The Orthohepadnavirus genus is a significant group of human viruses, and the Hepatitis B virus (HBV) is a prototype of the Hepadnaviridae family ( Summers et al., 1975). Globally, it is thought that more than 300 million people are infected with HBV, which can lead to chronic liver disease and hepatocellular carcinoma (HCC), which accounts for over 1 million annual fatalities ( Musyoki et al., 2015). The Baltimore virus taxonomy places HBV in the class VII of viruses ( Gust et al., 1986). It is an enclosed virus with 3200 nucleotide base pairs of partially antagonistic relaxed and circular double stranded DNA (RC-dsDNA), and it replicates through an intermediary ssRNA ( Liu et al., 2021). Hepatocytes are the cells that the hepatitis B virus infects to cause liver infection, which can then clinically show as symptomatic or asymptomatic acute hepatitis marked by liver inflammation to fulminant hepatitis. Acute HBV hepatitis that is not treated may progress to chronic HBV hepatitis, which can cause cirrhosis and HCC ( Liang, 2009). Many tests, including clinical, biochemical, serological, and molecular approaches, are used to detect HBV infection, illness associated with HBV, and to differentiate between acute and chronic infections. The hepatitis B surface antigen (HBsAg), the hepatitis envelope antigen (HBeAg), the antibody to the hepatitis surface antigen (anti-HBs), the antibody to the hepatitis B core antigen (anti-HBc), and the immuglubullin antibody sub-class of the anti-HBc are the HBV antibodies and antigens that are determined by the serological testing (IgM anti-HBc). The key clinical marker for acute or chronic infection, prevalence, and endemicity of HBV infection is the presence of HBsAg, which can be identified two weeks after exposure. By identifying the qualitative and quantitative HBV-DNA, molecular methods can diagnose HBV. Based on the entire genome genetic variability of roughly 8%, HBV genomes have been divided into ten primary genotypes designating A to J ( Tatematsu et al., 2009). The lack of a viral polymerase proofreading mechanism during viral replication causes the genetic heterogeneity ( Rahman et al., 2016). Moreover, immunization and antiviral medication have an external and internal impact on HBV sequence heterogeneity. Geographical differences in the distribution of the various HBV genotypes, however, influence the pathogenicity of HBV and the administration of treatment. Due to their shared method of blood-born transmission, HBV, and the human immunodeficiency virus (HIV) frequently co-infect individuals ( Audsley et al., 2010). Depending on where HBV is endemic, different geographic areas have different common pathways of transmission. Lamivudine (3TC) is used in the treatment of HBV and HIV ( Benson et al., 2009). However, due to the development of drug resistance after 4 years of single-drug treatment, HIV-infected persons should not receive 3TC or emtricitabine (FTC) monotherapy for HBV infection ( Di Martino et al., 1999). The emergence of the mutation rtm204 is a hallmark of 3TC resistance (YMDD mutation). The 3TC should only be administered to individuals on totally suppressive ART since it accelerates unchecked HBV DNA replication and raises the risk of death from HBV infection or illness. HBV prevalence has been documented in several studies among HIV-positive people from Durban, KwaZulu-Natal. However, investigations on the HBV genotype, HBV vaccine escape, and treatment resistance are still scarce in this field and most studies have concentrated on the seroprevalence. To ascertain the prevalence and genotypic characteristics of HBV among HIV-infected people in Durban, we undertook this study.

In the current study, the HBsAg seroprevalence in HIV-infected patients was 100%. The HBsAg prevalence in HIV-infected people is higher in this study as compared to previous studies in South Africa, with an estimation between 3% and 23% reported from the Western Cape, KwaZulu-Natal, and Gauteng provinces ( Andersson et al., 2013; King & Hagemeister, 2016; Lukhwareni et al., 2009; Maponga, 2012; Mphahlele, 2008; Msomi et al., 2020; Samsunder et al., 2019). The HBsAg seroprevalence from this study is not coherent with published data from South Africa; comparing with previous studies, they used larger sample cohort in their studies, whereas our study used a small sample size, which influenced the high prevalence rate. However, the study report supports previous studies that endemic HBV is still present in South Africa and that HBV seroprevalence among HIV-infected people is presently high among adults ( Amponsah-Dacosta, 2021; Maepa et al., 2022). All the HBsAg-positive samples were subjected to PCR amplification of the HBV DNA. The partially overlapping surface/polymerase gene region was successfully amplified in 78% (41/50), confirming the active replication of HBV and HBV/HIV co-infection on the input study samples. The presence of HBV/HIV co-infection is attributed to the shared route of viral transmission since both viruses share a similar route of transmission. Furthermore, it was also reported that females were the majority infected with HBV infection at 66% (N=33/50) compared to their male counterparts at 34% (N=17/50) in this population. This does not correlate with the findings of ( Kew et al., 2008), who reported a prevalence of 4% in women compared with 9% in men. Similarly ( Moonsamy et al., 2022), reported higher prevalence rates in males than females aged 25 to 49 years. The phylogenetic analysis shows the predominance of genotype A from our study sequences. The identification of HBV genotype A is coherent with previous studies in South Africa ( Kimbi et al., 2004; Kramvis, 2014). The patient’s sequences were further identified as sub-genotype A1 by Geno2Pheno, indicating that HBV showed significant diversity, suggesting an African origin. The HBsAg serves is part of the S region which is employed in the development of recombinant vaccines; this region consists of α, β and T–cell epitopes. The “α” epitope is the primary area that neutralizing antibodies attacks and it contains the β and T–cell epitopes ( Caligiuri et al., 2016). The “a” determinant, also known as the primary hydrophilic region of HBsAg, is the collection of B cell epitopes and is made up of the amino acids 124–147 ( Liu et al., 2017). The “α” epitope domain of HBsAg may undergo mutations due to nucleotide and amino acid substitutions, which may result in structural and functional alterations in the S protein ( Zuckerman & Zuckerman, 2003). This could alter the antigenicity of the vaccination and result in mutations that escape the immunization, allowing mutant HBV to replicate in the body of the vaccinated population ( Yan et al., 2017). We determined the mutations on the partial surface gene of HBV/HIV co-infected individuals from our study. A total of 18 nucleotide substitutions were observed within the HBsAg region in all 38 partial surface sequences. The study reports mutations at sequence locations at the at α, β and T–cell epitopes that have clinical ramifications. The primary hydrophilic region (aa79 to aa147) of the surface antigen had the “α” epitope and contained four mutations (K122R, F134L, T143S, and S117N). G145R, S207N, and Y200T variations were found in the "T" epitope, whereas P127T was found in the “β” epitope of the “α” determinant region. The following mutations were found to be absent from the “α” epitope: E164D, L209V, Y206H, A194V, P70H, P127L, T189I, S204R, and F129T. We found escape mutations from vaccines including P127T, G145R, S207N, Y200T, Y206H, and L209V. Among 27 samples, the S207N mutation was the most prevalent one. P127T has been linked to vaccination escape selection in the past ( Colagrossi et al., 2018); which contradicts the findings of Kuzin et al., (2012), who reported this mutation to causing diagnostic failure. The clinical implications of this mutations must further be explored in vitro. One patient sequence contained the mutation G145R, which has already been identified by ( Colson et al., 2007; Yan et al., 2017). In the “β”-cell epitope of HBsAg, the G145R mutation is a significant vaccine escape mutation that is linked to a point mutation that results in the substitution of arginine for glycine at position 145 (G145R). The HBsAg antigenicity is decreased because of the interference with antibody binding ( Kao & Chen, 2002; Su et al., 2008). Given that it was the first vaccine-escape mutation discovered in children who received vaccinations in Italy, the G145R mutation is a typical mutation ( Zanetti et al., 1988). Its occurrence and stability have increased over the years with an increase of HBV endemicity and use of universal immunization ( Caligiuri et al., 2016). This mutation is crucial in causing failure on the detection of HBV by serological routine assays ( Cooreman et al., 2001). The long-term success of mass vaccination, however, has been previously reported to be threatened by G145R, which has grown more dominant with vaccine escape selection ( Yan et al., 2017 ). Thus, it is advised that it be incorporated into the design of future vaccines ( Yan et al., 2017). It is said that this mutation has a possible horizontal transmission pattern ( Colagrossi et al., 2018). Globally, the G145R mutations have previously been linked to HBV breakthrough infections in the population who had received vaccinations ( Cooreman et al., 2001; Theamboonlers et al., 2001). On the other hand, mutations G145R and F143S have also been discovered in people with occult hepatitis B infection (OBI), and they have been associated to the clinical implications. On the other hand, mutations G145R and F143S have also been found in individuals with occult hepatitis B infection (OBI), and they have been linked to the clinical implications of diagnostic failure due to suboptimal HBsAg detection when using commercial ELISA kits ( Coppola et al., 2016). It was previously reported as a nucleos (tide) analog (NA-induced) immune escape mutation ( Colagrossi et al., 2018; Lai et al., 2003), demonstrated that the E164D result from the substitution change in the overlapping polymerase region caused by the mutation rtM204V, rtM204I, and rtV173L) ( Lai et al., 2003; Zöllner et al., 2001). E164D was another significant mutation discovered from this study. These mutations produce a decrease in anti-HBs and are linked to lamivudine resistance therapy ( Colagrossi et al., 2018). Additional mutations found in this study included K122R, T143S, Y206H, L209V, S207N, Y200T, and P70H. Nevertheless, there is little information available about these mutations’ effects on surface protein structure and activities, and more research is needed to determine how they may affect patients’ health. K122R and T143S mutations, on the other hand, have reportedly been linked to diagnostic adequacy ( Cooreman et al., 2001). With certain ELISA diagnostic methods failing to identify HBsAg from samples bearing the K122R in prior research ( Malik et al., 2012). All the HBsAg mutations mentioned above have clinical implications that are connected to immunological escape and have an impact on the antibodies’ ability to recognize HBsAg. whilst others are connected to diagnostic failure. Antiviral medications that prevent reverse transcription specifically target the HBV polymerase to prevent HBV DNA replication. The nucleoside/nucleotide analogues LMV, ADV, TDF, LdT, and ETV are antiviral medications that are used to treat HBV polymerase. Despite the introduction of TDF, lamivudine treatment is still favoured among patients with HBV/HIV co-infection in South Africa. Antiviral medications may potentially contribute to the development of drug resistance due to selection pressure during long-term use of the antiviral treatment, in addition to the high mutation rate caused by the HBV error-prone ( Fares & Holmes, 2002). The frequency of drug resistance-related mutations in the RT region was 50% (N=7/14). M204V, L180M, V163I, and S202K were mutations linked to LdT resistance; S202K also conferred ETF resistance. Combining L180M and M204V results in increased cross-resistance to other nucleosides and decreased sensitivity to ETV but not ADV. It may also result in vaccine escape mutations in the overlapping S-region, which would stop HBsAg from secreting ( Sheldon et al., 2005). ADV resistance mutations included the I253Y, I223V, and M250I mutations. A single sample from one patient that contained the mutations L180M, M204V, S202K, and M250I was shown to have several drug resistance mutations. All the drug resistance mutations found in the RT of the pol region were found in genotype A sequences. Moreover, the following compensatory mutations were found: S202K, Q125E, L217R, V124A, V204I, I253Y, T128A, and S105T. We still need to investigate how these changes affect RT functions. As this study found no mutations linked to tenofovir resistance, we advise using a tenofovir-based regimen to treat HBV in people who also have HIV. At P>0.05, there was no statistical difference between the mutations linked to medication resistance. The Pearson Correlation was used to conduct a correlation test between the SHB and RT mutations. The connection between SHB and RT mutations was not strong. Further in vitro studies must be carried out to investigate the effects of the detected alterations on the antibody neutralization and nucleoside/tide drug analogues. This does not rule out the possibility that these mutations have major clinical implications towards the diagnosis and therapy.

This study demonstrates the prevalence of HBV genotype A in HIV-positive patients as well as the presence of HBV mutations in HBV/HIV co-infected people. We discovered HBV mutations linked to immune escape and drug resistance that could have therapeutic repercussions by influencing the correct clinical diagnosis and HBV treatment. This study has improved our understanding of the genotypes and mutations of HBV that have clinical implications in those who have HBV and HIV co-infection in South Africa. To determine how immune escape and drug resistance-related HBV mutations affect diagnosis and drug resistance treatment in the context of HIV/HVB co-infection, we recommend testing the HIV samples for these mutations.