Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns

AbstractHepatitis B virus (HBV) is classified into genotypes A–F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV

A case-control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D

There are two subtypes of hepatitis B virus genotype A (HBV/A) and they are provisionally designated Aa (a standing for Africa/Asia) and Ae (e for Europe). In a case-control study, 78 HBV/Aa, 78HBV/Ae, and 78HBV/D carriers from several countries were compared. The prevalence of HBe antigen (HBeAg) in serum was significantly lower in carriers of HBV/Aa than in carriers of HBV/Ae (31% vs. 49%; P = .033), with a difference more obvious in the carriers aged 30 years or younger (34% vs. 67%; P = .029). HBV DNA levels in the carriers of HBV/Aa (median, 3.46 log copies/mL; 95% CI, 2.93-3.95) were significantly lower than those of carriers of HBV/Ae (6.09 log copies/mL; 95% CI, 4.24-7.64) or of carriers of HBV/D (5.48 log copies/mL; 95% CI, 4.06-7.02), regardless of the HBeAg status (P < .001). The most specific and frequent substitutions in 54 HBV/Aa isolates were double substitutions for T1809 (100%) and T1812 (96%) immediately upstream of the precore initiation codon, which would interfere with the translation of HBeAg in HBV/Aa infections. They were not detected in 57 HBV/Ae or 61 HBV/D isolates examined. The double mutation in the core promoter (T1762/A1764) was more frequent in both HBV/Aa (50%) and HBV/Ae (44%) than in HBV/D isolates (25%; P < .01), whereas the precore mutation (A1896) occurred in HBV/D isolates only (48%; P < .0001). In conclusion, the clearance of HBeAg from serum may occur by different mechanisms in HBV/Aa, HBV/Ae, and HBV/D infections, which may influence clinical manifestations in the Western countries where both genotypes A and D are prevalent

Genome-Wide Association Study Confirming Association of HLA-DP with Protection against Chronic Hepatitis B and Viral Clearance in Japanese and Korean

Hepatitis B virus (HBV) infection can lead to serious liver diseases, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC); however, about 85–90% of infected individuals become inactive carriers with sustained biochemical remission and very low risk of LC or HCC. To identify host genetic factors contributing to HBV clearance, we conducted genome-wide association studies (GWAS) and replication analysis using samples from HBV carriers and spontaneously HBV-resolved Japanese and Korean individuals. Association analysis in the Japanese and Korean data identified the HLA-DPA1 and HLA-DPB1 genes with Pmeta = 1.89×10−12 for rs3077 and Pmeta = 9.69×10−10 for rs9277542. We also found that the HLA-DPA1 and HLA-DPB1 genes were significantly associated with protective effects against chronic hepatitis B (CHB) in Japanese, Korean and other Asian populations, including Chinese and Thai individuals (Pmeta = 4.40×10−19 for rs3077 and Pmeta = 1.28×10−15 for rs9277542). These results suggest that the associations between the HLA-DP locus and the protective effects against persistent HBV infection and with clearance of HBV were replicated widely in East Asian populations; however, there are no reports of GWAS in Caucasian or African populations. Based on the GWAS in this study, there were no significant SNPs associated with HCC development. To clarify the pathogenesis of CHB and the mechanisms of HBV clearance, further studies are necessary, including functional analyses of the HLA-DP molecule

Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns

AbstractHepatitis B virus (HBV) is classified into genotypes A–F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV

Novel Type of Hepatitis B Virus Mutation: Replacement Mutation Involving a Hepatocyte Nuclear Factor 1 Binding Site Tandem Repeat in Chronic Hepatitis B Virus Genotype E

The genetic diversity of hepatitis B virus (HBV) strains has evolved through mutations such as point mutations, deletions or insertions, and recombination. We identified and characterized a novel type of mutation which is a complex of external insertion, deletion, and internal duplication in sequences from one of six patients with chronic hepatitis B virus genotype E (HBV/E). We provisionally named this mutation a “replacement mutation”; the core promoter upstream regulatory sequence/basic core promoter was replaced with a part of the S1 promoter covering the hepatocyte nuclear factor 1 (HNF1) binding site, followed by a tandem repeat of the HNF1 site. A longitudinal analysis of the HBV population over 6 years showed the clonal change from wild-type HBV/E to replacement-mutant type, resulting in a lower hepatitis B (HB) e antigen titer, a high HBV DNA level in serum, and progression of liver fibrosis. In an in vitro study using a replication model, the replacement-mutant HBV showed higher replication levels than the wild-type HBV/E replicon, probably mediated by altered transcription factor binding. Additionally, this HNF1 site replacement mutation was associated with excessive HB nucleocapsid protein expression in hepatocytes, in both in vivo and in vitro studies. This novel mutation may be specific to HBV genotype E, and its prevalence requires further investigation