In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes

Abstract

The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later hepatitis B e antigen (HBeAg) seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg-negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and/or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication-competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely, A2, B2, C2, D3, and the minor strain J, and compared their HBV replication phenotype using transient-transfection models. We identified striking differences in HBV replicative capacity as well as HBeAg and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity. IMPORTANCEThere have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication-competent 1.3-mer cDNA clones of the major genotypes A2, B2, C2, and D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may contribute to some of the observed differences in HBV natural history observed globally. Hepatitis B virus (HBV) is a major human health problem, with approximately 240 million people living with chronic hepatitis B (CHB) worldwide (1) and up to 1 million people dying each year from HBV-related liver conditions such as cirrhosis and liver cancer (2). HBV exists as nine genotypes worldwide (A to I), as well as a recently identified minor strain (designated J), with marked differences in natural history, pathogenesis, and treatment response observed across genotypes (3). These differences include the time to hepatitis B e antigen (HBeAg) seroconversion and the propensity for progression to serious liver disease such as liver cancer, both of which are greater for HBV genotype C (3). Genotypes differ by Ͼ8% in nucleotide sequence across the complete HBV genome, and within most genotypes multiple subtypes have also been identified that differ in nucleotide sequence by between 4 and 7.9% (3). The major genotypes worldwide are A to D, with genotypes...

Can quantitative hepatitis B surface antigen levels predict the severity of liver disease in genotype E Patients?

“…There may be several explanations for our findings. HBsAg generation is reflective not only of cccDNA transcriptional activity, but also originates from integrated DNA in hepatocyte . HBsAg production varies across genotypes with highest levels reported in genotype A individuals.…”

“…There have been reports of differential sensitivity of qHBsAg detection across HBV genotypes. A study focusing on the ability to detect and measure HBsAg levels in the different genotypes showed that in genotype D the detection was 97% lower than genotype A and that genotype F had 30% lower detection than genotype A. Mutations at T143M of the surface gene in genotype A might reduce the ability to detect HBsAg by 44%, in contrast to the mutation T125M in genotype D patients which increased detection by 34% . A study in HBeAg‐negative chimpanzees demonstrated that >90% of liver mRNA originated from integrated DNA in contrast to less than 10% from cccDNA .…”

“…Activation of the immune system in chronically infected patients may contribute to collateral damage by virus‐nonspecific immune cells leading to fibrosis and modulation of a decreasing pool of liver cells. This immune selective pressure could also contribute to higher frequency of changes in the pre‐S/S region . Thus there may be several drivers of HBsAg generation.…”

Characterization of Serum HBV RNA in Patients with Untreated HBeAg-Positive and -Negative Chronic Hepatitis B Infection

“…The finding was also verified in the subgenotype B2, C1, and C2 (Figure 3). It has long been known that different genotypes had marked differences in HBV natural history, disease progression, treatment response, and in vitro replication efficacy (22,23). In terms of genotype differences, the authors' previous study found that the core promoter transcriptional activity of genotype B was significantly higher than that of genotype C, hinting more production of pgRNA in genotype B (19).…”

HBV DNA genome co-transfection procedure for the evaluation of relative fitness

“…[17] Considering that there have been very few studies directly comparing the replication of different HBV genotypes this method could help evaluate some of the observed differences in natural history and disease progression.…”