Establishment Of Tree Shrew Animal Model For HHV-8 Latent Infection And Environmental Effects

Kaposi's sarcoma-associated herpesvirus?KSHV?,also named as Human herpesvirus 8?HHV-8?,belongs to the subfamily gamma-herpesirus monkey herpesvirus genus.As an important human tumor virus,KSHV infection may induce highly tumor-causing death in Kaposi's sarcoma?KS?,Primary Effusion Lymphoma?PEL?,Multicentric Castleman's Disease?MCD?,KSHV inflammatory cytokine syndrome?KICS?and high tumorigenicity particularly in HIV/AIDS patients.There are up to 34,000 KS cases caused by KSHV infection occurred each year worldwide.Especially in sub-Saharan Africa,the KSHV infection rate is as high as 95%and taken as the major cause of more than 50%of HIV/AIDS death.KSHV may infect a variety of human-derived cells,including endothelial cells,lymphocytes,epithelial cells and fibroblasts.The animal model of KSHV infection is a necessary platform to elucidate the transform of KSHV latent and lytic infection,the cytotropictiy of tissues and cells,the mechanism of KS carcinogenesis and the related immune response,the development of vaccines and therapy trchniques.Although immunodeficient mice and marmoset animal models of KSHV infection have been successfully established,the far phylogy relationship or high cost still impedes their using.Therefore,establishement of suit animal model of KSHV infection is essential to solve these problems.Tree shrew is a lower primate similar to squirrel,its genetic and physiological charecteristics are closely related to human.Thus,tree shrew has been widely used in the research of human virus infection.Here,we used tree shrew to establish an animal model of KSHV infection in vitro and in vivo.Firstly,the recombinant virus of rKSHV.219 was obtained by cell culture system of iSLK.219 virus strain.Six kinds of tree shrew primary cells were isolated by using collagenase and trypsin digestion,and identified as primary hepatic sinus endothelial cells,dermal fibroblasts,renal epithelial cells,vascular endothelial cells,pulmonary epithelial cells and peripheral blood lymphocytes.The intensity of cell fluorescence after KSHV infection was observed and flow cytometry analysis was performed.It was showed that KSHV virus can efficiently establish infection in primary renal epithelial cells of tree shrews with high GFP expression rate up to 97%.The infection rate of KSHV from high to low was:tree shrew primary renal epithelial cells?TSKEC?>HEK293T?TSKEC vs HEK293T,p<0.05?>peripheral blood lymphocytes of tree shrew?TSKEC vs TSPBMC,p<0.001?.In comparing with primary renal cells of rodent rat and rabbit,tree shrew primary renal epithelial cells have significant species advantages.Moreover,the infected renal epithelial cells of tree shrew showed that the robust virus replication,high gene expression and viral titer with maximum virus producing at 24 h post-infection.Fuerthermore,passage of KSHV infected primary renal epithelial cells of tree shrew showed that KSHV could long-term latent infect TSKEC with the stable expression of LANA protein in all passages.Comparatively,ORF26 protein expression,which means the occrence of lytic infection,could only be detected in the early P₀ and P₁ generations.In vivo,the young tree shrews of 3 to 5 months old were innoculated with 5×10⁷GFU KSHV virus for each animal by tail vein injecting.In the 119 days period of post infection,the viral DNA,RNA and virus specific protein could be detected in the blood and various tissues of tree shrews.Meanwhile,the expressed GFP fluorescence can also be observed in isolated and cultured lymphocytes.Pathologically,lymphocyte infiltration,focal aggregation,edema and necrosis were detected in spleen,lung and liver of infected animals.The results of immunohistochemical showed the distinct expression of LANA or ORF62 proteins in spleen,lung,liver and kidney,with the main LANA protein expression.These results indicated that KSHV could establish latent infection in tree shrews.Furthermore,the detection of antiviral immune response factors in infected tree shrew showed that the key inflammatory factors related to immune defence has significant increased.In particular,the expression of IFN-?was increased of 1000-fold in comaring with that of control animals.Additoanlly,the pathogenicity-related IL-6 and IL-8 inflammatory factors were also increased by nearly100-fold.It means a strong antiviral immune response was occurred in the infected tree shrews.In summary,tree shrew can be used as an animal model to support latent infection and replication of KSHV.The prilimanary establishment of tree shrew model for KSHV lantent infection has definitely provided a foundation for the further study on the pathogenicity of KSHV-infection and development of vaccine and drug.