Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

Histological characteristics of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1 in asthmatic murine model during A(H1N1)pdm09 infection.

Pandemic influenza virus A(H1N1)pdm09 infection occurred in healthy children and young adults, but asthmatic patients presented more rapid progression of respiratory distress and plastic bronchitis. To investigate the pathogenesis of worsening respiratory symptoms after A(H1N1)pdm09 infection, we focused on matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1). MMP-9 and TIMP-1 levels in bronchoalveolar lavage fluid and serum from mice with and without asthma were evaluated after A(H1N1)pdm09 or seasonal A(H1N1) infection. MMP-9 levels were more elevated in Asthma/A(H1N1)pdm09-infected mice than in non-Asthma/A(H1N1)pdm09-infected mice on both 3 and 7 days post-infection. Immunohistochemical findings in this pneumonia model showed that MMP-9 and TIMP-1 positive cells were observed in blood vessels and bronchus of lung tissue in severe pathological findings of pneumonia with asthma. Microscopically, shedding cells and secretions were conspicuous in the trachea on days 3 and 7 post-infection, in the A(H1N1)pdm09-infected mice with asthma. Our results suggest that MMP-9 and TIMP-1 expressions are related to severe pneumonia in the A(H1N1)pdm09 infection with asthma, leading to cause epithelial cell shedding.

Enhanced airway hyperresponsiveness in asthmatic children and mice with A(H1N1)pdm09 infection.

BACKGROUND: Severe asthma exacerbation is an important comorbidity of the 2009 HIN1 pandemic (A(H1N1)pdm09) in asthmatic patients. However, the mechanisms underlying severe asthma exacerbation remain unknown. In this study, airway hyperresponsiveness (AHR) was measured in pediatric asthma patients infected with A(H1N1)pdm09. We also evaluated AHR in asthmatic mice with A(H1N1)pdm09 infection and those with seasonal influenza for comparison. METHODS: AHRs in asthmatic children were defined as the provocative acetylcholine concentration causing a 20% reduction in forced expiratory volume in 1 s (PC20 ). To investigate the pathophysiology using animal models, BALB/c mice aged 6-8 weeks were sensitized and challenged with ovalbumin. Either mouse-adapted A(H1N1)pdm09, seasonal H1N1 virus (1 × 105 pfu/20 µl), or mock treatment as a control was administered intranasally. At 3, 7, and 10 days after infection, each group of mice was evaluated for AHR by methacholine challenge using an animal ventilator, flexiVent. Lung samples were resected and observed using light microscopy to assess the degree of airway inflammation. RESULTS: AHRs in the children with bronchial asthma were temporarily increased, and alleviated by 3 months after discharge. AHR was significantly enhanced in A(H1N1)pdm09-infected asthmatic mice compared to that in seasonal H1N1-infected mice (p < .001), peaking at 7 days postinfection and then becoming similar to control levels by 10 days postinfection. Histopathological examination of lung tissues showed more intense infiltration of inflammatory cells and severe tissue destruction in A(H1N1)pdm09-infected mice at 7 days postinfection than at 10 days postinfection. CONCLUSION: Our results suggest that enhanced AHR could contribute to severe exacerbation in human asthmatic patients with A(H1N1)pdm09 infection.

Nationwide Molecular Epidemiology of Measles Virus in Japan Between 2008 and 2017.

Genotyping evidence that supports the interruption of endemic measles virus (MV) transmission is one of the essential criteria to be verified in achieving measles elimination. In Japan since 2014, MV genotype analyses have been performed for most of the measles cases in prefectural public health institutes nationwide. With this strong molecular epidemiological data, Japan was verified to have eliminated measles in March, 2015. However, even in the postelimination era, sporadic cases and small outbreaks of measles have been detected repeatedly in Japan. This study investigated the nationwide molecular epidemiology of MV between 2008 and 2017. The 891 strains in the total period between 2008 and 2017 belonged to seven genotypes (D5, D4, D9, H1, G3, B3, and D8) and 124 different MV sequence variants, based on the 450-nucleotide sequence region of the N gene (N450). The 311 MV strains in the postelimination era between 2015 and 2017 were classified into 1, 7, 8, and 32 different N450 sequence variants in D9, H1, B3, and D8 genotypes, respectively. Analysis of the detection period of the individual N450 sequence variants showed that the majority of MV strains were detected only for a short period. However, MV strains, MVs/Osaka.JPN/29.15/ [D8] and MVi/Hulu Langat.MYS/26.11/ [D8], which are named strains designated by World Health Organization (WHO), have been detected in many cases over 2 or 3 years between 2015 and 2017. The WHO-named strains have circulated worldwide, causing outbreaks in many countries. Epidemiological investigation revealed repeated importation of these WHO-named strains into Japan. To demonstrate the elimination status (interruption of endemic transmission) in situations with repeated importation of the same strains is challenging. Nevertheless, the detailed sequence analysis of individual MV strains and chronological analysis of these strains provided sufficient evidence to show that Japan has still maintained its measles elimination status in 2017.

Multi-season analyses of causative pathogens in children hospitalized with asthma exacerbation.

BACKGROUND: Respiratory viral and mycoplasma infections are associated with childhood asthma exacerbations. Here, we explored epidemiologic profile of causative pathogens and possible factors for exacerbation in a single center over a three-year period. METHODS: Hospitalized asthmatic children with attack aged 6 months-17 years were recruited between 2012 and 2015 (n = 216). Nasopharyngeal mucosa cell samples were collected from the participants and examined by reverse transcription-polymerase chain reaction to detect rhinovirus (RV), respiratory syncytial virus (RSV), enterovirus (EV), parainfluenza virus (PIV), Mycoplasma pneumoniae, and others. Clinical features, laboratory data, asthma exacerbation intensity, and asthma severity were compared among participants. Epidemiologic profile of causative pathogens and possible factors for exacerbation were explored. RESULTS: Viruses and/or Mycoplasma pneumoniae were detected in 75% of the participants. Rhinovirus (48%) was the most commonly detected virus in the participants with single infection, followed by RSV (6%). The median age at admission in the RV group was significantly higher than that in the RSV group. Insufficient asthma control and allergen sensitization were significantly related to RV-associated asthma exacerbation. There was no seasonality of pathogen types associated with asthma exacerbation although a sporadic prevalence of EV-D68 was observehinovirud. Rhinovirus were repeatedly detected in multiple admission cases. CONCLUSION: Our three-year analysis revealed that patients with RV infection were significantly prone to repeated RV infection in the subsequent exacerbation and good asthma control could prevent RV-associated asthma development and exacerbation. Multiple-year monitoring allowed us to comprehend the profile of virus- and/or mycoplasma-induced asthma exacerbation.

The Association Between Documentation of Koplik Spots and Laboratory Diagnosis of Measles and Other Rash Diseases in a National Measles Surveillance Program in Japan.

Koplik spots are considered a disease-specific sign for measles, although comprehensive virological studies have not been conducted to date. In Japan, a national survey of 3023 measles and measles-suspected cases was conducted between 2009 and 2014 using polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) to detect various rash/fever-associated viruses. Koplik spots were observed in 717 of 3023 cases (23.7%). Among these, the measles virus was detected in 202 cases (28.2%), while the rubella virus was detected in 125 cases (17.4%). Other viruses were detected in 51 cases having the spots (7.1%). In some of the cases with spots, two or three viruses, such as the rubella virus, parvovirus, and human herpesvirus type 6 were also detected. The sensitivity and specificity of Koplik spots as a diagnostic marker for measles were 48 and 80%, respectively. The results suggested that Koplik spots might appear not only in measles but also in other viral infections, such as rubella, as a clinical sign.

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

Dissemination and genetic analysis of the stealthy vanB gene clusters of Enterococcus faecium clinical isolates in Japan.

BACKGROUND: VanB-type vancomycin (VAN) resistance gene clusters confer VAN resistances on Enterococcus spp. over a wide range of MIC levels (MIC = 4-1000 mg/L). However, the epidemiology and the molecular characteristics of the VAN susceptible VanB-type Enterococcus still remain unclear. RESULTS: We characterized 19 isolates of VanB-type Enterococcus faecium that might colonize in the gut and were not phenotypically resistant to VAN (MIC = 3 mg/L). They were obtained from two hospitals in Japan between 2009 and 2010. These isolates had the identical vanB gene cluster and showed same multilocus sequence typing (MLST) (ST78) and the highly related profiles in pulsed-field gel electrophoresis (PFGE). The vanB gene cluster was located on a plasmid, and was transferable to E. faecium and E. faecalis. Notably, from these VanB-type VREs, VAN resistant (MIC≥16 mg/L) mutants could appear at a frequency of 10- 6-10- 7/parent cell in vitro. Most of these revertants acquired mutations in the vanSB gene, while the remainder of the revertants might have other mutations outside of the vanB gene cluster. All of the revertants we tested showed increases in the VAN-dependent expression of the vanB gene cluster, suggesting that the mutations affected the transcriptional activity and increased the VAN resistance. Targeted mutagenesis revealed that three unique nucleotide substitutions in the vanB gene cluster of these strains attenuated VAN resistance. CONCLUSIONS: In summary, this study indicated that stealthy VanB-type E. faecium strains that have the potential ability to become resistance to VAN could exist in clinical settings.

Burden of Human Metapneumovirus and Respiratory Syncytial Virus Infections in Asthmatic Children.

BACKGROUND: Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are the leading causes of acute respiratory illness in children. Clinical burden of each infection on the respiratory distress in asthmatic patients remains unclear. The purpose of the study was to clarify the effect of these infections on the severity of asthmatic children in the seasonal outbreaks. METHODS: A total of 1,217 pediatric inpatients with hMPV (n = 114) or RSV (n = 1,103) infection in Yamaguchi prefecture, Japan, between 2011 and 2014 were enrolled. Bronchial asthma was defined as having more than 3 episodes of wheezing illness over 1 year of age. Infection was determined by the positive antigen test for each virus in the nasal specimens. RESULTS: The number of patients peaked at age 12-15 months in hMPV infection and at age 0-3 months in RSV infection. The proportion of hypoxic patients (40-50%) did not differ at any age between hMPV-infected and RSV-infected children. In the analysis of date from > 1 year old patients with hypoxia, hMPV-infection group was older (P = 0.036), and more frequently had history of asthma (P = 0.015) or abnormal chest roentgenogram (P < 0.001) than RSV-infection group. Multivariate analysis indicated that the hypoxia-associated factors were history of asthma in both hMPV (odds ratio [OR]: 15.8; P < 0.001) and RSV infections (OR, 2.2; P = 0.005), higher body temperature in hMPV infection (OR, 2.2; P = 0.009), and younger age in RSV infection (OR, 1.4; P = 0.004). CONCLUSIONS: Outbreaks of hMPV, rather than, RSV infection may have a greater impact on the development of hypoxic respiratory illness in asthmatic children.

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

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 VP1 gene and RdRp 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 VP1 gene and RdRp 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 VP1 gene and RdRp region were around 10-3 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 VP1 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.

Phylogeny and Immunoreactivity of Norovirus GII.P16-GII.2, Japan, 2016-17.

During the 2016-17 winter season in Japan, human norovirus GII.P16-GII.2 strains (2016 strains) caused large outbreaks of acute gastroenteritis. Phylogenetic analyses suggested that the 2016 strains derived from the GII.2 strains detected during 2010-12. Immunochromatography between 2016 strains and the pre-2016 GII.2 strains showed similar reactivity.

Pulmonary inflammation and cytokine dynamics of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during A(H1N1)pdm09 influenza infection.

Asthmatic patients present more rapid progression of respiratory distress after A(H1N1)pdm09 influenza infection than after seasonal infection. Here, we sought to clarify the pathophysiology of early deterioration in asthmatic patients after A(H1N1)pdm09 infection. Cytokine levels and virus titres in bronchoalveolar lavage fluid from mice with and without asthma after A(H1N1)pdm09 or seasonal H1N1 infection were examined. In asthma/A(H1N1)pdm09 mice, IL-6 and TNF-α levels peaked at 3 days post-infection and were higher than those in all other groups. IFN-γ levels in asthma/A(H1N1)pdm09 mice at 3 days post-infection were higher than in all other mice at any time point, whereas at 7 days post-infection, the levels were lowest in asthma/A(H1N1)pdm09 mice. Virus titres in asthma/A(H1N1)pdm09 mice were highest at 3 days post-infection, and decreased by 7 days post-infection, although the levels at this time point were still higher than that in any other group. Histopathological examination showed more inflammatory cell infiltration and lung tissue destruction in the asthma/A(H1N1)pdm09 group than in any other group. The distinct cytokine profiles in A(H1N1)pdm09-infected asthmatic mice indicated excessive inflammation and virus replication within a few days after infection. Thus, bronchial asthma could be a more exacerbating factor for pandemic influenza infection than for seasonal influenza infection.

Molecular evolution of the capsid gene in human norovirus genogroup II.

Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens.

BACKGROUND: An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. OBJECTIVES: To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. STUDY DESIGN: The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. RESULTS: The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. CONCLUSIONS: The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination.

Predicting genotype compositions in norovirus seasons in Japan.

Noroviruses cause acute gastroenteritis. Since multiple genotypes of norovirus co-circulate in humans, changing the genotype composition and eluding host immunity, development of a polyvalent vaccine against norovirus in which the genotypes of vaccine strains match the major strains in circulation in the target season is desirable. However, this would require prediction of changes in the genotype composition of circulating strains. A fitness model that predicts the proportion of a strain in the next season from that in the current season has been developed for influenza A virus. Here, such a fitness model that takes into account the fitness effect of herd immunity was used to predict genotype compositions in norovirus seasons in Japan. In the current study, a model that assumes a decline in the magnitude of cross immunity between norovirus strains according to an increase in the divergence of the major antigenic protein VP1 was found to be appropriate for predicting genotype composition. Although it is difficult to predict the proportions of genotypes accurately, the model is effective in predicting the direction of change in the proportions of genotypes. The model predicted that GII.3 and GII.4 may contract, whereas GII.17 may expand and predominate in the 2015-2016 season. The procedure of predicting genotype compositions in norovirus seasons described in the present study has been implemented in the norovirus forecasting system (NOROCAST).

Genetic analysis of human rotavirus C: The appearance of Indian-Bangladeshi strain in Far East Asian countries.

Rotaviruses C (RVCs) circulate worldwide as an enteric pathogen in both humans and animals. Most studies of their genetic diversity focus on the VP7 and VP4 genes, but the complete genomes of 18 human RVCs have been described in independent studies. The genetic background of the Far East Asian RVCs is different than other human RVCs that were found in India and Bangladesh. Recently, a RVC detected in 2010 in South Korea had genetic background similar to the Indian-Bangladeshi RVCs. This study was undertaken to determine the whole genome of eight Japanese RVCs detected in 2005-2012, and to compare them with other human and animal global RVCs to better understand the genetic background of contemporary Far East Asian RVC. By phylogenetic analysis, the human RVCs appeared to be distinct from animal RVCs. Among human RVCs, three lineage constellations had prolonged circulation. The genetic background of the Far East Asian RVC was distinguished from Indian-Bangladeshi RVC as reported earlier. However, we found one Japanese RVC in 2012 that carried the genetic background of Indian-Bangladeshi RVC, whereas the remaining seven Japanese RVCs carried the typical genetic background of Far East Asian RVC. This is the first report of the Indian-Bangladeshi RVC in Japan. With that observation and the reassortment event of human RVCs in Hungary, our study indicates that the RVCs are spreading from one region to another.

An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

[Fact-finding survey on regional healthcare services for patients with epilepsy based on a questionnaire administered to public health centers in Japan].

OBJECTIVES: Epilepsy is a common chronic neurological disorder characterized by recurrent unprovoked seizures. The prevalence of epilepsy is about 1%, and its incidence is increasing with the aging population. In addition to their medical problems, epilepsy patients face many social problems, including schooling, working, and maintaining their driver's licenses. However, these problems are not fully recognized by the regional healthcare centers (HCCs), and the inadequacy of collaboration between medical services, healthcare, and welfare is sometimes pointed out. Under these circumstances, this fact-finding survey was administered in the form of a questionnaire to HCCs across the nation for the purpose of improving the support system and educational activities for epilepsy in Japan. METHODS: A mail-back survey on regional healthcare services for epilepsy patients was sent out to 490 HCCs across the nation. Public health nurses (PHNs) responded to the self-completed questionnaire on behalf of each HCC. The questionnaire was comprised of the presence or absence of consultations on epilepsy, content of the consultations, and holding of workshops, lectures, or conferences in the community covered by the HCC. RESULTS: We obtained responses from 347 HCCs (response rate 71%). Seventy-three percent of the PHNs had experience with consultations regarding the medical and healthcare issues associated with epilepsy. However, only 10% of the PHNs responded that they could provide appropriate consultation for these issues. The content of the consultations mainly included medical services, clinical symptoms of epilepsy, and anxieties about their social life and their future. Workshops, lectures, or conferences on epilepsy were held for residents or health and welfare professionals in only 8% of the communities. This percentage is lower than those (21-70%) for other intractable or mental disorders that are mainly managed by HCCs (P<0.01). On the other hand, 76% of PHNs in the HCCs felt the need for knowledge about epilepsy, and 60% wanted to join the epilepsy educational programs. CONCLUSION: Although many PHNs belonging to HCCs conduct consultations regarding epilepsy-related issues, many feel they cannot adequately respond to these issues. Furthermore, they feel the need for further knowledge about epilepsy but are not able to gain such knowledge because of financial and geographical restrictions. To improve these situations, regional education programs for epilepsy should be established in each local municipality in the future with support provided by medical facilities, regional medical associations, the Japan Epilepsy Society, and the Government.