Analysis Of The Mutations In Enhancer â…  Of Hepatitis B Virus Genome And Their Implication

Human infection with the hepatitis B virus(HBV) will lead either to acute,self-limiting disease,or to chronic hepatitis B(CHB).Despite only 3 to 10% individuals who infected HBV will become chronic carriers. However, more than 2 billion people worldwide had been infected with hepatitis B virus, population of CHB is still huge.After infection with HBV, specific T and B lymphocytes response against viral proteins leads to clearance of the virus. However in CHB patients,HBV DNA levels were high during the acute phase after infection.Later,in the chronic phase,there were a strong decrease in serum HBV DNA levels,but failed to eliminate the virus,leading to a persistent infection with HBV.The switch mechanism between a high to a low HBV DNA levels during chronic HBV infection was very complex. One was selective mutation of HBV structural genes to evade the host immune surveillance. The relationship between HBV enhancer I (Enh I) DNA mutation with chronic HBV infection mechanism remained unclear. There are two enhancers in HBV genome. Studies showed that the HBV Enh I play an important regulatory role in the HBV genome transcription and replication, Site mutations at HBV Enh Iassociated with HBV chronic infection has not been addressed so far.In this paper, HBV Enh I region of CHB patients were amplified by PCR technology and sequenced, then typed using Genotyping software in Genbank , and analyzed of variations compared with the reference HBV sequence in Genebank with DNAMAN software, while serum marker such as HBV DNA, HBsAg and HBeAg titer were detected to analyze the relationship between the Enh I gene mutations.Objective:(1)To investigate the influence of Taq PCR MasterMix and Taq plus PCR MasterMix on the fidelity of PCR products of HBV DNA. (2)To investigate DNA Variations in HBV genome Enh I region of CHB patients in jiangxi province.(3)To study the influence of serum HBV DNA levels and HBsAg, HBeAg titer with HBV Enh I DNA mutations in CHB patients.Methods: (1)Two serum samples from patients infected with or without hepatitis B virus were collected for specific amplification of the gene of nt835 to nt1396 in HBV genome using two different PCR MasterMix respectively. The PCR products were sequenced and matched with DNAMAN software.(2)Serum samples were collected from 70 patients with CHB including 35 cases with seroconversion HBsAg(+)HBeAg(+)HBcAb(+), other 35 cases with seroconversion HBsAg(+)HBeAg(-)HBcAb(+) and 2 cases of healthy people. Enh I region DNA of HBV genome were amplified by PCR and sequenced, then HBV genotype were determined by Genotyping software in Genbank, and matched the standard strains by DNAMAN software to determine the relevant DNA mutations. Fluorescence quantitative PCR(FQ-PCR) was used to measure serum HBV DNA copies. Enzymer-linked immunosorbent assay (ELISA) was used to detect serum HBsAg, HBeAg titer by coubling dilution. The data were analyzed by SPSS 13.0 statistical software. HBV DNA copy number logarithmic mean show as ( x±s), antigen titers show as geometric mean, and positive rate was compared by the chi-square test , HBV DNA copy number of different groups was compared with multiple comparisons ANOVA and equal variance Not Vssumed Dunnett's T3 , and a P value less than 0.05 was regarded as significant.Results: (1) Two PCR Matermix could amplify the target gene in HBV genome specifically. Compared with the sequence of PCR product using Taq Plus PCR MasterMix, the amplified gene product using Taq PCR MasterMix showed deletion of nt1390 and two mutation sites of nt1389T→C and nt1392G→T. (2)The results with Sequence Alignment with Genotyping showed that all 70 sequenced HBV DNA belong to type B and were homologous to the reference sequence AF100309. Seventeen mutation sites were found in Enh I region and they were nt1010A→T,nt1013G→A,nt1031T→C,nt1034A→G,nt1053C→T,nt1082A→T,nt1133G→A,nt1167C→T,nt1223C→A,nt1227G→C,nt1230A→C,nt1241T→A,nt1244G→A,nt1246C→A,nt1251C→G,nt1289A→C,nt1349A→C.(3)Serum HBV DNA levels and HBsAg, HBeAg titer decreased obviously in CHB patients with HBV Enh I region nt1133G→A mutation.Conclusions: (1) The fidelity of the target DNA fragment amplification catalyzed by Taq and Pfu mixed DNA polymerase was higher than by Taq DNA polymerase. (2) HBV genotype B is the major genotype in jiangxi province. The mutation of nt1133G→A at HBV Enh I region could decrease Enh I function, down regulate HBV gene transcription and replication, mutations at HBV Enh I region DNA may be a possible mechanism for the progression to chronic hepatitis.