Research Of HCMV Infection And Reactivation In Patients With Chronic Serve Hepatitis B

Human cytomegalovirus (HCMV), is a double strand DNA virus. HCMV for allspecies are ubiquitous and have classic beta-herpes virus characteristics. Worldwidepopulations is susceptible, although different infection rate among developed areas andbackward regions. Following host control of primary lytic infection, HCMV establishes alife-long infection, becoming dormant in multiple end organs. Previous infection is mostoften confirmed by the presence of HCMVspecific IgG test. While an active infection,characteristics of the end organs, such as genitourinary system, respiratory system, centralnervous system and liver disorders and retinopathy, may be various and ranging from mildclinical manifestations asymptomatic latent infection to active infection causes severe organdysfunction and death. Generally, the host immune status differences in the reactivation oflatent HCMV infection play a critical role in the outcome of disease. Virulence of differentstrains of HCMV could be an important factor in the clinical and virological response to thetherapy. The difference of virulence between HCMV strains may result from geneticvariation. One of the most important potential virulence factors are glycoprotein B (gB),along with gH, gM, gL mediate virus invasion of host cells, and mainly mediate virus entryinto cells, cell to cell virus transmission, and fusion of infected cells. It may be an importanttarget of host cell and humoral immunity. According to the UL55gene encoding gBnucleotide polymorphisms, gB can be divided into four genotypes gB-1~gB-4.Recent research shows that the key point to distinguish reactivation from latentinfection is determined by the active viral infection proliferation-related genes (lytic gene)expression. Research report that some of the immediate early gene (IE) ofHCMVtranscription can be measured in the latent phase, but the gene IE1and IE2, locatedin UL123area, always can not be detecteted in the natural state of latent infected cells.Currently, detection of serum HCMV IgM antibody combined with quantitative of HCMVreal-time PCR, because of its high sensitivity and detection rate, has become a majoradjunct mesure in the diagnosis of reactivation from latent virus. However, due to diversity in primer selection, quality differences between different kits, clinically effective cut offvalues have not been conclusive.Patients with chronic HBV infection coexist chronic HCMV infection, studies inpatients with HBV infection progress to hepatocellular carcinoma found that higher serumand tissues positive rate of HCMV. Our pre-clinical test results also showed that HCMVIgM positive phenomenon in some severe hepatitis/hepatic failure patients. However, it isnot clear that HCMV infection under severe hepatitis/hepatic failure status. Little is knownabout chronic HCMV infection and the relationship between different HCMV gB type anddisease of patients with severe hepatitis B (CSH).This study collected in-patients with a diagnosis of CSH by Department of InfectiousDiseases of Southwest Hospital, Third Military Medical University, Chongqing. Serumsamples were collected to completely detect kidney function and virological indicators,using enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescenceimmunoassay (ECLIA) to test HCMV IgM, IgG, and nested-PCR and real time PCR to testDNA of the whole blood. Either HCMV IgM or DNA positive is defined as HCMV reactiveinfection and then classified into the group of CSH without HCMV reactivation and thegroup of CSH with HCMV reactivation. We collected their clinical characteristic andlaboratory test results, calculated MELD score. Meanwhile HCMV PP65antigen is testedfrom PBMC in reactivation patients, use electrochemiluminescence to detect HCMV IgMtiters of these cohort serum, explore the establishment of HCMV DNA quantitativedetection system, in order to observe the laboratory features of the reactivation of HCMVand research of its mechanism.We collected different clinical diagnosis blood samples as controls, and amplified gB,IE1gene fragment. For all positive samples, the PCR products were then sequenced usingABI PRISM Dye Terminator Sequencing Kit with Amplitaq DNA polymerase (ABI, FosterCity, CA) and loaded onto an ABI3700sequencer. And we used DNA Star Lasergene7.1-SeqMan program to do stitch bidirectional sequence results, EditSeq to translatenucleotide, and BioEdit to analyse the variation of nucleotide and amino acid. Meanwhile,according to the literatures, we downloaded various strains of HCMV IE1and gB sequencefrom Genbank, used ClustaX2and MEGA6.0software package to analysis, and constructedphylogenetic tree by Neighbor-joining method. Meanwhile homology comparison was blasted by NCBI, expect to explore the molecular epidemiology of HCMV in Sichuan andChongqing area clearly.Results:1. From April2011to December2012,132patients of CSH in the Department ofInfectious Diseases of Southwest Hospital were included, of which122cases were in thegroup without HCMV reactivation and10cases in the group with HCMV reactivation.HCMV IgM, IgG antibody detection positive rates were3.78%and100%respectively,HCMV DNA detection rate was5.30%, and higher than the serum IgM detection rate.Genotyping result display gB1type was100%.2. Seven point fifty-eight percent (7.58%,10/132) of CSH patients coexist HCMVreactivation. AFP median values of the two groups were101.75and27.19, the differencewas statistically significant (P=0.024), and the other laboratory parameters showed nostatistically difference. In HCMV reactivation group, the morbidity of spontaneous bacterialperitonitis, ascites, hepatic encephalopathy, acute kidney injury ratio shows higher thanHCMV reactivation group, but no significant statistically difference respectively, and3-month mortality of the former was19.68%, later was30%; the MELD score of the twogroups patients had no statistically difference (P=0.783).3. When HCMV activated, IgM titer in sera showed a continuous decline after thegradually rise and simultaneously peripheral blood displayed HCMV PP65antigenemia,HCMV DNA in PBMC was103or more.4. After BioEdit analysis, all4sequences of HCMV IE1nucleotide sequence showedhighly conserved regions except the sample FUO-2displayed T/C mutation at the153site(asparagine sites), but does not change the amino acid sequence (AAT/AAC), and so IE1isnot suitable for genotyping and molecular epidemiological analysis in the region.5. Thirteen patients’ serum samples by nested-PCR amplification arrived successfully,and got13HCMV gB sequences, in which six cases of fulminant hepatitis,4cases ofchronic hepatitis B patients,1case of primary biliary cirrhosis,1case of drug hepatitis,1case of Fever of Undetermined Origin(FUO).10sequences can be classified by molecularphylogenetic analysis as type gB1,3sequences as type gB3, and we did not find type gB2,gB4, all six cases of patients in the CSH group with active HCMV infection were type gB1,HCMV gB nucleotide and amino acid variation analysis showed consistent with the reference strain genotypes.6. Molecular epidemiological analysis found that HCMV gB1genotypes in the area ofChongqing and the area of Hunan Province were accounted for76.92%(10/13) and82.61%(19/23) respectively, and HCMV gB3type accounted for23.08%(3/10),17.39(4/23)respectively, and did not find type gB2, gB4. The differences in the genotype distribution ofthe two areas showed no significant statistically difference (P=0.683). Such genotypedistribution is higher similarly as that in the United States, Britain, and South Korea.Conclusions:1. In CSH patients, detection rate of HCMV DNA is higher than that of serum IgM,and IgG antibody positive rate is100%, indicating that these people were infected withHCMV before, and type gB1is the prodominate genotype in these people.2. CSH patients had a lower proportion of HCMV reactivation, and when reactivationthey may get worse, with increased complications, decreased survival, thus may affect theoutcome of the disease. When HCMV activated, IgM titer in sera showed a continuousdecline after the gradually rise and simultaneously peripheral blood displayed HCMV PP65antigenemia, quantitative detection shows thant HCMV DNA in PBMC was maintained atthe level of103~104. We established FQ-PCR detection system for HCMV DNA, thestability, sensitivity, specificity and reproducibility is better, and is expected to be widelyused to detect clinical samples.3. HCMV IE1is not suitable for HCMV genotypes and molecular epidemiologicalanalysis in the Chongqing region. In this area, HCMV type gB1predominate, and whenCSH patients cocurent with HCMV reactivation, HCMV type gB1is the only one genotype.Molecular phylogenetic tree differentiate varieties of HCMV gB genotype accurately andreliable, showed a true reflection of changes in genotype，and can be used as the standardfor analysis of gB genotypes.4. HCMV genotype distributed similarly in Chongqing and Hunan area, only see typegB1and gB3, and type gB1is the dominant type, Genotype distribution is higher similarlyas that in the United States, Britain, and South Korea.