Diversity of Hepatitis B genotypes in Nepal and updated Phylogenetic Tree: a Pilot Survey in 2012

Introduction: Hepatitis B virus (HBV) is a current global health problem. HBV genotypes influence the treatment and long term outcome of HBV infected patients. Moreover, HBV genotypes differ in various region of the world. Such data was reported haphazardly but yet to be comprehensive for Nepal. This study attempted to find out the diverse hepatitis B genotypes in Nepal. Methods: A convenient serum sample of 58 HBsAg positive patients from different parts of the country mainly from Nepalgunj, Palpa and Kathmandu were screened for hepatitis B genotype. Sequencing was done and Phylogenetic tree was created. Results: Among 58 samples, 23 were genotype D, 17were genotype A and B wereC/D recombinant. Phylogenetic trees were created by distance-matrix and neighbor-joining analyses after bootstrapping to 1000 replicates. Discussion: HBV genotypes A and D are the most common genotype in Nepal. Horizontal transmission is common in these genotypes. C/D recombinant genotype may be transmitted from Tibetan people living in Kathmandu. Prophylactic major controlling, horizontal and cross border transmission could be effective. Conclusions: Three major genotypes of HBV in Nepal were found to be A, C and D. Despite being a low prevalence area, Nepal has a diversity of hepatitis B genotypes _______________________________________________________________________________________


INTRODUCTION
Each year three million chronic infected patients and one million annual deaths by Hepatitis B(HBV) related liver disease, indicates an urgent necessity of better ways to prevent the complication of HBV. 1 It is the tenth leading cause of death and main contributor of hepatocellular carcinoma, which ranks fifth leading cause of cancer in the world. 2 Indefinite symptoms, delayed diagnosis and consequent progression to complication lead to death among silent HBSAg positive carriers. 3,4,5 Genotyping is critically necessary for therapeutic decision making as well as CC S for preventive and control measures.
Among HBV genotype A to I (9), genotype C, A, A, D and A+D is consecutively prevalence in Tibet Nepal, southern and northern India. 4,6,7,8 The report on Geographical distribution of HBV genotypes in Nepal is still lacking. The objective of the study is to find out the genotypes and to plot them in Phylogenetic tree.

METHODS
Site: This study was conducted as a multiple center research in Nepal Study design: This is a cross-sectional study based on different regions of Nepal.
Study population: Patients attending the Nepalgunj, Palpa and Kathmandu hospitals with the evidence of HBV infection by positive HBSAg were conveniently included. Patients gave informed consent and hospital authority allowed collection of samples.

Criteria for inclusion:
Steps of sample collections: --3ml each of peripheral venous blood from the routine collection of blood sample for medical check-up was used. Serum was separated and 1.5 ml serum sample were stored at -70˚C. Specimen transfer chain was maintained at -25˚C and sent to the Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, and Bangkok, Thailand. Sample: 1.5 ml each of serum samples stored in -70°C was used for the extraction of viral DNA.
Centre for genotyping: the Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, and Bangkok, Thailand.
Ethical clearance: This study was approved by institutional review board of Mid-Western Regional Police Hospital (MWRPH).The hospital director of Nepalgunj Medical College and, PalpaMedical College, and Sechen Hospital, Kathmandu, allowed collecting the samples. Informed consent was obtained from each patient after providing information concerning this research in the native language.

Extraction of HBV DNA and amplification
A total of 58HBSAg positive samples were collected from Nepalgunj, Palpa and Kathmandu under the supervision of experienced health personnel's.
HBSAg was tested by the micro particle enzymeimmunoassay. Positive serum was separated and kept frozen at -70˚C in each center. Viral DNA was extracted by using phenol/chloroform/isoamyl alcohol extraction method. Briefly, 100 µl plasma of each sample were incubated with lysis buffer (10 mMTris-HCl pH 8.0, 0.1 M EDTA pH 8.0, 0.5% SDS) and 20 mg/ml proteinase K. The DNA in the sample was subsequently extracted in phenol/chloroform/isoamyl alcohol followed by ethanol precipitation. The pellet was re-suspended with 30 µl distilled water. The polymerase chain reaction (PCR) was done under standard conditions: pre-cycle 94˚C/3 min, denaturing 94˚C/30 sec, annealing 55˚C/30 sec, extension 72˚C/1.30 min, 40 cycles, final extension 72˚C/7 min. a standard operating procedure was strictly applied to prevent cross contamination. First round PCR reaction mixture was comprised of 4 µl resuspended DNA, 10 µl PerfectTaq TM Plus MasterMix

Genotyping and sequencing
Nested PCR products were checked on 2% agarose gel electrophoresis. PCR products in gel slices were purified by HiYield TM Gel/PCR Fragments Extraction Kit (Real Biotech Corporation, Taipei, Taiwan). Purified products were subjected to determine the nucleotide sequences by direct sequencing method (

HBV DNA quantitative measurement
SYBR ® Green based quantitative real-time PCR was used to determine the HBV DNA concentration. The condition and reaction mixture followed to those described in the previous study. 18

RESULTS
All 58 samples were HBsAg positive. Mean age of the male was 39.9 years and of the female was 31.6 years. Twenty six samples were real time conventional PCR positive. After nested PCR, HBV DNA was successfully amplified from 42 samples. Among the 42 positive samples, complete S part of the viral DNA were easily obtained for 28 samples by direct sequencing using different primer pairs. For the rest of 14 samples complete scene was not amplified and was classified by viral genotyping tool on NCBI.

DISCUSSION
In our study we explored genotype A, D and C/D recombinant genotype which is slightly different from the finding in the previous study as authors explored A, B, C, and D genotypes. 4 The result is little bit various, may be due to the less number of distribution of Genotype b in Nepal.
Hepatitis B genotyping is important for the clinical management of the disease, because HBV genotype is correlated with the replication of the virus, its activity in the liver and treatment outcome. 9 In our study Hepatitis B is more prevalent in young men than in women the ratio of male to female is 1.13:1 Young female mean age less than male was found infected which is different picture than a study in neighboring country Pakistan. 10 Distribution of different genotype of HBV is different in both genders in our study. As huge percent of female was found genotype D and rest of the female has got the genotype A. There is no mix genotype isolated in female. Most of the male has genotype A, while less of them have Genotype D and mixed infection. Our study prevails that there is prevalence of single Genotype D and genotype A infection in Nepal ,which are the predominant genotype in India too but There is no mix infection of HBV genotype A+D or B+D, as in neighboring country India and Pakistan. 10 HBV genotype A and D correlation with age group was not significant in our study.
The geographical distribution of genotype intra-country, A and D is almost the same in ratio in Nepalgunj, the mid-western city of Nepal. Genotype D was dominant in Palpa, a western region of Nepal. The C/D recombinant genotype was found in Kathmandu, the central region of the country. The recombinant strain C/D in Kathmandu may be prevalent in Tibetan migrant as mentioned by the author. 19 A study conducted in New Delhi, India revealed that mix infection of A+D is prevalent in Northern India which is near by our one of the study site. The dominance of Genotype D in Nepal was reported in the previous study too. 12 HBV genotyping showed distinct geographical distribution within and between the regions. Although the sample size from Kathmandu and Palpa was small, distribution of the different genotypes in the mid -west Nepal(Nepalgunj) would be suggestive of all genotypes representing in the country including genotype A,B,C and D reported by previous publication. 13 In our study the mean age of the HBV patients is 35 years old, indicating that HBV is a disease of working population in Nepal. Young and sexually active female are more prone to the disease it may be due to their sexual behavior. 14 As genotype D can be transmitted by all routes (parenteral, perinatal, sexual) may be because of which it is distributed in all region.
This study confirmed that HBV genotype A and D are dominant in Nepal. 14 Shrestha et al. 2007 reported by the same observation Genotype of Nepal is more similar with India and Bangladesh. The HBV genotype A of Asian type has older Phylogenetic origin and have infiltrated more deeply in this region. 15 Philippines genotype Aa is more similar to Nepal than Bangladesh and south Africa 16 which suggest that there may be some ship trade between India and Philippines ,from where the disease was transmitted to neighboring country Nepal.
In Tibet, HBV genotype C is high prevalent .One of the famous Himalayan tribe known as Sherpa's are the ancestor of Tibetan, they have HBV genotype A reported by the previous study. Nepalese Sherpa who are settled for many 100 years in the base of Himalaya has Genotype A, which is more similar with Japanese genotype Aa 7 may be suggestive of the disease transmission intra country especially from the urban cities like as Kathmandu. 13 Asian type of genotype Aa has poor clinical outcome than genotype from Europe Ae. 17 HBV genotype A, which is prevalent in Africa, has 4.5 fold higher risks to get Hepatocellular carcinoma (HCC) than none A genotypes. 11 However, another group 9 reported a controversial result that HBV genotype A has the good clinical prediction than HBV genotype D. High mutant gene could be the early cause of HCC and Nepalese strain of HBV has high deletion mutant of s1 (about 27%). High prevalence of s mutant is in Nepalese HBV could be the cause of HCC, cirrhosis, chronic carrier or asymptomatic carrier of hepatitis B disease. 13 In our study C/D recombine genotype in Kathmandu may be suggestive that, the transmission of the disease in Kathmandu is horizontal and vertical both route as mention in previous study. 14 It could be suggestive that Nepal has less prenatal transmission (vertical) than other routes (horizontal).

LIMITATIONS
Even though we took the sample from different sites, the small sample in our study may not represent nation-wide distribution. We took HBSAg positive sample, the finding is not quite different from previous finding 4 represented by a little big sample size 121 from Kathmandu. Consequently, viral load was not sufficiently high to be sequenced in some samples.

CONCLUSIONS
HBV genotypes in Nepal. It is important for the prevention strategy. The authentic agency will have to focus on horizontal transmission to prevent HBV in Nepal. Moreover, the finding in this pilot study will guide to calculate the sample necessary for bigger nationwide study. A nation-wide, properly sampled molecular survey of HBV and a prospective trial or cohort study to see the treatment outcome among prevalent genotypes in Nepal are recommended