Evaluation of the pol/S Gene Overlapping Mutations in Chronic Hepatitis B Patients in Northern Cyprus

Hepatitis B virus (HBV) affects more than 2 billion people worldwide and approximately 247 million chronic individuals are known to be chronic carriers (Caligiuri et al. 2016; WHO 2018). HBV can cause severe liver infections and cirrhosis may develop in 15–40% of individuals if they are not treated (Tang et al. 2018). There are several novel treatment strategies that have been used for the treatment of chronic hepatitis B infection in order to prevent the risk of developing liver failure, cirrhosis, and cancer (Lapiński et al. 2013). Interferon alpha (IFN-α) 2a- 2b, PEGylated interferon-α-2a (Pegasys), PEGylated interferon-α-2b (Pegintron), and nucleos(t)ide analogues (NAs) are worldwide used for fighting chronic hepatitis B (CHB) infection by suppressing HBV replication (Ward et al. 2016). Lamivudine (LAM), telbivudine (LdT), entecavir (ETV), adefovir (ADV), and tenofovir (TDF) are antiviral agents that have been approved in Europe, America and many Asia-Latin America countries for the treatment of chronic HBV, however, there is a big concern of long term use of these agents as they are associated with the development of antiviral resistance mutations (Sun et al. 2016; Ozguler and Sayan 2016; Zhao et al. 2016; Ozguler and Sayan 2018). These mutations are associated with the amino acid substitutions in the reverse transcriptase (RT) domain and are classified as primary or secondary compensatory resistance mutations (Rugieri Pacheco et al. 2017). Mutations at the amino acid positions rt169, rt181, rt184, rt194, rt202, rt204, rt236, rt250 are classified as the primary resistance mutations which are associated with reducing the sensitivity to antivirals, whereas, the secondary compensatory resistance mutations occur at the amino acid positions rt80, rt173, rt180 and generally restore the replication capability of viral polymerase (Choi et al. 2018).

HBV is characterized by its high replication capacity (> 10¹² virion/day) and a lack of proofreading activity during replication (10⁻⁵ substitution/base/cycle). This means that each nucleotide on the HBV genome can be changed within a day and cause antiviral resistance-related mutations before treatment (Tezcan et al. 2015). Also, the S gene is completely overlapped by the pol gene. Because of this overlap between the S and the pol gene, changes that are related to antiviral resistance in the pol gene may also occur in the S gene as well (Sayan et al. 2012). This overlap leads to amino acids structure, found in surface antigen (HBsAg) of HBV to change and thus form naturally occurring antiviral resistance mutations. The vaccine escape, diagnosis escape, hepatitis B immunoglobulin (HBIg) escape, and immune escape variants against oral antivirals that are used in chronic HBV treatment may emerge. Another reason for the emerging of these mutations is the occurrence of the mutant viruses deriving from antiviral-treated patients (Zhao et al. 2016). These changes cause reactivation of HBV in vaccinated people and concerns during diagnosis and/or HBIg vaccination failure (Wang et al. 2017; Asan et al. 2018).

Cyprus is an island located in the Mediterranean Sea and has been divided into two communities (Northern and Southern Cyprus). Northern Cyprus, officially the Turkish Republic of Northern Cyprus, is a multinational society due to universities, casinos, luxury hotels, beaches, and other entertainment centers; thus, there are many people coming from foreign countries for education, business and/or tourism purposes. For HBV infection, the prevalence rate ranges from 1.2% (160/13 892) (Arikan et al. 2016) to 1.35% (339/25 442) (Guler et al. 2018). For Southern Cyprus, this rate was given as 0.77% and 1.01% (Altindis et al. 2016). Although there are few studies regarding the prevalence, up to now, there has been no publication on drug resistance in patients infected with HBV in Northern Cyprus (Altindis et al. 2006; Suer et al. 2014). Therefore, we aimed to show the presence of resistance mutations and their clinical significance in untreated chronic hepatitis B patients in Northern Cyprus.

The current study consisted of HBsAg positive-treatment naïve 100 CHB patients, of whom 13 were female and 87 were male, whose ages varied between 18–65 ages (with a median of 35 years). The origins of the patients were from Asia (68%), Africa (29%), North America (1%) and Europe (2%), and none of them have been taken antiviral therapy when their serum samples were collected. Ninety-six percent and 5% of the study group were positive for anti-HBc IgG and HBeAg, respectively. The serum HBV DNA level was calculated as a median of 1.0 + E7 (range: 1.9 + E1 – 2.8 + E8). ALT and AST levels were reported as 23 ± 19 and 28 ± 19, respectively. We sequenced only the samples of 68 patients in this study because HBV DNA was determined below 1000 IU/ml in 32 (32%) patients’ samples. The distribution of genotypes of the sequenced patients (68/100) was determined as D1 (n:48, 70.6%), D2 (n:4, 5.9%), D3 (n:1, 1.5%), A1(n:5, 7.3%), A2 (n:2, 2.9%), and E (n:8, 11.8%) (Arikan et al. 2016). Demographic and laboratory findings of the study are displayed in Table II.

In the pol region, there was no primary drug resistance mutation detected in this study. There was only one treatment-naïve patient (1.5%) who had the partial resistance mutation at rt173M amino acid substitution that may be related with LAM and LdT resistance. Twenty-five patients (36.7%) had secondary/compensatory mutations and the mutations patterns (rtL91I, rtQ149K, rtQ215H/P/S and rtN238D) were depicted in Table II.

In the S region, 30 (44%) patients had typical HBsAg escape mutations. Vaccine escape, diagnosis escape, HBIg, immune escape mutations were determined in 7/68 (10%), 3/68 (4%), 4/68 (6%), 16/68 (24%) samples of the patients, respectively and these mutations are presented in Table III.

There were also six different combined mutations. The mutation patterns were sI110L + sS193L, sP120L + + sT123N + sT126I + sA128V + sY134H + sD144E + +sG145A, sT118A+sP127T, sI110L+sP120T+sD144A+ +sG145R and sP120R+sC121Y+sT131I + sS132P. The resistance mutation patterns are given in Table III.

Even in treatment naïve patients because of the high replication capacity and the overlapping reading frames, antiviral resistance mutations may occur throughout the HBV genome (Yano et al. 2015). To our knowledge, this is the first study that focused on the resistance mutations in the HBV pol and S gene regions in treatment naïve CHB patients in Northern Cyprus. This information has confirmed that there was no primary drug resistance mutation in untreated patients. So, even if patients undergo long-term therapy with NA, they may not require periodic monitoring for the primary drug resistance. The previous studies indicated that primary resistance mutations could occur not only in patients undergoing an NA therapy but also might be detectable in patients receiving any treatment (Bottecchia et al. 2016; Zhao et al. 2016; Asan et al. 2018). The mutations that occur mainly at the amino acid positions 181, 204, 233 and 236 have been shown to be related with the primary resistance mutations in the treatment naïve patients (Asan et al. 2018). However, the mutations at the amino acid positions rt184, rt194, rt202, rt250 are mainly classified as partial resistance mutations and often act as compensatory mutations. The main functions of primary and partial resistance mutations are to reduce drug susceptibility and restore the viral replication defects, respectively (Asan et al. 2018). Thereof, the drug resistance should be analyzed during and/or before initiating the treatment to reduce the risk of liver damage and consequently progression of hepatocellular carcinoma. Sayan and his colleagues from Turkey have shown in many studies that acyclic phosphonate related mutations could occur in CHB patients (Sayan 2010b; Asan et al. 2018; Ozguler and Sayan 2018). Similarly, resistance mutations have been identified from other studies conducted in China and Pakistan (Mahmood and Anwar 2017; Zhao et al. 2016) which supports that primary resistance mutations can occur all over the world.

In the current study, we detected rtV173M partial resistance mutation in one patient according to Geno2pheno Drug Resistance Program, however, the rtV173L substitution has been reported more commonly in the literature (Lin et al. 2012; Gürsoy et al. 2019). Both amino acid substitutions are regarded as compensatory mutations and enhance viral fitness (Asan et al. 2018). The single compensatory mutation profiles are generally associated with low-level resistance; however, they may cause high-level resistance when they combine with other patterns (Lazarevic 2014). Thus, screening to detect resistance may be regarded to be a benefit in the control of CHB. Sayan has shown in one of his studies that rtV173L mutation combined with sD144E produced HBV vaccine escape +HBIG escape (Sayan and Bugdaci 2013). LAM-associated resistance triple mutation pattern (rtV173L + rtL180M + rtM204V) has also been shown to enhance viral replication compared with rtL180M + rtM204V (Sheldon and Soriano 2008).

In the current study, among 68 patients, there were 25 (37%) secondary/compensatory resistance mutations (rtL91I, rtQ149K, rtQ215H/P/S, rtN238D) which are related to LdT, LAM, and ADV resistance (Saran et al. 2017; Asan et al. 2018). It may be a result of naturally emerging mutations due to the biology of HBV. The most common compensatory mutations have been reported to be rtQ149K, rtQ215H/P/S and rtL91I (Asan et al. 2018). These compensatory mutations are important as they assist viral replication and fitness, and hence are associated with drug resistance (Ahn 2015). In our study, we determined one of the most commonly detectable compensatory mutation rtQ215/H/P/S, which is associated with LAM and ADV resistance (Shaw et al. 2006; Altindis 2016; Asan et al. 2018).

Mutations in the pol gene, due to the structure of HBV, also occur in the overlapping HBsAg (Zaaijer et al. 2007; Simon et al. 2013) and some typical HBsAg escape mutations emerge. The immune response escape, HBV vaccine escape, HBIg escape, and HBsAg diagnosis escape were detected in 44 patients in this study. sP120T, Sm133I, Ss143L, sD144A/E, sD145R and Se164D are the most frequently detected typical HBsAg escape mutations in CHB patients (Sayan et al. 2010b). Misdiagnosis in the HBsAg, failure to prevent with vaccination and HBIg, reactivation of hepatitis B, and re-infection in HBV infected recipients and/or orthotropic liver transplantations may be a consequence of the typical escape mutations (Asan et al. 2018). All patients that receive or not receive antiviral treatment should be monitored for these mutations.

In conclusion, these findings on drug resistance mutations show that treatment-naïve CHB patients have low HBV polymerase resistance mutation (1.5%) rate. Hence, primary drug resistance analysis may not be necessary to be performed before the initiation of antintiviral therapy; however, we have discovered a novel compensatory mutation, rt173M that may act as a primary mutation together with other patterns. To draw attention, we preferred to define this mutation as a partial resistance mutation rather than compensatory resistance mutation in this study. Additionally, HBsAg escape mutants in patients living in Northern Cyprus demonstrated us that early detection of possible S gene mutations may be beneficial and important to monitor the surveillance of S variants. Even though national compulsory HBV vaccination program, these mutants may develop in hepatitis B vaccinated and/or HBIG vaccinated individuals with CHB, in chronic carriers and/or patients undergoing antiviral treatment and may pose danger for public health. In addition, due to the arrival of people from many countries to North Cyprus for the reasons we have stated, drug resistance mutations should be analyzed as pol and S gene together for monitoring CHB.