ANALISIS SEKUEN PARSIAL GEN MAJOR CAPSID PROTEIN (MCP) INFECTIOUS MYONECROSIS VIRUS (IMNV) ISOLAT INDONESIA

ROZI, Prof. Dr. drh. Kurniasih, M.V.Sc.

2013 | Tesis | S2 Bioteknologi

Penelitian ini bertujuan untuk mengetahui Infectious myonecrosis (IMN) yang disebabkan oleh infectious myonecrosis virus (IMNV), merupakan agen penyebab penyakit sistemik serius yang dapat menyebabkan morbiditas dan mortalitas yang cukup signifikan pada budidaya Litopenaeus vannamei. Virus IMN di Indonesia dilaporkan pertama kali terjadi di Asia adalah daerah Situbondo, Indonesia tahun 2006 dan mortalitas pada udang vaname bisa mencapai 70%. Gen penyandi protein capsid IMNV diduga merupakan segmen gen spesifik yang digunakan untuk mempelajari variasi genetik virus IMN serta memperlihatkan hubungan antara evolusi molekuler dan distribusi geografisnya. analisis sekuen nukleotida dan asam amino, serta gambaran kekerabatan virus IMN antar pulau di Indonesia berdasarkan gen Major Capsid Protein. Isolat penelitian virus IMN berasal dari 3 pulau di Indonesia yaitu Lampung (Sumatra), Pontianak (Kalimantan), Kendal dan Banyuwangi (Jawa). Kemudian isolat Virus IMN tersebut dideteksi menggunakan metode OIE (2009) yaitu Nested RT-PCR dan berhasil teramplifikasi pada ukuran 329 dan 139 bp. Selanjutnya optimasi parsial gen MCP virus IMN pada region 9 dan 10 menggunakan metode one-step RT-PCR yang dimulai pada posisi nukleotida 3841 sampai posisi nukleotida 4345 (#ABN05324.1 GenBank) dan berhasil teramplifikasi 600 bp dan 633bp. Sekuensing dan analisis filogenetik digunakan untuk melihat hubungan kekerabatan virus IMN. Hasil alignment menunjukkan homologi nukleotida antar strain penelitian sebesar 98,90% -99.20% dan dengan strain pembanding sebesar 98.70% - 99.40%. Hasil translasi asam amino menunjukkan adanya homologi sebesar 98.30%-99.10% dan dengan strain pembanding sebesar 98.30%-99.70%. Analisis kladogram berdasarkan algoritma Neighbour-Joining dan Maximum parsimony menunjukkan bahwa Kekerabatan 3 isolat IMNV asal (Lampung, Pontianak, dan Banyuwangi) semua IMNV 100% valid satu group/kerabat, sampel IMNV asal Lampung identik dengan asal Banyuwangi, satu kluster dengan strain pembanding IMNV asal Indonesia dari genbank (47%) dan IMNV Pontianak berbeda dengan 2 sampel penelitian lain asal (Lampung, dan Banyuwangi) walau validitas rendah (60%). IMNV-Brazil menunjukkan hubungan kekerabatan berbeda kluster terhadap isolat IMNV-Indonesia.

Infectious Myonecrosis (IMN), which is caused by Infectious Myonecrosis Virus (IMNV), is rerecognized as causative agents of serious systemic diseases that cause significant morbidity and mortality in cultured Litopenaeus vannamei. In 2006 the first disease outbreaks caused by IMNV in Asia occurred in Situbondo district,, Indonesian and with the mortality of more than 70%. Gene encode sub major capsid proteinof this virus was predicted has a specific segment gene that used to study the genetic variation due to random mutations in a spaces uniform and it has been shown to reflect evolutionary relationships and geographic origins also of IMN virus strains. The aims of this study were to determine the nucleotide and amino acid sequences in Indonesia and phylogenetic relation of gene encode IMN virus sub major capsid protein. Gene encode IMNV sub major capsid protein was amplified using one-step RT-PCR followed by nested PCR. Amplification process began from nucleotide 3841 to 4345 (#ABN05324.1 GenBank) in the coat protein segment. Sequencing and phylogenetic analysis were used to obtain the phylogenetic relation of IMN virus. Homology of nucleotide in the studied strains were 98,90% to 99.20% and comparison strains were 98.70% to 99.40%. Amino acid translation homology ranged from 98.30% to 99.10% and strain comparison by 98.30% to 99.70%. Cladogram analysis constructed by neighbor-Joining and maximum parsimony algorithm showed that 3 isolates IMNV from (Lampung, Pontianak, and Banyuwangi) all IMNV 100% valid one group / family, samples from Lampung IMNV Banyuwangi identical to the original, a cluster with a strain comparison IMNV from Indonesia from GenBank (47%) and Pontianak IMNV different with 2 other sample origin (Lampung, and Banyuwangi) despite low validity (60%). IMNV-Brazil showed distinct phylogenetic relationship of the isolates of cluster-Indonesia.strain IMNV research closely related virus strains IMNV_Brazil and IMNV_Indonesia (100).

Kata Kunci : Gene major capsid protein, Infectious Myonecrosis Virus (IMNV), Litopenaeus vannamei, RT-PCR, nested PCR