Sophisticated viral quasispecies with a genotype-related pattern of mutations in the hepatitis B X gene of HBeAg-ve chronically infected patients

Abstract

Patients with HBeAg-negative chronic infection (CI) have not been extensively studied because of low viremia. The HBx protein, encoded by HBX, has a key role in viral replication. Here, we analyzed the viral quasispecies at the 5′ end of HBX in CI patients and compared it with that of patients in other clinical stages. Fifty-eight HBeAg-negative patients were included: 16 CI, 19 chronic hepatitis B, 16 hepatocellular carcinoma and 6 liver cirrhosis. Quasispecies complexity and conservation were determined in the region between nucleotides 1255 and 1611. Amino acid changes detected were tested in vitro. CI patients showed higher complexity in terms of mutation frequency and nucleotide diversity and higher quasispecies conservation (p < 0.05). A genotype D-specific pattern of mutations (A12S/P33S/P46S/T36D-G) was identified in CI (median frequency, 81.7%), which determined a reduction in HBV DNA release of up to 1.5 log in vitro. CI patients showed a more complex and conserved viral quasispecies than the other groups. The genotype-specific pattern of mutations could partially explain the low viremia observed in these patients.

Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?

“…Interestingly, these 3 conserved sequence fragments almost overlapped with hyper-conserved sequence fragments described in a previous study carried out by our group at circulating DNA quasispecies level (1255-1286, 1545-1573 and 1575-1603)[ 13 ]. Likewise, a recently published study by our group[ 14 ] also performed in HBV-DNA quasispecies, reported some of these hyper-conserved regions (1258-1286 and 1575-1605). In addition, these 2 previous studies[ 13 , 14 ] also reported as hyper-conserved the sequence stretch between positions 1519-1543, which we found among the 5% most conserved windows at HBV-DNA quasispecies level but not at HBV-RNA quasispecies level.…”

“…Likewise, a recently published study by our group[ 14 ] also performed in HBV-DNA quasispecies, reported some of these hyper-conserved regions (1258-1286 and 1575-1605). In addition, these 2 previous studies[ 13 , 14 ] also reported as hyper-conserved the sequence stretch between positions 1519-1543, which we found among the 5% most conserved windows at HBV-DNA quasispecies level but not at HBV-RNA quasispecies level. Conversely, we identified the sequence stretch 1559-1587 as hyper-conserved only in HBV-RNA quasispecies.…”

“…In this study, we analyzed the region between nucleotides (nt) 1255 to 1611, located at the HBX 5’ end. In this region, we previously identified hyper-conserved sequence stretches in the circulating HBV-DNA quasispecies[ 13 , 14 ], which we aimed at analyzing at circulating HBV-RNA level.…”

“…In order to interfere with the synthesis of all the viral proteins, these therapies should be directed to hyper-conserved sequence targets shared by all the HBV mRNAs. HBX thus provides an ideal target, as its sequence is located at the 3’ end of all HBV mRNAs[ 13 , 14 ]. Taking this into account, analysis of serum HBV-RNA quasispecies may be an especially useful tool when looking for hyper-conserved targets for this kind of antiviral therapy.…”

High prevalence of tryptophan-truncated S quasispecies in treatment-naïve chronic hepatitis B patients

“…Recent studies have revealed that different HBV subgenotypes show different patterns of mutations, including drug-resistant mutations [30,31]. For example, Li et al [31] demonstrated that lamivudine-and adefovir-resistance-associated mutational patterns were different between subgenotypes B2 and C2 in HBV-infected Chinese patients.…”