The Correlation Study Of HCMV Active Infection And Urothelial Carcinoma And The UL83、UL137and UL145Gene Sequence Analysis In The Clinical Isolate

Objective: Collecting cancerous tissues and anticoagulant specimens for HCMVisolation and identification, combining with the clinical data and comparing HCMVinfection and urothelial carcinoma and recurrence,and analysing the UL83, UL137,UL145gene and amino acid sequence in the HCMV clinical isolates, Understandingthe correlation of the genetic variation and urothelial carcinoma,to provide clues inthe pathogenesis of urothelial carcinoma.Methods: The tissue and blood samples are collected in33patients from the first andsecond hospital of Anhui Medical University, and are treated as follows:(1) theetiology detection of HCMV infection: Using cell culture to isolate virus;detectingHCMV UL55and UL83gene by PCR;HCMV pp65antigenemia;detecting HCMVIgG and IgM by ELISA;and analysing the results to identify the HCMV activeinfection rate in urothelial carcinoma patients. The laboratory evidence to diagnoseHCMV active infecton:virus isolation;the virus morphology and cytomegalicinclusion; the virus antigen;the specific viral gene (mRNA, DNA);the specificantibodies: paired serum anti HCMV-IgG titer is4times higher or anti HCMV-IgM ispositive. Combining with the related data,we think that meeting the above threepoints can be determined to be HCMV active infection.(2) The identification ofHCMV isolate: using electron microscope morphology to see the virus,using RT-PCRto identify the viral gene,using immunofluorescence to identify the viral protein,andeliminating VZV, HSV-1, HSV-2, HCMV AD169.(3)the UL83, UL137, UL145genesequence analysis in HCMV isolates: extracting the clinical isolates、 HCMVAD169、HCMV AD169(American strains)DNA for amplificating UL83、UL137、UL145,gene sequencing, and comparing and analying,using MEGA4to render thephylogenetic tree analysis. Results:(1)HCMV active infection in urothelial carcinoma: There is one clinicalisolate in33anticoagulant specimens,the specimen is via three generations and haveHCMV CPE,so it is identified as HCMV.the positive samples are6in HCMV UL55,UL83gene、HCMV pp65antigenemia and HCMV IgM,so we determined that HCMVactive infection clinical cases are6and the active infection rate was18.2%(6/33).Combinating with the clinical data to indicate that:low back pain, recurrence andpathological grade are related with HCMV active infection and the pathologicalgrading is elevated in HCMV active infector.(2) HCMV identification: observating virus inoculated in HF cells within thecytoplasm of typical herpes simplex virus particles and about the size of100~120nm virus by the electron microscopic;HCMV UL55and UL83gene are positiveby RT-PCR;virus protein IE1are positive by immunofluorescence; VZV, HSV-1,HSV-2are negative by PCR;HCMV UL137and UL145gene are positive by PCR.The results show that the isolate of HCMV is named:07-Hefei-CHN-12.(3) UL83, UL137and UL145gene sequence analysis in HCMV clinical isolate:UL83gene sequence is compared by Clustal W to find that: the sequence homology is97%~100%between HCMV clinical isolate and HCMV AD169, Towne, Merlin.compared with HCMV AD169standard strains,the eighteenth amino acid ischanged from V to F and the231amino acid is changed from G to R,areinduced by CTL responsiveness of the antigenic region, so we guess that CTL epitopevariants in UL83gene may be associated with urothelial carcinoma in HCMVinfected patients.Phylogenetic tree shows:07-Hefei-CHN-12and5strains of HCMVisolates in Genebank and Towne, Toledo, Merlin belong to a big branch,HCMVAD169standard strains, the chamber HCMV AD169strain and HCMV strainAD169(American Columbia University professor Chen Jingxian acknowledge)belonging to branch. UL137gene sequences in07-Hefei-CHN-12are compared with24strains in Genebank to find that variability are base substitution, insertion andwithout deletion mutations, are found that27nucleotide sequence of thesepolymorphisms.The phylogenetic tree showed:07-Hefei-CHN-12strain and8j,15j, 22m,25j and33j strains are confined in a branch, and in the same evolutionarydistance, the relationship is close. UL145gene sequences in07-Hefei-CHN-12arecompared with10strains in Genebank to find that variability are base substitution,insertion and without deletion mutations, are found that32nucleotide sequence ofthese polymorphisms.The phylogenetic tree showed:07-Hefei-CHN-12strain andMerlin、AF1、U11strains are confined in a branch, and the relationship is close.Conclusion:(1)HCMV active infection are6cases and the positive rate is18.2%;combinating with the clinical data to analysing that low back pain、recurrence and pathological grade elevate are related to infection.(2) a strain of HCMV clinical isolate is isolated from a blood leukocytesample,the specimen；observating virus by the electron microscopic;HCMV UL55and UL83gene are by RT-PCR;virus protein IE1are by immunofluorescence; VZV,HSV-1, HSV-2are negative by PCR;HCMV UL137and UL145gene are positiveby PCR. The results show that the isolate of HCMV is named:07-Hefei-CHN-12.UL137, UL145and UL83gene sequence alignment and phylogenetic tree analysisshowed that the mutations in UL83gene may be cause HCMV infection, and CTLepitope variants in UL83gene may be associated with urothelial carcinoma in HCMVinfected patients.UL137and UL145DNA sequence and amino acid sequence arehighly conserved.