Some Epitopes Conservation in Non Structural 3 Protein Dengue Virus Serotype 4

Background: Non Structural 3 (NS3) protein of dengue virus (DENV) is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify conservation ofT and B cell epitope in NS3 protein among DENV-4 strains and four serotypes DENV of Indonesia strains.Methods: Research was held at the Department of Microbiology, Faculty of Medicine, UniversitasIndonesia, June 2013 to April 2014. NS3 amino acid sequence of DENV-4 081 strain was obtained afterNS3 gene of DENV-4 081 PCR products were sequenced. T and B cell epitopes of NS3 protein of DENV-4081 strain were analysed and compared to NS3 proteins of 124 DENV-4 strains around the world and fourserotypes of Indonesia strains. World strains were isolated from America (i.e. Venezuela, Colombia, etc.)and Asia (i.e. China, Singapore, etc.). For the comparison, T and B cell epitope positions of NS3 proteinwere obtained from published report.Results: Eight positions of T cell epitopes and two positions of B cell epitopes of NS3 DENV-4 081 wereidentical and conserved to NS3 protein of 124 DENV-4 strains around the world. B cell epitope of NS3 DENV-4 081 protein at aa 537-544 was found identical and conserved to four serotypes DENV of Indonesia strains.Conclusion: This wide conservation of T and B epitopes in almost all DENV-4 strains around the worldand all serotypes of Indonesia strains. (Health Science Journal of Indonesia 2015;6:126-31)

Dengue virus (DENV) infection remains a public health problem, either in the world or in Indonesia. According to data from World Health Organization (WHO), 50 to 100 million infections were estimated to occur each year, including 500,000 cases of dengue fever and 22,000 deaths, mostly among children. 1 Based on data from Indonesian Ministry of Health, the number of patients with DHF increased annually, in 2013 there were 112,511 of DHF cases in 32 provinces (incidence rate of 45.85 per 100,000 population). 2 The virus has three structural proteins, the capsid protein (C), envelope (E) and membrane (M). These structural proteins along with seven non-structural proteins (NS) are encoded by the viral genome in the form of single-stranded RNA with positive polarity. DENV genome is a sequence of 5 'UTR-C-PRM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3' UTR. 3 NS3 protein plays an important role in DENV life cycle. 4,5 The functions of NS3 includes a viral protease that plays a role in cutting the DENV polyprotein after translation, and plays a role in viral RNA replication through the activities as nucleotide triphosphatase (NTPase), RNA 5' triphosphatase (RTPase) and helicase. 3,6 Non-Structural (NS) 3 protein has the ability to induce antibody responses, 7 CD4 + and CD8 + T cells. T cells activated by NS3 can cross-react between serotypes. 8 Anti-NS3 antibodies were proven in vivo to protect and delay the time of death (survival time) of mice challenged with DENV at doses of 100 LD 50 . 9 NS3 sized 618 amino acid/ 69 kD, is the second largest gene/protein of DENV and its primary amino acid sequence is the most conserved.
This study aimed to obtain information about the similarity of T cell and B cell epitopes of NS3 DENV-4 081 Indonesian strain in comparison to DENV-4 world strains, as well as the similarity to the NS3 of the other serotypes Indonesia strains. . After purification of the PCR products using QIAquick PCR purification Kits (Qiagen GmbH, Hilden, Germany), the samples were sent to PT Genetika Science Indonesia for direct sequencing. The sequencing processes used Sanger method with Taq Big Dye Deoxy Terminator Cycle sequencing kits (Applied Biosystems, Foster City, CA USA). Nucleotide and amino acid sequence analysis was performed using GENETIC MAC-and BLAST program. Codon positions described in this study were based on the data of DENV-4 Singapore strain (GenBank accession no.GQ398256.1). Primer sequences for DNA synthesis and sequence were designed using PRIMER program and obtained from the published data of DENV4 Singapore strain (GenBank accession no.GQ398256.1).

METHODS
Analysis of T and B cell epitope on NS3 protein were done by comparing the amino acid sequence of NS3 protein DENV4 081 to the NS3 protein of DENV-4 world strains and serotypes of Indonesia strains which obtained from the Genbank. Total world strains were 124 strains which 25 strains were from Asia (China, Singapore, India, Malaysia, Cambodia, India, Thailand, Philippines, Pakistan, and Taiwan), 99 strains were from America (Venezuela, Dominica, Colombia, US, Haiti, and Brazil) isolated between 1961 and 2011; and twelve Indonesian strains. The references for analyzing NS3 T 8, 10-17 and B cell epitopes [18][19][20] were listed in Table  1. NS3 protein epitopes of DENV-4 081, 124 DENV-4 world strains and other serotypes of Indonesia strains were analysed using Bioedit version 7.0.5.3 year 2005 (http://www.mbio.ncsu.edu/bioedit/bioedit.html).

RESULTS
Homology analysis of T cell epitopes of NS3 protein of DENV-4 081 to the NS3 protein of 124 DENV-4 world strains (25 strains from Asia and 99 strains from America) was shown in Table 2.
From fourteen T cell epitopes of the NS3 protein of DENV-4 081 compared to NS3 protein of 124 DENV-4 world strains, different amino acid at six epitopes were found in nine strains (Table 2), while the other eight epitopes were well conserved in all 124 world strains. T cell epitopes that have different amino acids were at aa 45-59, aa 145-159, aa 231-245, aa 495-509, aa 526-540 and aa 596-610 (Table 2).  Analysis of B cell epitopes of NS3 DENV-4 081 at aa 460-469 and aa 537-544 and several DENV-4 strains ( Table 2) showed different properties of amino acids as shown in Table 3.
The B cell epitope at aa 460-469 of NS3 protein DENV-4 081 have proline which is hydrophilic, compared to the same epitope position of several DENV-4 world strains ( Table 2) have leucine which is hydrophobic. The B cell epitope of NS3 protein DENV-4 081 at aa 537-544 have arginine (hydrophilic) compared to several 124 DENV-4 world strains, there is amino acid changing from arginine (hydrophilic) to lysine (hydrophobic).

DISCUSSION
From total fourteen T cell epitopes analysed, eight T cell epitopes were 100% identic and conserved to NS3 protein of 124 DENV-4 world strains ( Table 2). Two of four B cell epitopes were well also conserved in 124 DENV-4 strains around the world (Table 2). This analysis also showed that from total 25 strains from Asia and 99 strains from America, 68% of Asia strains and 98% of America strains have similarity in all fourteen T cell epitopes of NS3 protein DENV-4. In total, 84% of Asia strains and 93% of America The 68% similarity in all fourteen epitopes analysed in DENV-4 strains from Asia needs further study, because only 25 DENV-4 strains were obtained of total 124 world strains. The variations in the amino acid sequence of T cell epitopes of NS3 protein DENV-4 were about one or two amino acid difference ( Table 2 and Table 3).
It is known that the amino acid changes would not alter the preservation area epitope recognition by antibodies and lymphocytes as long as it still have same tertiary structure. Tertiary structure of a protein is determined by various factors, one of it is hydrophobic interaction of the protein-building amino acids. Amino acid changes can cause conformational change of the protein, which may change epitope recognition by antibodies. Mutated amino acids of T cell and B cell epitopes affect hydrophobicity of epitopes so further analysis is required to get threedimensional structure of NS3 protein.
Most T and B cell epitopes of NS3 protein (Table 1) were located in the NS3 helicase, only two of the T cell epitopes from total fourteen T and B cell epitopes were located in the NS3 protease. Helicase has three subdomains with significant sequence identity and structural similarity with helicase of another flavivirus. 21 NS3 helicase has RNA unwinding activity by disrupting the hydrogen bonds of double strands DNA.
In conclusion, this wide conservation of T and B epitopes were found in almost all DENV-4 strains around the world and all serotypes of Indonesia strains.