1. Introduction

For decades, hepatitis is considered a major significant public health problem and accounts for millions of deaths from chronic liver disease, cirrhosis, hepatocellular carcinoma (HCC) and even fulminant hepatitis.

Several hepatotropic viruses can target the liver and cause hepatitis, like viral hepatitis type A, B, C, D, E. Hepatitis B, C, and D, are transmitted parentally, sexually or through contaminated blood and can produce acute or chronic hepatitis ^{[1, 2, 3, 4]}, while hepatitis A and E viruses are transmitted by fecal-oral routes and cause self-limiting infections ^{[5, 6, 7]}.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the commonest causative agents of severe liver disease. They account for 60% of cirrhosis, 80% of HCC and cause around one million deaths every year, especially in developing countries ^[8]. HBV is a partially double- shelled circular DNA belonging to the family Hepadnaviridae ^[9] and HCV is an enveloped single stranded positive sense RNA virus of the genus Hepacivirus within the family Flaviviridiae ^{[10, 11]}. Infection with HBV can be either acute or chronic, ranging from an inactive carrier state to progressive state which leads to significant liver disease ^{[12, 13, 14]}. On the other hand 80% of HCV infected individuals will become chronically carriers with high risk to develop sever liver disease ranging from mild minimal histological changes to extensive fibrosis and cirrhosis, HCC which may progress to liver cancer ^{[15, 16]}. However, most HCC cases are due to HCV infection ^{[17, 18]}.

In the mid-90s, a novel viruses were identified by two independent groups; GB virus C (GBV-C) and hepatitis G virus (HGV) from patient with liver disease ^{[19, 20]}. Genome analysis showed that the two viruses shared 96% of the amino acid sequence and 86% of the nucleotide sequences and this suggest that they are different isolates of the same virus, and the term GBV-C is the commonly used ^{[19, 20, 21]}.

GBV-C is an enveloped RNA virus in the family Flaviviridae with HCV and proposed to be classified within the genus Pegivirus ^[11]. Although, GBV-C is the most closet virus to HCV and they are sharing approximately 30% amino acid homology, but unlike HCV which replicate in the hepatocytes, GBV-C is a lymphotropic virus ^[22]. This virus is a positive single stranded RNA of approximately 9,400 nucleotides. The 5´ untranslated region (5´UTR) contains an internal ribosomal entry site and is followed by a single open reading frame that encodes for a polyprotein of ~3,000 amino acids. This polyprotein is cleaved by viral and cellular proteases resulting in several structural and non-structural viral proteins ^[23].The structural proteins of GBV-C are two putative envelope proteins (E1 and E2), while the non-structural proteins include NS2 (a protease), NS3 (an RNA helicase and trypsin-like serine protease), NS4, NS5A and NS5B (an RNA-dependent RNA polymerase) ^[11].

So far, there are seven different genotypes identified from different geographical locations: Genotype 1 predominant in West Africa; genotype 2 in Europe and USA; genotype 3 in Asia; genotype 4 in the Southeast Asian countries; genotype 5 in South Africa; genotype 6 in Indonesia and genotype 7 isolated in China recently. Within a given genotype, additional intra genotype diversities could occur based on their sequence diversity of either full genome length or a particular genomic range ^{[24, 25]}.

GBV-C is mainly transmitted through parenteral exposure to blood or blood products ^[20], sexual contact ^{[26, 27]}, and less frequently through mother-to-child transmission ^[28]. GBV-C infection is common worldwide, with rate of approximately 1% to 6% among healthy blood donors in the US and Europe, whereas it may reach up to 18% among healthy population in developed countries ^{[29, 30, 31]}. GBV-C infection is common among patients with HIV, HCV or HBV due to share mode of transmission approaches 50% ^{[29, 32]}.

Several studies from different countries have reported the positive role of GBV-C in improving the clinical infection and treatment outcomes of HIV patients ^{[33, 34, 35]}. On the other hand the impact of GBV-C in liver disease is controversy. Although the virus has been isolated from patients with fulminant hepatitis ^[36], but other studies failed to prove any association ^[37]. Co-infection of GBV-C with one or more hepatotropic viruses like HBV or HCV may have negative or positive effect on the disease evolution ^[38].

So the aim of this study is to determine the prevalence and the role of GBV-C infection among patients with hepatitis B and C infections and healthy control group in Addis Ababa, Ethiopia.

2. Material & Methods

This case-control study was conducted in Addis Ababa University, Ethiopia, on frozen serum samples collected from adult patients with viral hepatitis seeking medical care and follow up in Adera Internal Medical Specialty Center. This center was purposely selected because it’s an internal medicine clinic that provides healthcare to a great number of patients especially in liver disease, which makes the study sample the best possible representative of the target population.

The study population comprised a total of 101 patients with viral hepatitis collected from Adera Internal Medical Specialty Center and 70 healthy individuals collected from the Ethiopian Public Health and Research Institute. Following collections, the serum samples were divided into 2 microtubes and transported in an icebox to the Immunology and Molecular laboratories at the department of Microbial Cellular & Molecular biology, Addis Ababa University. Demographic data, including age, gender, medical history and health status were collected from the patients cards and archives.

HCV and HBV markers (HBsAg, anti-HBs, HBeAg and anti-HCV) were carried out by using a rapid and qualitative commercial fourth generation two-site sandwich immunoassay test device (Qualpro Diagnostics, 88/89, phase IIC, Verma Industrial Estate, Verna, Goa-403 722, India, 2004) according to the manufacturer’s instructions. It uses a multi target region or epitopes that cross react with specific sites in the virus and the result interpretations were based on color formation.

The following parameters were measured: alanine aminotransferase GPT/ALT (normal <=40 IU/l), aspartate aminotransferase (GOT/AST, normal <=40 IU/l ), alkaline phosphatase (normal <=270 IU/l ), bilirubin total (normal <=1.2 mg/dl), and albumin (normal: 3.5-5.3 g/dL).

For the preparation of total RNA from human sera, we used the Ribo Virus kit (Sacace Biotechnologies, Italy) according to the manufacturer’s instructions. In brief, 150 μl of serum was incubated with 600 ml of lysis buffer containing guanidine thiocyanate at 70°C for 5 min. After the addition of 600 ml of ethanol, the precipitated RNA was applied onto a silica-based spin column for purification and was finally eluted with 50 ml of Rnase-free H₂O.

One-step reverse transcription of the RNA and Real Time amplification of the cDNA was performed by using (V2-FRT, Sacace, Italy), which include all the components to generate RT-PCR amplified products from GBV-C/HGV RNA. Briefly, the total reaction volume was 25 ul, containing 10 ul of extracted RNA and 15 ul of the master mix that was prepared based on manufacturer’s instructions. cDNA synthesis was performed at 50°C for 15 min, followed by inactivation of the reverse transcriptase enzyme at 95°C for 5 min. The two PCR rounds are the same. The amplification was done in 5 cycles for the first round (95°C for 5 s, 60°C for 20 s, 72°C for 15 s) and 40 cycles for the second rounds of the PCR (95°C for 5 s, 60°C for 30 s, 72°C for 40 s with a final extension at 72°C for 15 s). The fluorescence detection and analysis of the PCR product was performed during the final extension by using the FAM, JOE/HEX channels of the Mx3000P instrument. Negative control of amplification and positive control of amplification were maintained as provided by the kit. Internal control which serves as amplification control was used to identify possible reaction inhibition.

Ethical clearance was obtained from the Ethical Review committee of the College of Natural Science, Addis Ababa University and Ministry of Science and Technology.

Data were entered and analysis done by using SPSS v. 20. Student t- test was used to determine the differences of continuous variables. Chi-square tests were used to compare categorical variables. P value < 0.05 was considered statistically significant.

3. Results

Total of 151 subjects were tested, including 101 samples with liver disease and 50 samples of healthy individuals. Among liver disease patients eighty three (55%) were HBV positive, while the remaining 18 (12%) were HCV positive and two of them were co-infected with HBV and HIV respectively (Figure 1).

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Figure 1. Type of Hepatitis Among Liver Disease Patients

The socio-demographic characteristics of hepatitis patients as shown in (Figure 2), the majority 52 (62%) of HBV patients were male, and most of the participant 35 (42%) were age between 25 – 34 years, while among HCV the predominant patients were 11 (61%) females age between 45-60 years.

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Figure 2. Distribution of sex and age among hepatitis patients

We categorized hepatitis patients into four groups based on the level of the liver enzymes and the abdominal Ultrasound scan as shown in (Figure 3). Most of HBV patients were with mild liver injury because their level of the liver enzymes was in the upper limit and with normal liver scan. In contrast, most of HCV patients were suffering from cirrhosis and or hepatocellular carcinoma.

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Figure 3. Clinical Statutes of the hepatitis patients

The overall prevalence of GBV-C RNA were detected in 13 (12.9%) of the hepatitis patients, 2 (11.1%) HCV and 11 (13.25%) HBV respectively, while all the healthy subjects were negatives. The rate of GBV-C RNA was significantly higher among HBV patients.

Patients with HCV and GBV-C co-infection were in the age range 45-60 and there were no significant differences in sex and clinical status because all the HCV patients were in the end stage liver disease. Among the 83 patients with hepatitis B, there were no significant differences in sex, age and the clinical status between those with and without GBV-C infection. GBV-C infection had no influence on the severity of chronic liver disease, because there were no significant differences in the clinical picture between GBV-C positive and negative patients.

Table 1. Socio-demographic and clinical characteristics of HBV & HCV patients with and without GBV-C infection

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4. Discussion

GBV-C is a member of the Flaviviridae family and it is closely related to the Hepatitis C Virus (HCV). Although, this virus has been isolated from patients with fulminant hepatitis, but its impact in liver disease is still controversial.

In the present study, we investigated the prevalence and impact of GBV-C infection in 101 hepatitis patients and 50 healthy individuals. Analysis of the hepatitis patients samples showed that; 83(55%) were positive for HBV while only18 (12%) for HCV.

Most of the HBV patients were male’s age between 25 – 34 years, while among HCV the predominant patients were female age between 45-60 years.

Interestingly, we observed that most of HCV patients were females and older than HBV patients. Furthermore, among the 18 hepatitis C patients 11 were suffering from cirrhosis and hepatocellular carcinoma while among HBV patients there were only 4 with hepatocellular carcinoma or cirrhosis.

Among hepatitis patients, from 83 HBsAg positive samples, 11 (13.25%) were positive for GBV-C and from 18 anti-HCV positive samples, 2 (11.1%) were positive for GBV-C, while all the healthy subjects were negatives. Our study showed that the co-infection rate of (HGV/GBV-C) RNA among hepatitis patients was significantly higher (P <0.05) in HBV than in HCV patients. This is the first report showing the incidence of GBV-C in HBV and HCV patients in Ethiopia.

Our findings were consistent with other reports; Birgit et al. reported the incidence of co-infection with GBV-C in chronic hepatitis C to be 11% ^[39]. However, it has been reported from various regions that the prevalence of GBV-C among HCV patients range between 10-33.3% ^{[40, 41, 42, 43]}. While among HBV patients in other countries such as Taiwan, Egypt, Turkey, United Arab Emirates and Saudi Arabia have shown the prevalence of GBV-C to range between 4.1-29% ^{[42, 49, 50, 51, 52]}.

These differences in the prevalence of GBV-C infection may be due to sample size, the demographic and clinical features of patients. This controversy may be due to the number of patients and the laboratory systems in the detection of the virus RNA and the antibody which was one of our limitations. The detection of the viral RNA with reverse transcription polymerase chain reaction ^[44], indicated active infection, however the clearance of the virus is associated with the development of antibodies against envelope glycoprotein (E2) which are detected by immunoassays ^[45].

The prevalence of GBV-C in our study is higher among HBV than HCV; this might be duo to the impact of HCV treatment on GBV-C RNA clearance ^[46]. The presence of GBV-C infection among hepatitis patients was not associated with sex and age, which correlated with other reports ^{[42, 47]}. GBV-C incidence was relatively higher in the age group of 35 – 44 years and in male, but the difference was not statistically significant (p > 0.05).

Some studies have shown that GBV-C infection is associated with significantly less compensated and decompensated cirrhosis, and with improvement in cirrhosis-free survival among patients with HBV or HCV ^{[38, 48]}. Unfortunately, in agreement with previous reports GBV-C infection had no influence on the severity of chronic liver disease among HBV and HCV co-infection ^{[37, 47, 53, 54]}.

Our study was a preliminary survey to evaluate the incidence of GBV-C infection among hepatitis patients in Ethiopia. There were several limitations to our study including the small number of patients and measuring anti-GBV-C antibodies. Furthermore, there is no information regarding the predominant genotypes of GBV-C in Ethiopia which might have a different role on the disease progression as reported in various other geographical regions. Finally, wider studies with long follow ups are necessary to understand the role of GBV-C in liver disease.

Acknowledgments

The authors would like to thank the staff of Adera Internal Medical Specialty Center and the Ethiopian Public Health and Research Institutes for their help during this research.

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