The Study On The Mechanism Of Adenovirus Virus Pneumonia Complicating Central Nervous System Diseases

PART?CLINICAL CHARACTERISTICS OF TOXIC ENCEPHALOPATHY BY ADENOVIRUS 3/7 PNEUMONIA IN CHONGQING AREAObjective: Adenovirus is a common pathogen of respiratory tract infections in children.HAd V-3 and HAd V-7 are one of the main causes of severe pneumonia in children.Toxic encephalopathy is an important complication of adenovirus.Clinical data show that HAd V-7 infection is more severe than HAd V-3,but it is unclear whether there is a difference in the severity of toxic encephalopathy caused by HAd V-3 and HAd V-7 pneumonia.This part is retrospective analysis of the clinical feature of toxic encephalopathy in hospitalized children with HAd V-3 and HAd V-7 pneumonia.Methods: To collect nasopharyngeal secretions of hospitalized children with acute lower respiratory tract infection and diagnosed as having adenovirus infections in the respiratory ward of Children's Hospital of Chongqing Medical University from June 2009 to December 2014,DNA was extracted by PCR and sequenced.Differentiation of toxic encephalopathy caused by adenovirus pneumonia is analysed by gene sequencing,Gen Bank comparison typing,and clinical data.Results: 1.HAd V positive rate and typing: Totally in 4678 specimens from June 2009 to December 2014,adenovirus was detected positive in 307 specimens with the positive detection rate 6.56%,and it is successful classification of 280 cases.The principal adenovirus serotypes were HAd V-3(129,45.09%)and HAd V-7(122,43.41%).2.Clinical features of HAd V-3 and HAd V-7 pneumonia: the incidence of fever,wheezing of HAd V-7 were higher than HAd V-3 and other HAd V infected children In 280.Similarly,the incidence of severe pneumonia were higher in HAd V-7 than in HAd V-3.HAd V-7 pneumonia is more likely to be complicated with toxic encephalopathy and respiratory failure than HAd V-3.3.Clinical features of severe HAd V pneumonia complicated with toxic encephalopathy: HAd V-7(23/29 79.31%)is the main serotype of toxic encephalopathy.HAd V-7 children with severe pneumonia are more prone to toxic encephalopathy than other types of children(p<0.05).Conclusion: The HAd V positive rate was 6.56%,and HAd V-7 and HAd V-3 were the main serotypes.HAd V-7 pneumonia children were more likely to complicate toxic encephalopathy than HAd V-3.PART ? EXPERIMENTAL STUDY ON ADENOVIRUS VIRUS PNEUMONIA COMPLICATING CENTRAL NERVOUS SYSTEM DISEASESObjective: The results of Part ? suggest that there is a difference in the degree of central nervous system disease caused by HAd V-3 and HAd V-7.In this study,This section intends to establish an animal model of adenovirus infection to investigate the experimental study about two types of adenovirus pneumonia complicated by central nervous system diseases.Methods: 1.In vitro,the supernatant of 0h,2h,8h,12 h,24h,36 h,48h and 72 h of HAd V-3 and/or HAd V-7 infection of A549 cell was collected,Simultaneously,the supernatant of the rat neurons infected with the cells was collected for 24 hours.And the virus copy number of HAd V-3 and HAd V-7 were detected by q-PCR.2.In vivo,clinical HAd V-3 and HAd V-7 isolates were used to infect BALB/c mice and establish adenovirus infection animal models;Brain tissue,ganglia and blood were collected at days 3,5 and 7 post-infection for pathological analysis.Using immunohistochemistry and q-PCR,we compared the different CNS alterations the viral load of HAd V-3 and HAd V-7.Results: 1.In vitro,The virus copy number of HAd V-7 infected with A549 cells was significantly higher than that of HAd V-3(p<0.05);the virus copy number of HAd V-3 after infection of primary neurons of rat was significantly higher than that of HAd V-7(p<0.05).2.In vivo,animal experiments did not detect virus in the blood,ganglia and brain tissues of the HAd V-7 infection group,although the levels of inflammatory mediators(TNF-?,IL-1? and IL-6)were significantly increased(p<0.05).In contrast,virus was detected in the brain tissue and ganglia of the HAd V-3 infection group,although no virus was detected in the blood,and the levels of inflammatory mediators were normal.Conclusion: The virulence of HAd V-7 was higher than HAd V-3 in vitro infection of epithelial A549 cells.HAd V-3 causes encephalitis by viral invasion of the CNS,whereas HAd V-7 causes encephalopathy by releasing inflammatory mediators.Clinical treatment of the toxic encephalopathy caused by HAd V-7 viral infection should mainly comprise blocking the inflammatory response,whereas treatment of the viral encephalitis caused by HAd V-3 infection requires antagonism of viral replication.PART ? HMGB1 mediates HAd V-7 infection-induced pulmonary inflammation in miceObjective: The results of Part ?and Part ? suggest that HAd V-3 and HAd V-7 are important pathogens of severe pneumonia in children;Due to the large amount of inflammatory mediators released by HAd V-7,the hospitalized children with HAd V-7 pneumonia were significantly more severe than HAd V-3.Therefore,finding a therapeutic target for HAd V-7 severe pneumonia is the key to control adenovirus.In recent years,the high mobility group box-1(HMGB1)protein,released by necrotic cells,has been shown to play important roles in the pneumonia cause by viral infections.This study investigated the role of HMGB1 in adenoviral pneumonia and looked for therapeutic targets for HAd V-7 pneumonia.Methods: 1.In vitro,the supernatant of 0,2,8,12 h,24h,36 h and 48 h of HAd V-3 and/or HAd V-7 infection of A549 cell was collected.and the expression and level of extracellular HMGB1 were analyzed by immunofluorescence and ELISA.2.In vivo,lung tissue and BALF specimens from normal and anti-HMGB1 m Ab BALB/c mice infected with HAd V-7 on the 3rd,5th,and 7th days after infection were collected for,histopathological analysis,ELISA,and q-PCR.The levels of virus copy number,HMGB1,TLR4,TLR9,RAGE,and NF-?B,IL-1?,IL-6,and TNF-? were analyzed.Results: 1.In vitro,HMGB1 levels gradually increased in the media supernatants of HAd V-7 infected A549 cells,starting at 12 h post-infection.It was Significantly increased at 24 h,36h,and 48 h after infection compared with the Control group(p<0.05).In vivo,the level of protein in the lung tissue(after alveolar lavage)gradually decreased on days 3,5 and 7 after infection(p<0.05).2.In vivo,HMGB1 was elevated on day 3 of HAd V-7-infected mice(p<0.05),while TLR4 receptor and NF-?B were elevated on day 3 of HAd V-7-infected mice(p<0.05).3.In vivo,after blocking HMGB1,the copy number of lung tissue in the anti-HMGB1 m Ab HAd V-7 group was significantly lower than that in the HAd V-7 group on the 3rd day of infection,and the histological scores,differential counts,and inflammatory mediators of the lung tissue were significantly reduced in the HE staining(p<0.05).The levels of TLR4,TLR9,RAGE and NF-?B genes in Anti-HMGB1 m Ab HAd V-7 group were significantly lower than HAd V-7(p<0.05).Conclusion: HMGB1 Metastasizes to Extracellular After HAd V-7 Infection;Binding of HMGB1 to TLR4 receptor activates NF-?? on the third day of HAd V-7 infection triggers release of inflammatory mediators mediates inflammation and promotes virus replication;HMGB1 could be used as a therapeutic target in HAd V-7 infection.