Study On The Characteristics Of Adenovirus Infected Tree Shrew In Vitro And In Vivo

Objective:With the advent of China's aging society,the widespread use of immunosuppressive agents and the increasing number of AIDS cases,the incidence of adenovirus-induced viral pneumonia is increasing.At present,there is no safe and reliable new drug for the treatment of adenoviral infectious diseases,and the mechanism of persistent infection caused by the adenovirus is still unclear,a large reason is the lack of HAdV infection animal models for the study.Tree shrew is more closely related to primates than rodents.It has been proved that tree shrews can be infected by hepatitis B virus,tuberculosis bacillus,EV71,H1N1 and other pathogenic microorganisms.It is very suitable for the study of pathogen infection.At present,tree shrew adenovirus Kunming strain(TAV-KM)was isolated from tree shrew,which is close to human adenovirus genome.Whether tree shrew can be used as an animal model for adenovirus infection is worth exploring.In this paper,the characteristics of TAV-KM infection will be studied in vitro and in vivo.In addition,human adenovirus type 3(HAdV-3)and human adenovirus type 7(HAdV-7)were used to infect tree shrews.To explore the infection of different viruses in tree shrews,and the expression of cytokines interleukin-10(IL-10)and interferon-?(IFN-?)in lung after adenovirus infection.Methods:The optimum MOI value of TAV-KM on A549 was determined by experiment.TAV-KM was used to infect TKC,shrew lung fibroblasts,A549,H1299,Vero and TC-1 cells from different sources,respectively.One-step growth curve of TAV-KM on different cells was drawn.The cytopathic changes of TKC,A549 and H1299 infected with TAV-KM were observed.TAV-KM,HAdV-3 and HAdV-7 infected tree shrews by nose dropping.The control group was treated with saline of equal volume,4 animals in each group.The changes of anus temperature and viral load in blood,feces and different tissues of tree shrews were detected.Histopathological examination was performed by HE staining of paraffin sections.Detection of adenovirus hexon protein in lung tissue by immunohistochemical staining.The expression of IL-10 and IFN-? in lung tissue were detected by relative fluorescence quantitative PCR.Results:The optimal MOI of TAV-KM infect A549 was 0.5.Experiments in vitro showed that TAV-KM can infect not only tree shrew TKC and lung fibroblasts,but also could infect human cell lines A549 and H1299,and could replicate in human cell lines.In Vero and TC-1 cell lines,although the virus could enter the cell,but there was no significant virus proliferation.TKC aggregated into irregular grape clusters at 24 hours after TAV-KM infection,showing typical adenovirus CPE manifestations.However,no adenovirus typical CPE was observed in A549 and H1299 infected by TAV-KM.After 10 days of TAV-KM,HAdV-3 and HAdV-7 infection,tree shrew pulmonary congestion could be seen in general anatomy.HE staining showed pulmonary congestion and lymphocyte infiltration.The immunohistochemical results of hexon protein in lung tissue of experimental group were positive.Viral load was detected in the trachea of TAV-KM group on the 14th day of the experimental cycle,in lung tissue on the 6th and 10th day,in feces from the 2td to 12th day,and negative in blood.In HAdV-3 group,viral load was detected in trachea on 2th and 6th,lung tissue on day 2th and 10th,and no viral load was detected in feces and blood.In HAdV-7 group,viral load was detected only in lung tissues of 10th,and no viral load was detected in other samples.Compared with the control group,the early expression of IL-10 and IFN-? in HAdV-3 group and HAdV-7 group increased,and returned to normal level after 14 days.In TAV-KM group,IL-10 was still at a high level on the 14th day,and IFN-? level was lower than that in the earlier period.Conclusion:The vivo and vitro experiments show that tree shrew is the potential viral pneumonia animal of TAV-KM,HAdV-3 and HAdV-7 infection.TAV-KM can be used to stody adenovirus infection tree shrew model and is a potential zoonotic pathogen.