Association between quasispecies variants of hepatitis B virus, as detected by high‑throughput sequencing, and progression of advanced liver disease in Indonesian patients
- Authors:
- Published online on: May 16, 2019 https://doi.org/10.3892/mmr.2019.10250
- Pages: 16-24
Abstract
Introduction
Hepatitis B virus (HBV) infection is a significant health problem worldwide, with approximately 325 million people living with chronic HBV infection (CHB) (1). Approximately 10% of people carrying HBV die as a direct consequence of persistent viral infection (2). The chronic stage of this disease (i.e., CHB) can progress into liver cirrhosis (LC) or hepatocellular carcinoma (HCC) under the influence of several factors, including HBV DNA levels, genomic mutations, viral genotype, or viral subtype (3–5). Better understanding of these factors, especially the dynamic variations in HBV, should allow researchers to develop an effective strategy to reduce the burden of HBV infection.
HBV is a partially double-stranded DNA virus with four main overlapping open reading frames (ORFs) that encode the polymerase, surface, X, and pre-core/core proteins (6–9). The high mutation rate of HBV is a consequence of the lack of proofreading function of its polymerase gene (8). The estimated mutation rate in hepadnaviral genomes is 2×104 base substitutions/site/year (2,8). The high mutation rate of this virus produces diverse viral populations, commonly known as quasispecies (10,11). Many studies have indicated that the emergence of mutation-derived quasispecies is correlated with clinical outcomes and disease progression (9,10,12).
Viral quasispecies usually differentiate into minor (1–5% of the total population), intermediate (5–20% of the total population), and major (>20% of the entire population) populations (9). If variants in the major population account for >80% of quasispecies, it is likely that the modified mutants will ultimately replace the wild-type strain in a process that may take 50 years. Some of the variations are probably present at the time of HBV infection (9,13,14). Some variants identified in the major population are relatively stable in the absence of the wild-type strain (13,14). However, if modifications in 20–80% of the entire population, it is likely that there is a mixture of wild-type and variant strains in the viral population that may be selected as a consequence of antiviral drugs and the host's immune responses (9,15). It was reported that a high frequency of variants is probably driven by interactions between the host's immune responses and the virus itself during disease progression (9).
In Indonesia, variations in the HBV genome, including changes in BCP (A1762T, G1764A, and T1753V), the pre-S region, and the major hydrophilic region, have been correlated with advanced liver disease (ALD) (3,10,16,17). However, no studies have investigated whether variations in HBV quasispecies are associated with the progression of ALD. In the present study, we used high-throughput sequencing to detect whole-genome quasispecies variations in Indonesian patients with different types of liver disease. This study sought to determine the nature of the quasispecies variations across the entire HBV genome in vivo and whether they were related to the clinical diagnoses.
Materials and methods
Subjects
Twelve hepatitis B surface antigen (HBsAg)-positive patients were enrolled and their sera were collected at the General Hospital of Surabaya and Hajj Hospital (Surabaya, Indonesia). Eight of the patients had LC and/or HCC (ALD group) and four patients had CHB (CHB group). Chronic hepatitis was defined as being positive for HBsAg for >6 months with a raised or normal alanine aminotransferase (ALT) level in patients who did not meet the diagnostic criteria for LC or HCC. LC was diagnosed by either histology (stage IV fibrosis) or clinical evidence of cirrhosis as detected by liver biopsy, ultrasonography, computed tomography (CT), or magnetic resonance imaging (MRI). HCC was diagnosed by at least one of the following: positive liver biopsy, elevated α-fetoprotein levels, and imaging findings (ultrasound, CT, or MRI). All of the patients were treatment-naïve. None of the patients were co-infected with human immunodeficiency virus, hepatitis C virus, or hepatitis D virus.
Serologic testing
HBV DNA was detected with a TaqMan PCR assay (COBAS® AmpliPrep/COBAS® TaqMan® HBV tests; Roche Molecular Systems, Pleasanton, CA, USA) with a lower limit of detection of 2.1 log copies/ml. The HBsAg titer was quantified with a Lumipulse® HBsAg-HQ assay (Lumipulse, Fujirebio, Tokyo, Japan). Hepatitis B e antigen (HBeAg) levels were determined using a chemiluminescence immunoassay (Architect HBeAg; Abbot Japan Co., Ltd., Tokyo, Japan). ALT and aspartate aminotransferase (AST) levels were measured using standard procedures.
DNA extraction and PCR
HBV DNA was extracted from 200 µl of serum using QIAamp DNA Blood Mini kits (Qiagen, Tokyo, Japan). The whole genome (four genes) was amplified using the three primer pairs: W1F [GATTCCTGCTCAAGG AACC, nucleotides (nt) 529–547] and W1R (GCCTACAGC CTCCTAGTAC, nt 1770–1788); W2F (ACTGGGAGGAGT TGGGGGAG, nt 1729–1748) and W2R (GCTGTAGCTCTT GTTCCCAAG, nt 2827–2847); and HB10F (CGCAGAGAT CTCAATCTCGG, nt 2417–2437) and HB1R (GAAACATAG AGGTGCCTTGAGCAG, nt 557–534). The PCR protocol comprised pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min, and finally post-extension at 72°C for 5 min. The PCR products were analyzed on 2% agarose gel electrophoresis and visualized with ethidium bromide under a UV transilluminator. The target bands were purified with a QIAquick Gel Extraction kit (Qiagen). The purified PCR products were analyzed by direct sequencing and high-throughput sequencing to identify viral genotypes and viral quasispecies, respectively.
Direct sequencing and HBV genotyping
The PCR products were purified using ExoSAP-IT (USB Corporation, Cleveland, OH, USA) and sequenced on an ABI Prism 3100-Avant genetic analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using the BigDye Terminator version 3.1 cycle sequencing kit. The nt sequences obtained by direct sequencing were aligned using ClustalW (http://www.genome.jp/tools-bin/clustalw) and compared to reference sequences (genotypes A-G) retrieved from the National Centre for Biotechnology Information (https://www.ncbi.nlm.nih.gov). A phylogenetic tree was constructed using the minimum evolution and maximum likelihood algorithm with MEGA 5.2 software (http://www.megasoftware.net) to determine HBV genotypes. The viral subtypes were determined by S region analysis, as previously described (18).
Next-generation sequencing
A next-generation sequencer (Genome Analyzer; Illumina, Inc., San Diego, CA, USA) was used to detect variants in the whole genomes of HBV quasispecies. The concentrations of the purified PCR products were measured with a Qubit dsDNA HS Assay kit (Invitrogen; Thermo Fisher Scientific, Inc.). Next, a PCR product library encompassing the whole genome was prepared using the Nextera XT DNA Sample Prep kit (Illumina, Inc.). The PCR products were uniformly sheared into 500 bp fragments with the kits, and the PCR product libraries were mixed with 1% 8 pM PhiX sequencing control and run on a MiSeq sequencer (Illumina, Inc.) for paired-end targeted sequencing. Finally, the fluorescent signals were detected and analyzed using MiSeq Control Software (Illumina, Inc.). The resulting images were used to produce sequence data in the FASTQ format (11,15).
Sequence read mapping and data analysis
Illumina, Inc. paired-end sequencing generates overlapping read pairs (ORPs) from relatively short fragment sequence libraries combined with relatively long reads. Quality checks and data trimming were performed before assembling the sequences using Genomics Workbench version 6.0.1 software (CLC bio, Aarhus, Denmark). The sequencing results used in this study had read quality scores of 30 (Q30), according to the manufacturer's requirements. We used Q30 filtering and ORPs in order to eliminate false-positive variants generated by PCR errors during the sequencing process and to recover sequencing errors. The sequence reads could therefore be used to detect viral variants occurring at a low abundance with a high level of confidence. The aligned sequence reads were mapped against the reference HBV genome of genotype B3 and subtype adw (GeneBank accession no. AB713527). We then determined the prevalence of each viral quasispecies within the population. To achieve this, we used the setting ‘read conflict’ in Genomics Workbench. Any conflicts between the sequence reads were annotated on the consensus sequence after mapping was complete (9,15).
Variants were defined as nt modifications that resulted in an amino acid change (9,10,15,19). The percentage variation (mutation frequency) was determined as the proportion of nt changes in the total sequence reads that altered amino acids. All nts with quasispecies of ≥1% of the entire viral population were selected for analysis. A similar cut-off value was used in previous studies (9,10,11,15).
Statistical analysis
All statistical analyses were performed with SPSS software version 22 (IBM Corporation, Armonk, NY, USA). The Kolmogorov-Smirnov and Shapiro-Wilk tests were used to assess the normality of distribution of each variable. Differences between groups were determined using the nonparametric Mann-Whitney U test (for non-normally distributed variables) and Student's t-test (for normally distributed variables). P<0.05 was considered to indicate a statistically significant difference.
Results
Patient characteristics
The clinical characteristics of the patients with CHB and ALD are shown in Table I. There were no significant differences between the CHB and ALD groups in terms of age (53.5 vs. 53.13 years, respectively; P=0.922), AST levels (151.5 vs. 147.9 IU/l, respectively; P=0.836), or ALT levels (86.75 vs. 95.57 IU/l, respectively; P=0.956). However, the HBsAg titer was greater in the CHB group than in the ALD group (461,300 vs. 1,491 IU/ml, respectively, P=0.156). There were more males than females in both groups (Table I).
Based on direct sequencing of the whole genome and the S gene, all of the samples belonged to HBV genotype B3 (phylogenetic tree not shown) and subtype adw1, respectively.
High-throughput sequencing of HBV variants
The mean coverage depth per nt after quality control (ORP and Q30) was 45,769.92-fold in the CHB group and 53,921.85-fold in the ALD group. The number of mapping reads was 317,368.5±63,737 and 323,785.25±46,962 in the CHB and ALD groups, respectively (Table I). Mapping reads represent the nt annotation of the consensus sequence. The rate of gene variation represents the probability of change per position (9). The quasispecies variants detected in ≥1% of the total viral population were examined in four ORFs. The frequencies of modifications in the S gene (P=0.047) and X gene (P=0.044) were significantly greater in the ALD group than in the CHB group (Fig. 1A). The mutation rate in the S region was 89.53 and 50.69% in the ALD and CHB groups, respectively. The mutation rate in the X region was 76.95 and 35.88% in the ALD and CHB groups, respectively.
Variations in specific ORFs and domains
Further analysis revealed that the substitution rate in specific ORFs was significantly greater in the ALD group in RT box G (P=0.048), RNAseH (P=0.038), small S region (P=0.031), BCP (P=0.044) and B-cell epitope (P=0.042; Fig. 1B). Because the P-values indicate the order of significance for each ORF relative to the progression of severe disease, it seems that the small S region is the most strongly affected, followed by RNAseH, B-cell epitope, BCP, and RT box G. These specific ORFs might be strongly affected by the duration of infection, from chronic infection to the development of severe liver disease. Fig. 1C shows the quasispecies variants in the domain, a specific ORF in the same gene, and the inter-domain, the region between two domains (ORFs). Box G and RNAseH were classified as the polymerase domain, while B cell epitope and BCP were classified as X domain. The region between RT box G and RNAseH and the region between B cell epitope and BCP were classified as inter-domains. The mutation frequency in the P domain (72.52 vs. 158.70%, P=0.037) and the X domain (41.94 vs. 78.61%, P=0.015) were significantly greater in the ALD group than in the CHB group. By contrast, the mutation frequency in the inter-domain was not significantly different between the CHB group and the ALD group in either the P gene (49.11 vs. 72.38%, P=0.109) or the X gene (35.33 vs. 55.83%, P=0.214).
Mutations associated with progression to ALD
The quasispecies variants for each amino acid were compared between the CHB and ALD groups to understand the potential role of these variants on disease progression. The nt positions showing significant differences in quasispecies are shown in Table II. Because of the large number of mutations detected by the high-throughput sequencing method, we have only shown the mutations that were statistically significant. Several variations are already known, including rtV30F, rtD31V, pV697E, sK24Q, sE164A, sW196* I127N, K130M, and V131I. The present study confirmed that during disease progression, the proportion of quasispecies variants increased for most of the mutations, except for W196* in the small S gene (Table II).
RT box G and small S gene overlapping
This study found high rate of variants in the RT box G with emergence of major mutations (Fig. 2), even though the P gene is the most conserved gene in the HBV genome. The overlapping RT box G influenced the variations and in the small S gene and introduced major mutations in the small S gene. Major mutations with quasispecies in >20% of the population can usually be confirmed by direct sequencing, whereas mutations with quasispecies in <20% are only detected by high-throughput sequencing.
X gene alignment and mutation mapping
Fig. 3A shows the alignment of the HBV X protein (HBx). The quasispecies variants accumulated in the B-cell epitope and BCP region, in particular. Fig. 3B shows that the significant mutations within the overlapping X gene and RNAseH, and mutations in the pre-Core gene influenced other variations in each ORF (20).
Discussion
This study demonstrated that all the HBV strains isolated in Java, especially in Surabaya, belong to genotype B3. These results are consistent with previous studies showing that HBV genotype B3 and subtype adw are predominant in Indonesia and South East Asia (3,15,21,22). All of the HBV strains identified in this study belonged to subtype adw1, which is the predominant HBV subtype in Sumatra and Java (9,23). This subtype was strongly associated with surface gene variations and immune escape mutants in patients with HCC (3,9,23). Surface gene variations could modify the antigenicity of HBsAg and allow the virus to evade neutralizing anti-HBs, thus facilitating progression to severe disease (4,9,13,24). It was reported that the HBsAg titer decreases during disease progression but it may also increase during the re-activation stage of chronic infection (25). However, the HBsAg titer was not significantly different between the two groups in the present study (Table I). In this study, we ruled out age, sex, and other serological markers (ALT, AST, and HBeAg) as influencing factors because there were no significant differences in these variables between the CHB and ALD groups. Because of the comparable characteristics, we suggest that the progression towards severe liver disease was influenced by the duration of infection and quasispecies variation. The duration of infection indicates whether exposure occurred as a consequence of vertical or horizontal transmission (9).
In a population with high endemic HBV infection such as Indonesia, it is essential to understand HBV quasispecies variation in order to develop vaccines, immunodiagnostic reagents, and effective therapeutic strategies (26). Recently, several research groups in various countries have used high-throughput sequencing to analyze HBV variations. It was reported that quasispecies variations in the RT 1 motif region and the small S region were detected shortly after treatment with nt analogs (11). Next-generation sequencing was also compared with clone-based sequencing to analyze reverse transcriptase quasispecies complexity in China (27,28). The authors concluded that next-generation sequencing was clinically valuable for predicting treatment efficacy (28). Deep sequencing was useful to detect minor quasispecies variants in relation to emergence of drug resistance in Italy (29). The same method was also used to detect the accumulation of quasispecies variations in the ‘a determinant’ region in patients with vertically transmitted HBV infection in California, USA (30). Owing to its ability to identify quasispecies variants occurring at a low abundance, the high-throughput screening method used here is more sensitive and represents a more useful tool than conventional direct sequencing (9,15,31). By using high-throughput methods, it is possible to determine the actual prevalence of viral quasispecies variants that might be clinically relevant. Based on the present findings, high-throughput sequencing showed strong sensitivity for quasispecies variants occurring in <20% of the population. We observed a high variation in naturally occurring quasispecies in this population of Indonesian HBV patients, and most of these quasispecies increased during disease progression.
The present study revealed that the quasispecies variations mainly accumulated in the S and X regions of the HBV genome in patients with severe liver diseases. Variations in the S region can impair the secretion of HBsAg and virions. Furthermore, the accumulation of HBsAg or virions in hepatocytes can induce endoplasmic reticulum stress, exacerbate inflammation, and eventually lead to more severe liver disease (9). By contrast, variations in the X region are primarily associated with activation of signal transduction. The X protein is a nuclear coactivator that activates signal transduction by several pathways, including the s (NF-κB) signaling pathway. NF-κB is necessary for cell growth and viability. Recent studies showed the activation of NF-κB could prevent apoptosis (32–34). It was suggested that the activation of NF-κB by X protein might promote the survival of HBV-infected hepatocytes and could contribute to hepatocarcinogenesis (7,32,33). In this study, K130M and V131I were predominant in patients with ALD. Wild-type HBx protein was reported to activate hypoxia-inducible factor-1α (HIF-1α), which could contribute to HCC progression (35,36). Thus, the variations K130M and V131I in X protein might upregulate HIF-1α function.
In the present study, we observed a trend for increased frequencies of rtV27G, rtL29F, rtV30F, rtD31V, rtK32T, rtP34S, rtH35Y, and rtN36T during the period of infection. Interestingly, these mutations accumulated in RT Box G, especially the RT-1 motif, which is reportedly related to protein priming and an epitope on the polymerase that is targeted by antibodies (37). It was suggested that these mutations might enhance viral replication activity and thus affect the clinical outcome (37). The frequencies of the variants pV697E, pT702P, p710L, pE729K, and pK743T were also in the ALD group. These mutations were located in the RNAseH domain and were predicted to influence pgRNA degradation and enhance viral replication function. The frequencies of the variations sL21S/F, sT23I, sK24Q, sI25T, sE164A, and sV224A in the small S gene were also increased in the ALD group. These mutations altered the structural integrity of S protein and reduced its binding affinity to the anti-HBs antibody, to yield immune escape mutants. By contrast, the frequency of the vaccine escape variant sW196*, which is reportedly related to immune therapy failure, was decreased in this study. This change may indirectly contribute to the activity of another mutation (38–41). Because the envelope (S) gene is completely overlapped by the RT gene, the minor variant W196* may produce changes in the overlapping RT region and amino acid substitution rtM204I. Although we found no difference in the variant rtM204K/V between the two groups, we suggest that this substitution should be monitored, even in minor populations.
Two minor variations in the B cell epitope, E24D and A40G, were more frequent in the ALD group. These mutations are crucial for binding to neutralizing antibodies and to produce immune escape mutants. At the C terminal of the X region, we found that the frequencies of the variations I127N, K130M, and V131I increased with severe disease progression. These variations accumulated in the BCP region and can cause a substantial decrease in HBeAg expression, interfere with DNA repair, and enhance viral genome replication, which may contribute to liver disease progression via increased viral invasion and inflammation (20,42). These variations may also contribute to hepatocarcinogenesis via downregulated p21, a tumor suppressor protein, leading to rapid and uncontrolled cell proliferation (20,39). Similarly, a previous study reported that the variants K130M and V131I in genotype C were associated with the progression of ALD (10,21,43). The double mutation K130M/V131I was associated with severe clinical outcomes and progression of liver disease in chronic HBV patients in India (44).
A limitation of this study is the small number of patients and samples. A larger number of patients is needed to obtain more reproducible data.
In this study, we found that the accumulation of quasispecies variations in the S and X regions was predominant in patients with ALD. By using high-throughput sequencing, we found that quasispecies variants accumulated in the small S gene, RT box G, RNAseH, BCP, and B cell epitope genes, presumably in relation to increased disease severity. We suggest that viral quasispecies change dynamically during disease progression and might be related to progression of severe liver disease. Despite the limited number of samples, our results are clinically significant and provide new insight into the quasispecies variations of HBV isolated from Indonesian patients. Larger cohorts studies are needed to clarify the dynamics of viral quasispecies as well as their clinical implications.
Acknowledgements
Not applicable.
Funding
This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (grant no. 16H05826), and a Grant-in-Aid from the Japan Initiative for Global Research Network on Infectious Disease (J-GRID) supported by The Ministry of Education, Culture, Sports, Science and Technology.
Availability of data and materials
The datasets analyzed during the current study are available in the DDBJ under the following accession numbers: CHB group LC349872, LC349873, LC349875, and LC349879; ALD group LC349868, LC349869, LC349870, LC349871, LC349874, LC349876, LC349877, and LC349878.
Authors' contributions
WAP, YY, LNY, S, MIL and YH conceived and designed the study. WAP, YL and YM performed the experiments. WAP analyzed the data and wrote the study. YY and TU performed some of the experimental methods and techniques. YY, S, MIL and YH revised the manuscript and supervised the study.
Ethics approval and consent to participate
The research protocols were approved by the Institutional Review Board of the Institute of Tropical Disease, Airlangga University (Surabaya, Indonesia), and conformed to the Declaration of Helsinki (1975). Informed consent was obtained from all of the subjects involved in the study.
Patient consent for publication
Informed consent was obtained from all of the subjects involved in the study.
Competing interests
The authors declare that they have no conflicts of interest.
Glossary
Abbreviations
Abbreviations:
|
ALD |
advanced liver disease |
|
ALT |
alanine transaminase |
|
AST |
aspartate transaminase |
|
BCP |
basal core promoter |
|
CHB |
chronic Hepatitis B |
|
CT |
computed tomography |
|
HBeAg |
Hepatitis B e antigen |
|
HBsAg |
Hepatitis B s antigen |
|
HBV |
Hepatitis B virus |
|
HBx |
Hepatitis B × protein |
|
HCC |
hepatocellular carcinoma |
|
HIF-1α |
Hypoxia-inducible factor-1α |
|
LC |
liver cirrhosis |
|
MFI |
mutation frequency index |
|
MRI |
magnetic resonance imaging |
|
NF-κB |
nuclear factor-κB |
|
nt |
nucleotide |
|
ORF |
open reading frame |
|
ORP |
overlapping read pairs |
|
PCR |
polymerase chain reaction |
|
pgRNA |
pregenomic RNA |
|
RNAseH |
ribonuclease H |
|
RT |
reverse transcription |
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