Characteristics Of HCMV Infection In Primary Skin Dermal Fibroblasts Of Tree Shrew

Human cytomegalovirus(HCMV)belonging to the herpes virus family type 5,which is a widely transmitted infectious pathogens.HCMV infection may occurred in reproductive urinary system,central nervous system and liver system neonatal(immune development is not perfect)or immunodeficiency population,which results in congenital developmental defects or from minor asymptomatic infections to severe defects or death.Generally,HCMV is mostly harmful to the immune deficiency population.Up to now,there is no preventive vaccine and no effective drugs except for ganciclovir(GCV)for antiviral therapy.The ideal of small animal moodel of HCMV infection is essential to the development vaccine and drug as well as the research on its pathogencity.Due to the strict host-specificity of HCMV infection,the research on this virus is mainly carried out by using the humanized mouse and the corresponding cytomegalovirus of various animals(such as monkeys,mice,rats and guinea pigs).Compared with HCMV,these models have different degrees of difference,and can not be used as a suitable infectious disease model for related research.Tree shrews are expected to establish an HCMV cross-species infection model as a mammal,compare with rodents from relatively small size,evolutionary status and physiological and biochemical characteristics.In this study,a high titer HCMV-Towne strain was successfully cultured through human embryonic lung fibroblasts(HEL)and the virus titer was 5.8×106 PFU/mL by modified cell plaque method.Subsequently,it will be infection of the experimental strain.The skin dermal fibroblasts(TSDF)of tree shrews were successfully isolated by the way of collagenase I digestion.The culture conditions were optimized during the culture process,and the growth status of the cells and viability of the cells were further improved.The results showed that the dermal fibroblasts of tree shrew could be regarded as the prerequisite for the subsequent experiments of cell infection in the logarithmic growth phase on the 2?5th days.It infection of six primary cells of tree shrew was screened by Towne-Bac,and the dermal fibroblasts were finally established as HCMV infection.In the infection experiment,TSDF was used as the experimental group,HEL and Balb/c mouse embryonic fibroblasts(MEF)were used as positive and negative controls respectively,and then observed the lesion effect of the cells.It was found that TSDF was infected by Towne,which causing a large number of cell death.Viral load in the infected cells was detected by nested PCR and fluorescence quantitative PCR respectively.The results showed that ie-1 and ul83 remained at a low level 24 hpi of TSDF infection compared with HEL.According to the preliminary discovery TSDF can not support the effective replication of the virus and produce a large number of offspring viruses.Western blot was used to detect the expression and distribution of specific viral proteins in infected cells to reflect the replication activity of the virus.As a result,IE1 and UL44 in TSDF reached the maximum amount at 24 hpi,and then gradually decreased.At the same time,pp65 in TSDF remained almost at the same level throughout the infection relative to HEL,it indicating that there was no change in the amount of virus in the cell,and confirming that TSDF was not able to support viral replication.HCMV-Towne was indeed able to infect and enter the nucleus of TSDF cells,and the viral replication initiation activity occurred by indirect immunofluorescence detection of virus IE1,but the positive rate of IE1 decreased with infection time.The results indicated that virus replication is at a low level.In summary,HCMV-Towne was able to infect skin dermal fibroblasts of tree shrews across species,but the results of detected the virus from molecular and protein levels respectively,it confirmed that Towne had only a short progression in TSDF,but was low and unsustainable.Based on this,we initially thought TSDF was HCMV-infected semi-permitting cells.The results showed that HCMV could infect the skin fibroblasts of tree shrews and lay a preliminary foundation for the further study of HCMV infection mechanism of TSDF.