Construction And Application Of Human Coronavirus 229E(HCoV-229E) Pneumonia Model In Mouse

The past decades have witnessed the emergence of new coronavirus diseases(such as SARS,MERS,CovID-19),which forced mankind to find effective coping strategies and establish means and methods to control the epidemic situation.Appropriated animal models are the bases of vaccine research and drug screening.Establishment a lower-cost mouse model of coronavirus pneumonia that can be applied in P2 laboratory is of profound impact for epidemic control,drug screening and virus immunology research.Objective:To establish HCoV-229E mouse pneumonia model,we investigated the infection condition of different mice strains,different virus strains,different amount of infection and different times of infection.In this study,we established the model of pneumonia in mice infected with HCoV-229E,and observed the changes of CT,pathology,viral load and related immunology in the lungs tissue.In addition,the trial drugs recommended in the New Coronavirus pneumonia diagnosis and treatment plan(Trial Eighth Edition)were further studied:phosphate chloroquine,interferon a,Jinhua Qinggan granule,Lianhua Qingwen capsule,Shufeng Jiedu Capsule and related formula in the treatment of HCoV-229E mice pneumonia model.Methods:1.Establishment of the model of HCoV-229E in vitro and in vivoIn vitro,HCoV-229E virus was used to infect MRC-5,A549 cells,Raw264.7 and MDCK cells for susceptibility evaluation,CPE and TCID50 was determined.Three mouse strains,BALB/c mice,BALB/c-nude mice and ICR mice,were selected from the model of HCoV-229E infection.The lung index and viral titer in lung tissue were detected after virus intranasal infection.On this basis,we further investigated the condition that BALB/c mice infected twice,and detected the lung index,lung pathology and lung viral load.2.Establishment of HCoV-229E infected BALB/c mice pneumonia model and research of its detection indicatorIn this part,we established HCoV-229E infected BALB/c mice pneumonia model according to part 1.Lianhua Qingwen capsule served as positive control tested with 7.90 g/kg/d(high-dose)group and 3.80 g/kg/d(low-dose)group.Each group with 10 mice.Except for the normal control group,the rest of the mice were slightly anesthetized with isoflurane on the first and third days respectively,and then infected with 50 μL 100 TCID50 HCoV-229E virus solution by intranasal infection.The related indexes lung CT,lymphocyte typing ratio,lung index,histopathological examination of lung tissue and major organs of the whole body,viral load and inflammatory cytokines were detected at the fifth day.3.Application of mouse pneumonia model infected by HCoV-229EBALB/c mice were divided randomly by weight into normal control group,model control group,chloroquine phosphate group and interferon α2b group,Jinhua Qinggan granule group,Shufeng Jiedu Capsule group,formula 1-2 group,formula 2-2 group and 4-2 group with 10 mice in each group.Lymphocyte typing ratio,lung index and inflammatory cytokines were detected at the fifth day.Results:1.Establishment of the model of HCoV-229E in vitro and in vivoThe susceptibility of HCoV-229E to MDCK,A549,MRC-5 and RAW 264.7 cells were evaluated.Finally,A549 cells and MDCK cells were selected as virus proliferation vectors through the aspects of CPE,stability and preservation.The TCID50 of HCoV-229E virus in MDCK and A549 cells was 10’2.Three animal strains and two conditions of infection were explored:BALB/c mice,BALB/c-nude mice and ICR mice.The virus propagation in A549 was once infected by nasal drops at the concentration of 100 TCID50.Three days after BALB/c mice infected by HCoV-229E,the lung index of BALB/c mice and BALB/c-nude mouse model group increased significantly,with the mean value of lung index greater than 0.9(P<0.01),and the viral expression in lung tissue increased significantly(P<0.01),which reaching the standard of model establishment.However,5 days after infection,the lung index and nucleic acid expression decreased apparently,suggesting that the model showed a recovery trend.On this basis,the twice infection experiment was further investigated.On the fifth day after infection,pathological study showed diffuse focal alveolar necrosis and partial alveolar stent collapse.The lung index mean value of the model group mice was higher than 0.9 with high expression of virus load(P<0.01),which reached the standard of model establishment.2.Establishment of HCoV-229E infected BALB/c mice pneumonia model and research of its detection indicatorThe mice in HCoV-229E model control group were infected with HCoV-229E virus at a concentration of 100 TCID50 twice by nasal drops.Five days after infection,the lung index and the expression of HCoV-229E nucleic acid in lung tissue were significantly higher than those in normal control group(P<0.1).Compared with normal control group,the expression of inflammatory factors IL-6,IL-10 and TNF-α protein in lung homogenate of mice in model control group was significantly higher(P<0.1).The percentages of CD3+T cells,CD4+T cells,CD8+T cells and B cells in peripheral blood were significantly decreased(P<0.1).CT of the lungs of the model control group showed thickened texture and spot-like shadows.Diffuse alveolar necrosis of lung tissue,alveolar stent collapsed,and surrounding lungs widened.There were pathological changes consistent with clinical manifestations in the main organs of the whole body.These results indicate that the mild pneumonia model of HCoV-229E BALB/c mice can be successfully established by this method.After 4 days of treatment,Lianhua Qingwen Capsules had a significant improvement in lung index,lung tissue viral load within the treatment range.The above results show that mouse body weight,viral load,lung inflammation,inflammatory factor expression,lymphocyte typing,CT and pathological changes can be used as evaluation indicators for the HCoV-229E BALB/c mouse mild pneumonia model.3.Application of mouse pneumonia model infected by HCoV-229EBoth chloroquine phosphate and interferon α2b have certain therapeutic effects.Chloroquine phosphate can significantly reduce the content of TNF-α and increase the percentage of CD8+T cells.Chloroquine phosphate may antivirus through inhibit inflammation and improve immunity.Interferon α2b can significantly reduce lung index and inhibit the content of TNF-α,which may play a role by inhibiting inflammation.After 4 days of treatment,compared with the model group,formula 1-2 and 2-2 could significantly reduce the lung index,lung viral load,the content of inflammatory cytokines IL-6,IL-10 and TNF-α in the lung homogenate of BALB/c mice within the treatment range,and could significantly increase the percentage of B cells.In the treatment range,formula 4-2 can significantly reduce the lung index,HCoV-229E viral load in lung tissue,and the contents of inflammatory cytokines IL-6,IL-10 and TNF-αin the lung homogenate of mice.The low-dose group can also significantly increase the percentage of CD3+T cells,CD4+T cells,CD8+T cells and B cells.In the treatment range,Jinhua Qinggan capsule can reduce lung index,HCoV-229E viral load in lung tissue,and can reduce the contents of inflammatory cytokines IL-6,IL-10 and TNF-α in lung homogenate.The low-dose group can also significantly increase the percentage of CD3+T cells,CD4+T cells and CD8+T cells.Shufeng Jiedu Capsule in the treatment range can significantly reduce the lung index,lung HCoV-229E viral load,can reduce the content of inflammatory cytokines IL-6,IL-10 and TNF-α in the lung tissue of mice.The high-dose group can significantly increase the percentage of B cells,and the low-dose group can significantly increase the percentage of CD3+T cells and CD4+T cells.Conclusion:1 Successfully established the human coronavirus 229E infected model in vivo and in vitro.2 Constructed the evaluation system of mouse pneumonia model infected by HCoV-229E.3 Based on the HCoV-229E mouse pneumonia model,the pharmacodynamic effects of Jinhua Qinggan Granules,Shufeng Jiedu Capsules,Prescriptions 1-2,Prescriptions 2-2,and Prescriptions 4-2 were verified,and the feasibility,stability and repeatability of the model was also proved.Innovation:1.Established a BALB/c mice pneumonia model infected with HCoV-229E for the first time,and formed a set of model standard,which has not been reported at home and abroad.2.Our study clarified the effectiveness of Jinhua Qinggan granule,Shufeng Jiedu Capsule,formula 1-2,formula 2-2 and formula 4-2 in the treatment of pneumonia model of BALB/c mice infected with HCoV-229E.This study has laid a foundation for the screening and evaluation of drugs related to human coronavirus infection and the research of HCoV-229E infection.