Dynamic Transcriptomic Characterization Of Primary Epstein-Barr Virus Infection In Tree Shrew Model

Objectives Epstein-Barr virus(EBV)is a human herpesvirus that latently infects approximately 95%of adults and is associated with a spectrum of human diseases including Infectious Mononucleosis and a variety of malignancies.However,understanding the pathogenesis,vaccines and antiviral drugs for EBV-associated disease has been hampered by the lack of suitable animal models.Tree shrew is a novel laboratory animal with a close phylogenetic relationship to primates,which is a critical advantage for many animal models for human disease,especially viral infections.In this study,we intend to systematically evaluate the tree shrew model of EBV infection and the dynamic changes of biological processes in vivo during primary EBV infection by transcriptome sequencing to explore possible mechanisms of primary EBV infection.Methods We first compared the viral receptor CR2 protein sequences and the potential species-specific residues in CR2 protein that contribute to EBV entry between tree shrew and five other species.Thereafter,PBMC isolated from peripheral blood of tree shrew and EBV were co-cultured in vitro,and cell proliferation was observed by cell count,CCK8 and cell morphology;EBV gene and protein expressions were detected by immunofluorescence and RT-PCR.At the same time,we inoculated 6 healthy tree shrews with 1×10~8 copies of EBV suspension via the femoral vein,and dynamically monitored the general conditions(body temperature,body weight);blood routine;EBV DNA copies in whole blood,plasma,and saliva of the tree shrew;distribution of EBV-related proteins in blood and tissues,etc.In addition,we carried out RNA sequencing(RNA-seq)of blood samples to understand the dynamic changes in biological processes during the EBV primary infection in vivo.Finally,we explored the role of neutrophils in EBV primary infection in the tree shrew model and validated the findings using GEO datasets and retrospective case reviews.Results(1)we first identified the key residues in the CR2 receptor that bind the gp350 protein and facilitate viral entry.We found that tree shrew shares 100%sequence identity with humans in these residues,which is much higher than rabbits(50%)and rats(25%).(2)In vitro analysis showed that B lymphocytes of tree shrews are susceptible to EBV infection and replication,as well as EBV-enhanced cell proliferation,and the infected cells exhibited enhanced proliferation,showing lymphoblastoid cell lineage-like cluster growth;these characteristics were similar to those of human PMBC infected with EBV in vitro.(3)Results of in vivo experiments show the infected animals exhibited transient fever and loss of weight accompanied by neutropenia and high viremia levels during the acute phase of the viral infection.Thereafter,tree shrews acted as asymptomatic carriers of the virus in most cases that multiple EBV-related proteins could be detected in blood and tissues.(4)Nanopore transcriptome sequencing of the peripheral blood of EBV-infected animals showed different gene transcription patterns at different time points after EBV infection.Overall,the transcriptome changes were the most significantly in the early stage of infection,mainly manifested as host immune function suppression.Pathways related to neutrophil function were significantly downregulated on the 3-day post infection.PYCARD and NCSTN were the key genes that downregulated the neutrophil function were screened by DMNC algorithm combined with semantic similarity weight ratio.The data of patients with mononucleosis and the results of the retrieved datasets were consistent with the results of tree shrew model,and there was a strong negative correlation between the expression values of PYCARD and NCSTN and the copy number of EBV DNA in the blood of paired patients.Conclusions Our findings demonstrated that tree shrew is a suitable animal model to evaluate the mechanisms of EBV infection,and for developing vaccines and therapeutic drugs against EBV.Neutropenia and neutrophil dysfunction may play an important role in EBV escaping host innate immune response,leading to long-term latent infection.