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Many Escherichia albertii isolates, an emerging pathogen of human and birds, might have been misidentified due to the difficulty of differentiating this bacterium from Escherichia coli and Shigella spp. by routine biochemical tests, resulting in underestimation of E. albertii infections. We have developed a polymerase chain reaction (PCR) assay that targets E. albertii cytolethal distending toxin (Eacdt) genes, which include the genes previously identified as Escherichia coli cdt-II. This assay could generate a single 449-bp PCR product in each of 67 confirmed E. albertii strains but failed to produce PCR product from any of the tested non–E. albertii enteric strains belonging to 37 different species, indicating 100% sensitivity and specificity of the PCR assay. The detection limit was 10 CFU per PCR tube and could detect 105 CFU E. albertii per gram of spiked healthy human stool. The Eacdt gene-based PCR could be useful for simple, rapid, and accurate detection and identification of E. albertii.

In Escherichia coli , more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli .

Sapoviruses are associated with acute gastroenteritis. Human sapoviruses are classified into four distinct genogroups (GI, GII, GIV, and GV) based on their capsid gene sequences. A TaqMan probe-based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay that detects the representative strains of these four genogroups is widely used for screening fecal specimens, shellfish, and environmental water samples. However, since the development of this test, more genetically diverse sapovirus strains have been reported, which are not detectable by the previously established assays. In this study, we report the development of a broader-range sapovirus real-time RT-PCR assay. The assay can detect 2.5 × 107 and 2.5 × 10 1 copies of sapovirus and therefore is as sensitive as the previous test. Analysis using clinical stool specimens or synthetic DNA revealed that the new system detected strains representative of all the 18 human sapovirus genotypes: GI.1-7, GII.1-8, GIV.1, and GV.1, 2. No cross-reactivity was observed against other representative common enteric viruses (norovirus, rotavirus, astrovirus, and adenovirus). This new assay will be useful as an improved, broadly reactive, and specific screening tool for human sapoviruses.

The aim of this study was to report the emergence of a recombinant human mastadenovirus (HAdV) type 85 (HAdV-85) and to describe its genomic and clinical characteristics. The strains were detected and identified in Japan in cases of adenoviral conjunctivitis including epidemic keratoconjunctivitis (EKC). The type was designated as HAdV-85 based on the novel combination of penton base (P = HAdV-37), hexon (H = HAdV-19), and fiber (F = HAdV-8). The whole genome sequence determined for HAdV-85 was compared against sequences of other types in the same species. The results of the phylogenetic analysis suggested a recombinant origin between HAdV-53 and HAdV-64, which have been two major causes of adenoviral EKC in Japan over the past decade. During the period between 2008 and 2016 in Kumamoto city, southwest of Japan, 311 cases diagnosed with conjunctivitis were diagnosed as being the consequence of adenoviral infections. Among them, 11 cases were determined to have been caused by HAdV-85 since 2015. Thus, HAdV-85 could be an emerging causative agent of adenoviral conjunctivitis.

We performed detailed genetic analysis of the VP4/VP2 coding region in human rhinovirus species A to C (HRV-ABC) strains detected in patients with a variety of acute respiratory infections in Kumamoto, Japan in the period 2011-12. The phylogenetic tree and evolutionary timescale were obtained by the Bayesian Markov chain Monte Carlo method. Phylogenetic analyses showed that the present HRV-A, -B, and -C strains belonged to 25, 4, and 18 genotypes, respectively. Some new genotypes were confirmed as prevalent strains of HRV-C. An ancestor of the present HRV-ABCs could be dated back to about 20,000 years ago. The present HRV-A and -C strains have wide genetic divergence (pairwise distance >0.2) with rapid evolutionary rates (around 7 × 10(-4) to 4 × 10(-3)substitutions/site/year). Over 100 sites were found to be under negative selection, while no positively selected sites were found in the analyzed region. No evidence of recombination events was found in this region of the present strains. Our results indicate that the present HRV strains have rapidly evolved and subsequently diverged over a long period into multiple genotypes.

Since the 1980s, the Japanese encephalitis virus (JEV) variants with slightly short variable regions (VR) of the 3' non-translated region (NTR) have been found; however, the implications of these short VR remain unclear. We recently identified two novel types of short VR (5 and 9 nt shorter than that of major group of genotype I JEV strains) of genotype I JEV isolates. To elucidate the impact of these short VR on the replication and virulence of JEV, we generated five recombinant JEV viruses: M41-d5 and M41-d9 have deletions in the VR that correspond to those observed in some recent JEV isolates, M41-d5d9 has both the 5- and 9-nt deletions in the VR, M41-d27 has a large deletion that encompasses both the 5- and 9-nt deletion regions, and M41-a13 has a 13-nt sequence insertion of the genotype III JEV strain Beijing-1 into the parent genotype I JEV strain Mie/41/2002 genome. The recombinant viruses and the parent virus, except for the M41-d27 mutant, showed similar growth properties in mammalian and mosquito cell lines. Mouse challenge experiments indicated that no significant differences among the recombinant viruses M41-d5d9, M41-d27, M41-a13, and the parent virus. Our results suggest that the short VR in JEV 3' NTR do not affect its growth in vitro or its pathogenicity in mice.

Japanese encephalitis (JE) is a serious disease in Asia, but it can be prevented by vaccination. To evaluate the necessity for vaccination in areas with reduced numbers of vector mosquitoes, as well as patients, it is critical to understand the frequency of natural virus exposure. An antibody survey was recently conducted to estimate current natural infection rates in Japan, where the vaccination rate has dropped in recent years. Serum samples were collected in 2004-2008 from inhabitants of Kumamoto Prefecture in west Japan, and in 2004-2006 from the Tokyo Metropolitan area of east Japan. Average annual infection rates estimated from the prevalence of antibodies to the nonstructural 1 protein (NS1) of JE virus was 1.8% in Kumamoto and 1.3% in Tokyo. When estimated from percentages of populations with detectable neutralizing antibodies but with no vaccination history, the average annual infection rate was 2.6% in both survey areas. Thus, JE virus remains present and active in nature in Japan. Therefore, continuing a vaccination program is indispensable to prevent JE infection in humans.

To characterize Japanese encephalitis virus (JEV) strains recently prevalent in Japan, JEV surveillance was performed in pigs from 2002 to 2004. Eleven new JEV isolates were obtained and compared with previous isolates from Japan and other Asian countries. All of the isolates were classified into genotype 1 by nucleotide sequence analysis of the E gene. Two new isolates with different levels of neurovirulence and neuroinvasiveness, but with only one nucleotide difference in the E gene, Sw/Mie/34/2004 and Sw/Mie/40/2004, were isolated at the same farm on the same day. Sw/Mie/40/2004 displayed higher neurovirulence and neuroinvasiveness in mice than the other four new isolates. Another new isolate, Sw/Hiroshima/25/2002, was neutralized by antiserum to Beijing-1 at a level similar to the homologous Beijing-1 strain, whilst seven other new isolates were neutralized at 10-fold-lower titres. However, there were no amino acid differences in the E protein among these eight isolates. The present study indicated that the 11 new JEV isolates were genetically similar, but biologically and serologically heterogeneous.

More than 27 outbreaks per year of food poisoning caused by consuming horse meat were reported in Kumamoto Prefecture (including Kumamoto City) from January 2009 to September 2011. It was found that the causative agent of the outbreaks was a protein with a molecular weight of 15 kDa that had originated from bradyzoites of Sarcocystis fayeri parasitizing the horse meat. Rabit ileal loop tests showed that pepsin treatment of homogenates of frozen horse meat containing the cysts of S. fayeri induced loss of toxicity, presumably by digestion of the proteinous causative agent(s). Slices of horse meat containing the cysts were frozen at below -20°C for various periods. The cysts were collected after thawing the slices, then treated in an artificial stomach juice containing pepsin. The bradyzoites of the cysts kept at -20°C for 48 hr or more completely disappeared. Simultaneously, the 15 kDa protein also disappeared in the frozen cysts. After notifying the public and recommending freezing treatment of horse meat, no subsequent cases of food poisoning were reported. This indicates that freezing of horse meat is effective to prevent the occurrence of food poisoning caused by consuming raw horse meat containing S. fayeri.

Canine leptospirosis, which is caused by infection with pathogenic Leptospira species, occurs worldwide, but information regarding the causative Leptospira serotypes and genotypes and their effects on virulence in dogs remains limited. Monitoring acute leptospirosis in dogs as sentinels can also aid in estimating the risk of human leptospirosis, particularly when the disease is rare, as it currently is in Japan. Among 283 clinically suspected cases of leptospirosis diagnosed from August 2007 to March 2011 in Japan, 83 cases were laboratory diagnosed as leptospirosis by blood culture, a rise in antibody titres in paired sera using a microscopic agglutination test (MAT) and/or DNA detection using flaB-nested PCR. The infected dogs comprised hunting dogs (31 dogs) and companion animals (50 dogs) and two unknown; 63.4 % of the infected dogs were males. The mortality rate was 53.2 %. A rise of at least fourfold in MAT titre was detected in 30 dogs whose paired serum samples were obtained, and the predominant reactive serogroup was Hebdomadis (53.3 %), followed by Australis (16.7 %) and Autumnalis (16.7 %). Leptospira interrogans was isolated from 45 dogs of the following serogroups: Australis (16), Autumnalis (six), Canicola (one), Hebdomadis (21) and Icterohaemorrhagiae (one). All of these serogroups caused lethal infections (57.1-100 %). Genetic heterogeneity was demonstrated in serogroups Australis, Autumnalis and Hebdomadis by multilocus sequence typing (MLST) and/or RFLP analysis based on PFGE. In serogroup Hebdomadis, each genotype determined by MLST had a unique mortality rate in the infected dogs. Although classic canine leptospirosis is associated with serovars Canicola and Icterohaemorrhagiae, serogroup Hebdomadis has become the predominant serogroup causing high mortality in Japan. This study suggests that the virulence of members of serogroup Hebdomadis in dogs may be associated with the genotypes in this serogroup.

In recent years, the increase of traffic accident at the crossing where senior citizens are concerned has been a serious subject. Therefore, the driving support system which supports a driver is developed. This supporting system has various influences to a user. We observed about the influence it has to amind-and-body state. The mind-and-body change of state when using the biomarker of the heart and the circulatory system was investigated. The research was led for a youth and, senior citizens. It was analyzed how the influence of supporting system would appear in a living biomarker according to age. And senior citizen's characteristic was also examined.

Sapovirus (SaV) is an important pathogen that causes acute gastroenteritis in humans. Human SaV is highly diverse genetically and is classified into multiple genogroups and genotypes. At present, there is no clear evidence for gastroenteritis cases caused by re-infection with SaV. We found that two individuals were sequentially infected with SaVs of two different genogroups and had gastroenteritis after each infection, although in one of the subsequent cases, both SaV and norovirus were detected. We also found a genetic shift in SaVs from gastroenteritis outpatients in the same geographical location. Our results suggest that protective immunity may be at least genogroup-specific for SaV.

Infectious acute gastroenteritis is an important public health problem worldwide. A total of 639 stool specimens were tested for the presence of diarrhea pathogens. The specimens were from outpatients with acute gastroenteritis who consulted the pediatric clinic in Kumamoto Prefecture, Japan, from June 2002 to December 2007. Of these, 421 (65.9%) were positive for diarrhea pathogens. Among them were norovirus (NoV) in 260 (61.8%), sapovirus (SaV) in 81 (19.2%), rotavirus in 49 (11.6%), adenovirus in 19 (4.5%), enterovirus in 13 (3.1%), astrovirus in 9 (2.1%), kobuvirus in 1 (0.2%), and bacterial pathogens in 11 (2.6%). Mixed infection (co-infection of viruses) was found in 22 (5.2%) of the 421 pathogen-positive stool samples. NoV was the most prevalent pathogen throughout the study period; however, the SaV detection rate was unexpectedly high and was found to be the secondary pathogen from 2005 to 2007. Genetic analysis of SaV with 81 strains demonstrated that SaV strains belonging to genogroup IV emerged in 2007, and dynamic genogroup changes occurred in a restricted geographic area. This study showed that SaV infection is not as rare as thought previously.

Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified. Here, we sequenced the genome of 29 E. albertii strains (3 complete and 26 draft sequences) isolated from multiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1 Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors and a possibly active second T3SS known as ETT2 (E. coli T3SS 2) that is inactivated in E. coli. Although this organism has been observed to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based on these results, we have developed a nested polymerase chain reaction system to directly detect E. albertii. Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen.

Norovirus is an emerging pathogen that causes gastroenteritis outbreaks. Here, we reported an adult female case of norovirus-related encephalitis/encephalopathy (NvREE) with abnormal behaviour, apathy, motor aphasia, bradykinesia and gait disturbance. We treated the patient with intravenous methyl-prednisolone pulse therapy and she recovered quickly. There were slight abnormal signals in the cortex of the opercular part and insula on the MRI fluid-attenuated inversion-recovery (FLAIR) image, generalised slow wave as a background activity in her EEG and cerebrospinal fluid pleocytosis, which restored soon after her recovery. We successfully detected the norovirus genome in stool samples from all seven family members. This is a first case report of an adult NvREE with detection of pathogenic evidence. There could be more cases of NvREE with mild neuropsychiatric symptoms, considering the increasing outbreaks each year.

In order to establish a national reference system for Campylobacter serotyping in Japan, 7 local institutes of public health collaborated to prepare 30 serogrouping antisera including 26 antisera of Lior's serogrouping system and 4 antisera of TCK serogrouping system which was developed by the Tokyo Metropolitan Research Laboratory of Public Health. A total of 603 strains (92.2%) out of 654 isolates from 23 outbreaks of C. jejuni throughout Japan were serogrouped by the 30 antisera. Out of 1,198 strain isolated from sporadic cases of Camplyobacter gastroenteritis, 883 (73.7%) were typed and 298 strains (24.9%) were untypable. The remaining 17 strains belong to rough form were not used for serogrouping. A hundred thirteen out of 883 strains have reacted with more than one typing serum. Among C. jejuni isolated from 7 prefecture, Lior's serogroup 4 was most common followed by Lior's serogroup 4, 2, 11, 1 and TCK serogroup 1 and 12. We conclude from the experiment described above that this Lior's serogrouping system by combining with TCK serogroup for Campylobacter jejuni/coli is useful in epidemiological investigations in Japan.