Infection Of Mouse With Human Cytomegalovirus In Vitro And Vivo And Research The Genetic Variation Of Isolation Strain's UL83,UL54 Gene

Objective Analyze the genetic variation of isolation strain's UL83/UL54 gene,sieve the gene which influence it's cross-species infection,provide the clew to explore the mechanism of it further.Methods(1) Intraperitoneal inject 1.45×10~6 PFU/ml HCMV AD 169,3×10~5PFU/ml Shandong strain(Given by Shandong Academy of Medical Science) as experiment group,establish the inactivation and HF comparison group at same time,each group includes 12 mouses(6 male and 6 female) aged 6-8 weeks.All the mouses were sacrificed to obtain brain tissue for virus isolation and assessment(PCR detect HCMV UL83),reverse transcription PCR(RT-PCR) detect HCMV UL54 transcription in brain tissue a month later.We named the HCMV which isolated from the mouse vivo strain.(2) MF and HF cell monolayers were inoculated with the virus at an MOI of 100 PFU per cell and observed daily for characteristic cytopathic effect(CPE) of HCMV.Until most of the cells showed extensive cytopathic effects,the cells were harvested to detect HCMV UL83 and IE gene and their corresponding transcripts by PCR,RT-PCR.We named the HCMV which isolated from MF vitro strain.HF cell monolayers were inoculated with Shandong and Clinical isolate strain.(3) Take the cell cultures of the four isolates virus(Vivo strain,Vitro strain,Shandong strain,Clinical strain),extract DNA and amplificate the total length of UL83,UL54 gene,sequence them and align to other herpesvirus,phylogenetic tree is generated with MEGA version 4.0. Result(1) Intraperitoneal inject 1.45×10~6 PFU/ml HCMV AD169 to BALB/c mouse, all the mouses were sacrificed to obtain brain tissue for virus isolation,the femal of the experiment group showed extensive cytopathic effects.PCR and RT-PCR were also positive in the same mouse.3×10~5 PFU/ml Shandong strain,male mouse in the experiment group,inactivation and HF comparison group were all negative.(2) The MF cell monolayers showed the same viral cytopathic effect as HF cells after being inoculated with the virus.Both the UL83 and IE gene and their corresponding gene transcripts could be detected in the infected MF cells.(3) Aligned by the Culstal W,we found that the identity of UL83 between isolation strain and type strain is 98%-100%,the UL54 is 99%-100%.In the mutation point of UL83,nucleotide point 682-684 changed from CTG to TCT and leads the amino acid point 228 changed from leucine to serine.After analyzing the evolution relation by MEGA version 4.0,we found that:The HCMV which can adapt the vivo environment,and infect the mouse cerebral cortex,it's UL83 gene form a branch with other herpesvirus and MCMV,other strain's UL83 gene form a branch with type strain,Merlin and Towne.The UL54 gene of the four virus are in the same branch.Conclusion we have gained the strains which can infect MF and mouse cerebral cortex. After amplificating the total length of UL83,UL54 gene,sequencing and aligning,we found that:The mutation occurred at the amino acid point 228 which have the potential to induce the CTL response.The analysis of the evolution relation showed the HCMV which can adapt the vivo environment,and infect the mouse cerebral cortex,it's UL83 gene form a branch with other herpesvirus and MCMV,other strain's UL83 gene form a branch with type strain,Merlin and Towne.So it is a hint that the change of host may be related to the mutation of the UL83 and provide a clew to explore the mechanism of cross-species infection further.