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  Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 387-392
 

Hepatitis B virus X protein: The X factor in chronic hepatitis B virus disease progression


1 Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
2 Department of Medical Gastroenterology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
3 Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission05-Nov-2019
Date of Decision15-Nov-2019
Date of Acceptance22-Nov-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Prof. Padma Srikanth
Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai - 600 116, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_421

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 ~ Abstract 


Introduction: Hepatitis B virus (HBV) is the most common aetiological factor causing hepatocellular carcinoma (HCC). HBx gene plays an enigmatic role in HBV-related HCC. In this study we have analysed amino acid substitutions in HBx from HBV-infected individuals of different clinical stages. Materials and Methods: HBV-infected individuals (n = 93) were recruited in the study. DNA was extracted from plasma, amplified, and DNA sequencing was performed using specific primers targeting HBx gene (540 bp). Results: Among the study participants, 57% had chronic HBV infection, 30% had chronic liver disease (CLD) and 13% had HBV related HCC. Genotypes such as D1, D2, D3, A1, C2 and B2 were identified of which genotype D2 was predominant (78%). HBxC-terminal deletion was observed in four hepatitis B e antigen (HBeAg) negative participants with CLD. The frequency of aminoacid substitution in proapoptotic domain was higher in HBeAg negative participants including I127V (34%), K130M (34%), V131I (40%). The frequency of double mutation (K130M+V131I) and triple mutation (I127V+K130M+V131I) were found to be higher (32% and 36%) in HBeAg negative participants. Also, we identified L5M substitution (4.3%) in HBeAg positive participants with advanced liver disease. Conclusion: In HBx gene, aminoacid substitutions at positions 127, 130, 131 are associated with poor expression of HBeAg. We suggest screening for HBx aminoacid substitutions especially in patients with HBeAg negative chronic HBV infection to predict the clinical outcome and enable early treatment to prevent disease progression.


Keywords: Chronic liver disease, hepatitis B virus, hepatitis B e antigen negative, hepatocellular carcinoma, X gene


How to cite this article:
Mani M, Vijayaraghavan S, Sarangan G, Barani R, Abraham P, Srikanth P. Hepatitis B virus X protein: The X factor in chronic hepatitis B virus disease progression. Indian J Med Microbiol 2019;37:387-92

How to cite this URL:
Mani M, Vijayaraghavan S, Sarangan G, Barani R, Abraham P, Srikanth P. Hepatitis B virus X protein: The X factor in chronic hepatitis B virus disease progression. Indian J Med Microbiol [serial online] 2019 [cited 2020 Apr 2];37:387-92. Available from: http://www.ijmm.org/text.asp?2019/37/3/387/274089





 ~ Introduction Top


Globally, there are about 257 million chronic carriers of hepatitis B virus (HBV) as of 2017. India has the second largest burden with an intermediate prevalence of 4% chronic HBV carriers.[1] Two third of the burden is reported to be due to hepatitis e antigen (HBeAg) negative patients. HBV related hepatocellular carcinoma (HCC) burden in South India is about 42%.[2] There are many factors that contribute to the development of HCC such as HBV/hepatitis C virus (HCV), alcoholic liver disease and non-alcoholic fatty liver disease. Among these, HBV is the most common etiological factor of HCC in India. The prognosis of HBV related HCC is extremely poor with a survival rate of <16 months. The HBV genome comprises of an incomplete double stranded circular structure containing 4 overlapping open reading frames encoding proteins such as surface (S), core protein (C), polymerase protein (P) and X protein (X). X-gene is the smallest gene with a size of 540 bp encoding a small protein with 154 aminoacids. HBx protein is a multifunctional transactivator protein which regulates viral and cellular proteins. It is highly conserved and essential for viral replication. The X-gene overlaps the C-terminus of the polymerase gene that encodes for reverse transcriptase protein and N-terminus of the core-gene that encodes for core protein. Depending upon the site of mutation in X-gene, mutations can affect two genes such as core gene and a partial segment of polymerase gene simultaneously. The mutations in pre-core region encoding HBeAg is associated with an increased risk of HCC.[3] HBx gene point mutations can modulate the expression of HBeAg and either increase or decrease the viral replication capacity. T1762/A1764 double mutation is associated with the risk of HCC.[4] The core promoter (CP) and the enhancer II (Enh II) genes are located in the region that overlaps X gene. HBx proteins interfere with several signaling pathways associated with cell proliferation and invasion. HBxC-terminal truncation has been reported to impact the development of HCC.[5] As a transactivator, HBx can affect regulatory non-coding RNAs and mRNA. HBx interacts with various signal transduction pathways such as p53, Wnt, nuclear factor kappa B. HBx hastens the development of HCC. HBx function in HCC are nuclear translocation, protein–protein interactions, regulation of transcription factors, and induction of chromosome instability.[6] HBx involves in signal transduction and results in cell proliferation, transformation, invasion and metastasis. The present study aimed to investigate HBx mutations in chronic HBV carriers in different clinical phases of infection.


 ~ Materials and Methods Top


Ethics statement

This is a cross sectional study and was conducted from March 2016 to January 2019 after obtaining Institutional Ethics Committee approval (IEC-NI/16/AUG/55/55).

Study subjects

Adult chronic HBV carriers (n = 93) evidenced as chronic hepatitis and with plasma HBV DNA levels >2000 IU/ml. All the study subjects were seronegative for HIV and HCV. The participant's HBeAg, and serum alanine transaminase (ALT) levels were retrieved from the patient's hospital records.

Study settings

Patients attending medical gastroenterology clinic of our tertiary care center were directed to Molecular biology and clinical virology section under Department of Microbiology for the estimation of plasma HBV DNA levels by real time quantitative polymerase chain reaction (PCR).

Sample collection and processing

After obtaining written informed consent, 5 ml blood was collected in ethylenediaminetetraacetic acid vacutainers from 93 participants. Plasma was separated from the whole blood immediately after collection and stored at −80°C in multiple aliquots for further analysis.

DNA extraction and quantitative real time polymerase chain reaction

DNA was extracted from the plasma samples using QIAamp DNA Blood mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. Plasma HBV DNA levels were quantified using Artus HBV RG PCR assay (Qiagen, Hilden, Germany) in Rotorgene Q-5 plex system (Qiagen, Hilden, Germany).

Quality control measures

Appropriate quality control measures were taken including inclusion of sterile water as negative control and positive control in each assay. Estimate of HBV DNA of the samples was performed only after the interpretation of positive and negative controls. Positive control was an in-house control with a 2 log10 IU/ml HBV DNA (CV% - 3.6). The laboratory is accredited by national accredited board of laboratories (NABL) for detection of HBV by real time PCR and our laboratory participates in IAMM EQAS.

Hepatitis B virus X-gene nested polymerase chain reaction

First round of amplification was performed using hot start master mix kit (Qiagen, Hilden, Germany) along with outer primers 5'-TGC CAA GTG TTT GCT GAC GC-3' and 5'-ACG GGA AGA AAT CAG AAG G-3' and 10 μL of the extracted DNA. The reaction volume was adjusted to 50 μL by adding milli Q water. The amplification conditions included an initial activation at 95°C for 15 min; followed by 45 cycles of 94°C for 1 min, 45°C for 1 min, 72°C for 2 min and the final extension was 72°C for 10 min.[7] Round II was carried out with 2 μL of first round PCR product in hotstart master mix along with inner primers 5'-GCC GAT CCA TAC TGC GGA ACT-3' and 5'-GGC ACA GCT TGG AGG CTT GAA-3' with final reaction volume of 50 μL. The cycling conditions were 95°C 15 min; 94°C 1 min, 45°C 1 min, 72°C 2 min for 45 cycles; and finally 72°C for 10 min.[7]

Agarose gel electrophoresis

The amplified products were analyzed in ethidium bromide stained 2% agarose gel under UV transilluminator. The amplified product size of 540 bp was considered positive.

DNA purification and sequencing

The amplified DNA was filtered using vacuum in the MultiscreenHTS PCR plate (Millipore, USA). Cycle sequencing reactions was carried out using big dye terminator kit (V 3.1) and inner primers. Post-PCR purification was performed with Montage SEQ96 filtration (Millipore, USA). The purified products were sequenced by Sanger's method on ABI genetic analyzer 3730 platform (Applied Biosystems, USA).

Sequence analysis

The DNA sequences were obtained bi-directionally from forward and reverse primers. The consensus was prepared using sequencher software (Version 5.4.6, Gene Codes Corporation, Michigan, USA). Bioedit Version, 3.2.1, USA) was used to identify the aminoacid substitution.

Phylogenetic analysis

DNA sequences (HBV X gene) of all the genotype (A–H) were retrieved from the Genbank database. The study sequences (n = 93) along with Genbank sequences were included for the analysis. Multiple sequence alignment was performed using in-built ClustalW integrated in MEGA X using 1000 replicates bootstrap testing by neighbor joining method. The positions containing gaps and missing data were eliminated.

Statistical analysis

Data analysis such as median, inter quartile range were performed using Microsoft-excel version (14.0.4760.100). The correlation co-efficient calculation was performed between the plasma HBV DNA level and ALT levels. Kruskal–Wallis test was used to identify the frequency of mutations. All the analysis were carried out using Medcalc Statistical Software (Version 18.2.1, Ostend, Belgium).

Genbank submission

The DNA sequences obtained from the study were submitted to Genbank and the accession numbers are MN066375-MN066411, MN182657-MN182693.


 ~ Results Top


In all 93 study participants, 40 participants were HBeAg positive and 53 were HBeAg negative. All the study participants were analyzed for X mutations (1–132 aminoacids) using nested PCR. The median (interquartile range [IQR]) age of study participants was 49 (38–62) years. Among them 82% (n = 73) were male and the rest 18% (n = 20) were female. The median (IQR) plasma HBV DNA level was 5.99 log10 IU/ml (4.43 log10 IU/ml–6.88 log10 IU/ml).

Hepatitis B e antigen positive group

The median age was found to be 52 (35.7–64.2) years. The median plasma HBV DNA level was found to be 6.15 log10 IU/mL(4.64 log10 IU/mL–7.56 log10 IU/mL). The participants were in different stages of disease including 62.5% (n = 25) with chronic HBV infection, 22.5% (n = 9) with chronic liver disease (CLD), 15% (n = 6) were diagnosed with HCC [Table 1]. Amino acid substitutions in proapoptotic domain of HBx protein were seen 52Y (n = 1), S64T (n = 1) with participants with CLD. S101 L/P was noted in two individuals with HCC. A102V was detected in 15/40 (37.5%), of which 2 had HCC, 5 had CLD, 8 had chronic HBV infection. T105M was detected in one individual with CLD. M103A was identified in one individual with chronic HBV infection. L123S was observed in one individual with CLD. I127T was detected in 8/40 (20%) participants. K130M was detected in 9/40 (22.5%) participants, V131T was detected in 10/40 (25%).
Table 1: Characteristics of study participants (n=93) as per the hepatitis B e antigen status

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Hepatitis B e antigen negative group

Among the HBeAg negative study participants (n = 53), the median age was 49 (40–61) years. The median plasma HBV DNA level was 5.65 log10 IU/mL (4.26 log10 IU/m– 6.4 log10 IU/mL). The disease spectrum of the participants includes, chronic HBV infection seen in 53% (n = 28) participants, 36% (n = 19) with CLD, 11% (n = 6) HCC [Table 1]. In the proapoptotic domain, H94Y was detected in two participants with HCC. L98I was detected in one participant. S101P was detected in three (5.6%) participants. A102V, I127V, K130M were identified in 18/53 (34%) participants. V131T was identified in 21/53 (40%) participants.

The median plasma HBV DNA level was less in HBeAg negative participants (P = 0.029). The correlation between plasma HBV DNA levels and ALT levels were found to be statistically significant (r = 0.13, P = 0.0033) among the HBeAg positive participants. The frequency of mutations, I127T, K130M and V131 L were high among HBeAg negative participants and were statistically significant. F132Y was observed in two participants with HBeAg negative chronic hepatitis and one participant with CLD [Table 2]a.


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Phylogenetic analysis

Phylogenetic analysis of the study participants revealed the presence of D2 genotype in 73 participants (78%), primarily from Tamil Nadu and Andhra Pradesh. D3 was identified in six participants (6.4%) from West Bengal, A1 in seven participants (7.5%) from Tamil Nadu, Delhi and Bihar, C2 was detected in four participants (4.3%) from West Bengal, D1 was identified in two participants (2.1%) from Tamil Nadu and B2 (2.1%) was identified from two participants residing in Chennai [Figure 1].
Figure 1: Phylogenetic analysis was performed using neighbour joining method in MEGA X with 1000 bootstrap replicates. The analysis includes 105 nucleotide sequences. The Genbank sequences are shown with their accession numbers and their genotype. The study sequences were designated by study identification number and ▴ symbol

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Double and triple mutations

We have identified HBxC-terminal deletion (92–132 aminoacids) in four HBeAg negative participants with CLD. L5M which is an independent predictor of HCC[7] was identified in 3 participants with HCC and one participant with CLD [Figure 2]. We have also detected double mutations and triple mutations in HBx protein (K130M+V131I) in 26.9% (n = 24) participants of which 71% (n = 17) were HBeAg negative. Among the HBeAg positive participants (K130M+V131I) was detected among participants with CLD and HCC. (I127V+K130M+V131I) triple mutation was detected in 21.3% (n = 19), of which 79% (n = 15) were found among HBeAg negative participants. Other mutations (K130M+V131I) and (I127V+K130M+V131I) were also detected in HBeAg negative participants with chronic HBV infection. These double and triple mutations were not detected in HBeAg positive participants with chronic HBV infection [Table 2]b. Genotype specific aminoacid substitutions identified among the study participants are listed [Table 3]. The aminoacid substitution in the HBV X gene and their role are shown in [Figure 3].
Figure 2: Alignment of HBx amino acid codons from representative samples showing aminoacid substitutions identified in this study at positions 5, 102, 130, 131. We have observed 22 amino acid substitutions F30 L, S31P, T36A, S38P, S39P, P40A, S43P, A44V, S46P, S47T, H87G, F88N, A102V, L116V, K118T, D119E, E122D, I127V, K130M, V131I, C143R, A144S in an individual with genotype B2

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Table 3: Genotype specific mutations among the study participants with genotype A, B and D

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Figure 3: This model illustrates the tumourigenesis caused by amino acid substitutions in the regulatory and transactivation domain of HBx protein. We designed this model to demonstrate the role of amino acid substitutions in causing hepatocellular carcinoma. DR2: Direct repeat 2, NRE: Negative regulatory element, EBP: Enhancer binding protein, BH3-Bcl-2 homology region 3, DR1: Direct repeat 1

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 ~ Discussion Top


HBx protein is multifunctional and has a mysterious role in infection and replication of HBV in hepatocytes. It regulates virus and host genes since it does not have DNA binding activity.[8],[9] C-terminus of X gene is truncated for integration and promotes oxidative stress, damages the mitochondrial DNA which leads to HCC.[10],[11] We have noted a 40 amino acids deletion in 3'C-terminal proapoptotic domain among four HBeAg negative participants with CLD.

Five mutations such as H94Y in Enh II binding site of the HBx protein, I127T, K130M, V131I in BH3-like motif and F132Y/I/R are reported to be the associated risk factors of HCC and may promote progression of liver impairment.[12],[13] K103M and V131I mutations are present in CP region which decreases the expression of HBeAg. In our study, H94Y was identified in one participant with HCC and F132I in three participants of which two had chronic HBV infection and one CLD. H94 L and F132I/Y/R are reported to be higher in HCC.[14] Frequency of I127T in genotype D was higher in participants with HCC (70%) than in the CLD group (42.8%) which corroborates with earlier reports.[12]

In HBx protein, aminoacids from 52 to 65 and 88–154 domain are important in the transactivation, transcription and replication of HBV genome.[15] We identified H52Y and S64T from patients suffering from CLD. In our study T36A was noted in four participants with CLD. Among those four, three belonged to genotype D and one is genotype B. T36A and G50R aminoacid substitutions were associated with HCC.[16]

In our study 27% had (K130M+V131I) double mutation. We have also noted higher frequency (19%) of K130M+V131I among HBeAg negative participants. This was reported to be less (45%) in India than other countries like Taiwan (85%) and China (64%). This is may be due to the prevalence of different genotypes.[13] This double mutation impinge in cell cycle regulation, HBeAg expression and DNA repairing mechanism.[17] HBx 130, 131 sites overlap with the basal CP A1762T and G1764A sites, which are commonly reported substitutions in HCC. H94Y, I127T, K130M, V131I, and F132Y/I/R mutations are located in D and E functional domains of HBx protein that are associated with nuclear transactivation and signal transduction. Therefore, these mutations may be responsible for modulating the transactivation property of HBx. I127T+K130M+V131I triple mutation was noted in 21.3% and detected in participants with clinical progressive of liver disease.[7]

Among our study participants, genotype D was predominant (78%) which is concordant with earlier reports published from our region[18] and genotype A was detected in 7 (7.5%) participants. Genotype C (4.3%) was seen in participants from North Eastern part of India where the prevalence of genotype C is reported to be higher. We have also identified certain genotype specific amino acid substitutions. A102V, I127T, K130M were detected only in genotype D2 and B. P11S, R78C, S101 L, F132Y were seen only in genotype A1. In our study, amino acid substitutions such as C6Y, F30 L, S31A, P46S, T47S, Q87H, I88V, V131I which were reported to be unique to genotype A and D were also observed in genotype B.[19] A study from Southern India reported genotype specific mutations among participants with genotype A, however genotype specific mutations in D have not been documented previously.[19] Since these aminoacid substitutions are genotype specific and disease progression is genotype dependant, we believe that these amino acid substitutions may play a role in the genotype specific disease progression to HCC.

In our study, the median (IQR) age of HBe Ag positive participants was higher 52 (28.5) years and HCC was observed in males which is concordant with earlier reports.[18] There are reports on up regulation of androgen receptor by HBx protein which is likely to enhance the viral replication resulting in HCC among males.[20] As previously reported and in our study also we found a significant association between the plasma HBV DNA levels and ALT levels in the HBeAg positive group.[18]


 ~ Conclusion Top


The C-terminal deletion and aminoacid substitutions in HBx may be associated with CLD. We suggest that baseline analysis of HBV X gene be done to identify mutations which can be used to predict the disease progression. Screening for HBx mutations especially in HBeAgnegative individuals is important to carefully monitor the silent slow progression of HBV disease. The amino acid substitutions in X gene will be useful in predicting the clinical outcomes and to identify the risk of development of HCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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Wiktor SZ, Hutin YJ. The global burden of viral hepatitis: Better estimates to guide hepatitis elimination efforts. Lancet 2016;388:1030-1.  Back to cited text no. 1
    
2.
Ray G. Current scenario of hepatitis B and its treatment in India. J Clin Transl Hepatol 2017;5:277-96.  Back to cited text no. 2
    
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Wang Y, Zeng LI, Chen W. HBV X gene point mutations are associated with the risk of hepatocellular carcinoma: A systematic review and meta-analysis. Mol Clin Oncol 2016;4:1045-51.  Back to cited text no. 3
    
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Kim H, Lee SA, Kim BJ. X region mutations of hepatitis B virus related to clinical severity. World J Gastroenterol 2016;22:5467-78.  Back to cited text no. 4
    
5.
Ng KY, Chai S, Tong M, Guan XY, Lin CH, Ching YP, et al. C-terminal truncated hepatitis B virus X protein promotes hepatocellular carcinogenesis through induction of cancer and stem cell-like properties. Oncotarget 2016;7:24005-17.  Back to cited text no. 5
    
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Geng M, Xin X, Bi LQ, Zhou LT, Liu XH. Molecular mechanism of hepatitis B virus X protein function in hepatocarcinogenesis. World J Gastroenterol 2015;21:10732-8.  Back to cited text no. 6
    
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Barbini L, Tadey L, Fernandez S, Bouzas B, Campos R. Molecular characterization of hepatitis B virus X gene in chronic hepatitis B patients. Virol J 2012;9:131.  Back to cited text no. 7
    
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Murakami S. Hepatitis B virus X protein: Structure, function and biology. Intervirology 1999;42:8199.  Back to cited text no. 8
    
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Slagle BL, Bouchard MJ. Hepatitis B virus X and regulation of viral gene expression. Cold Spring Harb Perspect Med 2016;6:a021402.  Back to cited text no. 9
    
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Takaki A, Yagi T, Yamamoto K. Safe and cost-effective control of post-transplantation recurrence of hepatitis B. Hepatol Res 2015;45:38-47.  Back to cited text no. 10
    
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Salarnia F, Besharat S, Zhand S, Javid N, Khodabakhshi B, Moradi A. Mutations in hepatitis-B X-gene region: Chronic hepatitis-B versus cirrhosis. J Clin Diagn Res 2017;11:OC31-OC34.  Back to cited text no. 11
    
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Malik A, Singhal DK, Albanyan A, Husain SA, Kar P. Hepatitis B virus gene mutations in liver diseases: A report from New Delhi. PLoS One 2012;7:e39028.  Back to cited text no. 12
    
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Al-Qahtani AA, Al-Anazi MR, Nazir N, Ghai R, Abdo AA, Sanai FM, et al. Hepatitis B virus (HBV) X gene mutations and their association with liver disease progression in HBV-infected patients. Oncotarget 2017;8:105115-25.  Back to cited text no. 13
    
14.
Datta S, Ghosh A, Dasgupta D, Ghosh A, Roychoudhury S, Roy G, et al. Novel point and combo-mutations in the genome of hepatitis B virus-genotype D: Characterization and impact on liver disease progression to hepatocellular carcinoma. PLoS One 2014;9:e110012.  Back to cited text no. 14
    
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Tang H, Delgermaa L, Huang F, Oishi N, Liu L, He F, et al. The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication. J Virol 2005;79:5548-56.  Back to cited text no. 15
    
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Tuteja A, Siddiqui AB, Madan K, Goyal R, Shalimar V, Sreenivas V, et al. Mutation profiling of the hepatitis B virus strains circulating in North Indian population. PLoS One 2014;9:e91150.  Back to cited text no. 16
    
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Lin X, Xu X, Huang QL, Liu YQ, Zheng DL, Chen WN, et al. Biological impacts of “hot-spot” mutations of hepatitis B virus X proteins are genotype B and C differentiated. World J Gastroenterol 2005;11:4703-8.  Back to cited text no. 17
    
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Ismail AM, Samuel P, Eapen CE, Kannangai R, Abraham P. Antiviral resistance mutations and genotype-associated amino acid substitutions in treatment-naïve hepatitis B virus-infected individuals from the Indian subcontinent. Intervirology 2012;55:36-44.  Back to cited text no. 18
    
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Jagan OA, Manoj G, Jayaprakash NS, Nair RM, Chandy S. Molecular diversity of hepatitis B virus X gene in central Kerala, Southern India. J Clin Diagn Res 2018;12:10-14.  Back to cited text no. 19
    
20.
Yang WJ, Chang CJ, Yeh SH, Lin WH, Wang SH, Tsai TF, et al. Hepatitis B virus X protein enhances the transcriptional activity of the androgen receptor through c-Src and glycogen synthase kinase-3beta kinase pathways. Hepatology 2009;49:1515-24.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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