| BackgroundRespiratory viral infections are the most common causes of acute respiratory infections in children.Human adenovirus is an important pathogen causing acute respiratory infections in children under 5 years of age worldwide.HAdV-3 and HAdV-7 are the most common genotypes.Compared with HAdV-3 infection,patients with HAdV7 infection have a higher incidence of viremia and more likely to have severe pneumonia,acute respiratory distress syndrome,and multiple organ failure.This suggests that viremia is directly related to disease severity and death.Therefore,the mechanisms of HAdV-7 induced viremia is important to limit the systemic dissemination of the virus to multiple organs.The alveolar-capillary barrier plays an important role in the defense against pathogenic invasion.The polarity of the barrier results in directional release of offspring viruses.Viruses which bud apically tend to cause localized infections while basolaterally budding viruses are more likely to cause systemic infections.Our previous study found that HAdV-7 and HAdV-3 can infect alveolar epithelial cells and newly formed viruses were released from the apical surfaces of the alveolar epithelial cells,suggesting that HAdV-7 may have crossed the barrier into the blood system by other routes and caused viremia.The intercellular junctional structure of alveolar epithelial cells maintains the integrity of the barrier function.Virus infection damage to tight intercellular junctions,leading to disruption of barrier function and increased permeability,facilitating the entry of pathogens into the blood system and causing multiorgan failure.Therefore,the difference in disruption of tight junctions between HAdV-7 and HAdV-3 infected alveolar epithelial cells may be the key to HAdV-7 infection being more likely to cause viremia.ObjectiveThe infection rate,replication ability,barrier integrity and intercellular tight junction protein of polarized alveolar epithelial cells infected by HAdV-7 and HAdV-3 were compared to clarify the mechanism that HAdV-7 infection is more likely to cause viremia.Methods1.Coating the transwell chamber with Collagen Type Ⅰ Rat Tail(12.12μg/well)and inoculating alveolar epithelial cells(5×104/well)in the chamber to construct polarized alveolar epithelial cells model.On the third day of culture,dexamethasone(1μM/well)was added,and the TEER and FITC-dextran fluorescence intensity of lower chamber were measured daily.2.HAdV-3 and HAdV-7 infect polarized alveolar epithelial cells model at 10TCID50/cell.Indirect immunofluorescence assay was used to detect the expression of viral Hexon protein on days 0,1,2 and 3.The infection rate of polarized alveolar epithelial cells in different infection groups on days 1,2 and 3 was calculated using Image-J.The infection rate of polarized alveolar epithelial cells was equal to the number of Hexon positive cells divided by the total number of cells under DAPI staining multiplied by 100%.Viral titers were determined by collecting infected supernatants on days 0,1,2 and 3 by 50%tissue culture infective dose(TCID50).3.The experiment was divided into viral infection group(HAdV-3,HAdV-7),inactivated viral infection group(HAdV-3,HAdV-7 were inactivated at 56℃ for 30 min)and negative control group(1640 medium).The polarized alveolar epithelial cells were infected with 10TCID50/cell of HAdV-3 and HAdV-7 and inactivated HAdV-3 and inactivated HAdV-7,respectively.The transmembrane electrical resistance(TEER)and fluorescence intensity of FITC-dextran were measured on day 0,1,2 and 3 after infection in each group to compare the integrity and permeability of the barrier in different infection groups.4.The experiment was divided into viral infection group(10TCID50/cell of HAdV-3 and HAdV-7)and negative control group(1640 medium).Indirect immunofluorescence assay used to detect the expression of claudin 4,occludin,JAM-A on days 0,1,2 and 3 after infection,The average fluorescence intensity of each proteins were calculated by Image-J to compare the changes of tight junction proteins in different infection groups.The average fluorescence intensity is equal to the total fluorescence intensity of the field of view divided by the area of the field of view.5.Graphpad Prism 6.01 software were used for statistical analysis.The results of variables were expressed as mean ± standard deviation(Mean±SD).A difference of 0.05 was considered statistically significant.Results1.Viral Hexon protein expression was detected after HAdV-7 and HAdV-3 infection of polarized alveolar epithelial cells,indicating that HAdV-7 and HAdV-3 were able to infect alveolar epithelial cells.The infection rate of cells on days 1,2 and 3 after HAdV-7 infection was higher than that of HAdV-3 infection,with the infection rate on day 3(3.62±0.51)%being significantly higher than that of HAdV-3(1.97±0.15)%(P<0.05).The virus titer of supernant from HAdV-7 infected polarized alveolar epithelial cells at 1d(1×102.42±0.07TCID50/ml),2d(1×104.00±0.25TCID50/ml),3d(1×104.33±0.14TCID50/ml)significantly higher than HAdV-3 infected at 1d(1×101.92±0.14TCID50/ml),2d(1×103.33±0.14TCID50/ml),3d(1×103.92±0.14TCID50/ml)(P<0.05).2.The extent of TEER decrease and fluorescence intensity of FITC-dextran in the lower chamber of inactivated HAdV-3 and inactivated HAdV-7 infected polarized alveolar epithelial cells were not significantly different from those of the negative control group.The extent of TEER decrease of HAdV-7 infected polarized alveolar epithelial cells at 1d(16.10±0.52)%,2d(50.83±1.53)%,3d(72.85±0.35)%significantly higher than that of the negative control group(P<0.01).The extent of TEER decrease of HAdV-3 infected polarized alveolar epithelial cells at 1d(14.18±0.53)%,2d(34.40±1.24)%,3d(60.98±0.73)%,where the extent of TEER decrease on days 2 and 3 was significantly higher than that of the negative control group(P<0.01).The extent of TEER decrease of HAdV-7 infected polarized alveolar epithelial cells at days 2 and 3 was significantly higher than that of HAdV-3 infection(P<0.01).The fluorescence intensity of FITCdextran in the lower chamber of HAdV-7 infected polarized alveolar epithelial cells at 2d(1949.67±52.911),3d(3145.33±25.30)significantly higher than that of the negative control group(P<0.01).The fluorescence intensity of FITC-dextran in the lower chamber of HAdV-7 infected polarized alveolar epithelial cells at 2d(1716.17±36.94),3d(1940.00±23.19)significantly higher than that of the negative control group(P<0.01).The fluorescence intensity of FITC-dextran in the lower chamber of HAdV-7 infected polarized alveolar epithelial cells at days 2 and 3 was significantly higher than that of HAdV-3 infection(P<0.01).It indicates that HAdV-7 infection can cause more severe barrier damage.3.Compared with the negative control group,the mean fluorescence intensity of claudin 4,JAM-A,and occludin proteins of HAdV-7 and HAdV-3 infected polarized alveolar epithelial cells at days 2 and 3 were decreased(P<0.05).The mean fluorescence intensity of claudin 4 proteins of HAdV-7 infected polarized alveolar epithelial cells at days 2 and 3 was significantly lower than those of JAM-A proteins,occludin proteins(P<0.05).The mean fluorescence intensity of claudin 4 proteins of HAdV-3 infected polarized alveolar epithelial cells at day 2 and 3 were not significantly different from those of JAM-A and occludin proteins.These data suggest that HAdV-7 damages the epithelial barrier by damaging the epithelial tight junction,in particular claudin 4.ConclusionsHAdV-7 infects polarized alveolar epithelial cells with higher infection rate and replication capacity than HAdV-3,and causes more severe damage to the alveolar epithelial cell barrier.HAdV-7 infection of polarized alveolar epithelial cells decreased the expression of the tight junction proteins claudin 4,JAM-A,and ocludin,especially claudin 4.The claudin 4 protein may play a key role in the viraemia caused by HAdV-7 infection. |
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