Hepatitis B Virus Reverse Transcriptase Mutations Biology Essay

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Mutations in reverse transcriptase region of Hepatitis B virus genome lead to decreased susceptibility of nucleostide analogs approved for treatment of HBV infection. Hence aim of present study is to identify and analyse pre-existing HBV RT mutations in treatment naive HBV infected patients.

Material & methods: We enrolled 71 chronic hepatitis B treatment naive patients from January 2009 to June 2011. HBV RT sequence analysis was done by using direct bidirectional sequencing of semi nested PCR products. HBV genotypes were determined by using multiplex PCR.

Results: We found genotype D in 64 (90.14%) of cases followed by genotype A and C which were present in 4 (5.63%) & 3 (4.22%) cases respectively. The results of sequence analysis of RT region showed mutations in 34 (47.88%) patients. rtH248N mutation was most prevalent mutation and accounted for 47.05% (16/34) cases. Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D which were present in 26.47% (9/34), 29.41% (10/34), 20.58% (7/34), 20.58% (7/34), 20.58% (7/34), 17.64% (6/34), 14.74% (5/34), 14.74% (5/34), 11.76% (4/34), 11.76% (4/34) and 11.76% (4/34) respectively. Known primary drug resistance mutations were found in 3 (8.82%) patients.

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Conclusions: Present study shows presence of HBV RT amino acid substitutions in treatment-naïve CHB patients which may decrease susceptibility of available oral antivirals. On the basis of finding of this study, genotypic testing may be recommended before the start of therapy in naïve patients, so that proper antivirals can be prescribed.

Keywords: Hepatitis B virus, chronic hepatitis B, reverse transcriptase, genotype, nucleos(t)ide analogs, semi nested PCR

Introduction

Despite availability of hepatitis B virus (HBV) vaccine, HBV infection remains a major health problem. It is an etiological agent for acute & chronic liver disease. Chronic HBV infection, if left untreated may lead to the development of serious complications like cirrhosis and hepatocellular carcinoma. Currently, two types of drugs are approved for the treatment of chronic HBV infection which includes immunomodulatory agents (IFN-α & Peg IFN) and oral nucleos(t)ide analogues {Lamivudine (LMV), adefovir (ADV), tenofovir (TNF), Telbivudine (LdT) & entecavir (ETV)}[Chang et al., 2006; Lai et al., 1998, 2006, 2007; Marcellin et al., 2003]. The major goals of antiviral therapy include (i) the achievement of HBeAg seroconversion, (ii) undetectability of HBV DNA levels by real time polymerase chain reaction (PCR)(iii) persistent normalization of serum alanine transaminase (ALT) levels. HBsAg loss and seroconversion to anti-HBs is the best long-term goal of treatment which is difficult to achieve. Nucleos(t)ide analogues are well tolerated and very effective in suppressing viral replication. Long term treatment with oral antivirals leads to emergence of drug resistance mutations which nullifies the benefits of therapy and sometimes may be associated with hepatitis flares [Pawlotsky et al., 2008]. 

Because of the lack of a proof-reading function of the RNA dependent DNA polymerase, HBV has a high mutation rate [Fares and Holmes, 2002]. It has been shown that mutations in reverse transcriptase region of HBV genome are associated with resistance to antiviral drugs as all available oral antivirals target reverse transcriptase (RT) region of polymerase gene. Hence, presence of pre-existing drug resistant mutations may reduce the efficacy of treatment. These resistance mutations are classified into two categories- primary drug resistance mutations which directly reduces susceptibility to antivirals (rtM204I for lamivudine & telbivudine) and compensatory mutations (e.g rtV173L & rtL180M) contributing to restoration of RT activity associated with primary mutations [Lai et al., 2005; Lok et al., 2007]. Amino acid substitutions in HBV RT region at positions of 169, 180, 181, 184, 202, 204, 236 and 250 are associated with non response to available oral antivirals and considered as primary drug resistance mutations [Locarnini, 2008]. Mutations in HBV polymerase region have been reported in variable frequencies among treatment naive patients and clinical significance of some mutations is still unclear [Aberle et al., 2001; Huang et al., 2005; Lee et al., 2006; Ludwig et al., 2008; Pollicino et al., 2007]. These mutations are viral strategies to overcome the drug selection pressure, thereby escaping the antiviral action of drugs. Thus, understanding of the mechanisms of the evolutionary basis of the drug resistance mutants is important to prevent and control emergence of mutant virus.

Direct sequencing of PCR products is considered as gold standard in order to detect novel & already established drug resistance mutations. Hence, the aim of present study was to identify and analyse pre-existing HBV RT mutations in treatment naive HBV infected patients from North India by direct sequencing. Furthermore, we also determined mutations in ''a'' determinant portion (aa 124-147) of major hydrophilic region of small surface protein [Cooreman et al., 2001].

Material and methods

Sample collection

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A total of 71 chronic hepatitis B treatment naïve patients were enrolled between January 2009 and June 2011 from Liver clinic of department of Hepatology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. All the patients were HBsAg positive for at least 6 months with elevated or normal serum ALT levels. All subjects were positive for HBV DNA by polymerase chain reaction and negative for HIV, HDV & antibody for Hepatitis C Virus (AntiHCV). The study was approved by the ethics committee of PGIMER, Chandigarh and informed written consent was obtained from all of subjects. Blood samples were taken from all the recruited subjects. Tests evaluating serum bilirubin, serum total protein, albumin and globulin, alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and prothrombin time were performed in all cases. HBsAg, HBeAg, anti-HBe and anti-HCV were performed by enzyme linked immunosorbent (ELISA) assay using commercially available kits. HBV DNA levels were quantified by Cobas amplicor HBV monitor test (Roche Diagnostics).

Extraction of HBV DNA

Serum was separated from five millilitres of blood and HBV DNA was extracted from serum using QiaAmp (Qiagen, Hilden, Germany) according to the manufacturer's instruction. The quantity and quality of DNA was measured using UV spectrophotometer (Model-DU 640, Beckman Coulter, USA).

HBV Genotyping

In order to determine HBV genotype, multiplex PCR was performed which comprised genotype specific primers as described elsewhere [Kirschberg et al., 2004]. Multiplex PCR was performed with the Takara PCR Thermal Cycler Diceâ„¢ (Takara Bio Inc, Japan) and was carried out in a total volume of 50 l which contained a 200 µM concentration of dNTPs mix, 2.5 unit of HotStarTaq DNA Polymerase (QIAGEN GmbH, Germany), 5l of 10x PCR buffer containing 1.5 mM MgCl2, 2 l of each sense and antisense primer (10pmol/l), 5 l template and water for a total volume of 50 l. The thermocycler was programmed to first incubate the samples for 15 min at 95 â-¦C, followed by 35 cycles consisting of 94°C for 30 sec, 61°C for 30 sec, and 72°C for 1 min and final extension at 72°C for 10 min. The amplified fragments were electrophoresed on a 2.5% agarose gel, stained with ethidium bromide, and evaluated under UV light. The sizes of PCR products were estimated according to the migration pattern of a 50-bp DNA ladder (GeneDirex, Taipei, Taiwan). Random amplicons were directly sequenced with the amplification primers. Nucleotide sequences were aligned with reference sequences of HBV genotypes obtained from the GeneBank, using clustalW software.

HBV reverse transcriptase sequence analysis

In order to study mutational pattern in HBV polymerase, a 743 base pairs fragment was amplified before start of treatment of antivirals. To amplify the HBV RT, a semi-nested PCR was performed using the primers described by Sheldon et al (Table 1) [Sheldon et al., 2005].

The first round of PCR was carried out in a tube containing 25 l of a reaction mixture made up of the following components: 1 µl of each outer primer (10pmol/µl), a 200 µM concentration of dNTPs mix, 1 unit of highfidelity Taq DNA polymerase with 3'-5' proofreading activity (Fermantas life sciences), 2.5l of 10x PCR buffer containing 1.5 mM MgCl2, 3 l of extracted HBV DNA and water for a total volume of 25 l. The thermocycler was programmed to first incubate the samples for 7 min at 95°C, followed by 35 cycles consisting of 94°C for 30 sec, 54°C for 30 sec, 72°C for 1 min and final extension for 7 min. 2nd round of PCR was performed by using the 2µl of 1st PCR product in final volume of 50 µl. Annealing temperature for second PCR was 54°C.

The amplified products of second PCR were used for bidirectional sequencing after purification by using Qiagen purification kit (Qiagen GmbH, Germany) as per manufacturer's instruction. Nucleotide and amino acid sequences were analyzed with aid of nucleotide data of gene bank at www.ncbi.nlm.nih.gov/blast, clustalW software, Geno2pheno HBV drug resistance tool (Genafor, Bonn, Germany http://hbv.bioinf.mpg.de/index.php) and Stanford University HBV sequence analysis tool (http://hivdb.stanford.edu/HBV/HBVseq/development/HBVseq.html) Similarly HBV surface gene "a" determinant region mutations were analyzed.

Statistical Analysis

All statistical analysis were performed using SPSS software version 16. Kruskal-Wallis test was employed to compare number of HBVrt mutations & HBV DNA between the genotypes. Correlations among HBV DNA levels, ALT levels, RT amino acid substitution and age were analyzed using Spearman's correlation coefficient. Main characteristics of studied population were compared using Mann Whitney U test. A p value less than 0.05 was considered statistically significant.

Results

Patient's characteristics

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Total of 71 treatment naive chronic hepatitis B subjects were enrolled for mutational analysis of RT region of HBV genome. Out of them, 54 (76%) were male and 17 (24%) were female. HBeAg was negative in 42 (59.1%) cases (Table 2). Mean age of subjects was 33.07 years (±11.62), HBeAg positive cases being younger than HBeAg negative cases (26.5±6.50 vs 37.35±12.27years). Median HBV DNA level was 7.476 log10 copies/ml (range 3.777- 9.995 log10 copies/ml ) in eAg positive patients while it was 5.712 log10 copies/ml (range 3.559-8.806 log10 copies/ml ) in eAg negative cases. We found a positive correlation between HBeAg positivity and serum HBV DNA levels (r = 0.59, p < 0.001). A negative correlation was observed between age and HBeAg positivity (r = -0.517, p = 0.002).

HBV genotyping

Multiplex PCR was performed with genotype specific primers showing variation in length of amplicon according to HBV genotypes. Amplicon size of 147 base pairs associated with genotype D was observed in 64 (90.14%) patients. Samples with genotype A showed amplicon of 370 base pairs while samples with genotype C came with an amplified fragment of 701 base pairs as shown in figure 1. Genotype A was observed in 4 (5.63%) followed by genotype C which was present in 3 (4.22%).

Analyses of RT mutations

In order to study mutational changes in the HBV RT region, a 743-bp fragment was amplified and sequenced which includes all previously known mutational positions associated with nucleoside and nucleotide resistance. On comparison of observed RT nucleotide sequences with HBV GenBank sequences revealed amino acid substitutions at 50 different positions. Out of 71 patients, mutations in RT region were found in 34 (47.88%) patients as shown in table 3. The results showed rtH248N mutation as most prevalent which accounted for 47.05% (16/34) cases (Table 4). Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D which were present in 26.47% (9/34), 29.41% (10/34), 20.58% (7/34), 20.58% (7/34), 20.58% (7/34), 17.64% (6/34), 14.74% (5/34), 14.74% (5/34), 11.76% (4/34), 11.76% (4/34) and 11.76% (4/34) patients respectively. Mutations rtY54H, rtN53D/E, rtS78T, rtL80V and rtV253I were found in three patients (8.82%) each.

Known primary drug resistance mutations rtS202R and rtM250I was found in one patient that was responsible for entecavir resistance. Lamivudine, entecavir and telbivudine resistance mutation (rtM204I) was observed in two patients. Adefovir related resistance mutation (rtI233V) was established in two patients. One patient showed appearance of rtA181G mutation related to LMV, LdT, ADV and TNF resistance. Compensatory mutation to antivirals rtL80V, rtL180S, rtT128A, rtQ215S and rtN238H was identified in 3, 1, 1, 2, 1 patients respectively.

We also observed putative nucleos(t)ide analog resistance mutations rtN53D/E, rtN54H, rtH126R, rtH139H, rtP237H and rtN238H in few of our patients. Predefined pretreatment mutations rtH124Y, rtN139H and rtI224V were observed in one patient each. Upon analysis, we did not find any genotype associated amino acid substitution (Table 4). One patient of genotype A showed mutation rtM129L. Twenty five (73.5%) subjects were with 1 to 5 mutations in RT region of HBV genome (Table 5). There was no statistically significant difference between HBV genotypes & mean number of HBVrt amino acid substitutions between D and C genotypes. Moreover, we did not find any statistically significant difference in HBV DNA levels among different genotypes.

On comparison of overlapping HBV surface gene "a" determinant region with reference sequences, we observed amino acid substitutions only at one position. sA128V mutation was observed in 4 patients. All of these 4 subjects were with genotype D.

Discussions

Mutations in RT region are associated with resistance to antiviral drugs as all available oral antivirals target RT region of polymerase gene. The direct sequencing method based on semi nested PCR amplification has been employed in present study in order to detect both HBV RT mutations associated with drug resistance & corresponding 'a' determinant mutations in surface gene of HBV genome. Present study has described HBV RT mutations from 71 chronic hepatitis B treatment naive subjects. As described earlier, we found a positive correlation between HBeAg positivity & serum HBV DNA levels (r = 0.59, p < 0.001) and a negative correlation between age and HBeAg positivity (r = -0.517, p = 0.002) [Shao et al., 2007]. In the present study, genotype D was observed in 64 (90.14%) patients followed by genotype A which was present in 4 (5.63%). Previous studies have also shown that genotype D is most dominant genotype in India [Kumar et al., 2005; Vivekanandan et al., 2004]. Out of 71 cases, mutations in RT region had been found in 47.88 % (34) of cases, while 52.12% (37) cases were with wild type RT region. These results are in concordance with results observed by Pollicno et al and Aberle et al wherein they observed RT mutations in 38% and 48% of treatment naive cases respectively [Aberle et al., 2001; Pollicno et al., 2007].

Out of 34 subjects who have shown mutations in RT region of HBV genome, 25 (73.5%) subjects were with 1 to 5 mutations. Eight patients (23.5%) have shown 5 to 10 mutations in studied region of HBV genome while only one patient is with more than 10 mutations. Earlier study has shown association of amino acid substitutions with genotypes. Liu et al has shown genotype dependent amino acid polymorphic property at 8 sites , i.e. rtI53, rtI91, rtN124, rtD134, rtY221, rtV224, rtN238 and rtS256 for genotype A; rtN53, rtL91, rtH124, rtD134, rtF221, rtV224, rtN238 and rtC256 for genotype D [Liu et al., 2010]. We did not find any genotype associated amino acid substitution. This may be because of presence of genotype D in 90% patients and small sample size. rtH248N mutation has been found in 47.05% (16/34) cases. Stanford university HBV database has also shown rates of this mutation in 33.3% of CHB treatment naive cases with genotype D [Stanford University HBV sequence database and Ismail et al., 2012]. Other common mutations detected in our study include rtM129L, rtD263E/S, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D. Recent study from India has shown presence of these mutations in Indian CHB treatment naive patients [Ismail et al., 2012]. Primary drug resistance mutations rtS202R and rtM250I was found in one patient that was responsible for entecavir resistance. Lamivudine, entecavir and telbivudine resistance mutation (rtM204I) was observed in two patients. A study by Ludwig et al has found rtM204V/I mutation in 1.4% cases [Ludwig et al., 2008]. In contrast, Lampertico et al. found a pre-treatment prevalence of 3% for M204V⁄I and 8% for A181T in Italian CHB patients. They have also found mutations associated with ETV resistance such as rtS202 and rtM250 codon changes [Lampertico et al., 2008]. Ciancio et al suggested that the presence of rtL91 and rtC256 might represent helpful compensatory changes for restoring replication competence in viruses acquiring the drug-resistance rtM204V/I and/or rtL180M mutations by spatial structural analysis of HBV polymerase [Ciancio et al., 2004].

Adefovir related resistance mutation (rtI233V) was seen in two patients. A study by Schildgen et al has shown adefovir failure due to the pre-existing rtI233V mutation [Schildgen et al., 2010]. However, the role of the rtI233V mutation and adefovir response remains contradictory [Tan et al. 2008]. Many of the mutations found in present study may be naturally occurring mutations with no biological significance. These mutations may decrease susceptibility of antivirals whenever occur in conjunction with primary resistance. Compensatory mutations to antivirals rtL80V, rtL180S, rtT128A, rtQ215S and rtN238H have been identified in few patients. Earlier study has shown that rt215 and rt219 mutations in C-D interdomain of RT region can alter the nucleotide triphosphate binding active site [Bartholomeusz and Locarnini, 2006]. Study from China has also shown presence of rtN238H mutation in treatment naive patients [Zhong et al., 2012]. This mutation lies in close proximity to entecavir related resistance mutation.

Present study has also shown sA128V mutation which is present in 'a' determinant region of HBV surface gene, in four subjects with genotype D. Previous study from India has shown presence of sA128V mutation in treatment naive individuals [Ismail et al., 2012; Kazim et al., 2006].

Conclusions

Present study shows presence of HBV RT amino acid substitutions in treatment-naïve CHB patients which may decrease the susceptibility of available oral antivirals approved for treating CHB patients. On the basis of finding of this study, RT sequence analysis should be recommended before the start of therapy in order to determine drug resistance mutations in naïve patients, so that proper antivirals can be prescribed. Moreover it may also provide information regarding the presence of HBsAg immune escape mutations due to overlapping between P and S ORF (open reading frame) of hepatitis B. Thus, a study with large sample size is necessary to authenticate the need of routine resistance testing in drug-naïve patients.

Abbreviations

HBV: Hepatitis B virus; CHB: Chronic hepatitis B; HBsAg: Hepatitis B surface antigen; PCR: Polymerase chain reaction; Anti HCV: Antibody for hepatitis C virus; RT: Reverse transcriptase

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

This work was financially supported by Indian Council of Medical Research (ICMR), New Delhi, India. (ICMR No: VIR/28/2010-ECD-I). Authors are thankful to ICMR for providing Junior Research Fellowship to Bhupesh Singla (3/1/3JRF-2008/MPD,Dated 1/9/2008).