| Middle East respiratory syndrome coronavirus(MERS-CoV)is an emerging highly pathogenic coronavirus with 35% mortality,while SARS-CoV,which was prevalent in 2003,had only 10% mortality.Commonly used laboratory animal species such as Syrian hamsters,mice and ferrets are not susceptible to MERS-CoV,due to differences in the MERS-CoV receptor dipeptidyl peptidase 4(DPP4).To date,clinical autopsy data is sparse and the pathogenesis of this virus is poorly understood.Limited research data on the non-human primates showed that immune-mediated pathogenesis has been proposed as one of potential factors for the severe outcome of MERS-CoV-infected patients.In our previous study,highly pathogenic avian influenza H5N1 and H7N9 infection-induced aberrant complement activation could aggravate the lung damage,which suggest aberrant complement may be an important causative factor of MERS-CoV-induced lung injury.This study purposed to generate a MERS-CoV-susceptible human DPP4 transgenic mouse model to study the pathogenesis of MERS-CoV.1.Generation of a human DPP4 transgenic mouse model of MERS-CoV infection.A synthetic codon-optimized human DPP4 sequence was cloned into eukaryotic expression vector and then confirmed its expression by transfection of COS-7 cells detected by direct immunofluorescence assay with DPP4 antibody and MERS-RBD-Fc protein.After microinjection of linearized DNA,founder lines that transferred the hDPP4 gene to their progeny were established.Founder mice were then crossed with wide type C57BL/6 and their hDPP4 negative pubs detected by PCR were eliminated.HDPP4 transgenic mice were infected by MERS-CoV intranasally in an approved biosafety level 3 laboratory.Mice exhibited decreased activity and significant weight loss post MERS-CoV infection,and all of the infected mice died by day 10,suggesting this transgenic mouse model was susceptible to MERS-CoV and leading to a lethal end after infection.2.Characterization of a hDPP4 transgenic mouse model of MERS-CoV infection.Mice were intranasally inoculated with MERS-CoV in 20 ?L DMEM,and were monitored for weight loss and survival.Mice sera and tissues included lungs,kidneys,livers,spleens,intestines and brains were harvested on indicated time points.Histopathological analysis showed tissue damage and inflammatory responese in these tissues.Immunohistochemical staining and qPCR examination showed virus antigen expression and viral replication in lung,kidney and brain.Infiltration of neutrophils and macrophages in tissues and significant increase of cytokines in sera indicated an aberrant inflammatory responese.3.The mechanism of complement overactivation mediated multi-organ damage after MERS-CoV infection.To confirm complement activation after MERS-CoV infection,C3 aR,C5aR expression and C5 a level in sera was detected by IHC and ELISA respectively.Results showed aboundant expression of C3 aR and C5 aR in lung and a significant increase of C5 a level in sera.To explore the role of complement in MERS-CoV induced tissue injury,combination of C5 a and C5 aR was blocked by anti-C5 aR monoclonal antibody.Results showed that inhibition of complement activation with anti-C5 aR antibody will reduce systemic and local inflammatory responses,limit virus replication in the lung,alleviates lung and spleen damage,increase splenic cell regeneration and decrease splenic apoptosis.Taken together,we generated a hDPP4 transgenic mouse,to which MERS-Co V infection could be lethal.This model exhibited similar symptoms with clinical MERS patients and would play an important role in MERS-CoV pathogenesis study and vaccines and antiviral agents evaluation.Overactivation of complement and aberrant immune response were found after MERS-CoV infection,while blockade of C5a-C5 aR axis with anti-C5 aR antibody will restore normal immune response and alleviate dysregulated host immune response induced damage.Our study revealed a new mechanism of MERS-CoV-induced tissue injury and suggested a new effective clinical intervention for MERS-CoV infection. |
PDF Full Text Request