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.

Development of an RT-LAMP Assay for the Rapid Detection of SFTS Virus.

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >103.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease.

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.

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.

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.

Phylogenetic and Geographic Relationships of Severe Fever With Thrombocytopenia Syndrome Virus in China, South Korea, and Japan.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne acute infectious disease caused by the SFTS virus (SFTSV). SFTS has been reported in China, South Korea, and Japan as a novel Bunyavirus. Although several molecular epidemiology and phylogenetic studies have been performed, the information obtained was limited, because the analyses included no or only a small number of SFTSV strains from Japan. METHODS: The nucleotide sequences of 75 SFTSV samples in Japan were newly determined directly from the patients' serum samples. In addition, the sequences of 7 strains isolated in vitro were determined and compared with those in the patients' serum samples. More than 90 strains that were identified in China, 1 strain in South Korea, and 50 strains in Japan were phylogenetically analyzed. RESULTS: The viruses were clustered into 2 clades, which were consistent with the geographic distribution. Three strains identified in Japan were clustered in the Chinese clade, and 4 strains identified in China and 26 in South Korea were clustered in the Japanese clade. CONCLUSIONS: Two clades of SFTSV may have evolved separately over time. On rare occasions, the viruses were transmitted overseas to the region in which viruses of the other clade were prevalent.

Cytokine profile of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during seasonal H1N1 infection.

BACKGROUND: Several studies support the role of viral infections in the pathogenesis of asthma exacerbation. However, several pediatricians believe that influenza virus infection does not exacerbate bronchial asthma, except for influenza A H1N1 2009 pandemic [A(H1N1)pdm09] virus infection. We previously reported that A(H1N1)pdm09 infection possibly induces severe pulmonary inflammation or severe asthmatic attack in a mouse model of bronchial asthma and in asthmatic children. However, the ability of seasonal H1N1 influenza (H1N1) infection to exacerbate asthmatic attacks in bronchial asthma patients has not been previously reported, and the differences in the pathogenicity profiles, such as cytokine profiles, remains unclear in bronchial asthma patients after A(H1N1)pdm09 and H1N1 infections. METHODS: The cytokine levels and viral titers in the bronchoalveolar lavage (BAL) fluid from mice with and without asthma after H1N1 infection (A/Yamagata and A/Puerto Rico strains) were compared. RESULTS: The interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, IL-5, interferon (IFN)-α, IFN-ß, and IFN-γ levels were significantly higher in the BAL fluids from the control/H1N1 mice than from the asthmatic/H1N1 mice. The viral titers in the BAL fluid were also significantly higher in the control/H1N1mice than in the asthmatic/H1N1 mice infected with either A/Yamagata or A/Puerto Rico. CONCLUSIONS: A(H1N1)pdm09 infection, but not H1N1 infection, can induce severe pulmonary inflammation through elevated cytokine levels in a mouse model of asthma.

Sensitive and specific PCR systems for detection of both Chinese and Japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.

Analysis of bronchoalveolar lavage fluid in a mouse model of bronchial asthma and H1N1 2009 infection.

BACKGROUND: Bronchial asthma is known as a risk factor of admission to the intensive care unit. However, the mechanism by which pandemic 2009 H1N1 (A(H1N1)pdm09) infection increases the severity of symptoms in patients with bronchial asthma is unknown; therefore, we aimed at determining this mechanism. METHODS: Inflammatory cell levels in the bronchoalveolar lavage (BAL) fluid from the non-asthma/mock, non-asthma/A(H1N1)pdm09, asthma/mock, and asthma/A(H1N1)pdm09 groups were determined using BALB/c mice. Cell infiltration levels, cytokine levels, and viral titers were compared among the groups. RESULTS: Neutrophil, monocyte, interleukin (IL)-5, IL-6, IL-10, IL-13, and tumor necrosis factor (TNF)-α levels were significantly higher in the BAL fluid from the non-asthma/A(H1N1)pdm09 and asthma/A(H1N1)pdm09 groups than in the mock groups (p<0.05 for neutrophils and monocytes; p<0.01 for the rest). The number of eosinophils and CD8(+) lymphocytes and the level of transforming growth factor beta 1 (TGF-ß1) in BAL fluid in the asthma/A(H1N1)pdm09 group were significantly higher among all groups (p<0.05 for eosinophils and CD8(+) lymphocytes; p<0.01 for TGF-ß1). The levels of IL-6, IL-10, IL-13, and TNF-α were significantly higher in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group (p<0.05 for IL-6 and IL-10; p<0.01 for IL-13 and TNF-α). The level of IFN-γ in the asthma/A(H1N1)pdm09 group was significantly lower than that in the non-asthma/A(H1N1)pdm09 group (p<0.05). The viral titers in the BAL fluids were higher in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group (p<0.05). Histopathological examination showed more severe infiltration of inflammatory cells and destruction of lung tissue in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group. CONCLUSIONS: Severe pulmonary inflammation induced by elevated levels of cytokines, combined with increased viral replication due to decreased IFN-γ levels, may contribute to worsening respiratory symptoms in patients with bronchial asthma and A(H1N1)pdm09 infection.

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.

Detection of multiple sapovirus genotypes and genogroups in oyster-associated outbreaks.

This report describes multiple viruses in stool specimens from oyster-associated gastroenteritis. Eleven outbreaks of oyster-associated gastroenteritis were examined for enteric viruses between January 2002 and March 2006 in Japan. Multiple norovirus genotypes were detected in all outbreaks; moreover, kobuvirus, sapovirus, and astrovirus were also detected in 6, 3, and 1 of the 11 outbreaks, respectively. Notably, multiple sapovirus genogroups were detected in the stool specimens from subjects in two oyster-associated gastroenteritis outbreaks.