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Analysis of the HBV Small S Gene Partial Sequences and its Implications for Detection, Prevention and Treatment in Pakistani Patients

Hepatitis B virus (HBV) causes significant morbidity and mortality throughout the world, especially in developing countries. In Pakistan, the HBV infection rate is one of the highest in the world and about one third of infected population is co-infected with hepatitis C virus (HCV) and hepatitis D virus (HDV). In the present study, we isolated HBV from 49 HBV mono-infected and 25 HBV/HCV co-infected Pakistani patients and classified them based on the partial sequences of S gene. We further investigated mutations in these sequences that might result in the failure of hepatitis B surface antigen (HBsAg) detection, as well as vaccination and treatment failure. The D and D1 were identified as the most prevalent HBV genotype and subgenotype respectively in Pakistani samples. The same genotype/sub-genotype pattern was observed for the HBV/HCV coinfected patients. We identified several mutations in small S gene, which are previously reported to have roles in HBV diagnosis and treatment. Especially, the sT127P mutant, previously known to be implicated in vaccine escape, was prevalent with 98 and 96% frequencies in HBV mono-infected and HBV/HCV co-infected patients respectively. The findings of current study have implications with respect to prevention, diagnosis, and treatment of HBV infections in the Pakistani population.


Introduction
Hepatitis B virus (HBV) infection is a serious health issue contributing to significant morbidity and mortality worldwide, particularly in developing countries. It is a leading cause of acute as well as chronic hepatic infection, hepatocellular carcinoma, and liver cirrhosis. More than 2 billion people around the world have been infected by HBV, (Tan et al. 2021;Razavi-Shearer et al. 2018;Schweitzer et al. 2015) around 257-291 million of them chronically (Lim et al. 2020). In Pakistan, the infection rate of the HBV is increasing continually, with approximately 3-5% general population has been infected, according to a national survey (Ali et al. 2011). The HBV is classified into eight genotypes (i.e., A-H) (Norder et al. 2004), based on >8% variation over the entire genome (Arauz-Ruiz et al. 2002). These variations are partially due to host/virus interaction and partially due to their parallel evolution in distinct geographic regions (Kay and Zoulim 2007). HBV genotypes have been further divided into subtypes based on serological classification. Genotype A is subdivided into (A1-A4) which are predominant in Northwest Europe, North America, and Africa. Genotype B and C are mostly found in Asia and further subdivide into several sub-genotypes (B1-B6 and C1-C5, respectively). Genotype D is sub-divided into (D1-D10) and distributed worldwide. The genotype E, G, and H, have no sub-genotypes, while genotype F is divided into (F1-F4) sub-genotypes (Kramvis, Kew, and François 2005). Genetic variation among the HBV genotypes and sub-genotypes plays an important role in the severity of the disease, its complications, and resistance to treatment, and vaccination response (Chu and Lok 2002). In Pakistan, the most common genotype of HBV was found to be D which accounts for more than 70% HBV genotypes prevalent in Pakistan (Mahmood et al. 2016). The HBV genome is comprised of the four different genes, S (surface) including (Pre-S1, Pre-S2, small S), C (core), P (polymerase), and X gene which is involved in hepatocellular carcinoma. The small S protein, known as HBsAg, is one of the most important proteins because of its vital role in the viral binding to the host cell receptor and subsequent entry into the cell (Ganem and Prince 2004). Several mutations have been reported in the S gene. Most of the mutations are observed in the small S region, while some have also been found in the Pre-S1 and Pre-S2 regions. These mutations play important roles to in the failure of HBV vaccination, HBsAg detection, and immune escape (Coppola et al. 2015;Purdy 2007). The HBV coinfection with hepatitis C virus (HCV) is another important healthcare problem, contributing significantly to the burden of chronic liver disease (i.e., active hepatitis, cirrhosis, and hepatocellular carcinoma) (Konstantinou and Deutsch 2015). Globally, the HCV/HBV co-infection account for a substantial fraction of chronic liver disease patients (Liu and Hou 2006). Both viruses transmit vertically as well as horizontally (Wasley et al. 2010). The presence of both viruses increases the progression rate of liver disease and leads to a higher risk of the tumor than the presence of each of these viruses alone (Kurtz 1999). The objectives of the present study include identification of HBV genotypes and sub-genotypes isolated from Pakistani patients, based on the partial sequences of S gene. We also aimed to detect mutations in these regions that might result in the failure of HBV vaccination, HBsAg detection, and immune escape. Furthermore, we compared the genetic pattern at this locus between the HBV mono-infected and HBV/HCV co-infected cases.

Methods and Material Human Samples
This prospective study was approved by the Institutional Review Board & Ethics Committee of Dow University of Health Sciences, Karachi. Participating individuals were briefed about the project and informed consent was obtained from all the participants. All the experiments were performed in accordance with relevant guidelines and regulations including 'Declaration of Helsinki'. The study cohort included 49-mono-infection of HBV and 25 of HBV/HCV co-infected patients. Yellow cap tubes were used for blood collection, 200μl serum was separated and stored at -70 °C until further analysis.

DNA Extraction and Amplification
Total DNA was extracted from the patient serum using QIAamp DNA Mini Kit (Qiagen Germany), and quantified by 1% agarose gel electrophoresis, and spectrophotometric analysis. S-gene amplification was done by using the nested PCR technique. Sense primer, 5'-GTGGTGGACTTCTCTCAATTTTC-3' and antisense primer 5'-CGGTAWAAAGGGACTCAMGAT-3' were used first. The second round of PCR was performed with the sense primer 5'-CAAGGTATGTTGCCCGTTTG-3', and antisense primer 5'-AAAGCCCTGCGAACCACTGA-3'.

HBV DNA sequence analysis
The PCR products were purified and submitted for commercial DNA sequence services, Macrogen Korea. The quality of the sequences was checked by using the staden package and Finch TV. Trimming of sequences was performed by the Lasergene package v.7.1 (DNASTAR Inc UA) to yield high quality sequences draft.

Structure Analysis
The reference genomes (A-H) of (HBV were downloaded from the NCBI reference genotype tool (Rozanov et al. 2004). High quality trimmed sequence data was compared with the reference genomes. The DnaSP v6.0 package (Rozas et al. 2017) was used to calculate the sequence composition of parsimony informative sites and haplotypes. The Arlequin v3.5 software (Excoffier, Laval, and Schneider 2007) was used to find out the population genetic statistics, like Fst analysis (pairwise fixation index), analysis of molecular variance (AMOVA), and Nei's distance (DA) to estimate the nucleotide differences among the individual sequences.

Mutation Analysis of S gene sequences
The analyses of mutations that might be responsible for drug resistance, HBsAg detection, and vaccine escape were performed by the online tools, i.e. HIV-Grade: HBV-Drug Resistant Interpretation (DRI) (Neumann-Fraune et al. 2014) and Gen2Pheno HBV (Beggel et al. 2012a). The two tools differ in their approach to estimate whether a particular mutation is implicated in the detection, therapy, or vaccine escape.

Distance Correlation Matrix
A low genetic distance was observed between the sample sequences generated in present study against the reference genotype D through pairwise Fixation correlation index (Fst) ( Figure 1). Fst, the most widely used statistical method for studying population genetics, was employed to ascertain genetic structure of the sequences obtained from HBV mono-infected and HBV/HCV co-infected patients. A high correlation was observed between the sequences and the reference genotype D using Pearson construction matrix by R-package. Sequences from HBV mono-infected and HBV/HCV co-infected patients shared >98% similarity and clustered together with the genotypes D. However, other reference genotypes (i.e., A, B, C, E, F, G, H) showed less than <2% similarity with our sequences (Figure 1). Likewise, we found a high correlation of >90% between the sub-genotype D1 and our sequences from HBV mono-infected as well as HBV/HCV co-infected patients. Low genetic difference was also observed between our samples and reference sub-genotypes D3, and D9, showing more than >60% similarity. Other subgenotypes (D2, D4, D5, D6, D7, D8, D10) displayed significant distinction and exhibited <10% similarity with HBV mono-infected and HBV co-infected sample sequences ( Figure 2).

Figure 1. Correlation plot between various HBV genotypes and our samples based on the pairwise Fst values.
The Fst values for each genotype were calculated using the Arelquin software, and used the Pearson construction matrix for 'correplot' using R package. The plot shows hierarchical clustering and correlation constructed between the genotypes. The minimum genetic distinction value and high correlations are depicted by the dark brackets and large sizes. The Fst values for each genotype were calculated using the Arelquin software, and Pearson construction matrix for 'correplot' using R package. The plot shows hierarchical clustering and correlation between the subgenotypes. The minimum genetic distinction value and high correlations are depicted by the dark brackets and large sizes.
AMOVA was performed to elucidate the genetic structure to ascertain groups with the lowest variation. The AMOVA significances were interpreted with 99000 permutations. The low number of pairwise nucleotide difference (Nei's: DA) was found between the genotype D and HBV mono-infected, HBV/HCV co-infected sequences congruent to abovementioned pairwise analyses (Figure 3). The low genetic distance showed that Pakistani samples (both mono-infected and coinfected) belong to the HBV genotype D. Likewise, low mean pairwise population genetic differences (πxy) were observed between HBV mon-infected, HBV/HCV co-infected sequences and genotype D. Greater genetic differences (πxx) were found within the HBV monoinfected samples as compared the HBV/HCV coinfected group (Figure 3). Moreover, the HBV mono-infected and HBV/HCV coinfected sequences generated in current study displayed lowest genetic differentiation (i.e., DA, and πxy) to HBV sub-genotype, D1 (Figure 4). The genetic distance between our samples and the sub-genotype D3 was also found to be low, though it was higher than for D1 sub-genotypes. The sub-genotype D9 showed high similarity with our sequenced samples of HBV patients, although the genetic distance (DA) was higher than for sub-genotypes D1 and D3. Our samples showed high genetic distinction when compared to the other subgenotypes (D2, D4, D5, D6, D7, D8, D10) (Figure 4).

Mutational Analysis
A total of 24 samples (96%) showed mutations in HBV/HCV co-infected group as determined with the G2P tool, while mutations were found in all 49 samples in the HBV mono-infected group (Table 1). The most frequent mutation identified by G2P tool was T127P which was detected in 96% co-infected samples and 97.95% in mono-infected samples. The second most frequent mutation identified by the G2P tool in the mono-infected group was Y134F which was not detected in any samples of the co-infected group. Both I82S and P120S were the most common mutations in the co-infected group after T127P with a frequency of 8% each. P120S was absent in the mono-infected group while I82S was found at 4.08%. Likewise, the DRI identified mutations in 22 monoinfected HBV samples (45%), while mutations were found only in 7 out of the 25 sequences in HBV/HCV co-infected group (Table 2). E164G was the most frequent mutation observed in the mono-infected group at 6.12% which was not detected in the coinfected group. The most frequent mutations in the coinfected group were I82S (8%) and P120S (8%). While P120S was not detected in the mono-infected group, I82S was observed in the mono-infected group at 4.08%.  The graph compares genetic structures of various HBV reference sub-genotypes with our HBV mono-infected and HBV/HCV co-infected samples. It shows the genetic difference based on the three different statistical method, between samples-πxy (green above diagonal); within-samples-πxx (orange diagonal); and the net number of nucleotide difference among samples-DA (blue below diagonal).

Discussion
This study was conducted to determine the sequencebased genotypes/sub-genotypes and mutations in the small S gene of HBV mono-infected and HBV/HCV coinfected patients from Pakistan. The results show that the most common genotype and sub-genotype in Pakistani patients were D and D1 respectively (Figures  1 & 2). Previous studies from Pakistan also reported a high prevalence of genotype D in most of regions of Pakistan (Baig et al. 2007;Alam et al. 2007;Noorali et al. 2008), except in the Punjabi population where genotype C is mostly prevalent (M, S, and S 2004). About 1.6% infected patients with HBV in Pakistan show multiple genotypes (Zeng et al. 2004). The genotype D causes a more severe disease (Kao, Liu, and Chen 2002) and is less responsive to the interferon treatment as compared to other genotype such as A and B (Sablon and Shapiro 2004;Erhardt et al. 2000). Previous reports of HBV genotype prevalence shows that B and C are the predominant genotypes in South Asia outside Pakistan and Afghanistan. For example, the predominant genotype in Afghanistan is D (Amini- Bavil-Olyaee et al. 2006), while India shows a mixed genotypes pattern with mostly A, C, and D genotypes (Thakur et al. 2002;Gandhe, Chadha, and Arankalle 2003;Kumar et al. 2005). In fact, seven genotypes (A-G) of the HBV were found in Asia (Toan et al. 2006).

Mutations
Mono-infected Co-infected The genotype identification in our analysis was accomplished based on the small S gene because it is a more stable and conserved region as compared to the other Pre-S genes (Lindh et al. 1998). The S gene is also an important part of the coding region, helping in the formation of the HBV virions (Chotiyaputta and Lok 2009;Hsu and Yeh 2011). Our analysis of the small S gene sequences using pairwise genetic comparisons shows that the genotype D had low genetic difference among the investigated sequences. Likewise, the subgenotype D1 exhibited the minimum genetic difference against the HBV mono-infected and HBV/HCV coinfected sequences obtained in the Pakistani individuals while the genotype D3 and D9 showed moderate genetic differences (Figures 3 & 4). The S gene is responsible for HBsAg that includes a major hydrophilic region (MHR). Mutations in the MHR are associated with the vaccine escape. This happens due to the mutations in the MHR region leading to conformational changes in HBsAg, reducing its affinity towards antibody to HBsAg and resulting in the immune escape (Tian et al. 2007;Cooreman, Leroux-Roels, and Paulij 2001) The genotype identification in our analysis was accomplished based on the small S gene because it is a more stable and conserved region as compared to the other Pre-S genes (Lindh et al. 1998). The S gene is also an important part of the coding region, helping in the formation of the HBV virions (Chotiyaputta and Lok 2009;Hsu and Yeh 2011). Our analysis of the small S gene sequences using pairwise genetic comparisons shows that the genotype D had low genetic difference among the investigated sequences. Likewise, the sub-genotype D1 exhibited the minimum genetic difference against the HBV monoinfected and HBV/HCV co-infected sequences obtained in the Pakistani individuals while the genotype D3 and D9 showed moderate genetic differences (Figures 3 &  4). The S gene is responsible for HBsAg that includes a major hydrophilic region (MHR). Mutations in the MHR are associated with the vaccine escape. This happens due to the mutations in the MHR region leading to conformational changes in HBsAg, reducing its affinity towards antibody to HBsAg and resulting in the immune escape (Tian et al. 2007;Cooreman, Leroux-Roels, and Paulij 2001).
The G2P and DRI are the two most frequently employed tools to study drug-resistance, detection and vaccine escape mutations in the HBV genome (Neumann-Fraune et al. 2014;Beggel et al. 2012b). Analysis by G2P exhibited a prevalence of the HBsAg mutations in HBV/HCV samples at 96%, while DRI showed mutations in 14.3% samples. In HBV monoinfected samples, the G2P showed that 45% samples have mutations while DRI detected mutations in all samples. These results may have implications for patients who have a false-negative diagnosis of HBV due to these mutations. Such cases may contribute to the development of chronic carriers (Foy et al. 2012). Several HBsAg mutations (Table 1 & 2), observed in this study, have previously been shown to have a significant role in vaccine, and immune escape (i.e., sD144A, sY100C, sP70T, sY134N, sI110L, sQ129H, sY134F, sS143T, sV184A, sT189I, sT127P) (Sayan et al. 2010;Verheyen et al. 2012;Pal et al. 2013;Coppola et al. 2015;Hosseini et al. 2019). The frequency of some of the mutations such as sT127P was very high both in HBV mono-infected (48 out of 49 or 98%,) and HBV/HCV coinfected samples (24 out of 25 or 96%). While each of the mutations, sQ129H, sD144A, sP120S, and sY100C has been found in <3% samples, these mutations are reported to have clinical significance in vaccine escape (Ngui et al. 1997;Luongo et al. 2015;Ireland et al. 2000;Lee et al. 2001;Lazarevic 2014). Several mutations detected in our study are not previously reported for any role in vaccine escape and require further investigation to elucidate their role(s) in this phenomenon, if any. Around, 3-5% Pakistani population is infected with the chronic carrier HBV infection that incurs substantial treatment costs (Ali et al. 2011;Abdullah et al. 2019). The commonly used drugs for the treatment of HBV infection in Pakistan are Adefovir, Tenofovir, Entecavir, Telbivudine, and Lamivudine (Abbas et al. 2010). In our analysis, a single mutation (A181S) which is previously associated with drug-resistance to Adefovir in the HBV mono-infection individuals, was observed in 4% samples. No other mutation found in our analysis is previously linked with resistance to the actions of Tenofovir, Entecavir, Telbivudine, and Lamivudine. However, due to the limited sequenced sample size, these results may not be good enough for clinical decision-making. Additional studies with large sample size and proper patient follow up with respect to treatment response and efficacy are necessary before generalizing these results. We conclude that the HBV genotype and sub-genotype most prevalent in Pakistani patients with HBV monoinfection as well as HBV/HCV co-infection, are D and D1, respectively. Some of the mutations found in the small S gene during our analysis have roles in detection, treatment, and vaccine escape. However, further studies are necessary to reproduce and authenticate these findings.