Haplotype Network Analysis and Phylogenetic Tree Construction of Hepatitis C Virus (HCV) Isolated from Tuban, Indonesia
Supiana Dian Nurtjahyani1*, Rasyadan Taufiq Probojati2,3, Arif Nur Muhammad Ansori4, Mochammad Amin5,
Retno Handajani6
1Faculty of Teaching and Education, Ronggolawe University, Tuban, 62381, East Java, Indonesia.
2Division of Molecular Biology and Genetics, Generasi Biologi Indonesia Foundation,
Gresik, 61171, East Java, Indonesia.
3Faculty of Agriculture, Universitas Kadiri, Kediri, 64115, East Java, Indonesia.
4Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, 60115, East Java, Indonesia.
5Institute of Tropical Disease, Universitas Airlangga, Surabaya, 60115, East Java, Indonesia.
6Faculty of Medicine, Universitas Airlangga, Surabaya, 60115, East Java, Indonesia.
*Corresponding Author E-mail: diananin39@gmail.com
ABSTRACT:
This study aimed to analyzed the haplotype network and constructed the phylogenetic tree topology of hepatitis C virus (HCV) in Tuban, Indonesia and those from other countries, to determine the visualize genealogical relationship and inference about gene connected. The HCV isolates were collected from blood transfusion center of Tuban, East Java, Indonesia in 2015 and as a comparison, also the sequences of HCV isolates were retrieved from the GenBank®, National Center of Biotechnology Information (NCBI), USA database. To constructed of distribution map was performed through median joining analysis using Haplotype Network v4.6, whereas to constructed and phylogenetic model analyses were constructed based on the NS5B and 5’UTR regions using MEGA X, maximum-likelihood method based on the Tamura-Nei model. In the HCV NS5B region haplotype network showed high haplotype diversity (Hd=1.00) in 22 haplotypes. Based on phylogenetic analysis 3 sample isolates Tuban (BDT-55-p23, BDT-79-p23 and BDT-112-p23) were identified one group and closely related to isolates from Indonesia. However, isolates Tuban also have the same sequence characters as the isolates from Thailand. It was also confirmed in haplotype network that the three isolates are connected to each other. The identification of HCV genotypes circulating in blood donors in the Tuban of East Java confirmed were closely related to HCV isolates from Indonesia and Thailand. Therefore, this result might contribute in a better medical management towards HCV.
KEYWORDS: Haplotype Network Analysis, Hepatitis C virus, Phylogenetic Tree.
INTRODUCTION:
Hepatitis C virus (HCV) infection has through an important and spread concern the world. It is due to a global incident that has increased rapidly over the years1,2. HCV infection is known contributor to liver cells diseases and it can cause inflammation of the liver with multiple complications for a long time, including chronic hepatitis, liver cirrhosis (LC) and hepatocellular carcinoma (HCC)2,3.
HCV is categorized as a single stranded RNA virus and has been classified variability into 7 distinct HCV genotypes and over 70 subtypes4,5,6,7,8. The genetic diversity of the virus, especially HCV will increase as the virus spreads. The varied genotypes have been repeatedly sequenced at different geographical location, time points and by mode of transmission1,9,10. In recent years, the existence of genetic variations of HCV will make this virus to be resistant to combinations various types of antiviral drugs11,12,13. It is caused of biological response which shown to vary with host and each genotype variation have been shown particular features, for example genotype type 1 infected patient will have a different biological response from genotype type 2 infected patient2,13.
HCV identification supported by genomic research with assessment of HCV genotypes diversity are important to long-term evolutionary association between a new type of genotype. Nevertheless, the numerous recent reports of HCV genotype distribution14,15,16,17. Thus, resulted incongruent tree topologies being reported of HCV among recent studies. Therefore, it is important support by providing a haplotype network assessment of HCV genotype data. The regions of genome which can be used as basis of HCV classification is 50-UTR, C/E1 and NS5B4. NS5B is nonstructural proteins used in replication, other that relatively well conserved regions of the genome18,19.
This study aimed to describe the distribution of HCV in Tuban, Indonesia and constructed the topology phylogenetic tree and evaluated by haplotype diversity of Tuban HCV isolates from other regions and other HCV to determine their relationship. Haplotypes networks can visualize biogeographic relationships, history of evolutionary and make inferences based on gene flow. Haplotype networks considered more informative than visualize topology phylogenetic trees20,21. Previous studies were reported of visualize phylogenetic trees HCV in Indonesia, but still has not been evaluated by haplotype networks.
MATERIAL AND METHODS:
Distribution and sample collection:
The blood serum was obtained from 500 voluntary adult blood donors. The blood serum was collected from blood transfusion center of Tuban, East Java, Indonesia in 2015. Further separation of samples was conducted in the laboratory of Ronggolawe University in Tuban and tests of anti-HCV antibody and the HCV RNA detection were performed in the Institute of Tropical Disease, Universitas Airlangga, Surabaya, East Java, Indonesia. As a comparison, also the sequences of HCV isolates were retrieved from the GenBank®, National Center of Biotechnology Information (NCBI) database (Table 1).
Table 1: Hepatitis C Virus and Bat Hepacivirus from this study and NCBI databases.
|
Code |
Origin |
Host |
Isolation Source |
|
BDT-55-p23 |
Indonesia: Tuban |
Homo sapiens |
Serum |
|
BDT-79-p23 |
Indonesia: Tuban |
Homo sapiens |
Serum |
|
BDT-112-p23 |
Indonesia: Tuban |
Homo sapiens |
Serum |
|
AB103154.1 |
Myanmar |
Homo sapiens |
Unknown |
|
AB103153.1 |
Myanmar |
Homo sapiens |
Unknown |
|
MN807685.1 |
Thailand |
Homo sapiens |
Plasma |
GU441457.1 |
Indonesia |
Homo sapiens |
Serum of anti-HCV positive HCC patient |
GU441456.1 |
Indonesia |
Homo sapiens |
Serum of anti-HCV positive HCC patient |
GU441455.1 |
Indonesia |
Homo sapiens |
Serum of anti-HCV positive HCC patient |
GU441420.1 |
Indonesia |
Homo sapiens |
Serum of anti-HCV positive chronic patient |
GQ418381.1 |
Indonesia |
Homo sapiens |
Serum of anti-HCV positive chronic patient |
GQ418386.1 |
Indonesia |
Homo sapiens |
Serum of anti-HCV positive cirrhosis patient |
GQ418349.1 |
Indonesia |
Homo sapiens |
Serum of anti-HCV positive HCC patient |
AB714194.1 |
Indonesia: Yogyakarta |
Homo sapiens |
human serum obtained from hemodialysis patient |
AB714196.1 |
Indonesia: Yogyakarta |
Homo sapiens |
Human serum obtained from hemodialysis patient |
JN202397.1 |
Indonesia |
Homo sapiens |
Unknown |
JN202396.1 |
Indonesia |
Homo sapiens |
Unknown |
JN202368.1 |
Indonesia |
Homo sapiens |
Unknown |
KY624488.1 |
Singapore |
Homo sapiens |
Unknown |
KY624479.1 |
Singapore |
Homo sapiens |
Unknown |
KC796026.1 |
Kenya |
Bat hepacivirus |
Serum |
KC796031.1 |
Kenya |
Bat hepacivirus |
Serum |
Anti-HCV antibody detection:
Anti-HCV antibody detection was assessed by enzyme-linked immunosorbent assay (ELISA) using HCV Antibody EIA tool (Foresight, Acon Laboratories Inc., San Diego, USA). The inspection procedures carried out based on the ELISA kit’s manufacture protocol.
RNA extraction of HCV and reverse transcription:
HCV RNA was extracted from 140mL of serum anti-HCV positive using Qiagen RNA extraction kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s protocol. Furthermore, cDNA synthesis was performed using the enzyme reverse transcriptase by RT-PCR kit (Toyobo Inc., Osaka, Japan) with RNA extraction results as a template.
Nucleic acid testing using nested PCR and Direct nucleotide sequencing:
PCR amplification was done for positive anti-HCV samples obtained from ELISA test. Initially, nested PCR for NS5B region was done for all samples and followed by the nested PCR on 5’UTR region for the negative samples from previous nested PCR. First round PCR primers used to amplify the NS5B region22 and one set of primers for 5’UTR region23. Further, the positive amplicons were performed for sequencing. The purification of PCR products was performed by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), then directly sequenced by using Big Dye Terminator cycle sequencing Ready Reaction Kit ver. 1.1 (Applied Biosystems, Foster City, CA).
Figure 1: Schematic design of methods in this study.
Molecular Haplotype Network Analysis and Phylogenetic Analysis:
Sequences evaluation were evaluated using ABI sequences Scanner v10. Multiple sequences alignments were analyzed by ClustalW algorithm using MEGA X. Nucleotides variation were analyzed with Bioedit v7 and DnaSP v5. Haplotype diversity and reconstruction of distribution map was performed through median joining analysis using Haplotype Network v4.6. Phylogenetic reconstructions and phylogenetic model analyses were constructed based on the NS5B and 5’UTR regions using MEGA X, maximum-likelihood method based on the Tamura-Nei model, 1000 bootstrap replicates.
RESULTS AND DISCUSSION:
Haplotype network construction analysis used visualize the genealogical relationships among DNA sequences within a population or at the intraspecific level, or to make inference about biogeography and history of populations21,24.
In this study, haplotype network Maximum-likelihood (ML) phylogenetic analysis were used to map the genotypes of the HCV isolates that disseminated in the Tuban, Indonesia (BDT-55-p23, BDT-79-p23 and BDT-112-p23), as well as several countries that are publicly available on NCBI databases (Figure 2, 3).
Figure 2: Haplotype distribution map of HCV isolate.
In the HCV NS5B region haplotype network, using the bat hepacivirus as an out-group were resulted in 22 haplotypes with very high haplotype diversity (Hd=1.00). This result revealed the lineage pattern of HCV lineages from several country, with bat hepacivirus as out-group which were interconnected and classified according to their sequence’s DNA (Figure 2).
Figure 3: Molecular Phylogenetic analysis by maximum-likelihood method. Statistical support for phylogenetic nodes was assessed using a bootstrap approach 1000 replicates.
Based on NS5B region of HCV, 3 sample isolates Tuban (BDT-55-p23, BDT-79-p23 and BDT-112-p23) were identified one group and closely related to isolates from Indonesia (Figure 3). However, in the HCV NS5B region trees, 2 sample isolates Tuban (BDT-55-p23 and BDT-79-p23) formed a small distinct group and closely related to GQ418349.1 and GU441455.1 from Indonesia (Figure 3). It was also confirmed in haplotype network that the two isolates are connected to each other (Figure 2).
It is possible that the sample isolates Tuban also have the same sequence characters as the isolates from Thailand (MN807685.1). Isolate BDT-79-p23 (Haplotype 1) connected directly to MN807685.1 (Haplotype 20) and the two isolates were considered identical (Figure 2). Later, isolates from Myanmar (AB103154.1; AB103153.1) and Singapore (KY624488.1) formed a cluster of their own. However, the isolate (KY624479.1) from Singapore was not identically identified in the cluster, if based on the haplotype this isolate was classified as haplotype 18 and connected to the Bat hepacivirus outgroup sample (KC796026.1; KC796031.1) (Figure 2).
Hepatitis is an inflammatory disease of the liver that can be caused by various causes, including viral infection. This viral infection can cause injury, inflammation, and even death of infected cells in the liver. HCV is one of the viruses that cause hepatitis and is considered to have the greatest impact among other viruses that cause hepatitis. Most people who are infected with the HCV have no symptoms. In fact, many people do not know that they have been infected with the HCV until there is fatal damage to their liver (silent epidemic). This damage can include liver failure, cirrhosis, or liver cancer which can appear several years after infection (chronic hepatitis C). A number of studies have also shown that the incidence of hepatocellular carcinoma is closely related to HCV infection25.
Approximately 150 million people in the world who suffer from chronic hepatitis are infected with the HCV and more than 350 thousand people die every year from liver disease related to HCV infection. HCV can be found all over the world. As the fourth most populous country, Indonesia has one of the largest HCV epidemics in the world, with an estimated 2.5 million HCV infected people.
To our knowledge, no other published study has investigated treatment costs of a national treatment program to eliminate HCV in Indonesia or has proposed a framework for designing a national hepatitis elimination program. A limited number of studies have estimated the epidemiological burden. Sibley et al. estimated around 24,800 new cases of HCV in Indonesia occurred in 201426, Heffernan et al. estimated 37 00027, whilst Trickey et al. estimated around 34,00025. Sibley et al estimated around 20,000 people will have DC in 203026, whilst we estimated a much higher figure of 90,000. In 2030, Sibley et al. estimated around 8000 HCV‐related deaths will occur26, Heffernan et al. estimated 13,00027, and Trickey et al. estimated around 12,00025.
Countries with high rates of chronic hepatitis C include Egypt (15%), Pakistan (4.8%), and China (3.2%). The mode of transmission of the HCV in these countries is generally associated with the use of injection equipment (injection equipment) that has been contaminated with HCV. Approximately 75-85% of people who are newly infected with HCV can suffer from chronic disease and 60-70% of these sufferers can develop chronic hepatitis C sufferers. About 5-20% of people with chronic hepatitis C develop cirrhosis and about 1-5% are reported to have died from cirrhosis or liver cancer. About 25% of people with liver cancer, the cause is infection with the HCV28.
HCV genotype 1 is the most commonly found genotype in Indonesia. Genotype 1 is responsible for 60-65 percent of all HCV infections in Indonesia and this genotype is associated with a lower treatment response28. Patients with chronic liver disease in Indonesia are mostly infected by HCV subtype 1c and it has been proven that the virulence subtypes 1c are equal to 1b2. Based on the Humairah et al. genotype 1 and genotype 3 had the same prevalence in IDUs with HCV infection in Surabaya (40.9%), followed by genotype 4 (13.64%) and 6 (4.55%)28. This data shows that the spread and transmission of HCV in IDUs in Surabaya remains in accordance with the global distribution pattern in which are dominated by genotypes 1 and 3, however it was followed by an increase in genotypes 4 and 6. Genotype 6 is rarely found in Indonesia. However, there could be a possibility of the transmission of genotypes 6 which are mostly found in Myanmar and Thailand to Indonesia.
A study conducted by Utama et al. revealed that in the patients with chronic liver disease in Jakarta, it was found that HCV subtype 1b was found to be the most prevalent (47.3%), followed by 1c (18.7%), 3k (10.7%), 2a (10.0 %), 1a (6.7%), 2e (5.3%), 2f (0.7%) and 3a (0.7%)2. Humairah et al. demonstrated that genotype 1 and genotype 3 were both predominant28. Genotype 1 in this study was dominated by subtype 1a (31.82%) and 1c (9.09%), while no subtype 1b was found. Genotype 3 in this study consisted of subtype 3a (18.18%) and 3k (22.73%). In line with this, a study described by Amin et al. which took samples hemodialysis patients in Surabaya, 1a was the most common genotype (52.6%), followed by 1b (15,8%), 1c (15,8%), 2a (5,3%), and 3k (5,3%)5, except that in our study we found no subtype 1b29. In addition, differences in geographic location can cause differences in genotypic distribution so further research is needed to determine differences in genotypic distribution in each region in Indonesia.
Viral genotypes are also used to predict the response to administration of antiviral drugs. For example, it is not recommended to administer sovosbuvir in combination with ribavirin in genotypes 1, 4, 5, and 6. The high frequency of genotype 3 in a country is a separate problem because genotype 3 has a tendency to be difficult to treat and has a poor prognosis2.
CONCLUSION:
The research showed that HCV sequences of blood donors from Tuban confirmed to have similarities with isolate samples from Indonesia and Thailand (based on phylogentic dan haplotype). The sequence flow connection of the HCV genotype is expected to be able to provide valuable information for future medical management.
ACKNOWLEDGEMENT:
We thank to the Blood Donor Unit PMI Tuban who have helped in providing research samples and laboratory staff biology, Unirow Tuban and ITD Universitas Airlangga, Surabaya, Generasi Biologi Indonesia Foundation which has provided facilities and support research. As well as Kemenristek-Higher Education through Kopertis Region VII Surabaya for their grants for this research funds.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 06.10.2020 Modified on 12.11.2020
Accepted on 03.12.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(8):4231-4235.
DOI: 10.52711/0974-360X.2021.00734