Analisis filogenetik gen neuraminidase dan fingerprint RAPD RT-PCR virus avian influenza subtipe H5N1

IRIANINGSIH, Sri Handayani, Dr. drh. Aris Haryanto, M.Si

2010 | Tesis | S2 Bioteknologi

Penyakit Avian Influenza (AI) subtipe H5N1 pada unggas dilaporkan di Indonesia sejak tahun 2003 dan menular ke manusia sejak tahun 2005. Virus AI memiliki sifat mutasi yang tinggi, terutama gen Haemagglutinin (HA) dan Neuraminidase (NA). Protein NA berperan penting dalam replikasi virus yang terus dikaji dalam bidang epidemiologi molekuler dan pengembangan obat antiviral. Penelitian ini bertujuan untuk mempelajari karakteris tik sekuen nukleotida dan asam amino virus AI subtipe H5N1 berdasarkan gen NA, dan memanfaatkan analisis fingerprintRAPD RT-PCR untuk studi filogenetik berdasarkan genom virus AI subtipe H5N1. Sebanyak 29 isolat virus AI asal berbagai spesies unggas dan lokasi di Jawa tahun 2007–2010 digunakan dalam penelitian ini. Lima belas isolat virus teridentifikasi Influenza A subtipe H5N1 menggunakan One Step RT-PCR, dan enam isolat diantaranya disekuensing terhadap gen NA. Metoda RAPD RT-PCR menggunakan primer RAPD-1, RAPD-5, dan RAPD-6 dikerjakan untuk 15 isolat virus AI H5N1. Analisis sekuen nukleotida dan asam amino menggunakan program MEGA 4 sedangkan fingerprint RAPD RT-PCR menggunakan BioNumerics v6.1. Isolat sampel penelitian mempunyai keragaman genetik dengan membentuk 5 kluster. Perbedaan nukleotida gen NA sepanjang 483 bp antara enam isolat sampel dan isolat AI kasus pertama pada tahun 2003 menunjukkan 2,8–4,7%. Enam isolat sampel mempunyai kekerabatan genetik yang berjarak 1,3–3 % dengan isolat asal manusia. Asam amino pembentuk oseltamivir binding pocket yaitu R224, H274, E276, R292, dan N294 pada protein NA isolat sampel menunjukkan tidak ada perubahan. Isolat virus AI H5N1 yang dikaji berdasarkan gen NA dapat disimpulkan bahwa karakteristik nya menunjukkan perubahan dinamis sejak tahun 2003, terjadi perubahan hidrofobisitas asam amino namun tidak ada perubahan sensitifitas antiviral oseltamivir, dan metoda fingerprint RAPD RT-PCR dapat digunakan untuk studi filogenetik namun dibandingkan dengan sekuensing, kemiripan pola yang ditunjukkan rendah.

Avian Influenza (AI) subtype H5N1 disease in poultry has been reported in Indonesia since 2003 and transmit to human since 2005. The AI virus have a highly mutation , including Neuraminidase (NA) gene segment. The NA protein plays an important role in virus replication that needs continuing analyzed for molecular epidemiology and development of antiviral drugs. Aims of this study were to study the characteristic of nucleotides and amino acids sequence of AI virus subtype H5N1 based on NA gene, and to apply fingerprints RAPD RT-PCR technique in order to study the viral genome phylogenetic. Isolates of AI virus from various bird species and regions of Java between 2007 and 2010 were tested for their identification to Influenza A subtype H5N1 using One StepRT-PCR. Out of 29 AI virus isolates identified, 15 isolates were Influenza subtype H5N1 and 6 of them were further analyzed by sequencing of the amplified NA genes. Technique of fingerprints RAPD-PCR was carried out for 15 AIV H5N1 isolates. The analysis for sequence of nucleotides and amino acids using MEGA 4 software while fingerprints RAPD-PCR using BioNumerics v6.1. All isolates studied have genetic diversity that could generate 5 clusters. The difference in 483 nucleotide s of NA gene between 6 sample isolates and isolate of first outbreak in Indonesia 2003 showed 2.8-4.7%. Sequence analysis of six sample isolates have close genetic relationship with isolates from human about 1.3-3% in their genetic distance. Amino acids sequence of oseltamivir binding pocket in active sites R224, H274, E276, R292, and N294 of NA protein were still conserve. These findings suggest that studied AI virus isolates showed their characteristics were dynamics changes since 2003, the changes to amino acid hidrophobic ity while still conserve on their sensitivity to antiviral drug oseltamivir and fingerprint RAPD RT-PCR method could be applied in order to study their phylogenetics were compared to sequencing, the similarity pattern of fingerprint was low.

Kata Kunci : Avian influenza,Subtipe H5N1,Neuraminidase,RAPD RT,PCR,Filogenetik