The Mechanism Of Different Pathogenicity Caused By Fiber Of HadV-3 And HadV-7

PARTⅠEPIDEMIOLOGICAL STUDY AND CLINICAL CHARACTERISTICS ALYSIS OF ADENOVIRUS INFECTION IN HOSPITALIZED CHILDREN WITH ACUTE RESPIRATORY INFECTION IN CHONGQINGObjectives:Adenovirus(Ad V)is a viral pathogen causing severe pneumonia in infants and young children between June months and 2 years old.70 serotypes have been reported so far.Previous studies have shown that different severity of the disease caused by different serotypes of Ad V.The study is intended to retrospectively analyze Ad V infection in hospitalized children with acute respiratory infections from 2009 to 2015 in order to explore the epidemiological characteristics of Ad V and clinical features of different serotypes of Ad V.Methods:1.: A total of 4982 nasopharyngeal aspirate(NPA)samples from hospitalized children with respiratory adenovirus infections in Children’s Hospital of Chongqing Medical University from June 2009 ton May 2015 were dected.2.Firstly,the adenovirus DNA from NAPs were extracted.Secondedly,the viral DNA were amplified by polymerase chain reaction(PCR)and hexon,fiber gene were sequenced.Lastly,gene sequences were compared with sequences in Gen Bank by BLAST alignment.3.We retrospectively analyzed clinical characteristics of hospitalized children with acute respiratory infection caused by Ad V and compared different severity of disease caused by adenovirus type 3 and 7.Results:1.The detection rate of adenovirus infection in Chongqing was 5.8% for 5 consecutive years(289/4982).2.The main popular types are adenovirus type 3(43.9%,127/289)and adenovirus type 7(45.3%,131/289).3.Adenovirus is most prevalent throughout the year,with peak prevalence periods in summer and winter.4.Adenovirus infection is more common in males and children under 5 years old.5.Adenovirus type 7 caused a more serious disease than type 3.Conclusion:Adenovirus type 3 and type 7 are the most important epidemic types of respiratory adenovirus infection in children in Chongqing.Adenovirus type 7 caused a more serious disease than type 3.PART Ⅱ EFFECT ON THEIR PATHOGENICITY FOR DIFFERENT FIBER PROTEINS CAUSED BY ADENOVIRUS TYPE 3 AND 7Objectives:The results of the first part of this study have suggested that the pathogenicity of adenovirus type 7 is stronger than that of type 3,The previous study of our group found that the difference of the sequence of adenovirus type 3 and 7 are mainly on the fiber protein,it suggests that differences of fiber gene may be involved in the different pathogenicity of adenovirus type 3 and 7.The research of this aspect has not been reported in the relevant literature.Therefore,this section intends to further explore whether the differences of fiber of adenovirus type 3 and 7 affects their pathogenicity.Methods:1.A549,16 HBE and 293 cells were respectively infected with clinical strains of adenovirus type 3(CQ5291),type 7(CQ4411)and cell suspension was collected after 48 hours(h),virus copy number were quantified by quantitative polymerase chain reaction(Q-PCR).The culture media of A549 cells infected with either adenovirus type 3 or type 7,Ad3/H7(human adenovirus type 3 packaged by type 7 hexon)were harvested at 0,4,8,12,24,36,48,60,72 and 96 hpost-infection.TCID50 was used to titer the virus at each time point.Media supernatants of A549 cells infected with a mixture HAd V-3 and HAd V-7 at a ratio of 1:1 were harvested at 0,2,8,12,24,36,48,72 h post-infection.Virus copies were quantified at each indicated time point by Q-PCR.2.A549 cells were infected with adenovirus type 3,type 7 or Ad3/H7(MOI= 250 VPs/cell,or 25 VPs/cell)at 2,4,24,36,48 and 60 h post-infection.CCK-8 solution were added in A549 cells for 30 minutes.Absorbance value was read at wavelength 450 nm.Cell viability were assayed by the absorbance.3.Purified adenovirus type 3,type 7 and recombinant adenovirus Ad3/H7 strain were incubated with normal healthy human serum to detect complement C3 a product in vitro.4.BALB/c mice were intranasally inoculated with adenovirus type 3,type 7 and recombinant adenovirus Ad3/H7 strain to establish an animal model of adenovirus lower respiratory tract infection.The body weight changes of mice were monitored daily,specimens were respectively collected after 3,5,and 7 days of infection.The lung tissue homogenate of adenovirus infection were observed whether they showed cytopathic effect(CPE).Q-PCR was used to quantify pulmonary viral load.Inflammatory cells and differential counts in BALF were analyzed and counted after Wright’s staining.Lung tissue pathology were scored after hematoxylin and eosin.Cytokines in bronchoalveolar lavage fluid(BALF)were all measured using ELISA reagent kits.Results:1.Viral quantification showed that adenovirus type 7 was significantly higher than that of type 3.The growth curve indicated that the viral copies of adenovirus type 7 were significantly higher than that of type 3 and recombinant adenovirus Ad3/H7 at 24,36,60 and 72 h post-infection (p<0.05).Co-culture of adenovirus type 3 with type 7 indicated that viral replication adenovirus type 7 was significantly higher than type 3 at 12,24,36,48,and 72 h post-infection(p<0.05).2.The cytotoxicity showed that cell viability was no significant difference at all time points after A549 cells were infected at a low MOI.After A549 cells were infected at high MOI,viability of adenovirus type 7-infected cells were higher 26.5,42.6% and 33,37.7%,respectively than those of the HAd V-3-infected and recombinant adenovirus Ad3/H7-infected cells at 2 and 4 h post-infection(P<0.05,P<0.01).3.Complement assay showed that the C3 a product significantly increased at 2 minutes when adenovirus type 7 compared with adenovirus type 3 and recombinant adenovirus Ad3/H7.4.cytopathic effect appeared after A549 cells were infected with lung tissue homogenate.The viral copies of lung tissue of adenovirus type 7 were significantly higher than that of adenovirus type 3 at 3 days infection(p<0.05).The types of inflammatory cells in BALB/c mice caused by adenovirus infection were mainly macrophages and lymphocytes.The pathological damage of lung tissue was the most serious caused by adenovirus type 7 infection.The pathological grade of adenovirus type 7 infection were higher than those of the recombinant adenovirus Ad3/H7 strain and type 3 adenovirus(p<0.05).IL-1β,TNF-α,IFN-γ,and IL-6 were clearly elevated in BALF,the levels of IL-1β was highest at 7 days infection,adenovirus type 7 were most obvious compared with adenovirus type 3 and recombinant adenovirus Ad3/H7(p<0.05).Conclusion:Adenovirus type 7 cause a more severe than type 3,recombinant adenovirus Ad3/H7 strain,which suggestes that different fiber proteins lead to different severity of diseases.PARTⅢ THE STUDY ON THE MECHANISM OF FIBER PROTEIN OF ADENOVIRUS TYPE 3 AND 7 BINDING TO CD46 AND DSG-2 TO MEDIATE DIFFERENT PATHOGENICITYObjectives:The results of the second part of the study indicate that adenovirus type 3 and type 7 had different pathogenicity because they have different fiber proteins,but the mechanism of different fiber proteins leading to different clinical severity is still unclear.Current studies have confirmed that fiber of species B binding to its receptors CD46 and DSG-2 triggered infection.Therefore,this section explore whether fiber of adenovirus type 3 and type 7 combination with its receptors,CD46 and DSG-2 that lead to difference on disease development.Methods :1.Cell slide of 0.5,2h of adenovirus type 3,7 and/or type 7 and recombinant adenovirus Ad3/H7 infection of A549 cell were collected,the expression and level of adenovirus binding to its receptor were analyzed by laser confocal microscopy.2.In vitro,Transfection of CD46 si RNA and DSG-2 si RNA,Viral loads of adenovirus type 3 and type 7 were quantified by quantitative PCR,and the levels of IL-8 were measured using ELISA reagent kits.Results: 1.The results of laser confocal microscopyshowed that adenovirus type 7 and its receptors,CD46 and DSG-2 in the intracellular fluorescence was stronger than adenovirus type 3 and recombinant adenovirus Ad3/H7 strain.2.The results by Q-PCR showed that the copies of adenovirus type 3 respectively decreased by 5.8×104,7.9×104,16.6×104 and 34.2×104 at 2,6,12 and 24 h after CD46 si RNA.the copies of adenovirus type 7 was respectively reduced by 15.2×107,19.6×107,24.8×107 and 43.9×107.The extent of copies of adenovirus type 3 and type 7 decreased by 75.3% vs 65.8%;72.5% vs 69.3%;82.2% vs 67.6%;88.1% vs 74.2%.At 2,6,12,and 24 h after DSG-2 si RNA,the copies of adenovirus type 3 was respectively reduced by 6.7×104,6.6×104,12.1×104,and 21.4×104.The copies of adenovirus type 7 was respectively reduced by 4.5×107,13.4×107,21.7×107 and 33.7×107.The extent of copies of adenovirus type 3 and type 7 decreased by 88.2% vs 56.3%;75.9% vs 77.9%;86.4% vs 78.1%;87.7% vs 73.6%.At 6 and 24 h after CD46 si RNA,the amount of IL-8 induced by adenovirus type 3(pg/ml)was respectively reduced by 98.8,187.5.The amount of IL-8 induced by adenovirus type 7(pg/ml)was respectively reduced by 144.4,366.6.The extent of reduction of IL-8 induced by adenovirus type 3 and type 7 was 75.6% vs 73.7%;82.1% vs 84.3%.At 6 and 24 h after DSG-2 si RNA,the amount of IL-8 induced by adenovirus type 3(pg/ml)was respectively reduced by 69.9,136.5.The amount of IL-8 induced by adenovirus type 7(pg/ml)was respectively reduced by 105.2,186.1.The extent of reduction of IL-8 respectively induced by adenovirus type 3 and type 7 was 76.2% vs 77.9%;83.0% vs 83.3%.There was no significant difference on the amount and extent of IL-8 induced by adenovirus type 7 and type 3 before and after the interference.Conclusion : Viral entry and receptor gene silencing experiments suggest that the binding of ciliated proteins of different types of adenovirus to their receptor DSG-2 leads to the underlying mechanisms of different severity diseases.The immunofluorescence assay of adenovirus binding to their receptor and receptor gene silencing experiments suggest a potential mechanism that different types of adenoviral fiber proteins binding to their receptors,CD46 and DSG-2 lead to varying severity of disease.These results provide an important theoretical basis for understanding the virulence differences of different adenovirus and developing vaccines based on epitope of fiber protein and anti-adenovirus drugs in the future.