Vol 44, No. 6;Jun 2014
Detection of Hepatitis B virus (HBV) Infection in Jeddah, Saudi Arabia Sabir, J. S. M1, Mutwakil, M. H. Z1, Redwan, N. A1, Barnawi, M. BI1, Saini, K. S1 and Ahmed, M. M. M1, 2* 1 Department of Biological Sciences, Faculty of Science, P.O. Box 80203, King Abdulaziz University, Jeddah, 21589, Saudi Arabia. 2 Nucleic Acids Research Dept., Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research and Technology Applications. Alexandria, Egypt. *Corresponding author. Ahmed, M. M. M., Email:
[email protected]
Abstract HBV infection remains a serious global health problem, with two billion people infected worldwide and approximately 350 million chronic patients. The aim of this study was to examine the prevalence of HBV in Jeddah, Saudi Arabia, and further analyze mutations within the virulent genes of HBV. Serum HBV was detected using polymerase chain reaction (PCR) with three different primers for conserved area, HBX genes and HPS gene. Genetic sequence alignment and HBV from GenBank were compared to elucidate the relationships among genotypes in our patient population with the genotype of the HBV usually observed in other countries. Our results clearly show that the most prevalent was genotype D. This study provides valuable data that could be extrapolated to examine the cases of infection in the Saudi community and help us focus on those sequences, which might be useful in the development of diagnostic tests. It may further aid in the discovery and development of targeted therapies against HBV.
Keywords: HBV; HBX gene; HPS genes; PCR; DNA Introduction Liver metabolizes the absorbed nutrients, detoxify xenobiotics, fights infections, etc., however, its malfunctioning could lead to various disorders. Heavy alcohol use, toxins, certain medications and disease conditions could lead to hepatitis. In recent years, viral hepatitis has become a major global health concern. There are five well-known viruses that primarily infect the liver and cause hepatitis: hepatitis A, B, C, D and E (HAV, HBV, HCV, HDV, HEV). HBV, HCV, HDV infections could lead to chronic liver diseases, such as, cirrhosis and hepatocellular carcinoma (HCC). As per WHO estimates, approximately 350 million people worldwide are chronically infected with hepatitis B virus (HBV), varying widely from low (8% as in Africa, Brazil, Southeast Asia and China). Currently 8 genotypic variants, from A to H, and also 4 main surface antigen subtypes: adw, ayw, adr, and ayr are found to be normally distributed throughout the world. Epidemiologically, there has been significant interest in identifying the demographic distribution and prognosis associated factors for these genotypes and subtypes. PCR-based methods have gained popularity in HBV detection and commercialisation protocols (e.g. HBV Monitor, Roche Diagnostic Systems) and further led to their widespread adoption among the clinical and diagnostic labs (Ganem, and Varmus, 1987). The development of real time PCR methodology has obviously improved the ease with which HBV levels can be monitored and has increased the range over which such levels can be accurately quantified (Ahmed, et al., 2008a; Ahmed, et al., 2008b and Yeh, et al., 2004). With advances in DNA sequencing & PCR technologies (Metzker, 2010; Shendure & Ji, 2008; Tahaa et al., 2013 and Santos, et al., 2014), particularly next-generation sequencing & pyrosequencing of PCR products, the cost factor and the accuracy of generating longer sequence reads will be a great value addition for “personalized” diagnosis and prognosis. HBV is a DNA virus, which replicates almost exclusively in the liver (Gerken, el al., 1998). Despite the availability of effective recombinant vaccines, HBV infection remains a major global health problem causing about 1 million deaths every year (Kao, et al., 2000). Our study was designed to detect which genotype of HBV infection in Jeddah, Saudi Arabia with the aim of early detection and probable therapeutic interventions.
149
Pretoria, South Africa
Vol 44, No. 6;Jun 2014
Materials and Methods Samples collection Blood samples were collected from 638 patients randomly from AL-Thager General Hospital-Jeddah, and from Jeddah Regional Laboratory. All samples were collected from the Out Patient Department (OPD) in EDTA tubes and age of each patient was recorded. After centrifugation, serum or plasma samples were collected and transported on ice to the lab. All serum/plasma samples were stored at -20OC until analyses. The research work in this manuscript was carried out in accordance with the Helsinki Declaration (http://www.wma.net/e/policy/b3.htm) as adopted by the King Abdulaziz University guidelines for work on human tissue or blood samples and these guidelines were strictly followed. Oligonucleotide primers: The primers used for genetic detection of HBsAg were described earlier in our studies: a- Conserved regions 116 bp (Sitnik, et al., 2010): b- HBs gene 403 bp (Ahmed, et al., 2008a) c- HBx gene 591 bp (Ahmed, et al., 2008b)
Genomic DNA extraction: Genomic DNA extraction was accomplished using QIAamp DNA mini kit from Qiagen (Germany), as per instructions from the manufacturer. DNA samples were stored in 2 volume ethanol until analyzed.
Polymerase chain reaction (PCR) PCRs were accomplished using Taq PCR Master Mix kit from Qiagen, For conserved region primer (Sitnik, et al., 2006): a- initial denaturation at 94 °C for 5 min followed by 35 cycles each consisting of denaturation at 94 °C for 30 sec.; annealing at 63 °C for 1 min. and extension at 72 °C for 45 sec. The last cycle was linked to a final extension step at 72 °C for 10 min. For the HBs gene (Ahmed, et al., 2008a) and the HBx gene (Ahmed, et al., 2008b), the PCR conditions were the same as described in these 2 earlier publications from our lab. The amplification products were analyzed routinely after electrophoresis on 1% agarose gel followed by Ethidium bromide staining and the data recorded in Gel Documentation.
PCR product sequencing and BLAST analysis: PCR positive products displaying correct band size were collected, after visualizing under the UV light, and DNA was extracted. All three genes, conserved region, HBx gene and HBs gene along with the appropriate primers were sent for sequencing (GATC BIOTECH). The results from the sequencing data were compared with the HBV gene sequence in GenBank using BLAST program.
Statistical analyses: All the measurements were in triplicate and analysis of variance (ANOVA) was used to determine statistical significance.
RESULTS Prevalence of HBV by samples type: The patient population in our study based on seropositive, were stratified into two groups (positive samples & negative samples). The detection of HBsAg was tested using enzyme linked immunosorbent assay (ELISA) and results were calculated separately for each group. HBsAg results were found to be 6.11 % seropositive and 93.9% seronegative (Table 1).
150
Pretoria, South Africa
Vol 44, No. 6;Jun 2014
Table 1: Prevalence of Hepatitis B Virus Total Samples
Samples type +ve -ve
638
ELISA-Results 39 599
Ratio 6.11 % 93.9 %
PCR Amplification for Conserved region; HBs and HBx genes: DNA was extracted from all ELISA positive samples and from some ELISA negative (random) samples, and then amplified using PCR, with conserved region fragment primers. The results are shown in Figure 1. ELISA positive samples matched positively with our PCR results. The conserved region fragment from ELISA positive sample was detected in the positive sample, and was not detected in samples from ELISA negative controls. All results with HBs gene are shown in Figure 2; the results with HBx gene are shown in Figure 3.
M
1
2
C
2000 750 500 250 100 bp
Figure 1: Agarose gel electrophoresis showing PCR amplified product of the conserved region of the gene (116 bp). Lane 1: DNA markers, Lane 2 &3: DNA samples from plasma/blood and Lane 4: Negative control. In lane 3-a desired band of 116 bp was detected, while no amplification was seen in sample 1 (i.e. lane 2).
MM
1
2
33
4
5
CC
2000 1000 750 500 250 gel electrophoresis showing PCR amplified products of HBs gene (403 bp). Lane 1: DNA markers, Figure 2: Agarose Lanes 2 to 6: DNA samples from plasma and Lane 7: Negative Control. Samples in lanes 2, 5 & 6 show 100 bp amplification of 403 bp HBVs gene products while in lanes 3 and 4, no amplified fragment was detected.
151
Pretoria, South Africa
Vol 44, No. 6;Jun 2014
M
1
2
3
C
2000 1000 750 500
Figure 3: Agarose gel electrophoresis showing PCR amplified product of HBx gene. Lane 1: DNA markers, Lane 2 250 from plasma and lane 4: Negative Control. In lane 3, an amplified fragment of gene (591 bp) & 3: DNA samples was observed, whereas samples in lanes 2, 4 showed negative results for HBx gene DNA. 100 bp
DNA sequencing for Conserved region; HBs and HBx genes All PCR products from conserved region; HBs and HBx genes were sequenced, and BLAST analysis (Http://www.ncbi.nlm.nih.gov/BLAST/) was carried out and depicted as alignment percentage of similarity (98%), Gaps = 0/64 (0%).
Phylogenetic analysis We conducted phylogenetic analysis to reconstruct the evolutionary histories of related viral species found across the globe. The relationships were depicted in phylogenetic trees and are shown below in Figure 4.
Figure 4. Dendrogram showing average linkage (Between groups) based on the sequencing data analysis of the complete HBV genome (1); conserved region (2, 3, 4); HBs (5, 6, 7) and HBx (8, 9).
Discussion Acute and chronic hepatitis are the lethal endpoints of HBV infection across many Asian countries. Although a safe and effective vaccine has been available for the last two decades, HBV infection still represents a major global health burden (Block et al. 2007). Many epidemiological and molecular studies have clearly shown that chronic HBV infection represents the main putative risk factor for the development of hepatocellular carcinoma (Lok, 2004; Pollicino, et al., 2011; Shepard, et al. 2006). Three different segments within the HBV gene were amplified using PCR and positive products were sequenced to further validate the authenticity of our data. As observed earlier by other investigators, the prevalence of chronic HBV infection varies geographically, from high (>8%), intermediate (2-7%) to low (
