Studies On Pathogenesis Of Fatal Encephalitis Caused By Human Coronavirus OC43

BackgroundEncephalitis is a serious neurological disorder,usually caused by virus infection.Encephalitis is characterized by inflammation of the central nervous system,which sometimes could be fatal.Human Coronavirus OC43 is one of the main pathogens causing respiratory diseases.It mainly causes upper respiratory tract infection with main symptoms of cough and runny nose,accounting for 10-15%of acute respiratory diseases in children and adults.However,in infants,the elderly and immunocompromised people,it can also affect the lower respiratory tract,causing pneumonia and various respiratory distress syndromes.Human Coronavirus OC43also has neuroinvasive ability and can spread from the respiratory tract to the central nervous system.Once infected cells of the central nervous system,it can cause various central nervous system problems,such as epilepsy,encephalitis,and long-term neurological diseases.With the global outbreak of the novel Coronavirus Disease2019（COVID-19）,it has had an irreversible and devastating impact on all aspects of human life.With the increasing attention of the public to coronavirus,the symptoms of sensory,cognitive and motor functions after infected with novel coronavirus,as well as the sequelae such as diffuse symptoms of the nervous system such as headache or dizziness,it is urgent to solve the pathway of coronavirus invading the central nervous system and the pathogenic mechanism of encephalitis caused by the host after infection.As the operation of novel coronavirus needs to be carried out in a biosafety level 3 laboratory,human coronavirus OC43 can be carried out in a biosafety level 2 laboratory.Understanding the mechanism of encephalitis caused by HCo V-OC43 infection,can not only provide a valuable tool to study coronavirus,but also contribute to diagnosis,treatment,disease recovery,and guiding the vaccine development.ObjectiveThis study aims to develop a lethal model infected with human coronavirus OC43,and to explore the role of Toll-like receptors 7 in the process of lethal encephalitis.Using this mouse model to identify dominant epitopes of CD4 T cells and CD8 T cells,and functional characteristics of virus-specific T cells in infected mice.Method1.In this study,Human Coronavirus OC43（VR-759）was injected into brain of 4-7-day-old suckling mice.When the suckling mice were sick and dying,brain tissue was collected,and homogenized.The supernatant was diluted,and then injected into brain of 4-7-day-old suckling mice.After passaging in the brain of sucking mice for 23generations,using the adapted virus to infect six-week-old male mice.2.The titer of Human coronavirus OC43（MA-P23）was determined by using focus formation assay（FFA）.Six-week-old male C57BL/6 mice were infected with OC43（MA-P23）by intranasally to test half lethal dose and absolute lethal dose.3.To explore the role of Toll like receptors in the process of encephalitis,we compared weight loss,viral titers and pathological changes in brain of Toll-like receptor deficient mice and WT mice.4.To evaluate the role of T cells in process of encephalitis caused by OC43 infection,Rag1^-/-mice were infected with OC43 before adoptive transferred of total T cells,CD8 T cells and CD4 T cells respectively from spleen of WT mice infected with HCo V-OC43.The role of B cells in the process of OC43 infection was evaluated by comparing phenotypic changes ofμMT mice and WT mice after infection.After deletion of CD4 or CD8 T cells with GK1.5 or 2.43 antibodies,mice were infected with OC43.By comparing with weight,mortality and clinical symptoms,we evaluated the role of CD4 and CD8 T cells in process of encephalitis caused by OC43infection.5.We used XMG1.2 and XT3.11 blocking antibodies in vivo to verify the roles of IFN-γand TNF in the process of encephalitis caused by OC43 infection.6.Virus-specific CD4⁺and CD8⁺T cell epitopes were screened in OC43-infected mice by combining with intracellular cytokine staining（ICS）and flow cytometry.7.The response of virus-specific CD4⁺and CD8⁺T cell were analyzed.8.Mice were vaccinated with HE141 peptide-coated dendritic cells and boosted with recombinant vaccinia virus expressing CD8 T cell HE141 epitope.35 days after immunization,mice were infected with OC43.To explore the role of CO43-specific T cells in encephalitis caused by OC43 infection,we compared weight,mortality and virus titer of brain between DC-r VV mice and DC-no peptide mice.Result1.6-week-old male C57BL/6 mice were infected with OC43（MA-P23）by intranasally.Mice developed symptoms such as fried hair,hunched back,lethargy,and weight loss on the 5th day after infection.Hind limb paralysis and paralysis were observed in individual mice.Mice died on the 7th day after infection,and the peak of death occurred on the 9th to 10th day.Virus can be detected in brain and spinal cord.The results show that the mouse model of lethal encephalitis caused by OC43infection is successfully constructed.2.The whole genome of OC43（MA-P23）was sequenced and compared with the original OC43（VR-759）sequence.The results showed that there was a total of 44base mutations in the whole genome,including 15 mutations in ORF1ab,14mutations in Spike gene,2 mutations in HE gene and 2 mutations in N gene.Among14 mutations in the S gene,three mutations at positions 183,241 and 488,which were associated with increased viral virulence.3.OC43（MA-P23）was passaged in N2a cells and HRT-18 cells respectively.The progeny virus obtained in N2a was still neurotropic and could kill adult mice.While the progeny virus obtained in HRT-18 cells was still neurotropic,but virulence gradually decreased with the increase of passage times.By passaged third times in HRT-18 cells,the progeny virus no longer killed mice.4.TLR7^-/-mice were more susceptible to OC43（MA-P23）than WT C57BL/6 mice.After infection,TLR7^-/-mice showed symptoms with increased weight loss,more severe symptoms,hind limb hemiplegia,paralysis,and increased mortality.5.As the disease progresses,virus replicates in large quantities in the central nervous system.Virus titers in the brain and spinal cord of TLR7^-/-mice were higher than those in WT C57BL/6 mice,and the differences were significant（brain:5 d.p.i.,p=0.000002;7 d.p.i.,p=0.000002;Spinal cord:5 d.p.i.,p=0.000052;7 d.p.i.,p=0.000689）.These results indicate that TLR7^-/-mice infected with OC43（MA-P23）have a large amount of virus replication in the central nervous system,and viral load is maintained at a relatively high level.6.Transcriptome analysis showed that 1357 genes were up-regulated in brain tissue of WT C57BL/6 mice on day 5 of OC43 infection,while only 64 genes were up-regulated in TLR7^-/-mice.GO and KEGG pathway analysis were performed on the up-regulated genes in WT C57BL/6 mice and TLR7^-/-mice,respectively.The results showed that chemokines,cytokine-cytokine receptor interaction pathaway,TNF signaling pathway,NF-κB signaling pathway and other innate immune related pathways were down-regulated in TLR7^-/-mice compared with WT mice.In addition,TLR7 deficiency also can inhibit other pattern recognition receptor related pathways.Real-time PCR was further used to verify the results of differential gene analysis in mouse brain tissue transcriptome,including IFN-β,IFN-γ,TNF,IL-6,IL-10 were significantly down-regulated.7.Primary neuronal cells,astrocytes and microglia was isolated and cultured from WT C57BL/6 baby mice and TLR7^-/-baby mice.These cells were infected with OC43（MA-P23）separately.The results showed that virus titers on primary neuronal cells,astrocytes and microglia were higher than those in the WT group after the loss of TLR7.The level of IFN-βin TLR7^-/-group was significantly higher than that in WT group 24h after infection（p<0.001）.The difference between the in vitro and in vivo results suggests that TLR7 is an important pattern recognition receptor for OC43recognition,but it is not the only pattern recognition receptor for OC43 recognition.At the same time,it was also found that neuronal cells were more susceptible to OC43after the loss of TLR7,which showed that neuronal cytoplasm was more prone to vacuolization and cell body was more prone to disintegration.Detection of Caspase3expression also demonstrated that neuronal cells lacking TLR7 were susceptible to apoptosis when infected with OC43.8.OC43 infection of bone marrow macrophages induced by bone marrow differentiation of WT C57BL/6 mice and TLR7^-/-mice showed that macrophages derived from TLR7^-/-mice were more susceptible to OC43 infection,and the virus titer was significantly higher than that of WT group.The expression of type I and type II interferons was detected,and the results showed that macrophages with TLR7deletion infected with OC43 secreted higher type I interferons,especially IFN-β（p=0.001）.9.HE staining of brain tissue sections of WT C57BL/6 mice and TLR7^-/-mice on day3,5,and 7 of infection showed that.With the progress of the disease course,infiltration of lymphocytes and monocytes appeared in the brain tissue of WT mice.On the 7th day of infection,lots of lymphocytes infiltrated in the brain tissue and gathered around the cerebral blood vessels,forming a"vascular sleeve"phenomenon.In addition,glial cells gather around the necrotic cells to form glial nodules.However,TLR7^-/-mice had less lymphocytes and monocytes in central nervous system.10.The infiltration of lymphoid cells and myeloid cells in the brain tissue of WT C57BL/6 mice and TLR7^-/-mice after infection was detected.The results showed that the frequency and number of lymphoid cells and myeloid cells in TLR7^-/-mice were significantly lower than those in WT C57BL/6 mice.These results suggest that TLR7depletion decreases the migration of peripheral immune cells to the CNS.11.WT C57BL/6 mice and TLR7^-/-mice were intraperitoneally injected with Evans blue on days 3,5,and 7 of OC43 infection to detect changes in BBB permeability.The results showed that TLR7 deletion affected BBB permeability.12.The dominant T cell epitopes of OC43 in WT C57BL/6 mice were identified by intracellular cytokine staining and flow cytometry.13.Through tail vein injection of 2.43 antibody,mice were infected with OC43 after the deletion of CD8 T cells in the whole body.The results showed that after the deletion of CD8 T cells in the body,the weight loss of mice was reduced and the mortality rate was reduced.Titers in brain tissue and spinal cord from day 3 to day 5after infection did not differ from those in the non-deleted group.The expressions of type I interferon-αandβ,type II interferon-γand cytokine TNF were detected,and the expression of type II interferon-γin the non-deleted cell group was significantly higher than that in the deleted cell group（p=0.001）.14.After sorting total T cells,CD4 T cells and CD8 T cells in lymph nodes and spleen of naive WT C57BL/6 mice and mice infected with OC43 on day 7 by magnetic beads,Rag1^-/-mice were adoptive transferred these cells respectively by tail vein injection.One day later,Rag1^-/-mice were infected with OC43.Compared with Ctrl group,mice adoptive transferred cells showed more serious symptoms and died at.Mice of ctrl group developed late disease and had longer course of disease,but mice were also eventually died.15.Mice immunized with recombinant vaccinia virus expressing the dominant CD8 T cell epitope HE141 were infected with OC43 on day 7（effector phase）and day 28（memory phase）after booster immunization with r VV-HE141,and HE141 CD8 T cells showed protective effects,especially in weight loss.The survival rate of the mice increased.Significance1.In this study,the HCo V-OC43 mouse encephalitis model was successfully established by successive passage of HCo V-OC43 into the brain tissue of neonatal mice to 23 generations,and intranasal infection of male C57BL/6 mice aged 5-6weeks.This animal model is based on adult mice,which can better analyze the immune response and pathogenic mechanism caused by OC43 after infection.2.This study is the first to analyze the characteristics and functions of virus-specific T cell immune response after HCo V-OC43 infection in mice.This study will promote the study of cellular immune response to HCo V-OC43 infection and provide theoretical basis and support for the analysis of the pathogenic mechanism of HCo V-OC43 and the analysis of regional immune characteristics and diseases in the central nervous system.