HBV Genotype Distribution In Different Areas In Guangxi And Mutations In Wholegenome Of HBV

Background:HBV belongs to hepadnaviridae, which is incomplete double stranded DNA virus with envelope and of 3.2kb long. In all double-stranded DNA virus affecting human body and having independent replication capacity, HBV genome is minimal, but most efficient. It comprises 4 parts of overlapping open reading frame(pre S1/S2/S, pre C/C, P and X). HBV polymerase has no calibration function, so sequence of HBV has a strong heterogeneity. On the principle of total nucleotide heterogeneity exceeding 8%, 8 different genotypes(A-H) of HBV were divided, I and J as a kind of newly discovered genetype were still disputed. Meanwhile, because of the existence of viral quasispecies, it increases the complexity of HBV infection, brings some difficulties to the study of HBV.HBV can induce acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer, which influence human health seriously. HBV is the most common pathogens in the body. There are 2 billion people with previous infection or present infection. Among them, 25 million has been diagnosed as chronic hepatitis B and 75% of them live in South-East Asia and Western Pacific regions. China is a high incidence area of HBV infection, there are 12 million hepatitis B patients, and about 15%-40% patients develop HBV-related cirrhosis and liver cancer. 0.6 million people die from HBV-related disease. The distribution of HBV in china is: the north is dominated by genotype C, while the south is dominated by genotype B, the Southwest with genotype B and C equally. C/D recombinants are mainly distributed in the Northwest regions, C2 and B2 are the most common gene subgenotypes. The mortality of hepatocarcinoma is the second in China’s Rural Areas and following gastric cancer, while its mortality is the third in city following lung carcinoma and gastric cancer. The etiology, pathogenesis and prevention of hepatocarcinoma are the main contents in research.The incidence and mortality of liver cancer all have pasted 50/10000 in Fusui, Guangxi,which is much higher than national average of 27/100000 every year. But liver cancer with about 29/100000 incidence is close to the national level.In our previous studies, HBV infection, aflatoxin intake, pollution of drinking water source have been identified as the main three environmental risk factors to liver cancer development in Fusui, Guangxi. After controlling aflatoxin intake and pollution of drinking water source, liver cancer is sustained with high incidence in Fusui. Therefore, HBV genotype,Subtypes and variation become research focus as for its carcinogenic effects. In order to reveal the biological characteristics of HBV in Fusui, HBV infector and liver cancer patients were included into control study in low incidence area: Guilin. We expect to discover the primary causes of the continuing high incidence of liver cancer in terms of virology.Purpose:(1) PCR reaction system applied to a variety of HBV genotypes was produced and its reaction conditions were optimized as to accomplish high sensitivity amplification and ensure the specificity of the reaction to complete genomic sequence of each HBV genotype in The Guangxi Zhuang Autonomous Region. All of this would lay a good foundation for subsequent sequencing work.(2) The information of HBV sequence in serum was obtained by sequencing directly to PCR product on patients who were diagnosed as liver cancer, chronic hepatitis and HBV carriers in Fusui and Guilin regions. Bioinformatics analysis software was used to splice PCR product followed complete genomic sequence of HBV formation. Complete genomic sequence of HBV could be divided into different genotype and subtypes by timetree. In our study, mutations of HBV were studied between liver cancer patients and patients without developing to liver cancer, and independent risk factors to HBV-related liver cancer were identified.(3) Analyzing the unique HBV genotype in Fusui and Guilin by using bioinformatics methods.Methods:(1) PCR primers suitable for most of HBV genotypes were designed through document retrieval and primer design software, and then specificity PCR amplification was validated. After comparing difference on effect between single and two fragments PCR in HBV complete genomic sequence amplification, we chose optimized methods and definite PCR reaction system and reaction conditions. The samples which HBV DNA were accurately measured using fluorescence quantitative PCR were used to verify the detection sensitivity of the PCR reaction system.(2) Serum samples belongs to liver cancer patients and chronic hepatitis B patients and HBV carriers from Fusui and Guilin regions were collected. HBV DNA amplification was done using two segment method combined with seminested PCR. Under splicing PCR production by bioinformatics analysis software, HBV Complete genomic sequence was obtained. HBV Complete genomic sequences could be divided into different genotypes and subtypes by timetree and sequencing-based typing tool of NCBI online. Mutations of HBV were studied between liver cancer patients and patients without developing to liver cancer according to different genotypes. Sites of mutations associated with liver cancer was found through comparative analysis in SPSS17.0, Multivariate statistical analysis was performed to identity independent risk factors, and the sensitivity and specificity of these mutations in diagnosis of HCC were identified.(3) Recombinant gene sequence was obtained through gene analysis, and then “BLAST” in NCBI was used to find similar sequence. The origin and distribution of recombinant sequence was confirmed by original document acquirement. SIMPIOT was used to determine recombination sites and analyze differences between complete genomic sequences and standard sequence in different genotypes and expression of amino acid in S region. In the end, we cleared categorization in recombinant genotype.Results:(1) After detecting 30 clinical samples, two fragments PCR have much higher sensitivity than single fragments PCR. The sample with the level of HBV DNA exceeding 1.45×103 IU/ml could be detected production with clear strap. Therefore, two fragments method was selected to PCR in follow-up experiment. Meanwhile, matching and verifying primer sequences with HBV standard sequences performed in our study, good specificity and strong binding capacity were demonstrated. Concomitantly, the use of the nested PCR avoided nonspecific production.(2) There were 632 patients with serum samples collected in Fusui and Guilin regions. Patients with imcomplete data, low HBV DNA and samples sequenced failure were excluded. Eventually,183 subjects got complete HBV genome sequence. Reference sequences of HBV genotype B and C were obtained after identification and genotyping by genotyping tools in NCBI. 77.1% were HBV genotype C, 18.8% were HBV genotype B and 4.2% were recombinant genotype in Fusui. In Guilin, there were HBV genotype C and B accounting for 21.8%, 78.2% respectively. The distribution of HBV genotype in Fusui and Guilin was significant difference. In all subjects, B2 subtypes with 88.4%,B4 subtypes with 11.6% were in 86 HBV B genotype patients. C1 subtypes with 69.9%, C2 subtypes with 9.7%, C5 subtypes with 20.4% were in 93 HBV C genotype patients. C1 and C5 were predominant in Fusui followed by B2,C2,B4. However, B2 were predominant in Guilin followed by C1, C5, B4, C2. Complete HBV genome sequence in liver cancer patients and patients without developing to liver cancer were compared in B and C genotype separately. More than 10% subjects with the mutational sites, which would be defined as mutational hot spots. There were 147 mutational hot spots in B genotypes, and 249 mutational hot spots were found in C genotypes. The mutational hot spots placed into cancer patients and patients without developing to liver cancer were studied and analyzed. As above, 20 mutational hot spots of genotype B was considered statistically significant, and 17 mutational hot spots of genotype C was considered statistically significant. Through multivariate statistical analysis, 3 groups of mutation(T53C, A1762T/ G1764 A, G1775A) were identified the independent risk factors in the occurrence of HCC, and the combined diagnosis of the three would be helpful to improve the diagnostic efficiency of HCC.(3) In this study, we found 4 samples numbered 414,441,533,678 were identified as A, C, and G recombinant genotype by genotyping tool in NCBI, whose similar complete genomic sequence of HBV found in GENEBANK was performanced by phylogenetic analysis. Of interest, it doesn’t belong to existing classification, and was similar with the complete genomic sequence of HBV reported in recent years in Vietnam. Some scholars have named it as genotype“I”. Complete sequence and S region amino acid analysis both supported it as the new genotype. Timetree analysis result showed its divergence times was 1200 years ago.Conclusion:(1) We Combined existing literature with primer design tools, and found a high fidelity PCR method to complete genome sequence of HBV. The experimental method with a wide range of application prospects could ensure the detection of high sensitivity and high specificity.(2) We found distribution of HBV genotypes and subtypes in high and low prevalence area in Guangxi, which had significant differences. The establishment of B and C genotypes reference sequences provide reference for the follow-up study. The discovery of liver cancer-related mutation sites would provide help for early diagnosis and treatment of liver cancer.(3) In this study, 4 HBV recombinant genotypes determined as genotype “I” was discovered after multiple sequence alignment and biological evolution analysis. It is the first time to report genotype “I” in Fusui, Guangxi. Our discovery improved geographical information about the distribution of genotype “I”, and as supplement for genotyping tool in NCBI.