Molecular Epidemiology Of Human Metapneumovirus Infection And The Relationship Between Human Metapneumovirus Infection And Asthma Exacerbation

PART I:Molecular Epidemiology of Human Metapneumovirus Infection in Chongqing children with Actue Lower Respiratory Tract InfectionsObjectives:In this study, we describe the demographic and clinical characteristics associated with human metapneumovirus(hMPV) infection in patients presenting to a children's hospital retrospectively.Methods:878 Specimens were collected over a 2-year period from children hospitalized with acute lower respiratory tract infections (ALRTI) and analyzed for the presence of hMPV using real-time chain reaction assay. To verify the presence and sequence of the virus, traditional RT-PCR primers 450F and 450R were used to amplify the hMPV F gene to ensure reproducibility. GF and GR primers were used to amplify the hMPV G gene and the sequences were used for phylogenetic analysis. All hMPV-postive samples were sent for testing for respiratory syncytial virus (RSV) and human coronavirus NL-63 (HCoV NL-63) by use of polymerase chain reaction. Some of the samples were sent for testing for common respiratory viruses, including influenza A and B viruses, human parainfluenza virus types 1–3 and adenovirus by use of direct immunofluorescence assay (DFA) on cells present in the respiratory specimens.Results:The presence of hMPV was detected in 227 (25.9%) of the 878 children studied and may circulate year-round in the area, peaking during the winter-spring season. Younger children (aged less than 6 months) had the highest rate of positivity. Infections by hMPV showed similar epidemiology and clinical manifestations as for respiratory syncytial virus (RSV) and hMPV infection was related to wheezing, asthma exacerbations or bronchiolitis. HMPV was likely to be found in co-infections with common respiratory virus, especially RSV. HMPV and RSV co-infection might lead to a more severe disease. The phylogenetic tree of the hMPV isolates recovered during 2006-2008 showed two major groups or clusters. The majority of isolates grouped with subgroup A2 virus sequences in Chongqing.Conclusion:This study indicates that hMPV is one of the major respiratory pathogens found in children with ALRTI presenting to a children's hospital in Chongqing. Infant under six months of age seemed to be more susceptible to hMPV. PART II:Establishment of BALB/c mouse asthma model infected with hMPV or RSVObjectives:To establish a mouse asthma model infected with hMPV or RSV.Methods:BALB/c mouse asthma model: Female BALB/c mice were maintained on OVA-free diets and divided into 2 Groups (n=16). The mice of OVA group were sensitized by intraperitoneal injection with OVA on Days 1 and 14, and challenged via the airways with OVA on Days 28, 29, and 30 by ultrasonic nebulization. The mice of control group were received phosphate buffer solution (PBS) as the substitution of OVA. The two groups were assessed on Day 31 for airway hyperresponsiveness (AHR), anti-OVA IgE in serum, IFN-γand IL-4 in bronchoalveolar lavage fluid (BALF), categorization and count of leucocytes of BALF, and pulmonary histopathology. BALB/c mouse asthma model infected with hMPV/RSV: Female BALB/c mice were divided into 2 Groups (n=48, 24 for each group). Mice were sensitized with OVA on Days 1 and 14, and challenged with OVA from Days 28 to 37. The mice of OVA/hMPV group were infected intranasally with 107.0 TCID50/ml hMPV and the mice of OVA/RSV group were infected with 107.0 TCID50/ml RSV on Day 30. The two groups were assessed on Days 33, 35 and 37 for the hMPV/RSV replication.Results:BALB/c mouse asthma model: data showed that the mice of OVA group, as compared with the mice of control group, appeared symptoms of acute asthma, AHR,higher levels of OVA specific IgE in serum, lower levels of IFN-γand higher levels of IL-4 in BALF, greatly increased absolute numbers and percentages of leukocytes and eosinophils in the BALF, and significant change in Histopathology. These results indicated BALB/c mouse asthma model have been established successfully. BALB/c mouse asthma model infected with hMPV/RSV: two groups were observed with weight loss and abnormal praxiology. Cytopathic effects (CPE) were observed in Vero-E6 cells or Hep-2 cells which were inoculated the lungs of mice. Amplicons by RT-PCR were accordant with expectant. HMPV and RSV were obviously tested by use of immunofluorescence. These results indicated BALB/c mouse asthma model infected with hMPV/RSV have been established successfully.Conclusion: This successful BALB/c mouse asthma model infected with hMPV/RSV provides useful tools in the subsequent study of relationship between viral infection and asthma exacerbation.PART III:The relationship between human metapneumovirus or respiratory syncytial virus infection and asthma exacerbationObjectives: To observe the changes of airway hyperreactivity and immune response in a mouse asthma model infected with hMPV/RSV.Methods:Female BALB/c mice were maintained on OVA-free diets and divided into 4 Groups (n=96, 24 for each group). The mice of OVA group were sensitized with OVA on Days 1 and 14, challenged with OVA from Days 28 to 37, and infected with PBS on Day 30. The mice of control group were received PBS as the substitution of OVA. The mice of OVA/hMPV group were sensitized and challenged with OVA, and infected with hMPV. The mice of OVA/RSV group were sensitized and challenged with OVA, and infected with RSV. On Days 33, 35, 37, mice were assayed for airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell counts and differential counts, IFN-γand IL-4 cytokine measurements of BALF, lung histopathology and flow cytometric analysis for intracellular cytokine expression(IFN-γ, IL-4, IL-17 and Foxp3).Results:Clinical illness severity (illness score, weight loss, AHR, total cell number of BALF and histopathology), cytokine (IFN-γand IL-4), intracellular cytokine expression (IFN-γ,IL-4,IL-17 and Foxp3) of asthma mice infected with hMPV or RSV were significantly more severe than OVA group. HMPV infection, as compared with RSV infection, induced weak IFN-γresponse, and severe histopathology changes of lung during the acute phase. On day 3 afeter infection, hMPV and RSV infection increased proportion of Th1-type (IFN-γ), Th2-type (IL-4) cytokine-producing cells, and RSV infection induced more proportion of Th1-type (IFN-γ), Th2-type (IL-4) cytokine-producing cells than hMPV infection. On days 5 and 7 afeter infection, hMPV and RSV infection increased proportion of Th17 cells and Treg cell in the lungs. As compared with RSV infection, hMPV infection induced strong Treg response, and weak Th17 response.Conclusion: HMPV or RSV infections induced asthma exacerbation of asthma mice. As compared with RSV infection, hMPV infection induced severse histopathology changes of lung, strong Treg response, and weak Th1, Th2 and Th17 response.