Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

Abstract

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T-and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

Detection of Low Copy Number Integrated Viral DNA Formed by <em>In Vitro</em> Hepatitis B Infection

“…In previously published studies 19 , we have used 3 sets of primers that bind conserved HBV DNA sequences flanking the expected site of HBV DNA integration junctions. Other primer sequences and protocols have been described to determine the genomic sequence of HBV 44,45,46,47,48 and may work successfully.…”

“…Mix the tubes by pulse vortexing and briefly spin down the reaction mix. Add NaCl to a final concentration of 100 mM and dextran (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) kDa) to a final concentration of 90 µg/mL. Mix the tubes by pulse vortexing and briefly spin down the reaction mix.…”

“…The HBV DNA integrations formed in vitro resemble those found in patient tissues with respect to their distribution over the cellular genome and junction within the viral sequence that is integrated 13,16 , suggesting a comparable pathway to within an infected liver. Transfer the cell suspensions to a 6-well plate to induce one round of mitosis (which has been reported to induce loss of HBV cccDNA 44 and other HBV DNA intermediates that are amplifiable by invPCR). 11.…”

Characteristics of CpG Islands and their quasispecies of full-length hepatitis B virus genomes from patients at different phases of infection

“…Due to a high replication rate and lack of proofreading activity during reverse transcription, HBV exists as quasispecies (QS), including variants which are genetically distinct, but closely related (Ngui and Teo 1997 ). Because of the different adaptability, QS are related to the outcome of HBV infection (Cao et al 2014 ; Yang et al 2015 ) and antiviral response (Liu et al 2011 ; Chen et al 2009 ; Cheng et al 2013 ; Peveling-Oberhag et al 2013 ; Tong et al 2013 ). Collectively, the characteristics of CpG islands in HBV QS isolated from real-life patients remain largely unknown.…”

Relationships between low serum vitamin D levels and HBV “a” determinant mutations in chronic hepatitis B patients

“…High genetic variability is a characteristic feature of HBV because virus polymerase lacks proofreading activity and very high virion production per day (> 1,000 viruses) [4]. In addition to natural evolutionary changes that contribute to genetic variations, increased genome mutations might also be caused by the host immune system in order for the virus to evade immune clearance [5,6].…”