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Hepatitis B virus (HBV) is a highly infectious virus that remains one of the most important human pathogens [1]. ‘The virus is usually spread by exposure to infected blood and other blood products’ [2]. The virus has also been detected in peripheral mononuclear cells, tissues of the pancreas, spleen, kidney, skin, and other body fluids such as saliva, semen, sweat, breast milk, tears urine, and vaginal secretions [3]. The infection is highly endemic in Africa, with a significant portion of the population affected by chronic HBV [4,5].

Efforts to control HBV in Africa have made significant strides, particularly with the widespread implementation of vaccination programs aimed at newborns and children. However, challenges persist, including disparities in vaccine coverage, insufficient screening programs, and limited access to antiviral treatments. Nigeria is one of the countries with high incidence, the male to female predilection varies and children are not spared. Medical personnel are at the greatest risk of infection, while commercial sex workers and drivers have also been found to be at significant risk of getting infected [6]. Chronic HBV infection can lead to severe liver complications, including cirrhosis and hepatocellular carcinoma (HCC), contributing significantly to the burden of liver disease [7].

Hepatitis B virus has evolved with ten genotypes (A–J) so far identified with distinct geographical distribution [8]. This evolution is driven by a high mutation rate which leads to a high degree of genetic variation. ‘The dominant HBV genotypes differ by geographic location, transmission dynamics, disease progression, and response to antiviral therapy’ [9]. The entire genomic sequences within each group diverge from other groups by more than 8% [10]. Phylogenetic and phylogeographic evidence suggest that genotype E most likely originated from Nigeria [11]. In Africa, one of three genotypes, E, A, and D are predominant depending on the region [8]. Genotype A is distributed globally and is the main genotype found in Europe, North America, and India. Genotypes B and C are predominant in East and Southeast Asia. Genotype D is mainly found in the Middle East and Mediterranean countries but has also been reported globally [12]. End-stage liver disease and poor response to interferon therapy are commonly observed in chronic HBV infection associated with genotypes C and D. However, it has been observed that horizontal HBV transmission is more common with genotypes A and D.

Hepatitis B virus infection remains a significant health burden affecting millions of individuals worldwide with distinct variations in disease progression and treatment response linked to viral genotypes [13]. The classification of HBV genotypes indicates that these genotypes not only influence viral replication dynamics but also impact disease severity and response to antiviral therapy [14]. Despite advancements in understanding HBV genotypes, comprehensive genotype-specific epidemiological data are lacking particularly in regions with overlapping genotype distributions [15]. Thus, knowledge gaps persist regarding the specific impact of genotype variability on host immune response and the development of hepatocellular carcinoma [16]. Therefore, emerging genotypic variants and their implications for vaccine efficacy and antiviral resistance necessitate continuous surveillance and research efforts [17].

Hepatitis B virus genotyping is important because it provides information on the molecular epidemiology of the virus including phylogenetic and phylogeographic histories [18], it answers questions on the relation between the course of disease and viral strain [19], and it provides insight into the impact of human migration on viral evolution [20]. Although HBV infection is widely reported to be endemic in Nigeria [21,22,23], the molecular nature of the virus is poorly understood. Therefore, investigating HBV genotypes will provide critical insights into genotype-specific virulence factors which influence transmission, pathogenesis, disease progression, and response to antiviral therapy facilitating targeted interventions to reduce the burden of HBV in the study area, thus this study is designed to determine the circulating genotypes of Hepatitis B virus in Zamfara State, Northwestern Nigeria.

Materials and Method

Study Area

This study was conducted in Zamfara state northwestern Nigeria. The state is located at 12o 101 N 6o151E with an area of 39,762 km2 and an estimated population of 9,838,160 (2011 census). Until 1996, the area was part of Sokoto State. Zamfara comprises 14 Local Government Areas with three senatorial districts i.e., the Central senatorial district comprising four Local Government Areas (Tsafe, Gusau, Bungudu and Maru), the East senatorial district comprising four Local Government (Kaura, shinkafi, Zurmi, Birnin Magaji) and the West senatorial district comprising six Local Government (Mafara, Maradun, Bakura, Gummi, Bukuyyum and Anka). Most inhabitants of the area are predominantly Hausa whose major source of livelihood is from farming and rearing of domestic animals.

Ethical Approval

Ethical approval to carry out the study was obtained from ethical committees of Federal Medical Center (FMC) Gusau (FMC/2021/985/008/NHREC/TR/19/03/2016), Ahmad Sani Yariman Bakura Specialist Hospital (ASYBSH) Gusau (ASYBSH/SUB/205/VOL.1) and ethical committee of the Zamfara State Ministry of Health (ZSHREC04112022/101). Individual consent was obtained from all the participants. Adequate information was given to all participants for the purpose of the study and confidentiality of all information obtained was assured.

Study Population

This consists of individuals adults, males, females, outpatients, and patients who visited the hospitals during the period of the study, and who willingly gave consent for the study

Sample Size Determination

The sample size was calculated using a single proportion formula:

N=Z²PQ/D²

Where:

 N = Minimum sample size.

Z = Constant, standard normal deviation (1.96 for 95% confidence interval).

P = local prevalence rate of a previous study on [24] Hepatitis B virus among patients = 23.4% = 0.234.

Q = 1-P

D = acceptable margin of error (5% or 0.05).

N = 1.96² x 0.234 x 0.864

                   0.05²

N = 310

Inclusion and Exclusion Criteria

The inclusion criteria used include individuals who attended the hospitals during the period of the study and whose consent was obtained. However, persons who did not give consent to participate, children not older than five years, and persons who were too ill to understand the procedures involved in the study were excluded.

Sample Collections

Four (4) ml of blood samples were aseptically collected from each participant by venipuncture into an appropriately labeled sterile plain tube. This was allowed to clot at room temperature and spun for 5 min at 3000 rpm. The resultant serum was harvested and transferred into well-labeled sterile cryovials. Thereafter, the samples were transported from the sampling hospitals to the Antimicrobial Resistance (AMR) Fleming’s laboratory, Veterinary Teaching Hospital of Usmanu Danfodiyo University Sokoto. Subsequently, the sera samples were stored at −20 °C before analysis.

Nucleic Acid Extraction

The extraction was carried out using a Qiagen genomic DNA extraction kit, following the manufacturer’s instructions. All samples and reagents were equilibrated to room temperature for a few minutes to thaw prior to the commencement of the procedure. One hundred and fourty (140 µl) of the sera was pipetted into a new Eppendorf tube. Five hundred and sixty (560 µl) of binding buffer was added into the tube. Twenty (20 µl) of proteinase K was added to the mixture. The tube was closed and mixed by vortexing for 15 Seconds and incubated at room temperature for 10 min. The tubes were centrifuged for 5 minutes and 560µl absolute ethanol was added to the mixture. The mixture was then decanted into a DNA binding column, the cap was closed and centrifuged at 8000 rpm for 1 min. The QI-Aamp Mini column was transferred to a clean 2 ml collection tube, and the tube containing the flow-through was discarded. ‘Subsequently, 500 µl Buffer AW1 was added to each mixture and centrifuged at 14000 rpm for 1 minute’. The flow-through was then discarded. ‘Five hundred (500 µl) of wash Buffer AW2 was then added and centrifuged at 14,000 rpm for 1 minute’. The flow through discarded centrifuged for 3 minutes at 14,000 rpm to remove excess ethanol from the column. The QIA-amp Mini column was placed in a clean nuclease-free 1.5 ml microcentrifuge tube and the old collection tube containing the filtrate was discarded. About 30 µl elution Buffer AVE equilibrated to room temperature was added directly to the silica membrane of the spin column and incubated at room temperature for 5 min, centrifuged at 14000 rpm for 1 minute. The spin column was discarded and the microtube containing the eluted DNA was caped and stored at -20⁰ C until required for subsequent PCR and genotyping.

The Genotyping Procedure

The genotyping of HBV was carried out based on a rapid and specific genotypes system corresponding to six genotypes A through F by nested polymerase chain reactions method using a type-specific primer as described by Shuaibu et al. [1] with slight modifications in cycling profiles.

Hepatitis B Virus DNA Amplification

Multiplex-nested polymerase chain reaction (PCR) using a type-specific primer was used.  Primers were designed based on the conserved nature of the nucleotide sequences in regions of the pre-SI through the S genes. P1B and S1-2 being the universal outer primers and B2 the inner sense (forward) primer with a combination of BA1R, BB1R, and BC1R as anti-sense (reverse) inner primers for genotypes A, B, and C in a multiplex system tagged ‘Mix A. For genotypes D, E, and F, anti-sense primers B2R were used in combination with BD1, BE1, and BF1 as sense (forward) primers, also in a multiplex system tagged ‘Mix B’.

First Round Polymerase Chain Reaction: Detection of Hepatitis B Virus DNA

The HBV was detected by amplification of pre-SI region through the S genes using universal primers (P₁B) sense primer, (S_1-2) antisense primer. The total reaction mixture volume used for the first round of PCR was 20µl. All the premix tubes were labeled with sample Identification numbers. About 2µl of extracted DNA was added to a Master Mix (cocktail of 16µl of deionized water {D.H₂O}, premix of 250µM each of dNTP, 1X PCR buffer,15mM of Mgcl₂ and 1U of thermostable Taq polymerase) and 1µl each of P₁B (forward) and S_1-2 (reverse) outer primers. The PCR was performed using a thermal cycler (PTC-100^TM Programmable thermal controller, MJ Research, Inc.), and the reaction conditions were set as initial activation at 94^OC for 10 minutes, denaturation at 94^OC for 30 seconds, annealing at 53^OC for 30 seconds and extension at 72^OC for 1minutes. A total cycle of 35 from denaturation to extension was observed. The final extension was set at 72^OC for 5 minutes.

Second Round Polymerase Chain Reaction: Hepatitis B Virus Genotyping

 This was performed in two different tubes for each sample, one with universal sense primer B2 and type-specific primers for genotypes A, B, and C in ‘Mix-A’ and the other with universal antisense primer B2R and type-specific primers for genotypes D, E, F in ‘Mix B’. Seventeen (17µl) of deionized water (D.H2O) was added into each tube of premix A and B.  Two (2μl) of the cocktail primers (containing 0.5μl each of the four primers were added to the mixtures.  ‘One (1μl) of the first round PCR product was also added into each tube of the premix.  The mixture was gently agitated and then centrifuged at 3000rpm for 5 minutes. The PCR condition was set as initial activation at 94ºC for 3 min, followed by 30 cycles of denaturation at 94ºC for 1 minute, annealing at 50ºC for 1 minute, and extension at 72ºC for 1 minute for both “Mix A” and “Mix B”, with a final extension at 72ºC for 5 minutes.

Table 1: Sequences of primers for HBV amplification and genotyping

Agarose Gel Electrophoresis

The amplicons were resolved using Agarose electrophoresis. The agarose gel was prepared by weighing 0.5g of Agarose powder in 50 ml 1XTris- Acetate EDTA (TAE) buffer and subjected to heat until the Agarose was completely dissolved and appeared as a clear transparent solution. The molten agar gel was allowed to cool to 50°C and then 2µl of ethidium bromide (0.5µg/ml) dye was added and swirled until the color disappeared. Thereafter, the gel was poured into the gel casting tray held within the gel casting gates and the comb was placed into the slots on the tray. The gel was allowed to solidify for 30 min and then the comb was gently removed. The gel slab along with the running tray was submerged carefully into the electrophoresis tank containing 1X TAE buffer.  A total volume of 10µl amplicon was dispensed carefully into the wells of Agarose gel and electrophoresed for 40 minutes at 100V to determine the size of the amplified PCR product. The bands were visualized under the gel documentation system (BioRad Gel Doc-XR, USA) and screenshots were captured. The size of the separated bands (DNA fragments) was compared with the GeneRuler^TM100bp+ DNA ladder (MBI Fermentas, Life Sciences, Canada).

Phylogenetic Analysis

Phylogenetic analysis was used to compare the obtained sequence with corresponding sequences. Nucleotide sequences of the 6 genotypes (A-F) were obtained from the GenBank, European Molecular Biology Laboratory, and DNA Data Bank of Japan databases. A phylogenetic tree was constructed by using the neighbor-joining method component of the Molecular Evolutionary Genetics Analysis (MEGA) 3 program on the basis of the nucleotide sequences of the amplified pre-S gene of the HBV genome. Genetic distances were estimated by using the 6-parameter method, and phylogenetic trees were constructed by the neighbor-joining method. The principle of the neighbor-joining method is to find pairs of operational taxonomic units (i.e., neighbors) that minimize the total branch length at each clustering stage of operational taxonomic units starting with a star-like tree. The alignments for the complete genome were edited manually in GeneDoc.

Results

Out of the 21 Hepatitis B surface antigen (HBsAg) positive samples, Hepatitis B viral DNA (HBV-DNA) was detected in 13 samples at 1070bp. The HBV-DNA positive samples show bands representing identified genotypes (Plate I and Plate II).

Plate I: Agarose gel electrophoretogram of HBsAg positive HBV-DNA sample

Plate II: Agarose gel electrophoretogram of HBsAg positive HBV-DNA sample

Discussion