or mosaic BoNT-producing strains. A multiplex PCR method with primers for the light chain of boNT, ntnh, and ha70 gene detection was developed for typing of the boNT gene in type C and D strains. Upon applying this method, twenty-seven type C and D strains, including authentic strains and the isolates from avian and bovine botulism, were successfully divided into type C, C/D mosaic, type D, and D/C mosaic BoNT-producing strains. We then prepared an immunochromatography kit with specific monoclonal antibody showing high binding affinity to each mosaic BoNT. BoNT/CD and /DC in the culture supernatant were detected with limits of detection of 2.5 and 10 LD(50), respectively. Furthermore, we confirmed the applicability of the kit for BoNT/DC using crude culture supernatant from a specimen from a bovine suspected of having botulism. These results indicate that the genetic and immunological detection methods are useful for the diagnosis of avian and bovine botulism.

Stability of toxigenicity in proteolytic Clostridium botulinum type B upon serial passage.

Proteolytic Clostridium botulinum type B strains were investigated for stability of toxigenicity and bont/b gene upon serial passage. Strains with bont/b gene located on their plasmids showed loss or decrease of toxigenicity during serial passage. Some strains lost the bont/b gene-encoding plasmid. The stability of the plasmids varied between strains.

Increase of GII.2 norovirus infections during the 2009-2010 season in Osaka City, Japan.

During the 2009-2010 season, a significant numerical increase of genotype GII.2 norovirus (NoV)-associated outbreaks was observed in Osaka City, Japan. The most common genotype in that season was GII.2 (44.6%), followed by GII.4 (39.2%). Mostly, GII.2 strains were associated with outbreaks in children and with person-to-person contact. The National Infectious Disease Surveillance Center reported that GII.2 NoV infections were widespread in Japan in that season. Comparative phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) and capsid sequences revealed that this GII.2 epidemic resulted from two genetic strains. The first, GII.2p2 strains, had an identical genotype in the RdRp and capsid genes. GII.2p2 strains in the 2009-2010 season were a different genetic cluster from the strains of spring 2004, the previous epidemic of GII.2 NoV, but showed no unique amino acid change. The second, GII.2 chimera virus (GII.2p16), had GII.16 RdRp and GII.2 capsid genotypes, suggesting prior recombination at the junction of ORF1 and ORF2. GII.2p16 strains had four significant amino acid changes in the P2 subdomain, suggesting antigenic changes. Before the 2009-2010 season, GII.2 chimera viruses had been observed only sporadically. This spreading of GII.2p16 strains in the 2009-2010 season might be the first epidemic of GII.2 chimera virus. This study revealed that the NoV epidemic in the 2009-2010 season differed considerably from the prior season, when GII.4 was predominant. Furthermore, GII.2 strains persisted in human populations by drastic recombination and gradual accumulation of mutations, indicating a prevalent pattern of non-GII.4 genotypes with genetic evolution.

Molecular epidemiology of noroviruses detected in seasonal outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan, from 1996-1997 to 2008-2009.

In seasons from 1996-1997 through 2008-2009, noroviruses (NoVs) were detected in 505 outbreaks (71%) of nonbacterial gastroenteritis in Osaka City, Japan using molecular diagnosis with reverse transcription (RT)-PCR or real-time RT-PCR. The occurrences of NoV-associated outbreaks were related with the cold season during November-March (85.3%), and occasionally small epidemics of NoVs occurring during April-June were observed. Oyster-associated outbreaks were dominant transmission modes (25-61.1%) before the 2003-2004 season, and decreased (5-20.5%) from the 2003-2004 season, although outbreaks attributable to food-borne transmission (except for oysters) and person-to-person contact increased from the 2003-2004 season. The NoV strains were characterized into genotypes based on sequence analysis of partial capsid regions. Genotyping analyses identified at least 30 genotypes (12 in genogroup I [GI] and 18 in genogroup II [GII]) of NoV. The most common genotype was GII.4 (44.6%), followed in order by GII.3, GII.6, GII.2, and GII.5. The number of GII.4 NoVs increased greatly from the 2003-2004 season, eventually comprising a large share among the NoV- associated outbreaks (97.4%) of the 2006-2007 season. Occasional increased prevalence of genotypes other than GII.4 was observed during this study period. This study showed the appearance, spread, and disappearance of various genotypes and the change of NoV epidemic in a limited geographic region. Continuous NoV molecular surveillance is important for understanding NoV infections and for improving measures for their control and prevention.

A novel multiplex PCR method for Clostridium botulinum neurotoxin type A gene cluster typing.

A rapid, simple and sensitive multiplex PCR method for boNT/A gene cluster typing was developed by combining the results of BoNT/A subtype (boNT/A1 or /A2) gene detection with ha33 and/or p47 gene detection. Ten isolates associated with infant botulism in Japan were examined and divided into boNT/A gene cluster types 2 and 3 by origin (honey feeding or not) and period (1986-1987 or 1999-2007). It is suggested that this multiplex PCR method will be be useful for epidemiological studies of botulism.

Genetic characterization of Clostridium botulinum associated with type B infant botulism in Japan.

The 15 proteolytic Clostridium botulinum type B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of boNT/B gene sequences, genotyping, and determination of the boNT/B gene location by using pulsed-field gel electrophoresis (PFGE) for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a unique boNT/B gene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from a case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that the boNT/B gene is located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp, or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause.

Epidemic of genotype GII.2 noroviruses during spring 2004 in Osaka City, Japan.

Between March and May 2004, a GII.2 genotype norovirus strain caused an epidemic of acute gastroenteritis in Osaka, Japan. Phylogenetic analysis showed that this strain was distinct from all other GII.2 strains detected in Osaka City between April 1996 and March 2005.

Genetic analysis of the capsid gene of genotype GII.2 noroviruses.

Noroviruses (NoVs) are considered to be a major cause of acute nonbacterial gastroenteritis in humans. The NoV genus is genetically diverse, and genotype GII.4 has been most commonly identified worldwide in recent years. In this study we analyzed the complete capsid gene of NoV strains belonging to the less prevalent genotype GII.2. We compared a total of 36 complete capsid sequences of GII.2 sequences obtained from the GenBank (n = 5) and from outbreaks or sporadic cases that occurred in The Netherlands (n = 10) and in Osaka City, Japan (n = 21), between 1976 and 2005. Alignment of all capsid sequences did not show fixation of amino acid substitutions over time as an indication for genetic drift. In contrast, when strains previously recognized as recombinants were excluded from the alignment, genetic drift was observed. Substitutions were found at five informative sites (two in the P1 subdomain and three in the P2 subdomain), segregating strains into five genetic groups (1994 to 1997, 1999 to 2000, 2001 to 2003, 2004, and 2005). Only one amino acid position changed consistently between each group (position 345). Homology modeling of the GII.2 capsid protein showed that the five amino acids were located on the surface of the capsid and close to each other at the interface of two monomers. The data suggest that these changes were induced by selective pressure, driving virus evolution. Remarkably, this was observed only for nonrecombinant genomes, suggesting differences in behavior with recombinant strains.

Humoral immune responses against norovirus infections of children.

In 2 infants with gastroenteritis associated with Norovirus (NoV), serum immunoglobulin (Ig) G, IgM, IgA, and fecal IgA antibody responses against NoV were examined by enzyme-linked immunosorbent assay using 11 different antigenic and genetic types of NoV virus-like particles expressed in insect cells. These two cases were putative primary single NoV infections, because antibodies against NoVs were not detected in acute-phase serums. In one of two cases, long-term excretion of virus RNA for 33 days was observed. Serum IgG responses demonstrated strong seroresponse to the homologous type, and weak seroresponse to the heterologous types within the genogroup. After more than 2 years, the IgG antibody titer remained high to the homologous type and low to the heterologous type within the genogroup. IgM and IgA were specific to the homologous type. IgM was short lived and the serum IgA antibody titer remained low to the homologous type for a long period. These results improve our understanding of the humoral immune response to NoV infection.

Genetic and antigenic diversity among noroviruses.

Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1-14 and GII/1-17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.

Genotyping of Norovirus strains detected in outbreaks between April 2002 and March 2003 in Osaka City, Japan.

Noroviruses (NVs) are the major cause of food- and waterborne nonbacterial gastroenteritis in Japan. Between April 2002 and March 2003, a total of 111 fecal specimens from 40 outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan were subject to NV detection. Seventy-two samples (64.9%) from 31 outbreaks (77.5%) were NV positive by a real time reverse transcription (RT)-PCR assay. To further determine the genotype of individual NV strains, we sequenced the capsid N-terminal/shell (N/S) domain of some representative strains from each outbreak. The 51 NV strains detected in this study were segregated into 15 genotypes (6 in genogroup I and 9 in genogroup II), and GII/5 genotype NV was a dominant outbreak genotype.

Expression and antigenicity of virus-like particles of norovirus and their application for detection of noroviruses in stool samples.

Human noroviruses (NoVs), members of the genus Norovirus in the family Caliciviridae, are the leading agents of nonbacterial acute gastroenteritis worldwide. Human NoVs are currently divided into at least two genogroups, genogroup I (GI) and genogroup II (GII), each of which contains at least 14 and 17 genotypes. To explore the genetic and antigenic relationship among NoVs, we expressed the capsid protein of four genetically distinct NoVs, the GI/3 Kashiwa645 virus, the GII/3 Sanbu809 virus, the GII/5 Ichikawa754 virus, and the GII/7 Osaka10-25 virus in baculovirus expression system. An antigen enzyme-linked immunosorbent assay (ELISA) with hyperimmune serum against the four recombinant capsid proteins and characterized previously three capsid proteins derived from GI/1, GI/4, and GII/12 was developed to detect the NoVs antigen in stools. The antigen ELISA was highly specific to the homotypic strains, allowing assignment of a strain to a Norovirus genetic cluster within a genogroup.

Infection of different cell lines of neural origin with subacute sclerosing panencephalitis (SSPE) virus.

Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CDVLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.

Subcellular localization of Toll-like receptor 3 in human dendritic cells.

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.

Co-circulation of two genotypes of measles virus and mutual change of the prevailing genotypes every few years in Osaka, Japan.

Genotypes of 44 wild-type measles virus (MV) strains isolated in Osaka, Japan, during 1997-2001, were determined based on phylogenetic analyses of a 456-nt 3' terminal nucleoprotein gene sequence with the reference MV strains designated by the World Health Organization. The wild-type MV strains were classified into two genotypes, D3 and D5, recognized as indigenous in Japan. Six of 12 strains isolated in 1997 were classified into genotype D3 and the other 6 into D5. Eleven of 13 strains were D3, and 2 were D5 in 1998. There were no measles epidemics, and no strains were isolated in 1999. Nine of 10 strains were genotype D5, and only one was D3 in 2000, and 9 of 9 were D5 in 2001. These results indicate that the wild-type MV strains classified into genotypes D3 and D5 co-circulated without the complete change of the MV genotype in Osaka, except in 2001. Furthermore, the prevailing genotype was different between 1998 and 2000-2001. Together with a previous report about MV genotype in this area during 1993-1995, these results suggest that the mutual change of the prevailing wild-type MV genotypes between D3 and D5 occurs every few years in Osaka, Japan.

Prevalence of Norwalk-like virus infections in cases of viral gastroenteritis among children in Osaka City, Japan.

Surveillance of Norwalk-like virus (NLV) infections in cases of pediatric gastroenteritis between April 1996 and March 2000 showed that NLVs were an important causative agent in viral gastroenteritis cases among children between November and January in those years. The predominant type of NLV was closely related to Lordsdale virus in genogroup 2. During the 1999-2000 season, Arg320-like strains, which may be genetic recombinants, suddenly appeared and spread.

Molecular classification of enteroviruses not identified by neutralization tests.

We isolated six viruses from patients diagnosed with aseptic meningitis or hand, foot, and mouth disease. The cytopathic effect of these viruses on cultured cells was like that of enteroviruses. However, viral neutralization tests against standard antisera were negative. Phylogenetic analysis with the complete VP4 nucleotide sequences of these 6 viruses and 29 serotypes of enteroviruses classified 3 of the viruses as serotype echovirus type 18 (EV18) and 3 as serotype human enterovirus 71 (HEV71). These results were confirmed by remicroneutralization tests with HEV-monospecific antisera or an additional phylogenetic analysis with the complete VP4 nucleotide sequences. Phylogenetic analysis with complete VP4 genes is more useful than neutralization tests with enterovirus serotype-specific antisera in identifying enterovirus serotypes.

Prevalence of "Norwalk-like virus" infections in outbreaks of acute nonbacterial gastroenteritis observed during the 1999-2000 season in Osaka City, Japan.

We have investigated the incidence of Norwalk-like viruses (NLVs) associated with outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan, since April 1996 using reverse transcription (RT)-PCR and electron microscopy methods. From the results of the first 3 years, between April 1996 and March 1999, we previously reported that multiple genetic types of NLVs were detected in 71.9% of outbreaks using RT-PCR with Ando's primers except for one outbreak [Iritani et al., 2000]. However, during the 1999-2000 season, NLV outbreak strains, which could not be detected by RT-PCR with Ando's primers, were increased. From probe typing and sequence analysis, 76.9% of these undetectable outbreak strains were classified into the P1-B type and the others were untypable. These untypable strains were closely related with Alphatron type strains detected in the Netherlands. The P2-B probe type of the NLV outbreak strains was predominant (88.2%) in the 1999-2000 season. The phylogram based on the 81 nucleotide sequences from these P2-B outbreak strains formed 2 clusters closely related with Lordsdale virus. The dominant genetic type of the P2-B outbreak strains, during the 1996-1997 season in Osaka City, belonged in one of these 2 clusters. These findings of the emergence of NLVs escaping the RT-PCR method strongly indicated the importance of probe typing and sequence analysis to survey NLV infections. Our surveillance of NLV infection in the outbreaks, for these 4 years, showed that the predominant probe type and dominant genetic type of NLV outbreak strains changed each season.