Molecular study of Hepatitis B virus and detection of hepatitis genotyping as well as the relationship between interleukins and hepatitis in Anbar, Iraq

Hepatitis B virus (HBV) is a worldwide health problem. Cytokines play an important role in the elimination of HBV. Interleukins-6, 27 and 34 (IL-6, IL-27, IL-34) have recently been recognised as cytokines involved in the aetiology and pathogenesis of HBV. Genotyping of HBV is important because it can help guide treatment decisions. For example, some genotypes of HBV are more likely to respond to certain antiviral therapies than others. Furthermore, the risk of developing serious liver disease such as hepatocellular carcinoma and cirrhosis can be predicted using HBV genotyping. Studies have identified genotypes and sub-genotypes of hepatitis patients as well as variations in the genetic sequence. Furthermore, serum levels of IL-6, IL27 and IL-34 have also been determined in HBV patients and controls to understand their susceptibility role in disease development. Therefore, our study aimed to determine HBV genotype and allele frequencies in acute and chronic HBV patients living in Iraq. Our study included 139 hepatitis-infected patients (male and female) and 40 healthy (non-HBV-infected) individuals (male and female) of different ages between January and October 2023. HBs Ag ELISA kit was used to detect HbsAg in all serum samples and Anti-HBc IgM ELISA kit was used to identify acute HBV patients. The 112 samples tested were analysed by nested PCR to detect hepatitis B virus DNA. First round PCR products revealed that 45 (100%) of all acute hepatitis B patients showed a 1003 bp band, confirming that the present sample came from patients with hepatitis B infection with the virus. Of 67 chronic hepatitis B virus patients, 42 (62.7%) had a positive HBV-DNA test and 25 (37.3%) had a negative HBV-DNA test. The mean age ± SD of acute HBV patients was 39.25±2.17 years, chronic patients were 36.35±2.56 years, and the control group had a lower mean age (26.97±1.49 years). However, a highly significant difference was found between the patient and control groups (p=0.0002). While 50% of acute HBV patients were both men and women, 67.5% of chronic HBV patients were men and 32.5% were women. Finally, amplicons obtained from nested PCR were sequenced by Sanger sequencing method. Accordingly, it was determined that all 80 HBV patients whose DNA was analysed had a single type D genotype, 47 (58.75%) males and 33 (41.25%) females carried this genotype and the sub-genotypes were D1, D3 (92.5%, 7.5%) respectively. Seven nucleic acid variants were observed at different positions in the analysed samples: 348 T>C, 495 C>T, 501 A>C, 520 C>A, 522 G>A, 530 T>C and 535 A>C. The observed variation was found in all samples with the same position. Other variants were detected at different positions in different samples analysed but not in all samples. Amino acid alignment of these amino acid sequences with the HBV reference amino acid sequences (GenBank acc. NC 003977.2) was performed and ten variants were found at different positions, within the size of the amplified loci, showing an effect on the S protein. Four of these were detected in the majority of samples, namely p82.Pro>Leu, p84.Gln>Pro, p91.Gly>Glu and p33.Leu>Pro. Subsequently, a phylogenetic analysis was performed with the help of bioinformatics software tools to understand the actual distances between our studied samples and to identify relatives based on mutations and genotype. The boot strap method/Jukes-cantor model/neighbour-joining was used to construct the phylogenetic tree as a circular cladogram. Nested PCR, sequencing and phylogenetic analysis revealed that genotype D was the only genotype detected. As for our results on the relationship between interleukins and the studied group samples (acute, chronic and control), the results showed that serum levels of IL-6 and IL-27 were significantly increased in hepatitis cases, but higher in acute cases compared to chronic cases, and in both cases their levels were higher than in control groups. IL-27 levels had a highly significant value (p 0.0001**) compared to IL-6 levels. Interleukin 34 levels did not have a significant value (p 0.708 NS) in the comparison between patients and healthy control.