China has nearly 10% of the general HBV carrier population in the world; this infection is the most common cause of chronic liver disease. Understanding HBV epidemiology is essential for future infection control, evaluation, and treatment. This study determined the prevalence of HBV infection in Shenzhen by serological testing and analysis in 282,166 HBV screening cases for the following: HBcAb, indicative of previous HBV infection; HBsAg, indicative of chronic (current) infection; HBsAb, indicative of immunity from vaccination; and 34,368 HBV etiological screening cases for HBV-DNA, indicative of virus carriage, in which 1,204 cases were genotyped and mutation analyzed for drug-resistance evaluation. Shenzhen was a highly endemic area of HBV throughout the study period (prevalence 9.69%). HBV infections were almost entirely in the 20 and older age groups with a male-to-female ratio of 1.16:1 which is approximately the same as the male-to-female ratio of the general population in China. However, only 71.25% of the general population retained HBV immune protection. Genotype B and C were identified as the most common agents; recombinant B/C and B/D also existed; some cases, however, could not be genotyped. NAs resistant mutation occurrence patterns were multitudinous; single mutation patterns of rtM204I/V and rtL180M occurrences accounted for majority, followed by the combinational mutation pattern L180M + M204I/V. Drug-resistance was prevalent, mainly occurring in the cross resistance patterns LAM + LdT and LAM + LdT + ETV, and significantly more critical in males. These results demonstrate that all people free from HBV infection should obtain injections of the vaccine or booster shots, and conventional virologic detection in a clinical laboratory center should incorporate genotype and mutation alongside the serological factors for etiology and develop better classification methods, such as sequencing.

Hepatocyte damage, especially immunological liver injury, is a key process in the pathogenesis of hepatitis virus-induced liver diseases. The aim of this study was to investigate the protective effects of polyguluronate sulfate (PGS) against immunological liver damage. The results showed that PGS significantly reduced the H₂O₂-induced oxidative stress and increased the cell viability in HepG2 hepatocytes. PGS also suppressed the production of malondialdehyde (MDA), lactate dehydrogenase (LDH), TNF-α, and IL-6, while up-regulating the activity of SOD in HepG2 cells. Further, PGS (150 and 300mg/kg/day) significantly attenuated the elevation of serum glutamate pyruvate transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBiL), in addition to liver MDA and NO levels in Con A-induced immunological liver injury within mice (P<0.05). Significant improvements of organ indexes (liver, spleen, and thymus) were observed in PGS-treated mice. PGS also significantly reduced the disorganization of hepatocytes and decreased the inflammatory cell infiltration caused by Con A treatment, suggesting that PGS was able to attenuate Con A-induced liver injury. In conclusion, PGS possesses significant hepatoprotective effects on immunological liver injury in vitro and in vivo, and this may be related to its antioxidant activities.

The dynamics of resistance-associated mutations under combination therapy were explored.

A total of 46 patients were classified into adefovir (n=14) and entecavir (n=32) groups. In the adefovir (ADV) group, six patients receiving combined therapy were DNA-positive after more than 3 years of therapy. Ultra-deep pyrosequencing was used to analyze the dynamics of multi-drugs resistance mutations.

At baseline, all 46 treatment-naïve patients harbored rtA181V/T substitutions (1.2%-4.6%) and rtN236T substitutions (1.6%-6.1%). In the ADV group, eight patients with long-term treatment were consecutively HBV DNA-positive for more than 3 years. During treatment, the rtA181T resistance-associated site appeared with increasing frequency in six of eight patients (NOs. 1-6), and two patients (NOs.4 and 8) carrying the rtA181T resistance mutations increasingly showed high levels of rtN236T. One patient (NO. 8) experienced virological breakthrough. Other known pre-existing mutations showed no dynamic fluctuations, including in rtA194T, rtP177G, rtF249A, and rtD263E. In addition to the common substitutions, some previously unknown amino acid substitutions, such as rtD134N, rtL145M/S, rtF151Y/L, rtR153Q, and rtS223A, should be further studied.

HBV-resistance substitutions conferring to nucleoside analogs are present at baseline. The dynamics of the HBV RT-region quasispecies variation are heterogeneous and complex.

Adefovir dipivoxil (ADV) is used as first-line monotherapy or rescue therapy in chronic hepatitis B (CHB) patients. In this study, we sought to identify nucleotide changes in the reverse transcriptase (RT) of hepatitis B virus (HBV) at baseline and explore their predictive value for ADV antiviral response. Ultra-deep pyrosequencing (UDPS) was utilized to determine HBV genetic variability within the RT region at baseline and during a 48-week ADV therapy. According to the viral load at the end of ADV treatment, all patients were classified into responders (HBV DNA level reduction of ⩾ 3 log 10 IU/mL) and suboptimal responders (HBV DNA level reduction of <3 log 10 IU/mL). Based on UDPS data at baseline, we identified 11 nucleotide substitutions whose combination frequency was significantly associated with the antiviral response among 36 CHB patients in the study group. However, the baseline distribution and frequency of rt181 and rt236 substitutions known to confer ADV resistance was a poor predictor for the antiviral response. Compared with baseline serum HBeAg, HBV-DNA and ALT levels, the baseline HBV sequence-based model showed higher predictive accuracy for ADV response. In an independent cohort of 31 validation patients with CHB, the sequence-based model provided greater predictive potency than the HBeAg/HBV-DNA/ALT and the HBeAg/HBV-DNA/ALT/sequence combinations. Taken together, we confirm the presence of ADV resistance variants in treatment-naïve patients and firstly unravel the predictive value of the baseline mutations in the HBV RT region for ADV antiviral response.

Hepatitis B virus (HBV) polymerase and human immunodeficiency virus (HIV) reverse transcriptase are structurally related. However, the HBV enzyme has a protein priming activity absent in the HIV enzyme. Approved nucleoside/nucleotide inhibitors of the HBV polymerase include lamivudine, adefovir, telbivudine, entecavir and tenofovir. Although most of them target DNA elongation, guanosine and adenosine analogs (e.g. entecavir and tenofovir, respectively) also impair protein priming. Major mutational patterns conferring nucleoside/nucleotide analog resistance include the combinations rtL180M/rtM204(I/V) (for lamivudine, entecavir, telbivudine and clevudine) and rtA181V/rtN236T (for adefovir and tenofovir). However, development of drug resistance is very slow for entecavir and tenofovir. Novel nucleoside/nucleotide analogs in advanced clinical trials include phosphonates similar to adefovir or tenofovir, and new tenofovir derivatives with improved pharmacological properties.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus of the Hepadnaviridae family and is approximately 3.2 kb in length. Owing to a lack of proofreading capacity during reverse transcription and a high replication rate, HBV exhibits as quasispecies. To detect the genetic mutations of HBV, many methods with different sensitivities and throughputs were developed. According to documentary records, HBV mutation and evolution were important vial parameters in predicting disease progression and therapeutic outcome. In this review, we separately discussed the correlation between HBV genomic mutations in four open reading frames and liver disease progression. Since some of the results were controversial from different laboratories, it remains to be seen whether functional analyses will confirm their role in modifying the course of infection.

Deep sequencing technologies are currently cutting edge, and are opening fascinating opportunities in biomedicine, producing over 100-times more data compared to the conventional capillary sequencers based on the Sanger method. Next-generation sequencing (NGS) is now generally defined as the sequencing technology that, by employing parallel sequencing processes, producing thousands or millions of sequence reads simultaneously. Since the GS20 was released as the first NGS sequencer on the market by 454 Life Sciences, the competition in the development of the new sequencers has become intense. In this review, we describe the current deep sequencing systems and discuss the application of advanced technologies in the field of hepatology.

Many viruses, including the clinically relevant RNA viruses HIV (human immunodeficiency virus) and HCV (hepatitis C virus), exist in large populations and display high genetic heterogeneity within and between infected hosts. Assessing intra-patient viral genetic diversity is essential for understanding the evolutionary dynamics of viruses, for designing effective vaccines, and for the success of antiviral therapy. Next-generation sequencing (NGS) technologies allow the rapid and cost-effective acquisition of thousands to millions of short DNA sequences from a single sample. However, this approach entails several challenges in experimental design and computational data analysis. Here, we review the entire process of inferring viral diversity from sample collection to computing measures of genetic diversity. We discuss sample preparation, including reverse transcription and amplification, and the effect of experimental conditions on diversity estimates due to in vitro base substitutions, insertions, deletions, and recombination. The use of different NGS platforms and their sequencing error profiles are compared in the context of various applications of diversity estimation, ranging from the detection of single nucleotide variants (SNVs) to the reconstruction of whole-genome haplotypes. We describe the statistical and computational challenges arising from these technical artifacts, and we review existing approaches, including available software, for their solution. Finally, we discuss open problems, and highlight successful biomedical applications and potential future clinical use of NGS to estimate viral diversity.