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.
INTRODUCTION
Hepatitis B virus (HBV) infection causes a wide spectrum of clinical manifestations ranging from an asymptomatic carrier state (immunotolerant state) to acute or chronic hepatitis, with progression to severe liver disease (1, 2). It is therefore generally believed that the outcome of HBV infection and the severity of associated liver diseases are determined by the nature and strength of the host immune response against the virus (3).
It is well-known that HBV, a hepatotropic and noncytopathic DNA virus replicated by an error-prone polymerase through an RNA intermediate, exists as a quasispecies (QS) (4). HBV QS with genetically heterogeneous variants are thought to play a role in the progression of HBV-associated liver diseases. Selection pressures for the virus come from (i) competition between viral variants (differences in replicative efficiency) (5) and/or (ii) host immune activity (3). It was recently documented in a Chinese study that the coexistence of the HBV variants (A1762T/G1764A/G1896A mutation and preS deletion/S deletion at a 1:4 ratio) from an HBV-associated liver failure patient could increase, in vivo and in vitro, HBV replication, and this could significantly modulate specific host immune responses (6).
We previously reported that HBV QS complexity and diversity within the reverse transcriptase (RT) region in responders are lower than those in nonresponders during the early stage of oral antiviral treatment (7, 8), which may contribute to the prediction of long-term virologic responses (8). Other studies have also shown that dynamic changes in HBV QS complexity and diversity may predict a sustained virologic response to long-term nucleotide acid analogue treatment (9, 10). Similar, but not identical, studies from Singapore showed that the evolution of viral diversity increased in HBeAg seroconverters treated with lamivudine (11) and interferon (12).
Nevertheless, it remains unknown whether or not HBV QS complexity and diversity make significant variations through the natural history of chronic and acute disease progression without selective pressure of antiviral treatment. Coffin et al. (13) recently revealed the differences between HBV polymerase (Pol)/S variants from the plasma and peripheral blood mononuclear cell (PBMC) compartment in treatment-naive patients during different chronic hepatitis B (CHB) immune phases and the presence of drug-resistant and immune escape variants (“a” determinant mutation). There is some evidence that changes in HBV sequences can be selected due to host immune pressure (14) and that the HBV mutation rate is even higher in HBeAg-negative individuals (15), possibly due to increased immune pressure prior to the loss of HBeAg or higher levels of virus replication in the absence of HBeAg, which could be restored by basal core promoter (BCP) mutations (A1762T/G1764A dual mutations) (16). This suggests that the host immune response plays a critical role in viral evolution. During acute hepatitis B (AHB) disease progression, both innate and adaptive immune responses are effective, and the mechanism of HBV persistence is not fully understood but is likely multifactorial, including HBV-specific immunosuppression and virus factors (HBxAg and HBeAg) (17).
In an attempt to investigate HBV QS in treatment-naive patients with different levels of selective pressure (different phases of infection), HBV full-length genome (each clone revealed one complete HBV genome) QS and mutations by clonal sequencing were isolated from 40 individuals.
MATERIALS AND METHODS
Patients.
Forty treatment-naive HBV-infected inpatients from Shanghai Ruijin Hospital, The Sixth People's Hospital in Shanghai, and The Fifth People's Hospital in Wuxi, China, were enrolled in this study; comprising 10 patients with onset of acute hepatitis B (AHB), 9 chronic HBV carriers (immunotolerant [IT]), 11 patients with chronic hepatitis B (CHB), and 10 patients with HBV-associated acute-on-chronic liver failure (ACLF). Acute hepatitis B is defined as a transient presence of HBsAg within 6 months without history of chronic hepatitis B (2). The diagnosis of immunotolerant HBV infection, chronic hepatitis B, and ACLF was based on criteria recommended by the Asian Pacific Association for the Study of the Liver (APASL) (18, 19). In the present study, the IT phase was defined as being HBeAg seropositive with high HBV DNA levels (>2,000,000 IU/ml) but normal serum alanine aminotransferase (ALT) levels (18), while the inclusion criteria of the CHB group were being HBsAg positive for >6 months and having an HBV DNA level of >20,000 IU/ml with serum ALT level >2 the upper limit of normal (ULN). ACLF is defined as an acute hepatic insult manifesting as jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed chronic HBV infection (19). In our study, patients with chronic HBV infection (IT, CHB, and ACLF) had a history of being HBsAg seropositive for >8 years (8 to 20 years). Serum samples from AHB and ACLF patients were collected within 1 week of clinical onset. Spontaneous HBsAg seroconversion was observed within 3 months of clinical onset of the infections in all AHB patients. The exclusion criteria included human immunodeficiency virus, hepatitis C virus, or hepatitis D virus coinfection and liver disease due to other causes, such as autoimmune liver disease, alcoholic hepatitis, and drug-induced hepatitis.
Liver biochemistry, HBV serology, and HBV DNA tests.
Liver biochemical (ALT and total bilirubinemia [TB]) and coagulation (prothrombin time [PT]) parameters were tested using an automated chemistry analysis system (Beckman Coulter, Fullerton, CA, USA). HBV serologic markers were determined with a chemiluminescent microparticle immunoassay using the Abbott Architect immunoassay system (Abbott Laboratories, Abbott Park, IL, USA). The HBV DNA levels were measured by PCR using the Cobas Amplicor HBV monitor test (Roche Diagnostics, Mannheim, Germany), with a lower limit of quantification at 57 IU/ml. Patients with serum HBV DNA levels of >2,000 IU/ml were included in this study. The clinical features of all patients are presented in Table 1.
TABLE 1.
Demographic and clinical features of studied patients
| Characteristic | Acute infection (AHB) (n = 10)a | Chronic infection (n = 30)b |
||
|---|---|---|---|---|
| IT (n = 9) | CHB (n = 11) | ACLF (n = 10) | ||
| Age (mean ± SD) (yr) | 40.7 ± 2.6 | 28.6 ± 1.8 | 35.5 ± 3.9 | 43.8 ± 2.8 |
| Gender (no. of males/no. of females) | 9/1 | 5/4 | 6/5 | 10/0 |
| ALT (mean ± SD) (IU/ml) | 1,608.5 ± 199.3 | 29.7 ± 3.8 | 231.5 ± 131.8 | 1171.6 ± 184.5 |
| TB (mean ± SD) (μmol/liter) | 96.9 ± 16.5 | 16.0 ± 1.4 | 34.4 ± 10.1 | 301.8 ± 29.8 |
| PT (mean ± SD) (s) | 14.8 ± 0.6 | 12.3 ± 0.2 | 12.0 ± 0.2 | 26.3 ± 1.6 |
| No. of HBeAg(+)/No. of anti-HBe(+) | 10/4 | 9/0 | 10/1 | 2/8 |
| HBV DNA (mean ± SD) (log10 IU/ml) | 5.9 ± 0.5 | 7.4 ± 0.2 | 6.4 ± 0.4 | 5.7 ± 0.3 |
| No. with HBV genotype (B, C, C/D recombination) | 4/6/0c | 3/6/0 | 6/5/0 | 5/4/1 |
AHB, acute hepatitis B.
IT, immunotolerant; CHB, chronic hepatitis B; ACLF, acute-on-chronic liver failure.
For the four patients listed as infected with HBV genotype B, one patient was coinfected with genotypes B and C HBV for an acute onset.
Written consent was obtained from all patients. The study protocol was approved by the ethics committee of Ruijin Hospital in accordance with the Declaration of Helsinki.
Amplification of full-length HBV genomes and cloning of PCR products.
HBV genomes were extracted from 200-μl serum samples of treatment-naive patients using the QIAamp blood minikit (Qiagen, Hilden, Germany). HBV full-length genomic DNA was amplified by PCR in 100 μl of buffer containing 30 μl of HBV DNA template, 2 μl of PfuUltra Hotstart DNA polymerase (Agilent Technologies, Santa Clara, CA, USA), and primers F1-R1 (F1, 5′-TTTTTCACCTCTGCCTAATCA [nucleotides {nt} 1821 to 1841]; and R1, 5′-AAAAAGTTGCATGGTGCTGG [nt 1825 to 1806]), which were designed according to Günther et al. (20). AccuPrime Pfx SuperMix (Invitrogen, Carlsbad, CA, USA) was used to ensure high-fidelity PCR. The reaction parameters were as follows: 95°C for 15 min, 40 cycles at 95°C for 40 s, 56°C for 30 s, and 72°C for 190 s, with a final extension of 72°C for 7 min. PCR products of 3.5 kbp were purified using the QIAquick gel extraction kit (Qiagen), cloned into the pGEM-T vector after the addition of adenylate tails (Promega, Madison, WI, USA), and transformed into Escherichia coli strain TOP10 competent cells (Invitrogen) growing on ampicillin plates.
Sequencing.
An average of 15 (range, 14 to 17) positive clones per sample were sequenced by six primers (M13F sequence in vector, M13R sequence in vector, P60F [5′-CCTGCTGGTGGCTCCAGTT] [nt 56 to 74], P690F [5′-TGTTCAGTGGTTCGTAGGGC] [nt 691 to 710], P2300F [5′-AGACCACCAAATGCCCCTATC] [nt 2297 to 2317], and PRES [5′-ATGGGAGGTTGGTCTTCCAAACCTCG] [nt 2848 to 2873]) using an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). A total of 606 clones were sequenced from 40 treatment-naive HBV-infected patients.
Sequence analysis.
The quality of sequencing was analyzed using Codon Code Aligner 3.7.1 software. All mutations were checked manually. The following regions were evaluated with Clustal X (version 2.0) and the SeqVerter program aligned with the following reference sequences (genotype B, GenBank accession no. D00330; genotype C, GenBank accession no. AB033556): HBsAg region, nt 155 to 835; preS2-HBsAg, nt 3205 to 835; preS1-preS2-HBsAg, nt 2848 to 835; polymerase region, nt 2307 to 1623; RT region, nt 130 to 1161; HBxAg region, nt 1374 to 1838; HBcAg region, nt 1901 to 2452; core promoter region, nt 1591 to 1849; precore region, nt 1814 to 1900; core upstream regulatory sequence (CURS), nt 1643 to 1742; epsilon region, nt 1847 to 1908; negative regulatory element (NRE), nt 1611 to 1634; enhancer I/X promoter (Enh I/Xp), nt 950 to 1373; and enhancer II (Enh II), nt 1685 to 1773. The genotypes of the full-length sequences were determined using the HBV Star program online (21).
Viral QS heterogeneity was evaluated using two parameters (complexity and diversity). QS complexity, which is the number of variants identified in a single sample, was measured using normalized Shannon entropy (Sn), as previously described (7, 8). The viral diversity was defined as the average pairwise distance between the viral DNA sequences of a given time point of a patient (11). QS diversity was evaluated by three parameters: the mean genetic distance (d), the number of synonymous substitutions per synonymous site (dS), and the number of nonsynonymous substitutions per nonsynonymous site (dN). Calculations were conducted using the Tamura 3-parameter method or the JTT matrix-based method, or by analysis at the nucleotide or amino acid level, respectively, using the MEGA 5.2 software (22). The complexity of each nucleotide position (nt 1 to 3215) was calculated as the Shannon entropy [Sn = −∑iε[A,T,C,G,-](pi × lnpi)/lnN] where pi represents the relative frequency of nucleotide or deletion i at this position and N represents the total number of clones (23). The figure for the complexity was generated using the Circos visualization tool (24). The insertions were discarded when calculating the complexity.
The mutation frequency index (MFI) was calculated for three open reading frame regions (core, large HBsAg, and X) and compared to the reference sequences (genotype B, GenBank accession no. D00330; genotype C, GenBank accession no. AB033556) using the following formula (25): MFI = (x/[y × z]) × 100, where x represents the total number of mutations/deletions observed in the amino acid sequences, y represents the total number of samples in a group, and z represents the length of the amino acid sequence. Heat maps were generated for the individual amino acid data sets to graphically depict the distribution of mutation frequencies across the amino acid regions. The Perl programming language was used to calculate the MFI values for each patient, and the heat maps were generated using the ggplot2 package in R language. The number of mutations per 15 amino acids was plotted on the x axis, and regions across were color-coded with red (depicting the highest mutation frequency) and green (depicting the lowest mutation frequency) (25).
All clones were compared with the reference sequences (genotype B, GenBank accession no. D00330; genotype C, GenBank accession no. AB033556) to identify variations in the core promoter and precore regions at the nucleotide level and variations in HBcAg, large-HBsAg, and HBxAg at the amino acid level. Established nucleos(t)ide analogue (NA) resistance-related or compensatory mutations involved the following RT mutations: rtL80V/I, rtT128P (26), rtI169T, rtV173L, rtL180M, rtA181S/T/V, rtT184S/A/I/L/F/G, rtA194T, rtS202G/I, rtM204V/I/S, rtN236T, rtN/H238T/A, and rtM250V (27). All deletion/insertion mutations and stop codon mutations were also described in our study.
Statistical analysis.
The results of continuous variables are expressed as the mean ± standard error (SE) or the median and range. QS complexity or diversity was compared between the acute and chronic infection groups by unpaired t test and between four groups by one-way analysis of variance (ANOVA) (Bonferroni) or the Kruskal-Wallis test (Dunn). A P value of <0.05 indicated a statistically significant difference for complexity and diversity. The distribution of point variations within the core promoter and precore regions at the nucleotide level and within the large HBsAg, HBcAg, and HBxAg at the amino acid level among the four groups was analyzed by a chi-square test. The point variations (nucleotides or amino acids) with a P value of <0.001 and the number of patients with such variation in the highest group (not fewer than two) simultaneously was shown in our study. The statistical analysis was performed with SPSS 19.0 (Chicago, IL, USA).
RESULTS
Phylogenetic analysis.
Figure S1 in the supplemental material depicts the phylogenetic analysis performed on 14 HBV genome-length reference strains (genotypes A to J) and 41 isolated sequences (39 patients each, and one patient coinfected with genotypes B and C) from the studied patients. The viral isolates clustered in two branches corresponding to genotypes B and C.
Distribution of HBV QS and complexity and diversity.
The distribution of HBV QS for 40 patients is shown in Fig. 1. Fourteen to 17 clones were isolated from each patient infected with HBV. QS complexity (Sn) of 606 clones at the nucleotide and amino acid levels for HBV full-length (at the nucleotide level only), large HBsAg, middle HBsAg, HBsAg, HBxAg, HBcAg, polymerase region, RT region, and core promoter region (at the nucleotide level only) is shown as the mean ± standard error (SE) in Table 2. Generally, the QS complexity of HBV chronically infected patients was statistically higher than that of the patients with acute infection at large HBsAg, middle HBsAg, HBsAg, HBcAg, polymerase, and RT regions rather than at the HBxAg and core promoter region. Among the subgroups with chronic infection, the QS complexity of CHB and ACLF patients was higher than that of the IT patients at the HBcAg region at both levels but at the large HBsAg, middle HBsAg, and RT regions at the nucleotide level only and at polymerase at the amino acid level only, based on the statistics; however, no significant difference was found between CHB and ACLF in all studied regions. Thus, AHB, followed by IT, had lower QS heterogeneity than that of the other groups. In the ACLF group, five patients recovered from the acute phase and survived 90 days, but the other five patients died within 90 days. The survivors and nonsurvivors were comparable in age, gender, ALT level, TB, PT, HBeAg status, and HBV DNA level at onset, HBV genotype, cirrhosis status, and antiviral therapy; however, the QS complexity in the middle HBsAg (0.387 versus 0.741 in survivors and nonsurvivors, respectively; P = 0.017 at the nucleotide level; 0.284 versus 0.662 in survivors and nonsurvivors, respectively; P = 0.016 at the amino acid level) and the HBsAg (0.358 versus 0.699 in survivors and nonsurvivors, respectively; P = 0.022 at the nucleotide level; 0.238 versus 0.597 in survivors and nonsurvivors, respectively; P = 0.020 at the amino acid level) were lower in survivors than nonsurvivors, although no difference existed in other regions (data not shown).
FIG 1.
HBV full-length quasispecies in 4 groups of patients at different clinical phases of infection. The vertical bars indicate the proportion of viral QS within each patient. Each color represents one kind of QS. Generally, the QS distributions in CHB and ACLF patients were more complicated than those in AHB and IT, and AHB appeared to have the lowest complexity among the four groups. C/D Recom, C/D recombination HBV.
TABLE 2.
Quasispecies complexity (Sn) of 39 HBV-infected patients at different phases of infection
| Region of variation (level) | Acute infection (AHB) (mean ± SE) (n = 9)a | Chronic infection (mean ± SE) (n = 30)b |
P value (acute vs chronic)c | ||
|---|---|---|---|---|---|
| IT (n = 9) | CHB (n = 11) | ACLF (n = 10) | |||
| Full length (nt) | 0.4911 ± 0.0917 | 0.7466 ± 0.0629d | 0.9331 ± 0.0192e | 0.8687 ± 0.0619f | <0.001 |
| preS/S | |||||
| Large HBsAg (nt) | 0.2519 ± 0.0657 | 0.3788 ± 0.0704 | 0.7092 ± 0.0649e,g | 0.6774 ± 0.0825f,h | <0.001 |
| Large HBsAg (aa) | 0.2016 ± 0.0533 | 0.3422 ± 0.0723 | 0.5904 ± 0.0680i | 0.5881 ± 0.0799j | <0.001 |
| Middle HBsAg (nt) | 0.2199 ± 0.0545 | 0.3281 ± 0.0742 | 0.6112 ± 0.0718g,i | 0.5594 ± 0.0799k | 0.0034 |
| Middle HBsAg (aa) | 0.1723 ± 0.0549 | 0.3068 ± 0.0706 | 0.4898 ± 0.0674l | 0.4730 ± 0.0857k | 0.004 |
| HBsAg (nt) | 0.1103 ± 0.0287 | 0.2868 ± 0.0652 | 0.5188 ± 0.0735e | 0.5285 ± 0.0805j | <0.001 |
| HBsAg (aa) | 0.0896 ± 0.0296 | 0.2771 ± 0.0662 | 0.4503 ± 0.0658i | 0.4172 ± 0.0837j | <0.001 |
| X | |||||
| HBxAg (nt) | 0.1691 ± 0.0652 | 0.2764 ± 0.0809 | 0.3897 ± 0.0660l | 0.3674 ± 0.0566k | 0.0299 |
| HBxAg (aa) | 0.1089 ± 0.0441 | 0.2322 ± 0.0716 | 0.2585 ± 0.07819 | 0.2274 ± 0.0520 | 0.0898 |
| CP/C | |||||
| HBcAg (nt) | 0.1825 ± 0.0720 | 0.2609 ± 0.0565 | 0.5390 ± 0.0524g,i | 0.4421 ± 0.0859 | 0.0091 |
| HBcAg (aa) | 0.1046 ± 0.0640 | 0.2063 ± 0.0551 | 0.4274 ± 0.0401g,i | 0.3141 ± 0.0713 | 0.0052 |
| Core promoter (nt) | 0.0657 ± 0.0364 | 0.1127 ± 0.0612 | 0.1536 ± 0.0280 | 0.1643 ± 0.0472 | 0.1475 |
| P | |||||
| Polymerase (nt) | 0.3762 ± 0.0847 | 0.6796 ± 0.0655c | 0.8924 ± 0.0353e | 0.8384 ± 0.0617f | <0.001 |
| Polymerase (aa) | 0.2724 ± 0.0661 | 0.5249 ± 0.0712c | 0.7760 ± 0.0444e,g | 0.6508 ± 0.0614 | <0.001 |
| RT region (nt) | 0.1662 ± 0.0299 | 0.4796 ± 0.0701c | 0.6685 ± 0.0661e | 0.6727 ± 0.0739f | <0.001 |
| RT region (aa) | 0.1012 ± 0.0280 | 0.3412 ± 0.0573c | 0.4435 ± 0.0657e | 0.4797 ± 0.0599f | <0.001 |
An acute hepatitis case coinfected with genotypes B and C HBV was excluded. AHB, acute hepatitis B.
IT, immunotolerant; CHB, chronic hepatitis B; ACLF, acute-on-chronic liver failure.
Unpaired t test was performed when analyzing the genetic distance between acute and chronic infection groups.
P < 0.05, AHB versus IT.
P < 0.001, AHB versus CHB.
P < 0.001, AHB versus ACLF.
P < 0.05, IT versus CHB.
P < 0.05, IT versus ACLF.
P < 0.01, AHB versus CHB.
P < 0.01, AHB versus ACLF.
P < 0.05, AHB versus ACLF.
P < 0.05, AHB versus CHB.
QS diversity (d, dS, and dN), shown as the median and range, is shown in Table 3. The mean genetic distance (d) of CHB and ACLF patients was significantly higher than that of AHB patients at the large HBsAg, HBcAg, polymerase, and RT regions (P < 0.01) at both levels, while at the middle HBsAg and HBsAg, the difference existed at the nucleotide level only, compared to AHB patients. The dN of CHB and ACLF patients was also significantly higher than that in AHB patients at the large HBsAg, HBsAg, HBcAg, polymerase, and RT regions.
TABLE 3.
Quasispecies diversity (genetic distance, dS, and dN) of 39 HBV-infected patients at different phases of infection
| Region of variation (level) | Acute infection (AHB) (median [range]) (n = 9)a | Chronic infection (median [range]) (n = 30)b |
P value (acute vs chronic)c | ||
|---|---|---|---|---|---|
| IT (n = 9) | CHB (n = 11) | ACLF (n = 10) | |||
| Full length (nt) | 0.4150 (0–2.610) | 1.0437 (0.1–16.549) | 2.5683 (1.132–5.851)d | 3.4946 (0.4980–8.717)e | <0.001 |
| preS/S | |||||
| Large HBsAg (nt) | 0.4437 (0–2.227) | 0.9994 (0–12.248) | 2.4875 (0.666–5.798)f | 3.6670 (0.555–9.062)e | 0.0018 |
| Large HBsAg (aa) | 0.7083 (0.001–4.345) | 2.4736 (0.001–21.280) | 4.9905 (0.657–10.178)f | 7.1932 (1.417–21.823)e | 0.0021 |
| Middle HBsAg (nt) | 0.4733 (0–2.436) | 1.1066 (0–9.780) | 2.1213 (0.496–5.672)f | 3.4722 (0.473–9.182)g | 0.0046 |
| Middle HBsAg (aa) | 1.0177 (0.001–4.680) | 0.3057 (0.001–17.626) | 3.5885 (0.0001–12.033) | 2.0177 (0.946–22.343)g | 0.0070 |
| HBsAg (nt) | 0.2100 (0–2.430) | 1.1982 (0–6.342) | 2.1354 (0–5.669)f | 3.2015 (0.392–10.623)e | 0.0012 |
| HBsAg (aa) | 0.6460 (0.001–6.503) | 3.8971 (0.001–16.400) | 5.1188 (0.001–16.293) | 5.1773 (0.607–26.173) | 0.0110 |
| X | |||||
| HBxAg (nt) | 0.2872 (0–3.476) | 0.5746 (0–14.167) | 1.8369 (0–7.992) | 1.6993 (0.287–8.105) | 0.0612 |
| HBxAg (aa) | 0.001 (0.001–4.604) | 0.9339 (0.001–63.298) | 3.6503 (0.001–20.586) | 2.1101 (0.884–25.420) | 0.0956 |
| CP/C | |||||
| HBcAg (nt) | 0.2420 (0–4.158) | 0.8775 (0.227–18.596) | 4.1454 (1.002–9.231)d | 3.9384 (0.227–13.853)g | 0.0028 |
| HBcAg (aa) | 0.001 (0.001–10.539) | 1.4668 (0.001–19.510) | 8.7652 (2.294–16.285)d | 4.9007 (0.001–26.538)g | 0.0015 |
| Core promoter (nt) | 0 (0–7.534) | 0 (0–21.910) | 1.2462 (0–20.484) | 0 (0–10.005) | 0.1457 |
| P | |||||
| Polymerase (nt) | 0.4302 (0–2.250) | 1.1364 (0.158–14.027) | 2.1809 (1.009–5.553)f | 3.3477 (0.527–8.614)e | <0.001 |
| Polymerase (aa) | 0.6471 (0.001–3.133) | 2.5835 (0.484–18.226) | 3.6411 (1.642–9.845)f | 5.3645 (0.964–11.528)e | <0.001 |
| RT region (nt) | 0.3695 (0–2.656) | 1.7057 (0.259–10.913) | 2.3903 (0.388–4.341)f | 3.3810 (0.647–8.819)h | <0.001 |
| RT region (aa) | 0.4004 (0.001–3.264) | 2.3882 (0.397–12.443) | 3.1410 (0.001–5.823)f | 4.3656 (0.793–10.617)e | <0.001 |
| preS/S | |||||
| dS-large HBsAg | 0.4714 (0–3.734) | 0.4459 (0–24.288) | 2.768 (0–15.298) | 3.265 (0.446–9.188) | 0.0267 |
| dN-large HBsAg | 0.2963 (0–1.810) | 1.0349 (0–8.913) | 2.1112 (0.275–4.270)f | 3.0196 (0.593–9.236)e | 0.0024 |
| dS-middle HBsAg | 0.6296 (0–4.031) | 0 (0–17.249) | 1.293 (0–12.289) | 3.1386 (0.630–6.150) | 0.0996 |
| dN-middle HBsAg | 0.4517 (0–2.101) | 1.2783 (0–7.334) | 1.8789 (0–5.845) | 3.2502 (0.397–10.030) | 0.0320 |
| dS-HBsAg | 0 (0–3.376) | 0 (0–5.793) | 0.8111 (0–8.780) | 3.5278 (0.797–6.024)e,i | 0.0513 |
| dN-HBsAg | 0.2616 (0–2.155) | 1.5802 (0–6.597) | 2.0730 (0–6.550)f | 3.6194 (0.245–11.423)g | 0.0016 |
| X | |||||
| dS-HBxAg | 0 (0–6.667) | 1.1905 (0–29.077) | 0 (0–8.532) | 1.5901 (0–6.432) | 0.2865 |
| dN-HBxAg | 0 (0–2.370) | 0.4141 (0–31.588) | 1.8406 (0–9.058) | 0.9440 (0.366–11.044) | 0.0956 |
| C | |||||
| dS-HBcAg | 0.9390 (0–10.757) | 0.9442 (0–48.780) | 2.5675 (0.956–17.298) | 4.5237 (0.8993–19.194) | 0.0312 |
| dN-HBcAg | 0 (0–4.706) | 0.6536 (0–0.0087) | 3.897 (1.024–7.220)d | 2.3459 (0–13.521)g | 0.0015 |
| P | |||||
| dS-polymerase | 0.4204 (0–4.771) | 1.0600 (0.211–30.801) | 5.5203 (1.046–9.283)f | 6.3239 (0.837–21.659)h | 0.0004 |
| dN-polymerase | 0.2818 (0–1.355) | 0.5642 (0.141–8.510) | 1.6756 (0.715–4.321)f | 2.4147 (0.423–5.298)e | 0.0008 |
| dS-RT region | 0.5307 (0–6.052) | 2.0727 (0–27.998) | 5.7456 (1.587–11.462)f | 7.4535 (1.579–22.002)e | 0.0006 |
| dN-RT region | 0.1716 (0–1.404) | 1.0264 (0.171–5.646) | 1.3499 (0–2.496)f | 1.9659 (0.343–4.555)e | 0.0005 |
An acute hepatitis case coinfected with genotypes B and C HBV was excluded. AHB, acute hepatitis B.
Data are shown in units of 10−3 substitutions/site. IT, immunotolerant; CHB, chronic hepatitis B; ACLF, acute-on-chronic liver failure.
Kruskal-Wallis test was performed when analyzing the genetic distance between four groups, and the Mann-Whitney test was performed when analyzing the genetic distance between acute and chronic infection groups.
P < 0.01, AHB versus CHB.
P < 0.01, AHB versus ACLF.
P < 0.05, AHB versus CHB.
P < 0.05, AHB versus ACLF.
P < 0.001, AHB versus ACLF.
P < 0.01, IT versus ACLF.
The Enh I/X promoter region had less complexity and diversity in the acute group (acute versus chronic for mean Sn, 0.114 versus 0.413, respectively, unpaired t test, P < 0.001; for median d, 0.0003 versus 0.0022, respectively, Kruskal-Wallis test, P < 0.001), while there was no difference among the chronic subgroups. Furthermore, neither complexity nor diversity in the CURS, epsilon, NRE, and Enh II regions had any statistically significant differences between the acute and chronic groups or among the chronic subgroups (data not shown).
The complexity of each nucleotide position for the HBV full-length genome (nt 1 to 3215) is shown in Fig. 2. Generally, in AHB and IT, genotype B had fewer mutated nucleotide positions than genotype C; however, in CHB and ACLF, the core region appeared to be the most mutated region, followed by the preS2 region.
FIG 2.
HBV nucleotide acid (nt 1 to 3215) complexities for patients infected with genotype B or C. The colored bars indicate the complexity of each nucleotide acid for genotype B (blue lines) and genotype C (red lines) for the full-length HBV genome. One patient infected with C/D recombination HBV was excluded in this figure. The insertions were discarded.
MFI of the HBcAg, large HBsAg, and HBxAg regions.
The MFI distribution among the different clinical presentation groups is shown in Fig. 3. Overall, an increase in MFI was observed in ACLF and CHB patients compared to that in AHB and IT patients. Furthermore, the MFI was higher for HBV genotype B than genotype C. Heat maps were generated for the HBcAg, large HBsAg, and HBxAg regions to graphically depict the distribution of the mutation frequencies. Amino acids 90 to 135 of HBcAg in genotype C and amino acids 60 to 130 in genotype B had more mutations or deletions in ACLF patients, followed by CHB patients, compared to AHB or IT patients (Fig. 3A and B). Specific preS regions had a higher MFI in ACLF patients than in other patients (Fig. 3C and D). Amino acids 131 to 135 in the HBxAg region had more mutations in ACLF patients for genotype B, while the same amino acid sequence had more mutations in ACLF and CHB patients for genotype C (Fig. 3E and F).
FIG 3.
Heat map showing the distribution of amino acid mutation frequency index (MFI) in hepatitis B virus core of genotype B (A), large surface, and X region antigens of genotypes B and C. (A) HBcAg of genotype B. (B) HBcAg of genotype C. (C) Large HBsAg of genotype B. (D) Large HBsAg of genotype C. (E) HBxAg of genotype B. (F) HBxAg of genotype C. The number of mutations per 15 amino acids was plotted on the x axis, and the regions across are color-coded in red (depicting the highest mutation frequency) and green (depicting the lowest mutation frequency). The y axis indicates the MFI.
Point variations in HBV treatment-naive patients. (i) Nucleotide variations in core promoter and precore regions.
The frequency of potential influential variations associated with the clinical spectrum at the core promoter and precore regions, compared to the reference sequences (genotype B, GenBank accession no. D00330; genotype C, GenBank accession no. AB033556) in HBV treatment-naive patients, is summarized in Fig. 4 and Table S1 in the supplemental material. IT patients had the lowest frequency of mutations at the core promoter and precore regions, followed by AHB patients. Furthermore, of those with chronic infection status, ACLF patients had a higher frequency of mutations at T1753C (28.6%), A1762T (64.8%), G1764A (51.3%), T1800C (21.4%), A1846T/C (53.2%), G1862T (20.1%), and G1896A (61.0%) than that of the other groups. In addition, the number of A1762T/G1764A double mutations in ACLF patients (72/154 clones [5 of 10 patients]) was significantly higher than that in CHB patients (48/165 clones [4 of 11 patients]), followed by AHB patients (15/149 clones [1 of 10 patients]) and IT patients (10/138 clones [1 of 9 patients]; P < 0.001). Furthermore, the A1762T/G1764A/G1896A triple mutation was observed more frequently in genotype C (six of nine patients with A1762T/G1764A or G1896A mutations) than in genotype B (only one patient), all of them with the highest full-genome complexity. Approximately one-third (34.4%) of the clones from ACLF patients had triple mutations compared with <10% of the clones from other patients (2.0% for AHB, 7.2% for IT, and 9.7% for CHB; P < 0.001).
FIG 4.
Frequency (%) of nucleotide acid point variations of core promoter region and precore region (nt 1742 to 1900). The y axis indicates the frequency of point variations observed for all clones. The distribution of those point variations (x axis) presented in this figure exhibited significant differences among the four groups (P < 0.001). The numbers between parentheses indicate the number of patients with mutated clones/total number of patients in each group.
(ii) Amino acid variations in HBcAg, large HBsAg, and HBxAg.
Regarding variations in HBcAg, in genotype B, two or three of five ACLF patients had mutations or deletions in the region L60 to I97, which has been widely studied as being T- or B-cell epitopes (28, 29), but these were not observed in AHB and IT patients. E113, T114, A131, and P135 had more variation in frequency in the ACLF patients with genotype B, not only in CHB, but also in the ACLF patients with genotype C (see Fig. S2A and Tables S2 and S3 in the supplemental material).
Concerning amino acid changes in the large HBsAg in genotype C, approximately one-half (49.2% [31/63 clones]) of the clones in two of four ACLF patients had a mutation at I161, while 74.6% of the clones (47/63 clones) in three of four ACLF patients and 47.8% of clones (44/92 clones) in three of seven AHB patients had a mutation at N177. Nevertheless, in genotype B, six amino acid sites, five of which have known cytotoxic T lymphocyte (CTL) epitopes (30), had a higher frequency of variation in ACLF patients than that in IT or AHB patients (see Fig. S2B and Tables S2 and S3 in the supplemental material).
With respect to the distribution of HBxAg amino acid point variations, K130, V131, and C143 had the highest frequency of variation in ACLF patients (>40% of clones for two of five patients) infected with genotype C, while a similar frequency (more than one-half of the clones) was observed in patients infected with genotype B presenting as ACLF and CHB. In AHB patients, the highest frequency of variation at S101 was found in AHB patients (>60% of clones for five of seven patients) (compared to <10% in the others; see Fig. S2C and Tables S2 and S3 in the supplemental material).
NA resistance-related or compensatory mutations in the RT region.
None of the primary lamivudine-, telbivudine-, entecavir-, adefovir-, or tenofovir-related resistance mutations were found in 606 clones, but lamivudine compensatory mutations were found in 32 clones from three patients at rtT128P (AHB-10, 15/15 clones), rtV173L (AHB-17, 1/15 clones), and rtN238T (CHB-2441, 16/16 clones).
Stop codon, deletion, and insertion mutations.
Stop codon mutations due to nucleotide acid substitutions were observed in large HBsAg only these (shown in Table S4 in the supplemental material) rather than in 3 other open reading frames (core, X, and polymerase).
Deletion or insertion mutations were observed more frequently and were more complex in CHB and ACLF patients (five of 11 and five of 10 patients, respectively) than those in AHB and IT patients (2 to 3 for each), as summarized in Table 4. The deletion patterns at the nucleotide and amino acid levels in an AHB (AHB-10) patient are shown as a deletion of the first one-fifth of the nucleotide acids, leading to an amino acid deletion in the large HBsAg, middle HBsAg, and polymerase. In an ACLF patient (ACLF-401), numerous nucleotide acid deletions in different clones from nt 1997 to 2279 led to frameshift mutations, which resulted in a premature stop codon and truncated HBcAg.
TABLE 4.
Deletion and insertion of HBV quasispecies observed in the present study
| Clinical spectruma | No. of Del or Ins patients/total no. of patientsb | Patient ID (genotype) | No. of Del or Ins clones/total no. of clones | Del/Ins nt patterns (nt positions in full-length HBV genome) | Del/Ins aa patterns (aa positions of related region)c |
|---|---|---|---|---|---|
| AHB | 2/10 | AHB-10 (C) | 15/15 | Del 3215–2 and 10–50 | Del LHBsAg 126–142, Del MHBsAg 7–23, Del Pol 306–321 |
| AHB-13 (B + C) | 3/15 (genotype C) | Del 1769–1776 | Del and frameshift HBxAg at 130–135 with stop codon | ||
| IT | 3/9 | IT-2405 (C) | 1/15 | Del 2850–2864 | Del Pol 182–186 |
| IT-2520 (C) | 1/15 | Del 1752–1770 | Del and frameshift HBxAg at 127–128 with stop codon | ||
| 1/15 | Del 1770–1776 | Del and frameshift HBxAg at 133–177 with stop codon | |||
| IT-2553 (B) | 1/16 | Del 1133–1825 | Del and frameshift Pol at 682–683 with stop codon, Del start codon of HBxAg, Del start codon of PreCore | ||
| 1/16 | Del 1143–1819 | Del and frameshift Pol at 684–721 with stop codon, Del start codon of HBxAg, Del start codon of PreCore | |||
| CHB | 5/11 | CHB-2403 (C) | 1/15 | Del 11–55 | Del LHBsAg 127–141, Del MHBsAg 8–22, Del Pol 307–321 |
| Del 2849–2866 | Del start codon of LHBsAg, Del Pol 181–186 | ||||
| CHB-2409 (B) | 1/15 | Del 3145–22 | Del LHBsAg 100–130, Del start codon of MHBsAg, Del Pol 282–312 | ||
| CHB-2441 (C) | 6/16 | Del 25–54 | Del LHBsAg 131–140, Del MHBsAg 12–21, Del Pol 311–320 | ||
| 2/16 | Del 25–54 and 2161–2304 | Del LHBsAg 131–140, Del MHBsAg 12–21, Del Pol 311–320, Del HBcAg 88–135 | |||
| 3/16 | Del 2161–2304 and Ins 1768 | Del HBcAg 88–135 and Ins and frameshift HBxAg at 132–138 with stop codon | |||
| 1/16 | Del 3115–55 and Ins 1768 | Del LHBsAg 90–141 and Ins and frameshift HBxAg at 132–138 with stop codon, Del Pol 270–322 | |||
| 1/16 | Del 3039–3086 | Del and frameshift LHBsAg at 64 with stop codon, Del Pol 246–261 | |||
| 1/16 | Del 2161–2304 and 3175–3180 | Del HBcAg 88–135 and Del LHBsAg 110–111, Del Pol 290–291 | |||
| CHB-2447 (C) | 1/15 | Del 1950–1951 | Del and frameshift HBcAg at 17–21 with stop codon | ||
| CHB-2529 (B) | 1/15 | Ins 1789 | Ins and frameshift HBxAg at 139–140 with stop codon | ||
| ACLF | 5/10 | ACLF-2317 (B) | 2/14 | Del 2199–2325 | Del start codon of Pol, Del and frameshift HBcAg from 100 |
| 1/14 | Del 2905 | Del and frameshift LHBsAg at 20–108 with stop codon, Del and frameshift Pol at 200–581 with stop codon | |||
| ACLF-2453 (C/D) | 6/15 | Del 3037–19 | Del and frameshift LHBsAg at 63 with stop codon, Del start codon of MHBsAg, Del Pol 244–309 | ||
| ACLF-2642 (C) | 12/17 | Del 2165–2305 | Del HBcAg 89–135 | ||
| 3/17 | Del 23–55 | Del LHBsAg 131–141, Del MHBsAg 12–22, Del Pol 312–322 | |||
| 1/17 | Del 2207–2304 | Del and frameshift HBcAg at 101 with stop codon | |||
| ACLF-2644 (B) | 15/15 | Del 3204–3206 and Ins 2351–2356 | Del Pol 302 with Ins at 16–17, Del LHBsAg 120, Del start codon of MHBsAg | ||
| ACLF-401 (B) | 3/16 | Del 1965–2203 | Del and frameshift HBcAg at 22–37 with stop codon | ||
| 3/16 | Del 2066–2268 | Del and frameshift HBcAg at 55–65 with stop codon | |||
| 1/16 | Del 2104–2249 | Del and frameshift HBcAg at 68–84 with stop codon | |||
| 1/16 | Del 2002–2255 | Del and frameshift HBcAg at 35–48 with stop codon | |||
| 1/16 | Del 2006–2214 | Del and frameshift HBcAg at 36–47 with stop codon | |||
| 1/16 | Del 2064–2262 | Del and frameshift HBcAg from 55 | |||
| 1/16 | Del 2026–2279 | Del and frameshift HBcAg at 43–48 with stop codon | |||
| 1/16 | Del 2064–2263 | Del and frameshift HBcAg at 56–66 with stop codon | |||
| 1/16 | Del 1997–2015 and 2851–3060 | Del and frameshift HBcAg at 33–36 with stop codon and Del LHBsAg 2–71 and Del Pol 183–252 | |||
| 1/16 | Del 2851–3060 | Del LHBsAg 2–71, Del Pol 183–252 | |||
| 1/16 | Del 10–24 and 2851–3060 | Del LHBsAg 2–71 and 127–131, Del Pol 183–252 | |||
| 1/16 | Ins 1820–1987 | Ins HBxAg 150–205, Ins HBeAg 5–60 |
AHB, acute hepatitis B; IT, immunotolerant; CHB, chronic hepatitis B; ACLF, acute-on-chronic liver failure.
Del, deletion; Ins, insertion.
LHBsAg, large HBsAg; MHBsAg, middle HBsAg; Pol, polymerase.
DISCUSSION
HBV infection results in different clinical outcomes, such as acute self-limited infection, chronic hepatitis, or immunotolerant status, due to different levels of immune responses, such as complete or incomplete responses and immunotolerance (31). The HBV population in the host consists of genetically heterologous variants and exists in the form of a QS. It is proposed that QS may contribute to viral persistence and pathogenesis because QS contain a large number of mutated genes that serve as a reservoir for viral selection under the immune response and in antiviral treatment (32).
AHB is thought to be invariably associated with the control of HBV infection through the induction of an efficient HBV-specific T- and B-cell response or through an effective innate and adaptive immune response (33). Few studies have investigated QS in individuals with AHB because of the temporary presence of virus in serum. Our study demonstrated that the HBV QS had the lower complexity and diversity at the onset of AHB than those in the chronically infected patients. Thus, the HBV QS in AHB cases was less complicated and had weaker adaptability than those in the chronic infection cases. This might have resulted from not only the shorter time of infection but also efficient HBV-specific T- and B-cell responses of host immunity (34); the B-cell responses played a key role in the early stage of infection. Regarding the G1896A mutation in AHB patients, which was of interest, all mutations (three of six patients) were observed in genotype C only within 1 week of clinical onset. It seemed that patients infected with HBV genotype C might have earlier HBeAg seroconversion than that of those infected with genotype B. Furthermore, an amino acid substitution at HBx-S101 (T1674C/G) was significantly more frequent in AHB patients (60.9% of clones, 4 of 6 patients versus <10% in chronic patients), which is the first time this has been described in genotype C and could be further investigated.
Among HBV chronically infected patients, our present study showed that the HBV QS had a higher complexity in CHB and ACLF patients (stage of immunoactivation) than that in the IT patients (stage of immunotolerance). The IT phase has been characterized as having high levels of HBV replication and lack of clinical signs of liver inflammation at the early phase of chronic HBV infection. Wang et al. (35) recently investigated the HBV viral evolution from the immunotolerant to immunoactive phases of chronic HBV infection during a long-term follow-up, which suggested that immune selection plays a very important role in viral evolution in the course of chronic hepatitis B infection. Our study, concerning single amino acid variations (Fig. S2, and Tables S2 and Table S3 in supplemental material), also showed the low prevalence of amino acid variations in HBcAg and large HBsAg. Although five amino acid variations (at K7, Q10, H51, A81, and S96) at the preS1 region were observed in more than one-third of the clones in IT patients rather than in CHB and ACLF patients, these amino acids were not clustered in the T- or B-cell epitopes. An alternative hypothesis of immunotolerance has been proposed by Bertoletti and Kennedy (36) that a lack of clinical signs of liver inflammation in IT patients is not associated with a complete absence of HBV-specific T-cell response but with an antiviral noninflammatory immune response. Based on our viral descriptive results and the hypothesis of Bertoletti and Kennedy (36), we proposed that with the noninflammatory immune response in IT patients, the virus under immune pressure might result in amino acid mutations in nonepitope regions rather than in T- or B-cell epitopes. Whether HBV chronic infection with different clinical presentations (immunotolerance or immunoactivation) is an inflammatory rather than a virus-induced disease should be further evaluated (36).
The pathogenesis of ACLF remains unclear. It has generally been held that mutations of the viral genome and host immunologic deregulation underlie ACLF. During the natural course of HBV chronic infection, especially in the immunoactive phase, mutations in the core promoter (CP), precore, and core genes are most common (37). The association of hepatitis B virus mutations in the core promoter (T1753C, A1762T, G1764A, and A1846T/C) and precore regions (G1896A) with the severity of liver disease has been widely discussed (38–41). The G1896A mutation resulting in reduced or absent HBeAg production may be one of the factors contributing to severe hepatitis, as both of the above mutations have been shown to upregulate viral DNA replication and reduce HBeAg production in vitro (42). T1800C and G1862T comprised >20% of the mutations of all clones in ACLF but no mutations in the other groups. Whether or not these mutations are involved in HBV replication or other mutations has yet to be determined. Regarding those mutants in specific open reading frames (ORFs), the MFI was used to give an overall estimate of the conserved and variable regions. Core and HBx peptides have been shown to be more conserved than surface and polymerase peptides (25, 43). However, in our study, specific amino acid regions (aa 90 to 135 in genotype C, aa 60 to 130 in genotype B of HBcAg, and aa 131 to 135 in HBxAg) were observed to have a higher frequency of mutations or deletions in ACLF patients, followed by CHB patients. Furthermore, amino acid variations within the human leukocyte antigen (HLA) class II-restricted T-cell recognition (CD4+) epitopes (aa 50 to 69 and aa 117 to 131) and within the B-cell determinants (aa 74 to 89, aa 107 to 118, and aa 130 to 138) have been showed to accumulate during periods with frequent hepatitis exacerbation (44, 45). The beginning of the preS2 region had a higher rate of mutation as well, which was possibly due to host immune pressure. The MFI and single-amino acid variation results help us not only to get an overview of the changes in mutation frequency but also to speculate on the nature of the interaction of host immune with specific sites of peptides in different clinical phases and different genotypes.
Deletions and insertions are believed to be associated with the progression of viral hepatitis. Several nucleotide deletions and insertions at the region overlap the CP, and the precore region results in truncated HBxAg or additional amino acids, which potentially alters HBx functionality (46), was detected in our study (Table 4). Hot spots include the precore/core genes, preS region, and the region of the X gene overlapping the core promoter (47), which were more frequent and complex in CHB and ACLF patients than in AHB and IT patients. The coexistence of wild-type HBV, relative to deleted sequences, and mutants with deletions in the core gene have been shown to enhance viral replication (48). Deletions in HBsAg were rare; nevertheless, deletions in preS1/S2 were observed more frequently in patients with a different level of immunoactivation (ACLF, CHB, and AHB) than in IT patients, which was similar to another report from China (49) and what is seen in HBV/HIV coinfection (50–52). It appears that the amino acid deletions within the preS1/S2 and core regions might be associated with CHB or ACLF under different levels of inadequate selective pressure.
The existence of antiviral-resistant mutations in treatment-naive HBV-infected individuals has been reported for patients with chronic HBV infection (53). Our analysis of the HBV RT region, however, showed that no NA resistance-related primary mutations were found in our patients. By using ultradeep pyrosequencing in our recent published study, NA resistance-related mutations were identified more frequently in treatment-naive CHB patients than with clone-based sequencing (54). The basis for this absence of determining primary mutations might be the small number of clones (14 to 17 clones per patient) selected for sequencing, while up to 25 clones per patient were selected in our previous studies (7, 54).
Although next-generation sequencing technologies allow for massive parallel amplification and detection of individual DNA molecules and have been largely applied in viral research, the length of the sequence read (<500 bp) does not cover the full-length HBV genome. Clone-based sequencing has been used as the reference technique for studying the heterogeneity of viral QS. The present study is the first to describe the different characteristics of full-length genome QS patients with HBV acute and different phases of chronic infection; however, it still has some limitations, such as the number of patients enrolled and the limited number of clones selected for sequencing. Moreover, because HBV QS for all individuals was not longitudinally analyzed, the results obtained regarding the schema in the viral QS under the effect of selective pressure of the immune response require corroboration by further studies examining additional patients.
On the basis of the present study concerning different phases of HBV infection without antiviral therapy, HBV QS had a higher heterogeneity in ACLF and CHB patients than that in IT patients in the chronically infected groups. AHB patients had lower QS complexity and diversity at the onset than those in chronically infected patients. The HBV mutant spectrum may be associated with the duration of infection (acute versus chronic) and level of immune pressure. More HBV genome mutations, especially in epitope regions and in the form of more deletions, were observed in more severe diseases, from which we could conjecture that changes or adaptations in the HBV genome might be selected due to immune pressure and might be related to its pathogenesis.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science Foundation of China (grants 81171616 and 81371860), the Major National S&T Projects for Infectious Diseases (grants 2012ZX10002007 and 2013ZX10002001), and the National Program on Key Basic Research Project (973 Program) of China (grant 2012CB519000).
We declare no conflicts of interest.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00068-15.
REFERENCES
- 1.Bernal W, Auzinger G, Dhawan A, Wendon J. 2010. Acute liver failure. Lancet 376:190–201. doi: 10.1016/S0140-6736(10)60274-7. [DOI] [PubMed] [Google Scholar]
- 2.WHO. 2014. Hepatitis B. Fact sheet no. 204. World Health Organization, Geneva, Switzerland: http://www.who.int/mediacentre/factsheets/fs204/en/. [Google Scholar]
- 3.Guidotti LG, Chisari FV. 2006. Immunobiology and pathogenesis of viral hepatitis. Annu Rev Pathol 1:23–61. doi: 10.1146/annurev.pathol.1.110304.100230. [DOI] [PubMed] [Google Scholar]
- 4.Ngui SL, Teo CG. 1997. Hepatitis B virus genomic heterogeneity: variation between quasispecies may confound molecular epidemiological analyses of transmission incidents. J Viral Hepat 4:309–315. doi: 10.1046/j.1365-2893.1997.00066.x. [DOI] [PubMed] [Google Scholar]
- 5.Domingo E, Gomez J. 2007. Quasispecies and its impact on viral hepatitis. Virus Res 127:131–150. doi: 10.1016/j.virusres.2007.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Cao L, Wu C, Shi H, Gong Z, Zhang E, Wang H, Zhao K, Liu S, Li S, Gao X, Wang Y, Pei R, Lu M, Chen X. 2014. Coexistence of hepatitis B virus quasispecies enhances viral replication and the ability to induce host antibody and cellular immune responses. J Virol 88:8656–8666. doi: 10.1128/JVI.01123-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Liu F, Chen L, Yu DM, Deng L, Chen R, Jiang Y, Huang SY, Yu JL, Gong QM, Zhang XX. 2011. Evolutionary patterns of hepatitis B virus quasispecies under different selective pressures: correlation with antiviral efficacy. Gut 60:1269–1277. doi: 10.1136/gut.2010.226225. [DOI] [PubMed] [Google Scholar]
- 8.Chen L, Zhang Q, Yu DM, Wan MB, Zhang XX. 2009. Early changes of hepatitis B virus quasispecies during lamivudine treatment and the correlation with antiviral efficacy. J Hepatol 50:895–905. doi: 10.1016/j.jhep.2008.12.018. [DOI] [PubMed] [Google Scholar]
- 9.Tong J, Li QL, Huang AL, Guo JJ. 2013. Complexity and diversity of hepatitis B virus quasispecies: correlation with long-term entecavir antiviral efficacy. Antiviral Res 99:312–317. doi: 10.1016/j.antiviral.2013.06.020. [DOI] [PubMed] [Google Scholar]
- 10.Peveling-Oberhag J, Herrmann E, Kronenberger B, Farnik H, Susser S, Sarrazin C, Zeuzem S, Hofmann WP. 2013. Dynamics of hepatitis B virus quasispecies heterogeneity and virologic response in patients receiving low-to-moderate genetic barrier nucleoside analogs. J Viral Hepat 20:234–239. doi: 10.1111/jvh.12013. [DOI] [PubMed] [Google Scholar]
- 11.Cheng Y, Guindon S, Rodrigo A, Lim SG. 2013. Increased viral quasispecies evolution in HBeAg seroconverter patients treated with oral nucleoside therapy. J Hepatol 58:217–224. doi: 10.1016/j.jhep.2012.09.017. [DOI] [PubMed] [Google Scholar]
- 12.Lim SG, Cheng Y, Guindon S, Seet BL, Lee LY, Hu P, Wasser S, Peter FJ, Tan T, Goode M, Rodrigo AG. 2007. Viral quasi-species evolution during hepatitis Be antigen seroconversion. Gastroenterology 133:951–958. doi: 10.1053/j.gastro.2007.06.011. [DOI] [PubMed] [Google Scholar]
- 13.Coffin CS, Osiowy C, Gao S, Nishikawa S, van der Meer F, van Marle G. 2014. Hepatitis B virus (HBV) variants fluctuate in paired plasma and peripheral blood mononuclear cells among patient cohorts during different chronic hepatitis B (CHB) disease phases. J Viral Hepat 22:416–426. doi: 10.1111/jvh.12308. [DOI] [PubMed] [Google Scholar]
- 14.Maini MK, Reignat S, Boni C, Ogg GS, King AS, Malacarne F, Webster GJ, Bertoletti A. 2000. T cell receptor usage of virus-specific CD8 cells and recognition of viral mutations during acute and persistent hepatitis B virus infection. Eur J Immunol 30:3067–3078. doi:. [DOI] [PubMed] [Google Scholar]
- 15.Osiowy C, Villeneuve JP, Heathcote EJ, Giles E, Borlang J. 2006. Detection of rtN236T and rtA181V/T mutations associated with resistance to adefovir dipivoxil in samples from patients with chronic hepatitis B virus infection by the INNO-LiPA HBV DR line probe assay (version 2). J Clin Microbiol 44:1994–1997. doi: 10.1128/JCM.02477-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Hannoun C, Horal P, Lindh M. 2000. Long-term mutation rates in the hepatitis B virus genome. J Gen Virol 81:75–83. [DOI] [PubMed] [Google Scholar]
- 17.Chang JJ, Lewin SR. 2007. Immunopathogenesis of hepatitis B virus infection. Immunol Cell Biol 85:16–23. doi: 10.1038/sj.icb.7100009. [DOI] [PubMed] [Google Scholar]
- 18.Liaw Y-F, Kao J-H, Piratvisuth T, Chan HLY, Chien RN, Liu C-J, Gane E, Locarnini S, Lim S-G, Han K-H, Amarapurkar D, Cooksley G, Jafri W, Mohamed R, Hou J-L, Chuang W-L, Lesmana LA, Sollano JD, Suh D-J, Omata M. 2012. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2012 update. Hepatol Int 6:531–561. doi: 10.1007/s12072-012-9386-z. [DOI] [PubMed] [Google Scholar]
- 19.Sarin SK, Kumar A, Almeida JA, Chawla YK, Fan ST, Garg H, de Silva HJ, Hamid SS, Jalan R, Komolmit P, Lau GK, Liu Q, Madan K, Mohamed R, Ning Q, Rahman S, Rastogi A, Riordan SM, Sakhuja P, Samuel D, Shah S, Sharma BC, Sharma P, Takikawa Y, Thapa BR, Wai CT, Yuen MF. 2009. Acute-on-chronic liver failure: consensus recommendations of the Asian Pacific Association for the Study of the Liver (APASL). Hepatol Int 3:269–282. doi: 10.1007/s12072-008-9106-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Günther S, Li BC, Miska S, Kruger DH, Meisel H, Will H. 1995. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol 69:5437–5444. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Myers R, Clark C, Khan A, Kellam P, Tedder R. 2006. Genotyping hepatitis B virus from whole- and sub-genomic fragments using position-specific scoring matrices in HBV Star. J Gen Virol 87:1459–1464. doi: 10.1099/vir.0.81734-0. [DOI] [PubMed] [Google Scholar]
- 22.Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. doi: 10.1093/molbev/msm092. [DOI] [PubMed] [Google Scholar]
- 23.Zagordi O, Daumer M, Beisel C, Beerenwinkel N. 2012. Read length versus depth of coverage for viral quasispecies reconstruction. PLoS One 7:e47046. doi: 10.1371/journal.pone.0047046. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. 2009. Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–1645. doi: 10.1101/gr.092759.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Tuteja A, Siddiqui AB, Madan K, Goyal R, Shalimar, Sreenivas V, Kaur N, Panda SK, Narayanasamy K, Subodh S, Acharya SK. 2014. Mutation profiling of the hepatitis B virus strains circulating in North Indian population. PLoS One 9:e91150. doi: 10.1371/journal.pone.0091150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Mantovani N, Cicero M, Santana LC, Silveira C, do Carmo EP, Abrão PR, Diaz RS, Caseiro MM, Komninakis SV. 2013. Detection of lamivudine-resistant variants and mutations related to reduced antigenicity of HBsAg in individuals from the cities of Santos and São Paulo, Brazil. Virol J 10:320. doi: 10.1186/1743-422X-10-320. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Desmond CP, Bartholomeusz A, Gaudieri S, Revill PA, Lewin SR. 2008. A systematic review of T-cell epitopes in hepatitis B virus: identification, genotypic variation and relevance to antiviral therapeutics. Antivir Ther 13:161–175. [PubMed] [Google Scholar]
- 28.Chen CH, Lee CM, Tung WC, Wang JH, Hung CH, Hu TH, Wang JC, Lu SN, Changchien CS. 2010. Evolution of full-length HBV sequences in chronic hepatitis B patients with sequential lamivudine and adefovir dipivoxil resistance. J Hepatol 52:478–485. doi: 10.1016/j.jhep.2010.01.006. [DOI] [PubMed] [Google Scholar]
- 29.Alexopoulou A, Baltayiannis G, Eroglu C, Nastos T, Dourakis SP, Archimandritis AJ, Karayiannis P. 2009. Core mutations in patients with acute episodes of chronic HBV infection are associated with the emergence of new immune recognition sites and the development of high IgM anti-HBc index values. J Med Virol 81:34–41. doi: 10.1002/jmv.21337. [DOI] [PubMed] [Google Scholar]
- 30.Pollicino T, Raffa G, Costantino L, Lisa A, Campello C, Squadrito G, Levrero M, Raimondo G. 2007. Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma. Hepatology 45:277–285. doi: 10.1002/hep.21529. [DOI] [PubMed] [Google Scholar]
- 31.Chisari FV, Isogawa M, Wieland SF. 2010. Pathogenesis of hepatitis B virus infection. Pathol Biol (Paris) 58:258–266. doi: 10.1016/j.patbio.2009.11.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Pawlotsky JM. 2005. The concept of hepatitis B virus mutant escape. J Clin Virol 34(Suppl 1):S125–S129. doi: 10.1016/S1386-6532(05)80021-6. [DOI] [PubMed] [Google Scholar]
- 33.Prendergast AJ, Klenerman P, Goulder PJ. 2012. The impact of differential antiviral immunity in children and adults. Nat Rev Immunol 12:636–648. doi: 10.1038/nri3277. [DOI] [PubMed] [Google Scholar]
- 34.Bertoletti A, Ferrari C. 2012. Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection. Gut 61:1754–1764. doi: 10.1136/gutjnl-2011-301073. [DOI] [PubMed] [Google Scholar]
- 35.Wang HY, Chien MH, Huang HP, Chang HC, Wu CC, Chen PJ, Chang MH, Chen DS. 2010. Distinct hepatitis B virus dynamics in the immunotolerant and early immunoclearance phases. J Virol 84:3454–3463. doi: 10.1128/JVI.02164-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Bertoletti A, Kennedy PT. 2015. The immune tolerant phase of chronic HBV infection: new perspectives on an old concept. Cell Mol Immunol 12(3):258–263. doi: 10.1038/cmi.2014.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Hunt CM, McGill JM, Allen MI, Condreay LD. 2000. Clinical relevance of hepatitis B viral mutations. Hepatology 31:1037–1044. doi: 10.1053/he.2000.6709. [DOI] [PubMed] [Google Scholar]
- 38.Chen L, Zheng CX, Lin MH, Huang ZX, Chen RH, Li QG, Li Q, Chen P. 2013. Distinct quasispecies characteristics and positive selection within precore/core gene in hepatitis B virus HBV associated acute-on-chronic liver failure. J Gastroenterol Hepatol 28:1040–1046. doi: 10.1111/jgh.12109. [DOI] [PubMed] [Google Scholar]
- 39.Nordin M, Ingman M, Lindqvist B, Kidd-Ljunggren K. 2014. Variability in the precore and core promoter region of the hepatitis B virus genome. J Med Virol 86:437–445. doi: 10.1002/jmv.23839. [DOI] [PubMed] [Google Scholar]
- 40.Xu L, Chen EQ, Lei J, Liu L, Zhou TY, Gao Z, Tang H. 2012. Pre-core/basal-core promoter and reverse transcriptase mutations in chronic HBV infected-patients. Hepatogastroenterology 59:212–215. doi: 10.5754/hge10122. [DOI] [PubMed] [Google Scholar]
- 41.Zhang A, Wan Z, You S, Liu H, Zhu B, Chen J, Rong Y, Zang H, Li C, Wang H, Xin S. 2013. Association of hepatitis B virus mutations of A1846T and C1913A/G with acute-on-chronic liver failure development from different underlying chronic liver diseases. Hepat Mon 13:e12445. doi: 10.5812/hepatmon.12445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Jammeh S, Tavner F, Watson R, Thomas HC, Karayiannis P. 2008. Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol 89:901–909. doi: 10.1099/vir.0.83468-0. [DOI] [PubMed] [Google Scholar]
- 43.Datta S. 2008. An overview of molecular epidemiology of hepatitis B virus (HBV) in India. Virol J 5:156. doi: 10.1186/1743-422X-5-156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Homs M, Jardi R, Buti M, Schaper M, Tabernero D, Fernandez-Fernandez P, Quer J, Esteban R, Rodriguez-Frias F. 2011. HBV core region variability: effect of antiviral treatments on main epitopic regions. Antivir Ther 16:37–49. doi: 10.3851/IMP1701. [DOI] [PubMed] [Google Scholar]
- 45.Chuang WL, Omata M, Ehata T, Yokosuka O, Ohto M. 1993. Concentrating missense mutations in core gene of hepatitis B virus. Evidence for adaptive mutation in chronic hepatitis B virus infection. Dig Dis Sci 38:594–600. [DOI] [PubMed] [Google Scholar]
- 46.Homs M, Buti M, Quer J, Jardi R, Schaper M, Tabernero D, Ortega I, Sanchez A, Esteban R, Rodriguez-Frias F. 2011. Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome. Nucleic Acids Res 39:8457–8471. doi: 10.1093/nar/gkr451. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Günther S. 2006. Genetic variation in HBV infection: genotypes and mutants. J Clin Virol 36(Suppl 1):S3–S11. doi: 10.1016/S1386-6532(06)80002-8. [DOI] [PubMed] [Google Scholar]
- 48.Märschenz S, Brinckmann A, Nürnberg P, Krüger DH, Günther S, Meisel H. 2008. Co-replication analyses of naturally occurring defective hepatitis B virus variants with wild-type. Virology 372:247–259. doi: 10.1016/j.virol.2007.10.039. [DOI] [PubMed] [Google Scholar]
- 49.Zhang D, Dong P, Zhang K, Deng L, Bach C, Chen W, Li F, Protzer U, Ding H, Zeng C. 2012. Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment. BMC Microbiol 12:307. doi: 10.1186/1471-2180-12-307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Pult I, Chouard T, Wieland S, Klemenz R, Yaniv M, Blum HE. 1997. A hepatitis B virus mutant with a new hepatocyte nuclear factor 1 binding site emerging in transplant-transmitted fulminant hepatitis B. Hepatology 25:1507–1515. [DOI] [PubMed] [Google Scholar]
- 51.Taffon S, Genovese D, Blasi M, Pierotti P, Degli Esposti A, Catone S, Chionne P, Pulimanti B, Candido A, Dettori S, Tosti ME, Argentini C, Mazzotta F, Rapicetta M. 2014. HBV whole-genome mutation profile in HIV-1/HBV coinfected patients in a long-term follow-up study. Infection 42:675–687. doi: 10.1007/s15010-014-0616-2. [DOI] [PubMed] [Google Scholar]
- 52.Saha D, Pal A, Biswas A, Panigrahi R, Sarkar N, Das D, Sarkar J, Guha SK, Saha B, Chakrabarti S, Chakravarty R. 2014. Molecular characterization of HBV strains circulating among the treatment-naive HIV/HBV co-infected patients of eastern India. PLoS One 9:e90432. doi: 10.1371/journal.pone.0090432. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Baxa DM, Thekdi AD, Golembieski A, Krishnan PV, Sharif O, Kizy A, Shetron-Rama L, Jovanovich J, Chappell BJ, Snow-Lampart A, Borroto-Esoda K, Gordon SC. 2013. Evaluation of anti-HBV drug resistant mutations among patients with acute symptomatic hepatitis B in the United States. J Hepatol 58:212–216. doi: 10.1016/j.jhep.2012.09.014. [DOI] [PubMed] [Google Scholar]
- 54.Gong L, Han Y, Chen L, Liu F, Hao P, Sheng J, Li XH, Yu DM, Gong QM, Tian F, Guo XK, Zhang XX. 2013. Comparison of next-generation sequencing and clone-based sequencing in analysis of hepatitis B virus reverse transcriptase quasispecies heterogeneity. J Clin Microbiol 51:4087–4094. doi: 10.1128/JCM.01723-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
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