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.

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.

Pathogen profiles and molecular epidemiology of respiratory viruses in Japanese inpatients with community-acquired pneumonia.

BACKGROUND: The etiological profile of viruses among adult patients with community-acquired pneumonia (CAP) has not been characterized yet. The aim of this study was twofold: first, investigate the pathogen profiles and the molecular epidemiology of respiratory viruses among Japanese CAP patients; and second, explore the clinical significance of viral infections. METHODS: A cross-sectional observational study was conducted at Kyorin University Hospital. To identify respiratory pathogens, hospitalized CAP patients were enrolled, and reverse transcriptase-polymerase chain reaction technology was applied alongside conventional microbiological methods. Phylogenetic and pairwise distance analyses of 10 viruses were performed. CAP patients were divided into four etiological groups (virus alone, bacteria alone, co-detection of virus and bacteria, and not detected) and the clinical findings were compared. RESULTS: Seventy-six patients were enrolled. Bacteria alone were detected in 39.5% (n=30) of CAP patients. Virus alone or co-detection were found in 10.5% (n=8) and 11.8% (n=9) of cases, respectively. Streptococcus pneumoniae and human metapneumovirus were the most frequently detected bacterium and virus, respectively. Phylogenetic analyses of human metapneumovirus, human rhinovirus, and human respiratory syncytial virus showed that different subgroups and genotypes might be associated with CAP. Respiratory failure was more common when a virus was detected (both virus alone and co-detection groups; n=17, 100%, p<0.05) than when a bacteria alone was detected (n=17, 56.7%). CONCLUSION: Prevalence of respiratory virus infection in CAP inpatients was 22.3%. The detected viruses display high genetic divergence and correlate with increased respiratory failure.

Neuropathogenicity of Two Saffold Virus Type 3 Isolates in Mouse Models.

OBJECTIVE: Saffold virus (SAFV), a picornavirus, is occasionally detected in children with acute flaccid paralysis, meningitis, and cerebellitis; however, the neuropathogenicity of SAFV remains undetermined. METHODS: The virulence of two clinical isolates of SAFV type 3 (SAFV-3) obtained from a patient with aseptic meningitis (AM strain) and acute upper respiratory inflammation (UR strain) was analyzed in neonatal and young mice utilizing virological, pathological, and immunological methods. RESULTS: The polyproteins of the strains differed in eight amino acids. Both clinical isolates were infective, exhibited neurotropism, and were mildly neurovirulent in neonatal ddY mice. Both strains pathologically infected neural progenitor cells and glial cells, but not large neurons, with the UR strain also infecting epithelial cells. UR infection resulted in longer inflammation in the brain and spinal cord because of demyelination, while the AM strain showed more infectivity in the cerebellum in neonatal ddY mice. Additionally, young BALB/c mice seroconverted following mucosal inoculation with the UR, but not the AM, strain. CONCLUSIONS: Both SAFV-3 isolates had neurotropism and mild neurovirulence but showed different cell tropisms in both neonatal and young mouse models. This animal model has the potential to recapitulate the potential neuropathogenicity of SAFV-3.

MIP-1α level in nasopharyngeal aspirates at the first wheezing episode predicts recurrent wheezing.

BACKGROUND: Respiratory virus-induced wheezing, such as that induced by respiratory syncytial virus (RSV) and human rhinovirus, is an important risk factor for recurrent wheezing and childhood asthma. However, no biomarkers for predicting recurrent wheezing have been identified. OBJECTIVE: We searched for predictors of recurrent wheezing using nasopharyngeal aspirates obtained from patients during the first wheezing episode who were hospitalized with an acute lower respiratory tract illness. METHODS: We enrolled 82 infants during the first wheezing episode (median age, 5.0 months) who were hospitalized for acute lower respiratory tract illness between August 2009 and June 2012 and followed these patients for 2.5 years. Nasopharyngeal aspirates and blood samples were obtained on the first day of hospitalization. Viral genomes were identified by using RT-PCR and sequencing. Levels of 33 cytokines, tryptase, IgE, anti-RSV IgE, and anti-RSV IgG were measured by using ELISAs or the Bio-Plex multiplex assay. Predictors of recurrent wheezing were examined by using a stepwise logistic regression model with backward elimination. RESULTS: Sixty percent of the patients experienced recurrent wheezing episodes. One or more viruses were detected in the nasopharynxes of 93% of the patients during the first wheezing episode. IFN-γ, IL-2, IL-9, MIP-1α, and MIP-1ß levels were significantly higher among patients with recurrent wheezing than among those without recurrent wheezing (P < .05 or .01). The stepwise model demonstrated that the MIP-1α level (odds ratio, 7.72; 95% CI, 1.50-39.77; P = .015) was the strongest independent predictor of the occurrence of recurrent wheezing. CONCLUSION: An increased MIP-1α level in nasopharyngeal aspirates from patients with acute respiratory symptoms during the first wheezing episode caused by viral infections might predict recurrent wheezing.

Molecular Evolution of the Capsid Gene in Norovirus Genogroup I.

We studied the molecular evolution of the capsid gene in all genotypes (genotypes 1-9) of human norovirus (NoV) genogroup I. The evolutionary time scale and rate were estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also performed selective pressure analysis and B-cell linear epitope prediction in the deduced NoV GI capsid protein. Furthermore, we analysed the effective population size of the virus using Bayesian skyline plot (BSP) analysis. A phylogenetic tree by MCMC showed that NoV GI diverged from the common ancestor of NoV GII, GIII, and GIV approximately 2,800 years ago with rapid evolution (about 10(-3) substitutions/site/year). Some positive selection sites and over 400 negative selection sites were estimated in the deduced capsid protein. Many epitopes were estimated in the deduced virus capsid proteins. An epitope of GI.1 may be associated with histo-blood group antigen binding sites (Ser377, Pro378, and Ser380). Moreover, BSP suggested that the adaptation of NoV GI strains to humans was affected by natural selection. The results suggested that NoV GI strains evolved rapidly and date back to many years ago. Additionally, the virus may have undergone locally affected natural selection in the host resulting in its adaptation to humans.

Molecular evolution of haemagglutinin (H) gene in measles virus.

We studied the molecular evolution of the haemagglutinin (H) gene (full length) in all genotypes (24 genotypes, 297 strains) of measles virus (MeV). The gene's evolutionary timescale was estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also analysed positive selection sites. The MCMC tree indicated that the MeV H gene diverged from the rinderpest virus (same genus) about 250 years ago and that 24 MeV genotypes formed 3 lineages dating back to a 1915 ancestor (95% highest posterior density [HPD] 1882-1941) with relatively rapid evolution (mean rate: 9.02 × 10(-4) substitutions/site/year). The 3 lineages diverged in 1915 (lineage 1, 95% HPD 1882-1941), 1954 (lineage 2, 95% HPD 1937-1969), and 1940 (lineage 3, 95% HPD 1927-1952). These 24 genotypes may have diverged and emerged between the 1940s and 1990 s. Selective pressure analysis identified many negative selection sites on the H protein but only a few positive selection sites, suggesting strongly operated structural and/or functional constraint of changes on the H protein. Based on the molecular evolution of H gene, an ancestor MeV of the 24 genotypes emerged about 100 years ago and the structure of H protein has been well conserved.

Molecular evolution of the VP1, VP2, and VP3 genes in human rhinovirus species C.

Human rhinovirus species C (HRV-C) was recently discovered, and this virus has been associated with various acute respiratory illnesses (ARI). However, the molecular evolution of the major antigens of this virus, including VP1, VP2, and VP3, is unknown. Thus, we performed complete VP1, VP2, and VP3 gene analyses of 139 clinical HRV-C strains using RT-PCR with newly designed primer sets and next-generation sequencing. We assessed the time-scale evolution and evolutionary rate of these genes using the Bayesian Markov chain Monte Carlo method. In addition, we calculated the pairwise distance and confirmed the positive/negative selection sites in these genes. The phylogenetic trees showed that the HRV-C strains analyzed using these genes could be dated back approximately 400 to 900 years, and these strains exhibited high evolutionary rates (1.35 to 3.74 × 10(-3) substitutions/site/year). Many genotypes (>40) were confirmed in the phylogenetic trees. Furthermore, no positively selected site was found in the VP1, VP2, and VP3 protein. Molecular modeling analysis combined with variation analysis suggested that the exterior surfaces of the VP1, VP2 and VP3 proteins are rich in loops and are highly variable. These results suggested that HRV-C may have an old history and unique antigenicity as an agent of various ARI.

Molecular evolution of human respiratory syncytial virus attachment glycoprotein (G) gene of new genotype ON1 and ancestor NA1.

We conducted a comprehensive genetic analysis of the C-terminal 3rd hypervariable region of the attachment glycoprotein (G) gene in human respiratory syncytial virus subgroup A (HRSV-A) genotype ON1 (93 strains) and ancestor NA1 (125 strains). Genotype ON1 contains a unique mutation of a 72 nucleotide tandem repeat insertion (corresponding to 24 amino acids) in the hypervariable region. The Bayesian Markov chain Monte Carlo (MCMC) method was used to conduct phylogenetic analysis and a time scale for evolution. We also calculated pairwise distances (p-distances) and estimated the selective pressure. Phylogenetic analysis showed that the analyzed ON1 and NA1 strains formed 4 lineages. A strain belonging to lineage 4 of ON1 showed wide genetic divergence (p-distance, 0.072), which suggests that it might be a candidate new genotype, namely ON2. The emergence of genotype NA1 was estimated to have occurred in 2000 (95% of highest probability density, HPD; 1997-2002) and that of genotype ON1 in 2005 (95% HPD; 2000-2010) based on the time-scaled phylogenetic tree. The evolutionary rate of genotype ON1 was higher than that of ancestral genotype NA1 (6.03×10(-3) vs. 4.61×10(-3) substitutions/site/year, p<0.05). Some positive and many negative selection sites were found in both ON1 and NA1 strains. The results suggested that the new genotype ON1 is rapidly evolving with antigenic changes, leading to epidemics of HRSV infection in various countries.

Novel aspects on the pathogenesis of Mycoplasma pneumoniae pneumonia and therapeutic implications.

Mycoplasma pneumoniae (Mp) is a leading cause of community acquired pneumonia. Knowledge regarding Mp pneumonia obtained from animal models or human subjects has been discussed in many different reports. Accumulated expertise concerning this critical issue has been hard to apply clinically, and potential problems may remain undiscovered. Therefore, our multidisciplinary team extensively reviewed the literature regarding Mp pneumonia, and compared findings from animal models with those from human subjects. In human beings, the characteristic pathological features of Mp pneumonia have been reported as alveolar infiltration with neutrophils and lymphocytes and lymphocyte/plasma cell infiltrates in the peri-bronchovascular area. Herein, we demonstrated the novel aspects of Mp pneumonia that the severity of the Mp pneumonia seemed to depend on the host innate immunity to the Mp, which might be accelerated by antecedent Mp exposure (re-exposure or latent respiratory infection) through up-regulation of Toll-like receptor 2 expression on bronchial epithelial cells and alveolar macrophages. The macrolides therapy might be beneficial for the patients with macrolide-resistant Mp pneumonia via not bacteriological but immunomodulative effects. This exhaustive review focuses on pathogenesis and extends to some therapeutic implications such as clarithromycin, and discusses the various diverse aspects of Mp pneumonia. It is our hope that this might lead to new insights into this common respiratory disease.

Epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus.

Viral respiratory infections may be associated with the virus-induced asthma in adults as well as children. Particularly, human rhinovirus is strongly suggested a major candidate for the associations of the virus-induced asthma. Thus, in this review, we reviewed and focused on the epidemiology, pathophysiology, and treatment of virus-induced asthma with special reference on human rhinovirus. Furthermore, we added our preliminary data regarding the clinical and virological findings in the present review.

Triplex real-time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus.

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT-PCR method was established. This triplex real-time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real-time PCR across a broad dynamic range of 10(2) -10(7) copies/assay using plasmid DNA standards. The limit of detection was 10(2) copies/assay. The quantitative value was comparable with that of monoplex real-time PCR of stool samples. Our triplex real-time PCR is useful for detection of NoV and SaV infections.

Novel reassortant influenza A(H1N2) virus derived from A(H1N1)pdm09 virus isolated from swine, Japan, 2012.

We isolated a novel influenza virus A(H1N2) strain from a pig on January 13, 2012, in Gunma Prefecture, Japan. Phylogenetic analysis showed that the strain was a novel type of double-reassortant virus derived from the swine influenza virus strains H1N1pdm09 and H1N2, which were prevalent in Gunma at that time.

Severity of respiratory signs and symptoms and virus profiles in Japanese children with acute respiratory illness.

Associations between the severity of respiratory signs and symptoms and the respiratory viruses identified in 214 Japanese children with acute respiratory illness (ARI) enrolled between January and December 2012 were studied. Respiratory rate, wheezing, cyanosis, and the use of accessory muscles were used as indices of respiratory severity and phylogenetic analysis of the viruses identified in these children was performed. Respiratory viruses such as respiratory syncytial virus (RSV), human rhinovirus (HRV), human parainfluenza virus (HPIV), and human metapneumovirus (HMPV) were prevalent, being detected in approximately 70% of the patients (151/214 patients). Co-detection of viruses occurred in about 9% of patients. RSV was identified more frequently in cases scored as moderate/severe than in those scored as mild (P < 0.05). Severity scores of patients with RSV were significantly higher than those of cases with HPIV. Moreover, severity scores in patients with mild disease and co-detections were higher than in those in whom only HPIV or adenovirus was detected. Phylogenetic analysis showed that many genotypes of HRV-A and -C with wide genetic divergence were associated with acute respiratory illness (ARI). On the other hand, only a limited number of genotypes of RSV were associated with ARI. HPIV and HMPV were associated with ARI at similar frequencies. These results suggest that different respiratory viruses with unique genetic characteristics can be found in patients with mild to severe ARI.

Molecular epidemiology of respiratory viruses in virus-induced asthma.

Acute respiratory illness (ARI) due to various viruses is not only the most common cause of upper respiratory infection in humans but is also a major cause of morbidity and mortality, leading to diseases such as bronchiolitis and pneumonia. Previous studies have shown that respiratory syncytial virus (RSV), human rhinovirus (HRV), human metapneumovirus (HMPV), human parainfluenza virus (HPIV), and human enterovirus infections may be associated with virus-induced asthma. For example, it has been suggested that HRV infection is detected in the acute exacerbation of asthma and infection is prolonged. Thus it is believed that the main etiological cause of asthma is ARI viruses. Furthermore, the number of asthma patients in most industrial countries has greatly increased, resulting in a morbidity rate of around 10-15% of the population. However, the relationships between viral infections, host immune response, and host factors in the pathophysiology of asthma remain unclear. To gain a better understanding of the epidemiology of virus-induced asthma, it is important to assess both the characteristics of the viruses and the host defense mechanisms. Molecular epidemiology enables us to understand the pathogenesis of microorganisms by identifying specific pathways, molecules, and genes that influence the risk of developing a disease. However, the epidemiology of various respiratory viruses associated with virus-induced asthma is not fully understood. Therefore, in this article, we review molecular epidemiological studies of RSV, HRV, HPIV, and HMPV infection associated with virus-induced asthma.

Neurite outgrowth of PC12 cells by 4'-O-ß-D-glucopyranosyl-3',4-dimethoxychalcone from Brassica rapa L. 'hidabeni' was enhanced by pretreatment with p38MAPK inhibitor.

The cellular effects of eleven compounds including chalcone glycosides isolated from Brassica rapa L. 'hidabeni' and their synthetic derivatives were studied in rat pheochromocytoma PC12 cells. Of the compounds tested, 4'-O-ß-D-glucopyranosyl-3',4-dimethoxychalcone (A2) significantly increased the levels of the phosphorylated forms of extracellular signal-regulated kinases 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38MAPK), and stress-activated protein kinases/Jun amino-terminal kinases (JNK/SAPK), but it did not affect Akt. Nerve growth factor (NGF), a well-known neurotrophic factor, increased the levels of phosphorylated ERK1/2, JNK/SAPK, and Akt but not p38MAPK, which may mediate marked neurite outgrowth. Signals evoked by A2 shared common characteristics with those induced by NGF; therefore, we evaluated the neuritogenic activity of A2 and found it induced only weak neurite outgrowth. However, this effect was enhanced by pre-treatment with a p38MAPK inhibitor, suggesting that the phosphorylation of p38MAPK down-regulated neurite outgrowth. From the results of this study, it was found that A2 in combination with a p38MAPK inhibitor can induce NGF-like effects. Hence, a combination of chalcone glycosides containing A2 and a p38MAPK inhibitor increases the likelihood that chalcone glycosides could be put to practical use in the form of drugs or alternative medicines to maintain neural health.

[Training programs for staff at local Infectious Disease Surveillance Centers: the needs and usefulness].

OBJECTIVES: The objective of this study was to assess the need for and usefulness of training programs for Local Infectious Disease Surveillance Center (LIDSC) staff. METHODS: A structured questionnaire survey was conducted to assess the needs and usefulness of training programs. The subjects of the survey were participants of a workshop held after an annual conference for the LIDSC staff. Data on demographic information, the necessity of training programs for LIDSC staff, the themes and contents of the training program, self-assessment of knowledge on epidemiology and statistics were covered by the questionnaire. RESULTS: A total of 55 local government officials responded to the questionnaire (response rate: 100%). Among these, 95% of participants believed that the training program for the LIDSC staff was necessary. Basic statistical analysis (85%), descriptive epidemiology (65%), outline of epidemiology (60%), interpretation of surveillance data (65%), background and objectives of national infectious disease surveillance in Japan (60%), methods of field epidemiology (60%), and methods of analysis data (51%) were selected by over half of the respondents as suitable themes for training programs. A total of 34 LIDSC staff answered the self-assessment question on knowledge of epidemiology. A majority of respondents selected "a little" or "none" for all questions about knowledge. Only a few respondents had received education in epidemiology. CONCLUSION: The results of this study indicate that LIDSC staff have basic demands for fundamental and specialized education to improve their work. Considering the current situation regarding the capacity of LIDSC staff, these training programs should be started immediately.

Genetic analysis of attachment glycoprotein (G) gene in new genotype ON1 of human respiratory syncytial virus detected in Japan.

We studied the evolution of the G gene in the new genotype ON1 of RSV detected from patients with acute respiratory infection in Japan. Phylogenetic analyses and the evolutionary timescale were obtained by the Bayesian MCMC method. We also analyzed p-distance and positive selection sites. A new genotype ON1 emerged around 2001. The evolution rate was rapid (3.57 × 10(-3) substitutions/site per year). The p-distance was short and no positive selection site was found in the present strains. These results suggested that a new genotype ON1 of RSV-A emerged approximately10 years ago and spread to some countries with a high evolution rate.

Elevated macrophage inflammatory protein 1α and interleukin-17 production in an experimental asthma model infected with respiratory syncytial virus.

BACKGROUND: Respiratory syncytial virus (RSV) infection is associated with both the development and exacerbation of bronchial asthma. We examined eosinophil infiltration and the cytokine profiles of both airway and peripheral blood in antigen-sensitized mice infected with RSV to investigate the pathogenesis of exacerbations of asthma due to RSV infection. METHODS: Ovalbumin (OVA)-sensitized mice were challenged by OVA inhalation 3 times and then infected with RSV [10(5) TCID50 (50% of tissue culture infectious dose)/25 g body weight] or mock infection immediately after the last challenge. Animals from each group, namely, the control (PBS instead of OVA inhalation plus mock infection), RSV (PBS plus RSV), OVA (OVA plus mock) and OVA/RSV (OVA plus RSV) were analyzed. Analysis included evaluation of airway responsiveness to methacholine, pathological findings in the airway by hematoxylin and eosin (HE) and Luna staining, bronchoalveolar fluid (BALF) and peripheral leukocytes counts, and concentrations of multiple cytokines/chemokines in both BALF and serum. RESULTS: Airway responsiveness was significantly enhanced in the OVA and OVA/RSV groups compared with the control group. Levels of tissue and BALF eosinophils were higher in the OVA and OVA/RSV groups than in the RSV or control group. Significantly higher levels of macrophage inflammatory protein (MIP)-1α in BALF were observed in the OVA/RSV group compared with the 3 other groups. Production of serum IL-17 was also significantly elevated in the OVA/RSV group compared with the control or OVA group. CONCLUSIONS: These findings suggest that MIP-1α and IL-17 may play important roles in acute exacerbation of asthma induced by RSV in an animal model.

Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008-2011.

We investigated the evolution of the C-terminal 3rd hypervariable region of G gene in the prevalent human respiratory syncytial virus (RSV) subgroups A (RSV-A) and B (RSV-B) in Japan in 2008-2011. Phylogenetic analysis and the evolutionary timescale was obtained by the Bayesian Markov Chain Monte Carlo method. All 38 RSV-A strains detected were classified into genotype NA1 and the 17 RSV-B strains detected belonged to genotypes BA and GB2. NA1 subdivided around 1998 in the present phylogenetic tree. Genotype BA subdivided around 1994. The evolutionary rates for RSV-A and RSV-B were estimated at 3.63×10⁻³ and 4.56×10⁻³ substitutions/site/year, respectively. The mean evolutionary rate of RSV-B was significantly faster than that of RSV-A during all seasons. The pairwise distance was relatively short (less than 0.06). In addition, some unique sites under positive selection were found. The results suggested that this region of the RSV strains rapidly evolved with some unique amino acid substitutions due to positive pressure.