ANALISIS KEKERABATAN VIRUS DENGUE SEROTIPE 1BERDASARKAN SEKUEN NUKLEOTIDAE/NS1 GENE JUNCTION

NUR FAIDAH MUNIR, Dr. biol.hom. Nastiti Wijayanti, S.Si., M.Si.; dr. Tri Wibawa, Ph.D

2012 | Tesis | S2 Bioteknologi

Penyakit demam berdarah dengue (DBD) yang disebabkan oleh infeksi virus Dengue masih menjadi masalah kesehatan penting di sebagian besar negara tropis dan subtropis dalam dua dekade terakhir. Hal ini ditandai dengan penyebaran geografis dari virus Dengue maupun vektornya, yang diikuti dengan peningkatan epidemi, hiperendemisitas dan munculnya demam berdarah dengue di daerah baru. Vektor pembawa virus Dengue adalah nyamuk Aedes aegypti dan Aedes albopictus. Genom virus Dengue tersusun atas tiga protein struktural (protein nukleokapsid, protein envelope, dan protein pre-membran) dan tujuh protein nonstruktural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, dan NS5). E/NS1 gene junction merupakan segmen gen spesifik yang digunakan untuk mempelajari variasi genetik virus Dengue serta memperlihatkan hubungan antara evolusi molekuler dan distribusi geografisnya. Penelitian ini bertujuan untuk mengetahui sekuen nukleotida dan asam amino di Indonesia, serta gambaran kekerabatan virus DEN-1 berdasarkan E/NS1 gene junction. Segmen E/NS1 gene junction virus Dengue diamplifikasi menggunakan metode one-step RT-PCR yang dimulai pada segmen envelope pada posisi nukleotida 1869 (#AB074760.1 GenBank) sampai posisi nukleotida 2643 (#AB074760.1 GenBank) pada segmen NS1. Kemudian dilanjutkan dengan nested PCR. Sekuensing dan analisis filogenetik digunakan untuk melihat hubungan kekerabatan virus DEN-1. Hasil alignment menunjukkan homologi nukleotida antar strain penelitian sebesar 96,8% - 100% dan dengan strain pembanding sebesar 96,1% - 98,5%. Hasil translasi asam amino menunjukkan adanya homologi sebesar 94,7 - 100% dan dengan strain pembanding sebesar 93,6% - 97,8%. Analisis kladogram berdasarkan algoritma Neighbor-Joining menunjukkan bahwa strain virus penelitian berkerabat dekat dengan strain virus DEN-1 asal Thailand, Hawaii, dan Jepang. Namun, memiliki kekerabatan yang jauh dengan strain virus DEN-1 asal Indonesia yang telah ditemukan sebelumnya.

Dengue hemorrhagic fever (DHF), which is caused by Dengue virus infection remains a significant health concern in most tropical and subtropical countries in the last two decades. It is characterized by the geographic distribution of Dengue virus and its vector, followed by the epidemic increase, hyperendemicity and the emergence of dengue hemorrhagic fever in new areas. Vector of Dengue virus are Aedes aegypti and Aedes albopictus mosquito. Dengue virus genome composed of three structural proteins (nucleocapsid protein, envelope protein, and pre- membrane protein) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4B, and NS5). E/NS1 gene junction is a specific segment gene that used to study the genetic variation due to random mutations in a uniform spaces and it has been shown to reflect evolutionary relationships and geographic origins also of dengue virus strains. The aims of this study were to determine the nucleotide and amino acid sequences in Indonesia and phylogenetic relation of DEN-1 based on E/NS1 gene junction. E/NS1 Dengue virus gene junction was amplified using one-step RT-PCR followed by nested PCR. Amplification process began from nucleotide 1869 (#AB074760 GenBank) in the segment envelope to 2643 (#AB074760 GenBank) in the NS1 segment. Sequencing and phylogenetic analysis were used to obtain the phylogenetic relation of DEN-1 virus. Homology of nucleotide in the studied strains were 96.8% to 100% and comparison strains were 96.1% to 98.5%. Amino acid translation homology ranged from 94.7 to 100% and strain comparison by 93.6% to 97.8%. Cladogram analysis constructed by Neighbor-Joining algorithm showed that virus strains closely related with DEN-1 virus strains from Thailand, Hawaii, and Japan. Moreover, it has a distant phylogenetic with DEN-1 virus strains from Indonesia which had been found previously.

Kata Kunci : demam berdarah dengue, DEN-1, E/NS1 gene junction, one-step RT- PCR, nested PCR