Distribution of hepatitis B virus genotypes in general population of Myanmar via Nation wide study

Hepatitis B virus (HBV) infection is a severe health concern worldwide. HBV is a DNA virus with a rapid rate of mutation. Based on the heterogeneity of the HBV nucleotide sequence, the HBV strains are divided into ten genotypes, A to J, with a characteristic geographical distribution. Identifying and tracking the changes of HBV genotypes is important in epidemiological and transmission studies, predicting the risk for the development of severe liver disease and response to antiviral treatment. The present study was conducted to detect HBV genotypes and sub-genotypes in general population of different states and regions in Myanmar.


Conclusion
Genotype HBV/C, sub-genotype C1 was the most predominant genotype distributed in all states and regions of Myanmar. This study was first report on Nation-wide distribution of HBV genotype and sub-genotypes in Myanmar and the findings will be a huge support for hepatitis disease surveillance programme which is the one of the National Priority Prevalence was the highest in the African (6.1%) and Western Pacific regions (6.2%) and followed by Asia [2]. Myanmar has a moderate to high endemicity of hepatitis B infection.
According to the nation-wide seroprevalence survey in 2015, 6.5 % of general population was infected with viral hepatitis B. The prevalence was found to be varied with geographic variation with highest prevalence in Yangon Region (10%) and lowest in Kayah State HBV is grouped into many genotypes, according to genome sequence. Ten well-known genotypes (A-J) of the HBV genome have been defined. Some HBV genotypes are further classified as sub-genotypes. HBV sequence is characterized by more than 8% nucleotide differences for genotype, and 4%-8% nucleotide differences for sub-genotype. Over 30 related sub-genotypes belonging to HBV genotypes have been determined to date [5][6]. An earlier classification system divided the HBs Ag into four major serological subtypes, adw, adr, ayw and ayr. There is a correlation between HBs Ag subtypes and HBV genotypes. In general, HBV genotypes of A, B, F, G or H have HBs Ag subtype adw, those with genotype C have adr; and those with HBV/ D and HBV/E have ayw [7][8]. Genotype A and D have global distribution but genotype B and C are found predominantly in east and southeast Asia. Genotype E prevails in West Africa. The most divergent genotype F is found exclusively among indigenous peoples in central and south America. Genotype G, found in the USA and France, exhibits some unique molecular structures [6].
Myanmar is one of the most ethnically diverse countries and bordered by Bangladesh and India at the western border and China, Laos and Thailand at the eastern border, Thailand at the southern border and China at the northern border. The major genotype in China and Thailand is genotype C and Genotype D is the most dominant genotype in India [9][10][11].
There were limited studies in Myanmar on HBV serotypes and genotypes. Previous studies in HBV serotypes and genotypes distribution in Myanmar was carried out but mainly on specific population. A study in 2012 reported the distribution of HBsAg subtypes among HBV carriers in Yangon as adr (93.2%), adw (4.85%) and ayw (1.94%) [12]. A hospital-based study showed prevailing HBV genotypes in chronic liver diseases patients was HBV/C followed by HBV/A and there were also mixed genotypes and un-typable genotypes [13]. Sa-Nguanmoo et al., (2010) also found that HBV/C (97.5%), HBV/B and HBV/D (1.25% each) among Myanmar migrant workers [14]. Recently, Latt AZ and group Moreover, HBV genotype/ sub-genotypes and genetic variability of HBV are also useful in epidemiological and transmission studies, tracing human migrations, in predicting the risk of development of severe liver diseases and response to antiviral therapy [16]. There is no large scale study on geographical distribution of HBV genotypes in Myanmar and this is the first report on Nation-wide distribution of hepatitis B genotypes and sub-genotypes.

Sampling procedure and recruitment
Two-stage cluster sampling method was used and selection of primary sampling units (PSUs) was performed that one township was randomly selected, which is considered to have average level of viral hepatitis B infection in all States and Regions of Myanmar.
Selection of secondary sampling units (SSUs) was performed that from each selected PSU (township), 10 wards and villages were selected according to probability to population size. From each selected SSU (ward/village), 30 households were selected using systematic random sampling. The sampling frame for this sampling is the list of households available from the Basic Health staff. One eligible participant aged between 15 to 80 years in the selected households was recruited by random sampling.

Screening of HBV infection and sample collection for genotyping study
HBV screening was carried out at the field sites using SD Bioline HBsAg WB

Confirmation of HBs Ag positive serum samples
HBs Ag positive serum samples by ICT (Immuno-chromatographic Test) were further confirmed by commercially available immunoassay kit, HBs Ag ELISA 3.0 assay (Cat. No 01EK10, Standard Diagnostic Test Kit, SD, Korea). The tests were performed according to the manufacturer's instruction.

Viral DNA extraction
The ELISA confirmed HBs Ag positive were undergone viral DNA extraction which was performed with the QIAamp DNA Mini kit (Qiagen, Inc., Hilden, Germany), according to the manufacturer's instructions.

Amplification of pre-S gene of HBV by PCR
The HBV PreS gene was amplified with nested PCR using PF-PR and NF-NR primers sets (PF Negative samples after first round PCR were amplified in nested PCR using second round primer set and a thermal profile like the first round but repeated for 35 cycles instead of 30 with annealing 54ºC. After confirming by gel electrophoresis, the products were purified with SV column PCR purification kit (GeneAll Biotech, Korea) according to the manufacturer's instruction.

Determination of HBV Genotypes by direct sequencing of preS gene
The purified PCR products were subjected to sequencing by chain termination method using commercially available Kit (Big Dye Terminator Cycle Sequencing Kit, Applied Biosystems). Briefly, 2 μl of purified DNA was mixed with 1.85 ul of 5x sequencing buffer, 0.25 μl of Big dye terminator, 0.5μl of 0.125 μM primer (Forward or Reverse) and 5.4μl of water. The thermal profile used was; 35 cycles of 60 sec at 96ºC; 05 sec at 50ºC and 3 min at 60ºC. The 3500 XL Genetic Analyzer (Applied Biosystems) was used for Sanger sequencing method [17].

Determination of HBV genotypes and sub-genotypes
HBV genotypes were determined using the sequences of pre-S/S genes with NCBI Web based HBV Genotyping Tool (http://www.ncbi.nlm.nih.gov/projects/genotyping/ formpage.cgi) [18]. HBV DNA sequences were aligned with reference sequences using CLUSTAL method (MedAlign, Lasergene, DNASTAR Inc., Madison, WI) and manually edited the sequences with BioEdit Sequence Editor (version 7.2.5) and phylogenetic relationships were estimated by neighbor-joining method [19]. For determination of sub-genotype, the study sequences were aligned with published sequences representing all known HBV subgenotypes. Multiple sequence alignment was performed using the built-in ClustalW integrated in MEGA 4.20 [20]. HBV sub-genotypes were determined by phylogenetic analysis in MegAlign software using the neighbour-joining method with a bootstrap test.

Distribution of HBV genotypes among the study population
All 353 HBs Ag positive serum samples were positive by HBV ELISA. Of confirmed 353 HBs Ag positive samples, 153 (43.3%) were PCR positive and proceeded for genotyping procedures. The above mentioned 153 PCR positive samples (69 males and 84 females), mean age 34.0 ± 11.54 years were included for genotype analysis. Three major genotypes-C, D and B were found in this study population. HBV genotype C (n = 102; 66.7%) was found to be the predominant circulating genotype (p=0.002), followed by genotype D (n = 49; 32%) and B (n = 2, 1.3%) shown at table 1.

Distribution of HBV genotypes in five geographical areas
Distribution of different HBV genotypes was described in different regions of Myanmar (Table 2, Figure 2). HBV genotype C was predominant in all areas except western area. Genotype B was found in two areas (north and central area) with only 1.3 percent of HBV isolates. Genotype D was major genotype (59%) for western part of Myanmar which is bordered with India and Bangladesh. Among the 35 subjects from the eastern area, HBV genotype C was predominant, identified in 26 (74.3%) of subjects. Differential genotype distributions are seen on western and eastern areas of Myanmar.
Genotyping of 153 HBV isolates (Accession number: MH816993-995 and MH925817-925683) were determined by constructing phylogenetic tree (Fig.3). The test sequences were grouped with reference sequences (additional file 1) according to their genotypes and sub-genotype. The genotypes of these sequences were also determined by NCBI genotyping tool which gave complete fidelity findings with the phylogenetic results.
Genotype D study sequences were clustered into sub-genotype D3 by reference sequences of genotype HBV/ D sub-genotype D1 to D8 retrieved from GenBank data base together with genotype D study sequences to construct phylogenetic tree by neighbor joining Method using the maximum composite likelihood method to calculate evolutional distance (Fig.4) and Genotype C sequences were clustered into sub-genotype C1, C5 and C7 by subgenotype reference sequences (Fig.5).

Discussion
The genotyping of HBV is important for clarifying the route of infection with and virulence of the virus. In particular, examination of sequence diversity among different isolates of the virus is important since variants may differ in their patterns of serological reactivity, replication of the virus, activity of liver disease, prognosis and response to treatment.
A total of 353 subjects with hepatitis B infection from general population of Myanmar were enrolled in this study. Previously, there is no information regarding the regional prevalence of HBV genotypes from Myanmar. A multi-country study on chronic liver diseases patients, the most common genotype identified in Myanmar was type C [21]. The recent study findings, the major genotype was C which is in accordance with previous HBV studies in Myanmar.
HBV genotypes have been shown to have a distinct geographic distribution. The predominant genotypes reported from Southeast Asian countries are genotype HBV/ C from Thailand, genotypes HBV/C and HBV/ B from Indonesia. In China, HBV/C and HBV/ B were found to be predominant among the Negrito and Mongoloid tribes of these islands.
Genotype HBV/A and HBV/ D were the most prevalent genotypes in India.
In this study, genotype HBV/D is predominant genotype in western area of Myanmar and type C mostly found in Eastern border area bordered with China and Thailand as well as central and southern area. It's indicated that genotype C is major genotype of Mongoloid tribes and might be spread of root of infection.
Moreover, Paraskevis et. al [22] reported that genotype C is the oldest HBV genotypes and it has highest numbers of sub-genotypes, C1-C16 [23][24], reflecting the long duration of its endemicity in humans. In this study, a few numbers of sub-genotypes circulate in different parts of Myanmar but majority was sub-genotype C1. It was quite similar subgenotypes found in United States-bound refugees from Myanmar and adult immigrant in Australia from Myanmar [25][26].
Regarding the sub-genotypes of HBV/ C is at least two subtypes in Asia and HBV/C1 was found only in Southeast Asia including Vietnam, Myanmar and Thailand, while HBV/C2 was found in east Asia including Japan, Korea and China. In this study, most of the Genotype C was found to be sub-genotype C1 which was equally distributed all geographical areas of Myanmar. Some strains from study subjects showed sub-genotypes C5 and C7 which was in few percentage of study population and found mainly on central area of Myanmar.
Moreover, this result was quite similar to the previous study on 15 isolates from hepatitis carriers which showed that all the HBV isolates were sub-genotypes C1 [15] and it was seemed to be present in quite long time in Myanmar [21]. Presence of multiple subgenotypes HBV/C indicated that HBV has been spread for a very long duration in Myanmar. According to recent system and comparative analysis of sub-genotypes D, at least six (D1-D6) can be classified. Among the genotype HBV/D, the sub-genotypes HBV/ D3 was mostly found in this study and it was followed by D6 (Table.1). This is the first report on second most prevalence of genotype HBV/D, sub-genotypes D3 in Myanmar because few percentages of genotype D were reported previously on clinical case study [13]. However, the prevalence of genotype D was higher than previous findings and it might be due to frequent travelling of people from one country to another and migration. In addition, there is no large scale study of HBV genotypes in Myanmar with sequencing method for reliable data and it might be also associated with regional variation. Genotype D was mostly found in western area of Myanmar which was quite near to India and Bangladesh in which Genotype D is more prevalence [27,28].
In this study, a relatively lower portion (1.3%) of study population is found genotype HBV/B, sub-genotype HBV/B4 and HBV/B5. The previous study in Yangon region also showed that the absence of B genotype was found in their study population [13] and discrepant findings with adult immigrants study in Australia where there was shown 10.5% of adult immigrant from Myanmar was characterized as genotype B [26]. This study showed only 1.3% of study population was found to be HBV/B genotype which indicated that low prevalence of this genotype was circulating in the country.
Geographic distribution of HBV genotypes may be related to route of exposure. For example, genotypes B and C are more common in high-endemic regions of perinatal or vertical exposure, which plays an important role in viral transmission. Other genotypes are primarily observed in regions of horizontal exposure [9][10][11]29]. Therefore, genotyping provides an epidemiological clue in the investigation of acquisition and the geographical distribution of HBV [9][10][11]29]. In this study, genotype C is predominant in most regions of Myanmar and vertical transmission is seemed to be the main mode of transmission. As there is no documented study on transmission pattern of HBV in Myanmar, further study will be needed to be clarified.
In an era of frequent international travel and human migration, introduction of new HBV genotype to a community might have far reaching effects, including recombination between genotypes [30] or replacement of one genotype by another [31]. HBV genotype C is associated with delayed hepatitis B e antigen (HBeAg) seroconversion [32], more-active hepatitis [33], lower response to antiviral therapy [31], more advanced liver disease and a higher risk of hepatocellular carcinoma [33], compared with HBV genotype B.
In addition, 102/153 (66.7%) of study population are genotype C isolates. Thus, the patients infected with genotype C are needed to be carefully monitored to assess their clinical outcome in future. In the case of the genotype C, in particular C1, for example, an increased tendency for the development of cirrhosis and hepatocellular carcinoma (HCC) has been found for patients of over 50 years of age [37,38] obtained from the study participants who were at or more than 16 years and from the parents/guardians of the study participants who were under 16 years.

Consent for Publication
Not applicable.

Availability of data and material
The partial sequences of the 153 HBV isolates were submitted to Gene Bank. The accession numbers of this study isolates were MH816993-995 and MH925817-925863.
Original data may be obtained by email to corresponding author.  HBV genotype distribution in five geographical parts of Myanmar. The map that was described in figure 2 was drawn by ourselves, own creation.

Figure 3
Phylogenetic tree of 153 HBV sequences with NCBI eight major genotypes.
Phylogenetic tree was constructed using 578 bp nucleotide sequences (2860-222) PreS1/ PreS2 region of reference genome of hepatitis B genotype A-_H representing the standard genotypes throughout the world were used for analysis.  Genbank reference sequences are shown by HBV sub-genotype and accession number. Study sequences were designed by study number.