Kunihisa Kozawa

Detection, Quantitation, and Phylogenetic Analysis of Noroviruses in Japanese Oysters

ABSTRACT Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 102 copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation‐induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB‐irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0–400 mJ/cm2 UVB irradiation. UVB at a dose 400 mJ/cm2 induced the release of various cytokines; interleukin (IL)‐1β, IL‐6, IL‐8, interferon (IFN)‐γ, granulocyte‐colony stimulating factor (G‐CSF), macrophage inflammatory protein (MIP)‐1β, and tumor necrosis factor (TNF)‐α. These results suggest that UVB irradiation‐induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.

Isolation and phylogenetic analysis of Arcobacter spp. in ground chicken meat and environmental water in Japan and Thailand

Prevalence of Arcobacter spp. in chicken meat samples and environmental water samples in Japan and Thailand was investigated. Arcobacter was isolated from 48% of chicken meat samples (20/41) and 23% of river water samples (4/17) from Japan, and 100% of chicken meat samples (10/10) and 100% of canal water samples (7/7) from Thailand. A. butzleri was among the species isolated from all positive samples. About 10% genetic diversity was seen in the rpoB‐rpoC in Arcobacters, and phylogenetic trees were divided into two clusters. In both countries, the results suggested that chicken and environmental water were highly contaminated with a genetically diverse population of Arcobacter.

Elevation of cytokine concentrations in asphyxiated neonates

Background: Various cytokines are reportedly associated with many neonatal diseases. Asphyxia is considered to result in ischemia-reperfusion injuries and induces abnormal inflammatory responses involving excessive cytokine production. Objectives: To evaluate alteration in sera levels of various cytokines/chemokines in case of perinatal asphyxia at birth. Methods: In orderto determine the concentrations of various cytokines/chemokines in sera, we used a highly sensitive fluorescence microsphere method. We measured the concentration of 8 types of cytokines/chemokines in sera obtained from 17 cases of asphyxia, 10 normal neonates, and 6 healthy adults. Results: The concentrations of IL-6, IL-8, and IL-10 in the sera of asphyxiated neonates were higher than those in the normal neonates. Irrespective of the presence or absence of asphyxia, sera concentrations of IL-2, IL-4, IFN-γ, and TNF-α were higher in the neonates than those in the adults. The concentration of IFN-γ in the asphyxiated neonates was lower than that in the normal neonates. Sera levels of IL-10 were higher in the asphyxiated cases than those in the normal neonates. The sera levels of IL-6, IL-8, and IL-10 in asphyxiated neonates with either a poor outcome or death were higher than those without poor outcomes. Conclusions: The concentrations of various types of cytokines/chemokines were different in neonatal sera and some of them increased drastically during asphyxia. The concentration of an anti-inflammatory cytokine IL-10 was elevated in asphyxiated neonates immediately after birth, thereby suggesting that IL-10 might be associated with neuroprotective functions.

Clarification of the predominant emission sources of antimony in airborne particulate matter and estimation of their effects on the atmosphere in Japan

Environmental context. The remarkable enrichment of potentially toxic antimony (Sb) in inhalable airborne particulate matter has become of great environmental concern among recent air pollution issues. The present study clarifies the predominant sources of Sb by focusing on the similarities in elemental composition, particle size distributions, and microscopic images found in ambient airborne particles and several potential sources. We identify automotive brake abrasion dust and fly ash emitted from waste incineration as dominant sources of atmospheric Sb in Japan. These results will contribute towards an in-depth understanding of the cycles and fates of Sb in the environment. Abstract. By focusing on the similarities in elemental composition, particle size distributions of elemental concentrations, and microscopic images between ambient airborne particulate matter (APM) and several potential sources, we discuss the predominant sources of antimony (Sb) in APM in Japan. The distribution of Sb concentration in size-classified ambient APM showed a characteristic bimodal profile in which peaks were found in coarse (3.6–5.2 μm) and fine (0.5–0.7 μm) fractions. Elemental ratios, particle sizes, and microscopic images observed in the coarse APM fractions were found to be in good agreement with those of brake abrasion dust. However, in the fine APM fractions, fly ash originating from waste incineration was identified as the most probable source of Sb. Chemical mass balance analysis was performed to determine the effects of the emission sources of Sb, and it was revealed that brake abrasion dust and waste fly ash were the dominant sources of Sb in the coarser and the finer fractions of APM, respectively. The present study provides important clues to understanding the cycles and fates of Sb in the environment.

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.

Molecular epidemiological study of human rhinovirus species A, B and C from patients with acute respiratory illnesses in Japan

Recent studies suggest that human rhinovirus species A, B and C (HRV-ABCs) may be associated with both the common cold and severe acute respiratory illnesses (ARIs) such as bronchiolitis, wheezy bronchiolitis and pneumonia. However, the state and molecular epidemiology of these viruses in Japan is not fully understood. This study detected the genomes of HRV-ABCs from Japanese patients (92 cases, 0-36 years old, mean±sd 3.5±5.0 years) with various ARIs including upper respiratory infection, bronchiolitis, wheezy bronchiolitis, croup and pneumonia between January and December 2010. HRV-ABCs were provisionally type assigned from the pairwise distances among the strains. On phylogenetic trees based on the nucleotide sequences of the VP4/VP2 coding region, HRV-A, -B and -C were provisionally assigned to 14, 2 and 12 types, respectively. The present HRV-A and -C strains had a wide genetic diversity (>30 % divergence). The interspecies distances were 0.230±0.063 (mean±sd, HRV-A), 0.218±0.048 (HRV-B) and 0.281±0.105 (HRV-C), based on nucleotide sequences, and 0.075±0.036 (HRV-A), 0.049±0.022 (HRV-B) and 0.141±0.064 (HRV-C) at the deduced amino acid level. Furthermore, HRV-A and -C were the predominant species and were detected throughout the seasons. The results suggested that HRV-A and -C strains have a wide genetic divergence and are associated with various ARIs in Japan.

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.

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.

Serotypes, Antimicrobial Susceptibility, and gyr A Gene Mutation of Campylobacter jejuni Isolates from Humans and Chickens in Thailand

In Thailand, 51% (36/70) Campylobacter jejuni isolates from humans and 68% (47/69) isolates from poultry were classified into 10 Penner serotypes (serotype B, C, R, E, G, A, K, D, I, and L) and 9 serotypes (serotype A, C, I, K, B, E, S, D, and L), respectively. The rate of antimicrobial drug resistance to nalidixic acid, ciprofloxacin, ampicillin, tetracycline, and erythromycin shown by human isolates were 96%, 96%, 29%, 57%, and 14%, while that shown by poultry isolates were 77%, 77%, 22%, 26%, and 17%, respectively. All quinolone‐resistant strains contained a mutation in the gyrA gene (T86→I86), suggesting that the strains were already widespread in Thailand.