Toshiaki Ojima

Genoepidemiology and its relationship to clinical features in patients infected chronically with hepatitis B virus (HBV)

The route of hepatitis B virus (HBV) infection and subsequent clinical course vary widely. It is not clear if the prevalence of HBV genotypes differs in the different clinical features of HBV carriers. The genotype of HBV was determined by direct sequencing of HBV DNA amplified with a PCR method from 310 Japanese HBV carriers (189 male and 121 female, aged from 14 to 82 years). Genotype A was detected in eight (2.6%) of 310 HBV carriers, genotype B was detected in 92 (29.7%) and genotype C was detected in 210 (67.7%). None of them had genotype D, E or F. Among the eight patients infected with genotype A, one patient had been infected with HBV in his adulthood. Furthermore, both of one married couple also had genotype A. On the other hand, a HBeAg-negative state and HBeAg-negative healthy carrier state were significantly more common in patients infected with genotype B than those with genotype C by univariate analysis (P<0.0001 and 0.0058) and multiple logistic regression (P<0.0001 and 0.0029). Hepatocellular carcinoma was present in two of eight patients with genotype A, 11 of 210 patients with genotype C and none of the 92 patients with genotype B. These results suggest that the genotype of HBV may be a determinant of the outcome after acute HBV infection and of chronic HBV infection.

Analysis of the entire nucleotide sequence of hepatitis B virus: characteristics of HBeAg-positive asymptomatic carriers, HBeAb positive asymptomatic carriers and patients with liver cirrhosis.

Entire nucleotide sequences of the HBV genome typical for various stages of HBV carriers are currently unknown. Comparison between conserved sequences in HBeAg-positive asymptomatic carriers (HBeAg ASCs) and mutations characteristic for HBeAb-positive asymptomatic carriers (HBeAb ASCs) are of clinical importance. In this study, we determined the entire nucleotide sequences of the HBV genome of patients infected with genotype C (HBeAg ASCs, 11 cases; HBeAb ASCs, seven cases; patients with liver cirrhosis (LC), five cases). Mutations in the entire nucleotide sequences were found more frequently in HBeAb ASCs than in HBeAg ASCs. In the precore/core (preC/C) region, amino acid mutations were more frequent in HBeAb ASCs (3.03%) than in HBeAg ASCs (0.00%) and patients with LC (0.69%). It was suggested that the mutations in the preC/C region had a close relationship with clinical status of HBV carriers. Mutations of leucine to isoleucine at a.a. 100 of the core region and of threonine to serine at a.a. 340 of the polymerase region were found frequently in HBeAb ASCs. In patients with LC, it was suggested that defective interfering particles (DI particles) play a role in the progression of stages. We conclude that attention should be given to mutations at a.a. 340 of the polymerase protein in addition to core protein.

HBe seroconversion and HBs mutation among Japanese hepatitis B virus carriers

It has been reported in Germany that seroconversion to anti-HBe or anti-HBs is frequently associated with genotype changes of hepatitis B virus (HBV) from genotype A to genotype D. We previously reported that the HBeAg-negative state in Japan was significantly more common in patients infected with genotype B HBV than those infected with genotype C HBV. To determine whether the high prevalence of genotype B in the HBeAg-negative state is due to a change from genotype C to genotype B, 72 pairs of serum samples before and after HBe seroconversion were examined for nucleotide sequences in the S gene (amino acids 42-164) among Japanese HBV carriers. No one was identified to have undergone genotype change during HBe seroconversion. A total of 71 codon mutations were observed. Sixty-two of these 71 codon mutations (87.3%) were non-synonymous. Genotype B HBV had no mutational hot spots. In genotype C, there was a mutational hot spot at amino acid 126 of the S protein, and it was suggested that Thr126 before HBe seroconversion was more susceptible to becoming an asymptomatic carrier after HBe seroconversion than Ile126. In conclusion, genotype changes during HBe seroconversion were not found to be common in Japan.

Genoepidemiology of TT virus infection in hepatitis B virus carriers with high sensitivity PCR

Abstract We employed a PCR assay system TaKaRa Ex Taq™ (heat-resistant DNA polymerase), which has 3′→5′ exonuclease activity to increase the sensitivity for TT virus (TTV) DNA detection. Sera obtained from 95 hepatitis B virus carriers without hepatitis C virus coinfection were tested for TTV DNA and the sensitivity of this assay system was compared with the PCR systems reported previously. Of the 95 individuals, TTV DNA was identified in 14 (14.7%) with the PCR reported by Nishizawa et al., in 66 (69.5%) with the PCR reported by Okamoto et al., in 80 (84.2%) by our assay system, and in 86 (90.5%) with the PCR reported by Takahashi et al. Phylogenetic analysis of nucleotide sequences amplified by the PCR revealed that genotypes 1a, 1b, 2, 3, 4, 5 were amplified efficiently by our assay system, while only a part of TTV DNA clone of genotype 1a was amplified by the PCR reported by Nishizawa et al. The prevalence of circulating TTV DNA became higher with age. These results indicate that our assay system with TaKaRa Ex Taq™ has confirmed high prevalence of TTV infection and that at least five genotypes prevail in Japan. In addition, acute TTV infection is supposed to cause long-standing viremia.