Chronic hepatitis is among the most common viral diseases. In endemic regions, it can cause cirrhosis and HCC [1–2]. Higher DNA levels are associated with a greater risk of liver disease in CHB patients [3]. Since DNA replication is continuously suppressed during therapy for chronic hepatitis, the goal is to delay or halt the progression of liver disease [4].

The development of viral resistance presents a major challenge to ongoing therapy with nucleotide analogs [NAs], despite the drugs' high potency in blocking HBV [5, 6]. Mutations in the reverse transcriptase [RT] domain of the polymerase gene lead to NA resistance [7]. While entecavir and tenofovir effectively suppress DNA replication with minimal drug resistance, adefovir and telbivudine are better than LAM because they have a lower rate of viral resistance [8].

Lamivudine, the first of these medications to receive global approval, inhibits viral replication by disrupting RT activity, reduces disease activity, improves liver histology, and slows disease progression [9, 10]. Resistance to NAs results from mutations in the RT region of the polymerase gene. It has been demonstrated that Lamivudine lowers DNA levels, increases seroconversion rates [11], and boosts serum ALT levels and liver histological markers [12]. The drug's effectiveness against mutated strains may decrease due to these mutations [13, 14]. Additionally, the reverse transcriptase region of the polymerase gene contains the FLLAQ motif, which, along with YMDD, may contribute to lamivudine resistance [15].

Even in untreated CHB patients, natural RT mutations have been observed [16]. HBV genotypes may influence the effectiveness of antiviral treatments and response to Lamivudine in CHB patients [17]. As a result, examining the YMDD motif in CHB patients may provide clinicians with important information for developing appropriate medications. The current study was conducted to assess polymerase gene mutations in three generations of CHB patients and to identify similar mutations related to their susceptibility to antiviral drugs.

Transient elastography was used to assess liver stiffness at the end of the study using the Fibro Scan 502 device [EchoSense, Paris, France, and 5 MHz]. At least ten measures were taken for each subject, and their median value was recorded. Values were regarded as valid if the interquartile range [IQR] was less than 30% of the median value [18].

HBV DNA was extracted from 200 µl of each plasma sample using the High-Pure Viral Nucleic Acid Extraction Kit according to the manufacturer's instructions. Real-time PCR measured HBV DNA levels in serum samples [AltonaStar® HBV PCR Kit, Germany]. Amplification components for the PCR process, such as DNA polymerase, buffer, magnesium salt, probes, and primers, were added to 25 µL of each sample [eluate from the nucleic acid extraction]. The Rotor-Gene QIAGEN was used to perform the thermal cycling, which included the initial denaturation of the extracted DNA at 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 s, 58°C for 45 s, and 72°C for 15 s [18].

PCR and Partial Amplification of the polymerase gene

following primers produced 645bp at nucleotide positions 376–395 (5՛-GATGTGTCTGCGGCGTTTTA-3՛) for the forward primer and 1000–1021(5՛-CAGCAAAGCCCAAAAGACCCAC-3՛) for the reverse primer. The PCR method was conducted based on the last study [Applied Biosystems Simpliamp Thermal Cycler]. PCR products were processed and sent for direct sequencing by an automated system [3130 Genetic analyzers, ABI/HITACHI]. Using the reference sequence registered in the Gene Bank database [AB033559] and the Blast, Chromas, and MEGA version 11 software, sequences were checked, and then statistical analyses were carried out by SPSS software [version 20] [18, 20].

It was also shown that the children of the three-generation and two-generation groups had the highest levels of DNA. The average HBV DNA in patients showed that mothers in the three-generation group (6526 ± 769 IU/ml) had the lowest average DNA. In addition, the patients' ages and HBV DNA showed a statistically significant difference (P-value = 0.04) [Table 1]. The positive HBeAg Individuals had significantly higher baseline levels of DNA than patients who tested negative (P-value = 0.02).

Based on sequence analysis, the three-generation group had the highest percentage of sequence similarity [91.2%]. However, grandmothers also had drug-resistant mutations despite not using antiviral drugs and not being vaccinated. When the sequences of the patients were compared to the standard sequence, it was found that 9% of CHB patients had no mutations. Our research shows that in none of the three groups is there a statistically significant connection between viral load or LSF and mutations in the P gene. In 42.2% of patients, DNA polymerase drug resistance mutations were identified, including rtM204I/V, rtN236T, rtM250V, rtL180M, rtT184G, rtM207I, rtS202I, and rtM204V/I. Among all variants, rtM204V/I had the highest prevalence rate at 4.5%, which was higher than the prevalence rates of rtL180M, rtN236T, rtM250V, rtT184G, rtS202I, and rtV207I.

Many anti-HBV drugs act by blocking polymerase. Therapy of LAM for an extended period of time may result in YMDD mutations and drug resistance, reducing treatment efficacy [22]. Mutations in RT resulting in amino acid changes, known as primary and secondary drug resistance, are strongly linked to developing viral resistance to NAs [23]. While secondary mutations result in amino acid changes that repair the replication defect caused by primary resistance and may result in a moderate degree of reduced susceptibility, primary mutations impair susceptibility to an antiviral drug [16, 24]. The current research revealed that the two-generation group had the highest rate of YMDD drug resistance mutations (26.7%). Even though they had never been vaccinated or taken antiviral drugs, the grandmothers in the three-generation group also had mutations linked to treatment resistance, such as YMDD. The two-generation group exhibited the greatest reported rates of treatment resistance mutations (50%) and had received immunizations and antiviral medications in the previous 1–3 years.

Pretreatment DNA levels and HBeAg status are independent variables linked to rtM204 I/V mutations, according to a multivariate study that showed ALT and baseline genotype were also related to a faster incidence of YMDD mutations [13, 25]. One of the most important variables in lamivudine resistance mutations is the association between the types of mutations and the genotype of the virus in the population [25]. Pan et al. discovered that the prevalence of reverse transcriptase mutant rt204I was lower in genotype B than in genotype C among 53 HBV-resistant individuals, but rt204V mutations were greater in genotype B than in genotype C. HBV DNA levels, HBeAg status, and HBV genotype were found to be independent factors associated with shorter lamivudine resistance time in this study [26]. Unlike in the previous research, the genotype of all CHB patients was D/ayw2, and the rt204I mutation was more particularly in the two-generation group. Furthermore, DNA levels were greater in HBeAg-positive individuals, and this mutation was also seen in these patients. It should be mentioned that genotype D is one of the genotypes with the YMDD mutation.

Lazarevi et al. and Delaney et al. discovered three major resistance-associated variants: rtM204V, rtM204I, and rtA181T, as well as two compensatory mutations, rtV173L and rtL180M. The existence of resistance-associated mutations was linked with age, the presence of clinically significant HBsAg mutations, and higher viral loads [3, 27]. Suppiah et al. showed that L180M/V and M204I were the most often found (80%) and linked with Lamivudine in conjunction with emtricitabine and telbivudine drug resistance in another study [14]. rtA181T and rtV173L mutations were not found in any of the analyzed cases, while rtM204V, rtM204I, and rtL180M were detected, with the greatest frequency in the two-generation group. This mutation at codon rtA181T was not particularly common in the studied population, but because it is also selected during adefovir therapy, it may be responsible for cross-resistance between these two NAs. In addition, the mutations described in the research by Suppiah et al. were associated with the concurrent use of Lamivudine and telbivudine, which contradicts the findings of our investigation. They were related to the use of Lamivudine alone.

A survey on HBsAg-positive patients has reported that all patients with lamivudine resistance have the YMDD and FLLAQ mutation, which affects the RT domain of the polymerase gene [29]. Dolati et al. found that among the analyzed samples, drug-resistant variations were identified in the majority of chronic Hepatitis B and HIV co-infected individuals. In the majority of cases, YIDD and FLMAQ mutations were observed [20]. Lee et al. and Rezanezhadi et al. demonstrated that YMDD mutations were found in HBeAg-positive/negative patients, some of whom had YMDD mutations after therapy [29–31]. Consistent with the research, the current investigation found that HBeAg-negative patients in three groups had resistance mutations YMDD, L180M, and FLLAQ. Also, M204I and L180M mutations; M204V + L180M and M204I + M204V mutations were found in the majority of patients. Additionally, rtL180M, rtN236T, rtM250V, rtT184G, rtS202I, and rtV207I mutations were reported. In the study carried out by Zhao et al., 8.9% of patients had mutations in DNA polymerase, and those mutations included rtM204I/V, rtN236T, rtM250V, rtL180M, rtT184G, rtM207I, rtS202I, rtM204V/I + rtL180M, and rtM204I + rt250V [32]. In our findings, the L180M mutation alone provides minimal resistance to Lamivudine. Despite this, the combination of M204V/M204I causes resistance owing to enhanced RT and transcription activity. In addition to sharing the mutations with other family members and the infectious source with them, grandmothers who had resistance mutations may have acquired these mutations over time due to a long history of infection.

In conclusion, the majority of chronic Hepatitis B patients had identifiable drug-resistant variations, and more than half of those receiving long-term therapy were resistant to Lamivudine (1–3 years). Additionally, the presence of similar mutations in the grandmother, mother, and child, especially similar treatment resistance mutations, indicated that these individuals were infected from the same source. As a result, we suggest that clinicians create a distinct HBV treatment protocol before beginning therapy and that drug resistance mutations in HBV be evaluated in a broader statistical population.