Background
Hepatitis B virus (HBV) infection is a major health problem leading to significant morbidity and mortality worldwide. Approximately, two billion people in the world have been infected by HBV [
1]. The majority of acute HBV infections are self-limiting, whereas chronic HBV infection can cause chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma. It is well known that Indonesia has a moderate to high endemicity of hepatitis B virus (HBV) infection [
2], due to the lack of proper health facilities or poor economical status and less public awareness.
HBV, a member of the
Hepadnaviridae, is a relaxed circular double-stranded DNA virus, and is currently classified into eight genotypes (A-H) based on a comparison of the entire HBV genomic sequence [
3]. HBV genotypes appear to show varying geographic distribution. For instance, HBV/A is prevalent in Europe, Africa, and India [
4,
5]. HBV/B and HBV/C are predominant in most part of Asia, including China and Japan [
4,
6]. The HBV/D is common in the Mediterranean area, the Middle East and India, whereas the HBV/E is localized in sub-Saharan Africa [
4‐
10]. The HBV/F and HBV/H is only identified in Central and South America [
4,
11‐
13]. The HBV/G has been found in France, Germany and United States [
14‐
16].
Molecular epidemiological studies of HBV, either a nationwide study or study on particular areas in Indonesia, have shown that HBV/B and HBV/C are the most prevalent genotypes in Indonesia [
2,
17‐
20], although HBV/A and HBV/D have also been found in Eastern Indonesia, such as Moluccas and Papua [
19,
20]. Particularly in South Moluccas, the prevalence of HBV/D is high, ranging from 50–88% [
20]. In the same study, analysis of 12 samples from Makassar demonstrated that only genotype B and C were found in the samples [
20]. To date, there is no comprehensive study about HBV genotype prevalence as well as the genetic analysis of HBV circulated in Makassar. In this study, we have analyzed HBV genotype/subgenotype and mutations in BCP region from voluntary blood donors in Makassar, which is located in Wallace territory and the biggest city in Eastern Indonesia.
Discussion
The present study demonstrates that HBV/B (61.21%) and HBV/C (25.23%) were the most prevalent among HBsAg-positive blood donors in Makassar, South Sulawesi, although HBV/D was also rarely found in the samples. Analysis of pre-S sequence of 58 samples revealed that in HBV/B the percentage of HBV/B3 was much higher than HBV/B5. In HBV/C, on the other hand, HBV/C1 and HBV/C2 were detected with similar frequency.
HBV/B3 is widely distributed in Indonesia, but has not been reported in other countries, suggesting that HBV/B3 is indigenous to Indonesia [
20]. The HBV types we found in Makassar are similar to those reported in previous studies from several areas in Indonesia [
2,
17‐
19], but the frequencies are different to Papua and Moluccas in where HBV/C is more dominant [
19,
20]. In addition, no HBV/A was found in Makassar, although it was found in Balikpapan and Kupang [
20].
Several studies have reported the prevalence of HBV genotype from blood donors in the southern Asian region. A study from Thailand demonstrated that HBV/A, HBV/B and HBV/C were detected among blood donors, where HBV/C (89.3%) was the most prevalent compared to HBV/B (7.4%) and HBV/A (0.5%) [
29]. Although this study did not identify the subgenotype, complete genome analysis of HBV/C from Thailand and some other countries which are geographically close to Indonesia such as Vietnam and Myanmar showed that most of HBV/C was classified into HBV/C1 [
28], whereas in Makassar HBV/C1 and HBV/C2 were dominant. On the other hand, analysis of asymptomatic HBV carriers from the Philippines showed that the prevalence of HBV/B, HBV/C, HBV/D, mix of HBV/B and HBV/D, and HBV/A were 53.4, 21.4, 14.3, 7.1, and 3.6%, respectively [
30]. In general, the percentage of HBV/B in the Philippines was similar to that in Makassar, although it is not possible to compare the subgenotypes with the available data. However, all HBV/C strains in the Philippines samples were classified into HBV/C5, which is different with subgenotypes circulated in Makassar (HBV/C1 and HBV/C2). A study from Malaysia using small number of healthy blood donors and chronic hepatitis B carriers demonstrated that HBV/C, HBV/B, and HBV/D were identified in the samples [
31], which is quite similar to our findings in Makassar.
Studies from India demonstrated that the distribution of HBV genotype in blood donors was slightly different between Eastern and Southern India. In Eastern India, beside the major HBV genotypes transmitted in India such as HBV/D (56.0%) and HBV/A (20.6%), quite high percentage of HBV/C (23.4%) was also found in the blood donors [
32]. Phylogenetic analysis revealed that those HBV/C strains that clustered with HBV/C found in South-East Asia including Indonesia were classified as HBV/C1 [
32]. On the other hand, in Southern India, only HBV/D (76.11%) and HBV/A (11.94%) were detected [
33], similar to the HBV genotype distribution pattern in Northern and Western India [
5].
In China, HBV/C was detected in high percentage (68.0%) from blood donors along with HBV/B (25.8%), HBV/A (2.1%) and mix HBV/B and HBV/C (4.1%), although the subgenotype was not identified in this study [
34]. Thus, in general, the distribution of HBV genotype from blood donors in Makassar is different to that in India and China.
Prior to our study, in Indonesia HBV/D has only previously been found in Papua and Moluccas [
19,
20]. Therefore, we were interested to analyze the complete genome of the two HBV/D strains (22.165.07 and 22.252.07) found in our samples from Makassar. Phylogenetic analysis of complete genome, partial S, and pre-C/C regions confirmed that the two HBV/D strains were subgenotype D6 (Fig.
2). The present HBV/D strains were then compared with two references recently published from Papua (AB493846 and AB493846) and one reference which found in France (AM422939). Based on the complete genome, divergences between the present strains and Papua strains (HBV/D6) were lower than the divergence between the two strains and France strain (HBV/D3) (Fig.
2). Thus, it is confirmed that the HBV/D found in Makassar was the same with the HBV/D strains present in Papua (HBV/D6) [
19]. Because a previous study found HBV/D strains in Moluccas were HBV/D1 and HBV/D3 [
20], it is possible that the HBV/D found in Makassar has been imported from Papua, not Moluccas, even though Moluccas is very close to Makassar.
Many studies have demonstrated that HBV mutations, including mutations in BCP region, are linked with the severity and outcome of HBV infection [
35‐
37]. Our recent data also revealed that mutations in BCP were associated with clinical outcome of liver disease [
38]. We therefore looked for mutations in the BCP region. Double mutation (A1762T/G1764A) was only found in 1.96% of HBV/B (Fig.
3). Likewise, the double mutation was only observed in 5.36% of HBV/C (Fig.
3). Analysis of the nucleotide at position 1753 showed that a T-to-V (A/G/C) mutation, which has also been associated with HCC development [
39], was found to be less frequent in both HBV/B and HBV/C (2.94% and 1.79, respectively). None of these mutations was identified in HBV/D (Fig.
3). Hence, our results demonstrated that the frequency of mutations in BCP in blood donors in Makassar was low. In our previous study, we included 15 HBV-associated liver disease samples from Makassar [
38], and found that the prevalence of T1753V and A1762T/G1764A mutations were 40.0% and 60.0%, respectively, suggesting that those mutations were associated with severity of liver disease. Since both T1753V and A1762T/G1764A mutations were less common in blood donors, the chance of developing severe liver disease might be relatively low in the blood donors in Makassar.
In comparison, a study from Indian blood donors showed that the occurrence of double mutation in BCP was extremely high in HBV/C (72.4%) and relatively high in HBV/A (24.1%) and HBV/D (21.5%) [
32]. Another study also reported that K130M and V131I substitutions, which are corresponding to double mutation in BCP, from Indian inactive carrier were generally high (36.0%) [
40]. However, they found that the occurrence of other mutations suggested to have associated with HCC was low [
40]. If BCP mutation is strongly associated with clinical outcome of liver disease including HCC, the incidence of HCC must be high in India. In fact, the HCC incidence was very low in India [
41], thus the association of BCP mutations as well as other mutations with severity of liver disease might be not depend only on virus mutations, but also host factors, and therefore needs further investigation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Conceived of the study, participated in its design and coordination, drafted the manuscript and coordinate the whole work team: AU. Carried out the molecular genotyping study: TIO. Responsible for sample and clinical data collection, and contributed in data analysis: RD. Participated in sample and clinical data collection: UAM. Participated in the editing the manuscript and clinical data: IY. Coordinated the research effort and participated in manuscript preparation: ST. All authors have read and approved the final manuscript.