Karakter Genetik Protein Membran Virus Avian Influenza Subtipe
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DHARMAYANTI . et al. Karakter genetik protein membran virus avian influenza subtipe H5N1 Karakter Genetik Protein Membran Virus Avian Influenza Subtipe H5N1 N.L.P. INDI DHARMAYANTI1, D.A. HEWAJULI1, A.K RATNAWATI1, R. INDRIANI1 dan DARMINTO2 2 1 Balai Besar Penelitian Veteriner JL RE Martadinata 30, Bogor Pusat Penelitian dan Pengembangan Peternakan JL Raya Pajajaran, Bogor (Diterima dewan redaksi 26 Juli 2010) ABSTRACT DHARMAYANTI, N.L.P.I., D.A. HEWAJULI, A. RATNAWATI, R. INDRIANI and DARMINTO. 2010. Genetic characteristic of protein membran of avian influenza viruses H5N1 subtype. JITV 15(3): 231-239. In 2006-2008 there were findings about the antigenic drift on AI virus due to vaccination and the AI H5N1 subtype viruses which was similar to H5N1 viruses in human. The findings indicated that the AI viruses continue and undergoing to mutate and try to adapt with their environment. The objective of this study to characterize the mutation of recent AI viruses (2009) on the membran protein namely Hemagglutinin (HA), Neuraminidase (NA) and Matrix 2 (M2). In this study RT-PCR – sequencing methods and genetic analysis for the protein membran of AI viruses were used. Result revealed that there were specific mutation belong to AI 2009 viruses on HA and NA protein such as AI virus mutation in 2008 which isolated from backyard chicken. The mutations were non synonimous and not caused by immunological pressure. Furthermore, M2 analysis indicated that the viruses were resistant to amantadine. Key Words: Mutation, AI Subtype H5N1 Viruses, Membran Protein ABSTRAK DHARMAYANTI, N.L.P.I., D.A. HEWAJULI, A. RATNAWATI, R. INDRIANI dan DARMINTO. 2010. Karakter Genetik Protein Membran virus avian influenza subtipe H5N1. JITV 15(3): 231-239. Ditemukannya virus AI yang mengalami antigenic drift akibat vaksinasi pada tahun 2006, 2007 dan 2008 serta beberapa virus yang mempunyai kemiripan dengan virus H5N1 pada manusia memperlihatkan virus AI subtipe H5N1 di Indonesia sedang dan terus bermutasi dan berusaha beradaptasi dengan lingkungannya. Penelitian ini bertujuan untuk mengetahui karakter mutasi pada tiga protein membran yaitu Hemagglutinin (HA), Neuraminidase (NA) dan Matrix 2 (M2) pada virus AI subtipe H5N1 yang diisolasi pada tahun 2009. Metode yang digunakan dalam penelitian ini adalah propagasi virus pada telur specific pathogen free (SPF) dan karakterisasi dilakukan dengan RT-PCR sekuensing pada tiga protein membran virus AI subtipe H5N1 tahun 2009 yaitu HA, NA dan M2 dan analisis genetika. Hasil penelitian memperlihatkan mutasi spesifik terjadi pada virus AI tahun 2009 yaitu pada gen HA dan pada gen NA seperti yang dimiliki oleh virus AI tahun 2008 asal ayam buras. Virus yang dianalisis menunjukkan bahwa mutasi yang terjadi bersifat non sinonim dan tidak disebabkan karena tekanan vaksinasi. Enam virus tahun 2009 yang dianalisis juga menunjukkan resisten terhadap amantadin yang ditunjukkan oleh adanya mutasi pada protein M2. Kata Kunci: Mutasi, Virus AI Subtipe H5N1, Protein Membran PENDAHULUAN Virus influenza adalah virus yang mempunyai materi genetik RNA untai tunggal berpolaritas negatif dengan genom bersegmen. Virus influenza A da B mempunyai delapan segmen, sedangkan virus influenza C mempunyai tujuh segmen (MCGEOCH et al., 1976; DESSELBERGER et al., 1980). Segmen-segmen tersebut secara bebas diselubungi oleh nukleoprotein dan dihubungkan dengan polimerase kompleks. Partikel virus terdiri dari RNA viral (vRNA), Nukleoprotein (NP) dan komplek polimerase yang disebut dengan partikel ribonukleprotein (RNP) (KATES et al., 1962). Tiga protein virus terdapat dalam membran adalah dua glikoprotein permukaan (spike) yaitu hemaglutinin (HA) dan neuraminidase (NA) dan protein membranchannel, (M2) (ZEBEDEE dan LAMB, 1988). Haemagglutinin (HA) homotrimer yang mempunyai aktifitas pengikatan terhadap reseptor dan aktivitas fusi membran yang diaktivasi dengan pH rendah dalam endosom selama masuknya virus ke dalam sel (WHARTON et al., 1990). Neuraminidase homotetramer yang mempunyai aktifitas menghancurkan reseptor untuk membebaskan virus baru dari permukaan sel yang terinfeksi (COLMAN, 1989), sedangkan M2 adalah protein membran protein yang memiliki domain 231 JITV Vol. 15 No. 3 Th. 2010: 231-239 membran-spanning yang juga menyediakan sinyal untuk transpor ke permukaan sel yang berbentuk tetramer membran channel (SUGRUE dan HAY, 1991; PINTO et al., 1992; WEBSTER et al., 1992). Setelah lebih dari enam tahun bersirkulasi di Indonesia, karakter molekuler virus AI di Indonesia telah mengalami perubahan. Virus AI yang diisolasi dari unggas yang divaksinasi AI mengalami antigenic drift yang cukup ektenstif jika dibandingkan dengan virus AI yang diisolasi dari ayam yang tidak divaksinasi AI (DHARMAYANTI, 2009). Penelitian DHARMAYANTI (2009) mengidentifikasi adanya motif tertentu yang dimiliki oleh virus AI yang kemungkinan dapat ditularkan ke manusia. Mutasi yang diamati pada virus AI sepanjang tahun 2003-2008 adalah mutasi non sinonim yang terjadi pada gen HA dan M. Penelitian ini bertujuan untuk mengetahui karakter mutasi pada tiga protein membran yaitu HA, NA dan M pada virus AI subtipe H5N1 yang diisolasi pada tahun 2009. Hasil penelitian diharapkan dapat memberikan gambaran dan informasi terbaru tentang karakter genetik virus AI subtipe H5N1 tahun 2009. MATERI DAN METODE Virus AI Virus AI subtipe H5N1 yang digunakan untuk studi ini dipropagasi pada telur specific pathogen free (SPF) berembrio umur 9-11 hari. Cairan alantois hasil panen dari telur SPF berembrio yang telah diinfeksi, diuji lebih lanjut yaitu dikarakterisasi dengan RT-PCR dan sekuensing. Ekstraksi RNA dan RT-PCR Ekstraksi RNA virus yang berasal dari cairan alantois terinfeksi dilakukan dengan menggunakan QIAmp RNA viral mini kit (Qiagen) (DHARMAYANTI, 2009). Reaksi RT-PCR dilakukan dengan menggunakan Supercript III one step RT-PCR system (Invitrogen). Sekuensing DNA Strategi dan set primer untuk mengamplifikasi gen HA dan M sesuai dengan HOFFMAN (2003); KOMADINA (2007, komunikasi pribadi) dan DHARMAYANTI (2009). Fragmen DNA hasil amplifikasi dipurifikasi dengan QIAquick PCR purification (Qiagen). Reaksi sekuensing dilakukan dengan menggunakan reagen Big Dye Terminator v 3.1 (Applied Biosystem) dan DNA sekuensing dilakukan dengan mesin Genetic Analyzer 3130 (Applied Biosystenm). Hasil sekuensing dianalisis dengan menggunakan Finch TV (http://www.geospiza.com/Products/finchtv.shtml) dan pembuatan multiple aligment dengan menggunakan BioEdit, versi 7 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Pohon filogenetika dihasilkan dengan MEGA 4 (http://www.megasoftware.net). HASIL DAN PEMBAHASAN Sebanyak enam virus yang berhasil dianalisis menunjukkan bahwa hasil analisis genetika pada gen HA1 memperlihatkan bahwa virus AI tahun 2009 mempunyai mutasi asam amino yang tidak dimiliki oleh virus AI tahun sebelumnya (2003-2008). Mutasi asam amino terjadi pada urutan 9 yaitu asam amino A menjadi S (A9S). Mutasi lainnya adalah asam amino pada urutan 69 yaitu asam amino R menjadi K (R69K) dan asam amino R menjadi M pada posisi 205 (R205M). Mutasi yang terjadi adalah mutasi non sinonim (Gambar 1). Protein HA virus influenza mempunyai glikoprotein trimerik yang mempunyai 3-9 N-linked glycosylation sequons perunit, tergantung pada galur virus (SCHULZE, 1997). Pada penelitian ini virus mempunyai sebanyak 7 tempat glikosilasi pada protein HA1 yaitu pada posisi 11, 23, 84, 154, 165, 193 dan 286 (Gambar 1). Hal berbeda jika dibandingkan dengan virus yang mengalami antigenic drift yaitu virus yang Tabel 1. Isolat AI subtipe H5N1 yang digunakan pada penelitian Nama Isolat Patogenesitas Asal sampel A/Chicken/West Java/Bgr-Cmgg/2009 Mortalitas tinggi Wabah unggas, disekitar kasus manusia terinfeksi H5N1 A/Chicken/West Java/Smi-Dmn/2009 Mortalitas tinggi Wabah unggas A/Chicken/West Java/Smi-Endo/2009 Mortalitas tinggi Wabah unggas A/Chicken/West Java/Smi-Mgg/2009 Mortalitas tinggi Wabah unggas A/Chicken/West Java/Indramayu-Indr/2009 Mortalitas tinggi Wabah unggas A/Chicken/West Java/Smi-Emn/2009 Mortalitas tinggi Wabah unggas 232 DHARMAYANTI . et al. Karakter genetik protein membran virus avian influenza subtipe H5N1 diisolasi dari unggas yang divaksinasi (Smi-Hj18/07, Smi-Sud1/07, SMI-M1/08, SMI-M6/08, SMI- Biot/08 termasuk virus Pwt-Wij/06 dan Smi-Pat/06) hanya memiliki sebanyak 4 tempat glikosilasi karena tidak mempunyai tempat glikosilasi pada posisi 84, 165 dan 193 (DHARMAYANTI, 2009). Hasil penelitian ini menunjukkan bahwa seluruh virus AI 2009 yang dianalisis tidak mengalami penurunan atau peningkatan jumlah glikosilasi sehingga kemungkinan tidak menciptakan populasi virus yang mengalami peningkatan afinitas terhadap reseptor dan tidak menghasilkan populasi virus yang lebih tahan terhadap netralisasi daripada parentalnya (SCHULZE, 1997), tidak seperti halnya virus yang diisolasi dari ayam yang divaksinasi AI yang telah terbukti lebih tahan terhadap netralisasi daripada induknya. Berdasarkan sekuen asam amino pada cleavage site HA, enam virus yang digunakan pada penelitian ini mempunyai rangkaian asam amino yang menandakan virus termasuk kelompok highly pathogenic dan mempunyai substitusi pada posisi -6 protein HA1 yaitu Arginin (R)ÆSerin (S) (Gambar 1). Variasi pada motif dapat terjadi berkaitan dengan geografi asal isolat, tidak dihubungkan dengan perubahan virulensi dan infeksi pada manusia (WRITTING COMMITTEE OF SECOND WORLD ORGANIZATION CONSULTATION, 2008). Virus yang dianalisis pada penelitian ini tidak mengalami mutasi pada asam amino posisi 222 dan 224 sehingga masih mengenal avian receptor (α2,3) dan belum mengenal human receptor (α2,6) (STEVENS et al., 2006). Substitusi sebuah asam amino pada protein hemaglutinin dapat mengubah spesifisitas pengikatan sialyl-linkage Neu5Ac2-3Gal (Ac2-3) menjadi Neu5Ac2-3Gal (Ac2-6) atau sebaliknya. Substitusi Ser205Tyr yang terletak pada antigenik D dari hemaglutinin virus H3 (jaraknya jauh dari reseptor binding site), menghasilkan perubahan spesifisitas pengikatan reseptor dari 2-3 ke 2-6. Perubahan Leu226Gln pada pocket receptor binding site juga mengubah spesifisitas pengikatan reseptor 2-6 ke 2-3. Perubahan ini sangat penting karena berpengaruh pada kemampuan infeksi virus pada inangnya. Pada penelitian ini, dikarenakan virus masih mengenal avian receptor (Ac2-3) sehingga infeksi pada manusia kemungkinan tertular dari unggas yang terlebih dahulu terinfeksi virus H5N1. Pada multiple alignment protein NA menunjukkan kekhasan virus AI 2009 yaitu perubahan asam amino pada posisi 163 yaitu V menggantikan L (V143L). Selain itu mutasi juga terjadi pada posisi 189 yaitu asam amino S digantikan oleh G (S189G), asam amino E digantikan oleh asam amino K pada posisi 259 (E259K), dan asam amino G menggantikan E (G382E) (Gambar 2). Tiga mutasi ini serupa dengan virus AI tahun 2008 yang diisolasi dari ayam buras tanpa vaksinasi AI yaitu isolat A/Ck/West Java/Smi-Acl/08 dan A/Ck/Banten/Srg-Fadh/08. Pada protein NA, semua virus H5N1 Indonesia mempunyai delesi 20 asam amino pada regio stalk yaitu pada posisi 48-67. Tempat glikosilasi pada regio stalk dari protein neuraminidase berperan dalam menjaga struktur tetramer dari protein (LUO et al., 1993). Semua virus pada penelitian ini tidak mempunyai tempat glikosilasi pada stalk protein neuraminidase karena delesi di daerah ini, sehingga pada protein NA hanya memiliki 3 tempat glikosilasi yaitu pada posisi 88, 146 dan 235. Delesi pada daerah ini akan meningkatkan retensi virion pada membran plasma (MATROSOVICH et al., 1999). Mutasi yang terjadi tidak seperti virus yang diisolasi dari ayam yang divaksinasi AI secara intensif dan tidak terjadi pada epitop pengenalan antibodi (DHARMAYANTI, 2009), sehingga besar kemungkinan mutasi yang terjadi bukan akibat tekanan vaksinasi sehingga asal virus diperkirakan adalah virus yang bersirkulasi di lingkungan. Mutasi asam amino pada protein permukaan virus yaitu HA dan NA yang terjadi pada virus tahun 2009 mengindikasikan bahwa virus terus berusaha beradaptasi dengan lingkungan sekitarnya. Indikasi adanya mutasi pada gen NA adalah hal baru, karena sebelumnya DHARMAYANTI (2009) tidak menemukan adanya mutasi pada gen NA pada virusvirus tahun 2003-2008, sehingga paparan mutasi hanya terbatas pada gen HA saja serta beberapa virus memperlihatkan mutasi pada gen internal. Hal ini tentunya sangat menarik karena paparan mutasi pada virus AI tahun 2009 telah mengakibatkan perubahan asam amino pada glikoprotein permukaan virus yaitu Neuraminidase selain Hemagglutinin. Dengan perkataan lain bahwa hasil penelitian ini menggambarkan virus AI sedang dan terus bermutasi. Hasil analisis filogenetika, enam virus 2009 yang dianalisis pada aras gen HA (Gambar 3) dan NA (Gambar 4) menunjukkan bahwa virus tahun 2009 membentuk kelompok tersendiri meskipun masih dalam kelompok besar virus AI subtipe H5N1 asal Indonesia, dan berbeda dengan kelompok virus yang mengalami antigenic drift yang berasal dari flok ayam yang divaksinasi AI. Substitusi asam amino tunggal pada asam amino 26(LeuÆPhe), 27 (ValÆAla atau Thr), 30 (AlaÆThr atau Val), 31 (SerÆAsn atau Arg) dan 34 (GÆE) dalam domain transmembran M2 diimplikasikan dengan hilangnya sensitivitas bloker M2 yang mengakibatkan resisten terhadap amantadin (HAY et al., 1985; PINTO et al., 1992; SUZUKI et al., 2003). Analisis pada protein M2 memperlihatkan bahwa enam virus tahun 2009 yang digunakan pada penelitian ini memiliki substitusi pada asam amino posisi 27 yaitu A (ValÆAla; V27A) yang menunjukkan virus tersebut resisten terhadap amantadin (Tabel 2). DHARMAYANTI et al. (2010) dalam penelitiannya memperlihatkan 233 JITV Vol. 15 No. 3 Th. 2010: 231-239 10 20 30 40 50 60 70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Ck/East Java/BL-IPA/03 Ck/West Java/1074/03 MD/Jakarta/DKI-Uwit/04 MD/West Java/Bgr-Cw/05 Ck/Jakarta/DKI31/05 MD/West Java/Bgr-Cw/05 Ck/West Java/Smi-Hay/05 MD/Jakarta/Sum106/06 Duck/Jakarta/Slmt306/06 MD/West Java/Bks3/07 Ck/Pessel/BPPVRII/07 Ck/Inhu/BPPVRII/07 Ck/Jakarta/DKI-Nurs/07 Ck/West Java/Smi-Hj18/07 Ck/West Java/Smi-Sud1/07 Ck/West Java/Smi-Acul/08 Ck/Banten/Srg-Fadh/08 Ck/West Java/Smi-M1/08 Ck/West Java/Smi-M6/08 Ck/West Java/Smi-Biot/08 Ck/West Java/Bgr-Cmgg/09 Ck/West Java/Smi-Dmm/09 Ck/West Java/Smi-Emn/09 Ck/West Java/ Smi-Endo/09 Ck/West Java/Indramayu/09 Ck/West Java/Smi-Mgg/09 DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKS ..............................................................................................L......................... .............................................................................................S.......................... .......................................................................K.....................S.......................... ........................................................................................................................ .......................................................................K.....................S.......................... .....................................................................................T.......S.......................... .....................................................................................T.......S.......................... .............................................................................................S.......................... .....................................................................................T.......S.......................... .....................................................................................T.......S.......................... .............................................................................................S.......................... .......................................................................K.Q.........S.T.......S..............K.....R..... .......................................................................K.Q.........S.T.......S..............K.....R..... .......................................................................K.Q.........S.T.......S..............K.....R..... .....................................................................................T.......S.......................... ....................................................K................................T.......S.......................... ..........................................N............................K.Q.........S.T.......S..............K.....R..... ..........................................N............................K.Q.....G...S.T.......S..............K.....R..... .H........................................N............................K.Q.........S.T.......S..............K.....R..... ....................................................K................................T.......S.......................... ...........T......................................L.K................................T....D..S.......................... ..............................................E.....K................................T.......S.......................... .......................D............................K................................T.......S.......................... ....................................................K................................T.......S.......................... ..................................R.................K................................T.......S.......................... 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Ck/East Java/BL-IPA/03 Ck/West Java/1074/03 MD/Jakarta/DKI-Uwit/04 MD/West Java/Bgr-Cw/05 Ck/Jakarta/DKI31/05 MD/West Java/Bgr-Cw/05 Ck/West Java/Smi-Hay/05 MD/Jakarta/Sum106/06 Duck/Jakarta/Slmt306/06 MD/West Java/Bks3/07 Ck/Pessel/BPPVRII/07 Ck/Inhu/BPPVRII/07 Ck/Jakarta/DKI-Nurs/07 Ck/West Java/Smi-Hj18/07 Ck/West Java/Smi-Sud1/07 Ck/West Java/Smi-Acul/08 Ck/Banten/Srg-Fadh/08 Ck/West Java/Smi-M1/08 Ck/West Java/Smi-M6/08 Ck/West Java/Smi-Biot/08 Ck/West Java/Bgr-Cmgg/09 Ck/West Java/Smi-Dmm/09 Ck/West Java/Smi-Emn/09 Ck/West Java/ Smi-Endo/09 Ck/West Java/Indramayu/09 Ck/West Java/Smi-Mgg/09 FESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIA ................................................................................................. ................................................................................................. ................................................................................................. ................................................................................................. ................................................................................................. ....................................................................................S............ ....................................................................................S............ ................................................................................................. ....................................................................................S............ ................................................................................................. ....................................................................................G............ ................................D...................................................S............ ................................D...................................................S............ ................................D...................................................S............ ....................................................................................S............ ....................................................................................S............ ................................D........I..........................................S............ ................................D........I..........................................S............ ...........................D....D........I..........................................S............ ................................D...................................................S............ ...............................S....................................................S............ ....................................................................D...............S............ ....................................................................D...............S............ ....................................................................................S............ .............................................................................G......S............ 130 140 150 160 170 180 190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 250 260 270 280 290 300 310 320 330 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.. Gambar 1. Multiple alignment protein HA1 virus AI subtipe H5N1 tahun 2003-2009 Keterangan: Tempat glikosilasi ditandai dengan kotak tertutup dan residu asam amino didaerah cleavage site HA ditunjukkan dengan kotak tertutup dengan warna abu-abu. Penomoran asam amino berdasarkan virus BL-IPA/03. Ck : Chicken; MD : Muscovy duck 234 DHARMAYANTI . et al. Karakter genetik protein membran virus avian influenza subtipe H5N1 10 20 30 40 50 60 70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Goose/Guangdong/1/96 Ck/East Java/BL-IPA/03 Ck/West Java/1074/03 MD/JakartDKI-Uwit/04 Ck/Banten/Pdgl-Kas/04 Ck/JakartDKI31/05 MD/West Java/Bgr-Cw/05 Ck/West Java/Smi-Hay/05 MD/JakartSum106/06 Duck/JakartSlmt306/06 MD/West Java/Bks3/07 Ck/Pessel/BPPVRII/07 Ck/Inhu/BPPVRII/07 Ck/JakartDKI-Nurs/07 Ck/West Java/Smi-Hj18/07 Ck/West Java/Smi-Sud1/07 Ck/West Java/Smi-Acul/08 Ck/Banten/Srg-Fadh/08 Ck/West Java/Smi-M1/08 Ck/West Java/Smi-M6/08 Ck/West Java/Smi-Biot/08 Ck/West Java/Smi-Dmn/09 Ck/West Java/Smi-Emn/09 Ck/West Java/Smi-Endl/09 Ck/West Java/Smi-Mgg/09 Ck/West Java/Indramayu/09 MNPNQKIITIGSICMVVGIISLMLQIGNIISIWVSHSIQTGNQHQAEPCNQSIITYENNTWVNQTYVNISNTNFLTEKAVASVTLAGNSSLCPISGWAVHSKDNGIRIGSKGDVFVIREP ................I..V........M..................S--------------------T....P....................R.........S............... ................I..V........M..................S--------------------.....P....................R.........S............... ................I..V........M............D.....S--------------------.I.N.P...N................R.........S............... ................I..V........M............D.....S--------------------.I.N.P...N................R.........S............... ................I..V........M....I.............S--------------------.....P....................R.........S............... ...............AI..V........M..................S--------------------.....PP...................R.........N............... ................I..V........M..................S--------------------.....P....................R.........N............... ................I..V........M..................S--------------------.....P....................R.........N............... ................I..V........M..................S--------------------.....P....................R.........N............... ................I..V........M..........K.......S--------------------.....P....................R.........N............... ................I..V........M..........K.......S--------------------.....P.........I..........R.........N............... ................I..V........M..........K.......S--------------------.....P.........I..........R.........N............... ................I..V........M..................S--------------------.....P....................R.........N............... ................I..V........M..T...............S--------------------.....P.N..................R.........N............... ................I..V........M..................S--------------------.....P.N..................R.........N............... ........AT......I..V........M..........K.......S--------------------.....P....................R.........N.....R......... ........A.......I...........M..........K.......S--------------------.....P......T.............R.........N............... ................I..V........M..................S--------------------.....S.N..T...............R.........N............... ................I..V........M..................S--------------------.....S.N..................R.........N............... ................I..VG.......M..................S--------------------.....S.N..................R.........N............... ................I..V........MR...I.....K.....T.S--------------------.....P....................R.........N............... ................I..V........M....I.....K.....T.S--------------------.....P....................R.........N............... ................I..V........M....I.....K.....T.S--------------------.....P....................R.........N............... ................I..V........M....I.....K.....T.S--------------------T....P....T...............R.........N............... ........S.......I..V........M..........K.....T.S--------------------.....P...........T........R.........N............... Goose/Guangdong/1/96 Ck/East Java/BL-IPA/03 Ck/West Java/1074/03 MD/JakartDKI-Uwit/04 Ck/Banten/Pdgl-Kas/04 Ck/JakartDKI31/05 MD/West Java/Bgr-Cw/05 Ck/West Java/Smi-Hay/05 MD/JakartSum106/06 Duck/JakartSlmt306/06 MD/West Java/Bks3/07 Ck/Pessel/BPPVRII/07 Ck/Inhu/BPPVRII/07 Ck/JakartDKI-Nurs/07 Ck/West Java/Smi-Hj18/07 Ck/West Java/Smi-Sud1/07 Ck/West Java/Smi-Acul/08 Ck/Banten/Srg-Fadh/08 Ck/West Java/Smi-M1/08 Ck/West Java/Smi-M6/08 Ck/West Java/Smi-Biot/08 Ck/West Java/Smi-Dmn/09 Ck/West Java/Smi-Emn/09 Ck/West Java/Smi-Endl/09 Ck/West Java/Smi-Mgg/09 Ck/West Java/Indramayu/09 FISCSHLECRTFFLTQGALLNDKHSNGTVKDRSPHRTLMSCPVGEAPSPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFT ........................................................................................................................ ........................................................................................................................ ........................................................................................................................ ........................................................................................................................ ........................................................................................................................ ........................................................................................................................ ........................................................................................................................ ...................................................................N.........S..E....................................... ...................................................................N............E...................K................... ................................................................................E....................................... ................................................................................E....................................... ................................................................................E....................................... ................................................................................E....................................... ................................................................................E....................................... ................................................................................E....................................... ....................................................................G...........E....................................... ....................................................................G...........E....................................... ................................................................................E....................................... ................................................................................E....................................... ................................................................................E....................................... ..........................................L.........................G...........E....................................... ..........................................L.........................G...........E......................................A ..........................................L.........................G...........E......................................A ...............H..........................L.........................G...........E......................................V ..........................................L.........................G...........E....................................... Goose/Guangdong/1/96 Ck/East Java/BL-IPA/03 Ck/West Java/1074/03 MD/JakartDKI-Uwit/04 Ck/Banten/Pdgl-Kas/04 Ck/JakartDKI31/05 MD/West Java/Bgr-Cw/05 Ck/West Java/Smi-Hay/05 MD/JakartSum106/06 Duck/JakartSlmt306/06 MD/West Java/Bks3/07 Ck/Pessel/BPPVRII/07 Ck/Inhu/BPPVRII/07 Ck/JakartDKI-Nurs/07 Ck/West Java/Smi-Hj18/07 Ck/West Java/Smi-Sud1/07 Ck/West Java/Smi-Acul/08 Ck/Banten/Srg-Fadh/08 Ck/West Java/Smi-M1/08 Ck/West Java/Smi-M6/08 Ck/West Java/Smi-Biot/08 Ck/West Java/Smi-Dmn/09 Ck/West Java/Smi-Emn/09 Ck/West Java/Smi-Endl/09 Ck/West Java/Smi-Mgg/09 Ck/West Java/Indramayu/09 VMTDGPSNGQASYKIFKMEKGKVVKSVELNAPNYHYEECSCYPDAGEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSPNGAYGVKGFSFKYGNGVWIG .............................D...................................................................M...................... .............................D...................................................................M...................... .............................D...................................................................M...................... .............................D...................................................................M...................... .............................D...................................................................M...........A.......... .............................D...................................................................M...................... .............................D...................................................................M...................... .............................D...................................................................M...................... .............................D.T.................................................................M...................... ..................K..........D...................................................................M...................... ...........................K.D...................................................................M...................... .............................D...................................................................M...................... .............................D...................................................................M.S.................... .............................D...................................................................M.S.................... .............................D...................................................................M.S.................... ..................K..............................................................................M...................... ..................K..........D...................................................................M...........L.......... .............................D..................I................................................M.S.................... .............................D..................I................................................M.S.................... .............................D..................I................................................M.S.................... ..................K..........D...................................................................M...................... ..................K..........D...................................................................M.....S................ ..................K..........D...................................................................M.....S................ ..................K..........D....................................D..............................M...................... ..............V...K..........D...................................................................M...................... Goose/Guangdong/1/96 Ck/East Java/BL-IPA/03 Ck/West Java/1074/03 MD/JakartDKI-Uwit/04 Ck/Banten/Pdgl-Kas/04 Ck/JakartDKI31/05 MD/West Java/Bgr-Cw/05 Ck/West Java/Smi-Hay/05 MD/JakartSum106/06 Duck/JakartSlmt306/06 MD/West Java/Bks3/07 Ck/Pessel/BPPVRII/07 Ck/Inhu/BPPVRII/07 Ck/JakartDKI-Nurs/07 Ck/West Java/Smi-Hj18/07 Ck/West Java/Smi-Sud1/07 Ck/West Java/Smi-Acul/08 Ck/Banten/Srg-Fadh/08 Ck/West Java/Smi-M1/08 Ck/West Java/Smi-M6/08 Ck/West Java/Smi-Biot/08 Ck/West Java/Smi-Dmn/09 Ck/West Java/Smi-Emn/09 Ck/West Java/Smi-Endl/09 Ck/West Java/Smi-Mgg/09 Ck/West Java/Indramayu/09 RTKSTNSRSGFEMIWDPNGWTGTDSSFSVKQDIVAITDWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVGWSWPDD .............................................................................................S.....G .............................................................................................S.....G ...........................................................................S.................S.....G ....................................N........................................................S.....G ......................P......................................................................S...... .............................................................................................S.....G ....................................................................................L........S..C... .............................................................................................S..R..G .............................................................................................S.....G .............................................................................................S.....G .............................................................................................S.....G .............................................................................................S.....G ..................................................................................K..........S.....G .............................................................................................S.....G .....................................................................................G.......S.....G .....................E...G...................................................................S.....G .....................E.......................................................................S.....G .............................................................................................S.....G ....................................................................E........................S.....G ..................................................................T..........................S.....G .....................E.......................................................................S.....G .....................E.................................................Q.....................S.....G .....................E.................................................Q.....................S.....G .....................E.......................................................................S.....G .....................E.......................................................................S.....G 130 140 150 160 170 180 190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 250 260 270 280 290 300 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| 370 380 390 400 410 420 430 440 450 460 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Gambar 2. Multiple alignment protein NA virus AI subtipe H5N1 tahun 2003-2009 Keterangan: Glikosilasi ditandai dengan kotak tertutup. Delesi 20 asam amino ditunjukkan dengan kotak tertutup warna abu-abu. Penomoran asam amino berdasarkan virus Gs/Guangdong/1/96. Ck : Chicken; Md : Muscovy duck 235 JITV Vol. 15 No. 3 Th. 2010: 231-239 A /chicken/Kupang-3-NTT/B P P V6/2004 A /chicken/B angli B ali/B B P V6-1/2004 A /chicken/Jembrana/B P P V6/2004 A /chicken/M angarai-NTT/B P P V6/2004 A /Ck/Indonesia/B L/2003 A /Chicken/East Java/B L-IP A /2003 A /Ck/Indonesia/P A /2003 A /Chicken/West Java/HA M D/2006 77 A /chicken/Indo nesia/11/2003 A /Goo se/Guangdo ng/1/96 A /chicken/Indonesia/7/2003 97 A /quail/Tasikmalaya/B P P V4/2004 A /Chicken/West Java/1074/2003 A /Chicken/Jakarta/DKI31/2005 66 A /Chicken/P adang/B B P VII/2006 A /chicken/Dairi/B P P VI/2005 A /Chicken/M edan/B B P V1-576/2005 98 A /chicken/Tebing Tinggi/B P P VI/2005 65 A /Chicken/P idie/B P P V1/2005 A /chicken/Deli Serdang/B P P VI/2005 75 99 A /Quail/Central Java/SM RG/2006 A /Chicken/West Java/GA RUT-M A Y/2006 A /Chicken/Indonesia/M agelang1631-57/2007 92 A /Chicken/Gunung Kidul/B B VW/2006 61 92 A /Indo nesia/6/2005 A /M uscovy Duck/Jakarta/DKI-Uwit/2004 A /M ucso vy duck/West Java/B gr-Cw/2005 99 A /Chicken/P apua/TA 5/2006 98 A /Chicken/P apua/TB 1/2006 A /duck/P arepare/B B VM /2005 71 A /Duck/Jakarta/Slmt306/2006 A /Chicken/Inhu/B P P VRII/2007 A /Chicken/Indonesia/B angka SelA tan1631-2 75 A /Chicken/P alembang/B P P V-III/2005 94 A /Chicken/Way Kanan/B B P VIII/2006 A /Chicken/B andar Lampung/B B P VIII/2006 A /Duck/Indramayu/B B P W109/2006 A /Indo nesia/CDC7/2005 88 A /Chicken/West Java/Smi-Hay/2005 A /Chicken/West Java/SM I-ENDRI1/2006 A /M ucso vy duck/West Java/B ks3/2007 99 A /Indonesia/CDC1047/2007 A /Indonesia/CDC1046/2007 87 A /Chicken/B anten/Srg-Fadh/2008 A /Chicken/West Java/B o gor-Cmgg/2009 99 A /Chicken/West Java/Smi-Dmn/2009 89 A /Chicken/West Java/Indramayu-Indr/2009 A /Chicken/West Java/Smi-M gg/2009 95 97 A /Chicken/West java/Smi-Emn/2009 75 A /Chicken/West java/Smi-Endl/2009 63 A /Quail/Jakarta/JU1/2006 A /Chicken/West Java/TA SIKSOL/2006 A /Indo nesia/5/2005 A /Indonesia/CDC370/2006 89 A /Chicken/West Java/TA SIK1/2006 96 A /Chicken/West Java/TA SIK2/2006 A /chicken/West Java/TA SIKSOB /2006 A /Chicken/M urao Jambi/B B P V-II/2005 A /Chicken/Indonesia/P ekenbaru1631-11/200 61 87 A /Chicken/Indonesia/P adang1631-1/2006 80 A /M uscovy Duck/Jakarta/HA B WIN/2006 A /Chicken/West Java/Smi-A cul/2008 87 A /M uscovy duck/Jakarta/Sum106/2006 85 A /Chicken/P essel/B P P VRII/2007 A /Chicken/Indo nesia/Semerang1631-62/2007 A /Swan/Indo nesia/M alang1631-61/2007 A /chicken/West Java/SM I-CSLK-EB /2006 A /Chicken/West Java/SM I-CSLK-EC/2006 A /Chicken/West Java/SM I-P A T/2006 A /Chicken/West Java/P WT-WIJ/2006 A /Chicken/Jakarta/DKI-Nurs/2007 A /Chicken/West Java/Smi-Sud1/2007 A /West Java/Smi-Hj18/2007 99 A /Chicken/West Java/Smi-B io t/2008 99 A /Chicken/West Java/Smi-M 1/2008 64 A /Chicken/West Java/Smi-M 6/2008 Virus AI H5N1 tahun 2009 Virus antigenic drift th. 2006 Virus antigenic drift th. 2007-2008 0.05 Gambar 3. Filogenetika virus AI subtipe H5N1 pada aras gen HA1 Keterangan: Virus yang berhasil diisolasi pada penelitian ditandai dengan warna biru. Nukleotida yang dianalisis pada aras gen HA1 adalah posisi 49-1059 236 DHARMAYANTI . et al. Karakter genetik protein membran virus avian influenza subtipe H5N1 75 A /Dk/Indo nesia/M S/2004 A /Chicken/East Java/B L-IP A /2003 A /Duck/IB ufeleng/B P P V1/2005 87 A /Duck/P ali/B B VW/2005 A /Ck/Indo nesia/P A /2003 A /Chicken/Indo nesia/Kulo n1631-47/2006 74 A /Chicken/Jakarta/DKI31/2005 A /Chicken/West Java/1074/2003 A /M usco vy Duck/Jakarta/DKI-Uwit/2004 81 93 A /Chicken/B anten/P dgl-Kas/2004 A /Chicken/Indo nesia/Wates83/2005 65 A /Duck/M adiun/B B VW1358/2005 90 A /Turkey/Langkat/B B P V1/2005 90 A /Chicken/M edan/B B P V1-498/2005 A /Chicken/Karo /B B P V1/2006 65 A /Chicken/Ro kan Hilli/B P P V11/2005 75 A /Chicken/P adang/B P P V11/2006 99 90 A /Chicken/P aulau Rampang/B P P V11/2006 A /M ucso vy duck/West Java/B gr-Cw/2005 A /Duck/Indramayu/B B VW109/2006 A /Chicken/B andar Lampung/B P P V111/2006 99 A /Chicken/P alembang/B P P V111/2005 A /Chicken/M urao Jambi/B P P V11/2005 A /Chicken/West Java/Smi-Hay/2005 84 A /Chicken/Indo nesia/So ppeng1631-71/2007 89 A /Swan/Indo nesia/M agelang1631-57/2007 A /Chicken/Indo nesia/Lampung1631-23/2006 96 A /Chicken/P essel/B P P VRII/2007 77 A /Chicken/Inhu/B P P VRII/2007 A /Chicken/Indo nesia/P ekenbaru1631-11/200 A /Ck/West Java/B ks 2/2007 A /Indo nesia/CDC1031/2007 A /M ucso vy duck/West Java/B ks3/2007 79 A /Chicken/West Java/Smi-A cul/2008 A /Chicken/B anten/Srg-Fadh/2008 A /Chicken/West Java/Smi-Dmn/2009 91 A /Chicken/West Java/Indramayu-Indr/2009 69 Virus AI H5N1 93 A /Chicken/West Java/Smi-M gg/2009 th 2009 A /Chicken/West java/Smi-Emn/2009 93 99 A /Chicken/West java/Smi-Endl/2009 70 A /Chicken/Indo nesia/Gunung Kidul1631-33/ A /M usco vy duck/Jakarta/Sum106/2006 96 A /Duck/Jakarta/Slmt306/2006 A /B ird cucak wilis/Jakarta/Walko t 4/2007 A /Chicken/Jakarta/DKI-Nurs/2007 69 A /Ck/Jakarta/Jakbar-o nh/2007 A /Ck/Jakarta/Walko t 1/2007 A /Chicken/West Java/Smi-Hj18/2007 Virus antigenic drift A /Chicken/West Java/Smi-Sud1/2007 th. 2007-2008 99 A /Chicken/West Java/Smi-B io t/2008 A /Chicken/West Java/Smi-M 6/2008 A /Chicken/West Java/Smi-M 1/2008 A /Go o se/Guangdo ng/1/96 A /Ho ng Ko ng/483/1997 A /Ho ng Ko ng/514/97 99 63 A /Ho ng Ko ng/542/97 0.2 Gambar 4. Filogenetika virus AI subtipe H5N1 pada aras gen NA Keterangan: Virus yang berhasil diisolasi pada penelitian ditandai dengan warna biru. Nukleotida yang dianalisis pada aras gen NA adalah posisi 1-1157. Tabel 2. Posisi asam amino yang bertanggung jawab terhadap sensitivitas amantadin pada protein M2 Posisi asam amino pada protein M2 Virus 26 27 30 31 34 A/Chicken/West Java/Bgr-Cmgg/2009 L A A S G A/Chicken/West Java/Smi-Dmn/2009 L A A S G A/Chicken/West Java/Smi-Endo/2009 L A A S G A/Chicken/West Java/Smi-Mgg/2009 L A A S G A/Chicken/West Java/Indramayu-Indr/2009 L A A S G A/Chicken/West Java/Smi-Emn/2009 L A A S G 237 JITV Vol. 15 No. 3 Th. 2010: 231-239 bahwa dari 147 data sekuen asam amino M2 virus influenza asal Indonesia yang dianalisis, diketahui bahwa sebanyak 62,58% atau 92 isolat virus influenza H5N1 di Indonesia telah mengalami resistensi terhadap amantadin, bahkan 10 virus diantaranya mempunyai mutasi ganda yaitu pada posisi V27A dan S31N. Data penelitian pada studi ini memperkuat penelitian DHARMAYANTI et al (2009) yaitu virus AI subtipe H5N1 di Indonesia mengindikasikan terdapatnya peningkatan resistensi terhadap amantadin. KESIMPULAN Hasil penelitian menunjukkan bahwa virus AI subtipe H5N1 yang dianalisis pada penelitian ini menunjukkan bahwa virus AI di Indonesia terus bermutasi terutama pada protein membran virus. Mutasi yang terjadi adalah spesifik pada virus AI tahun 2009 yaitu pada gen HA dan pada gen NA, virus AI tahun 2009 memilki mutasi yang spesifik seperti yang dimiliki oleh virus AI tahun 2008 asal ayam buras. Virus yang dianalisis menunjukkan bahwa mutasi yang terjadi bersifat non sinonim dan tidak disebabkan karena tekanan vaksinasi. Enam virus tahun 2009 yang dianalisis juga menunjukkan resisten terhadap amantadin yang ditunjukkan oleh adanya mutasi pada protein M2. Meskipun wabah avian influenza subtipe H5N1 pada unggas telah berkurang, namun virus ini terus bermutasi sehingga monitoring sirkulasi virus ini masih harus terus dilakukan untuk mengetahui kemungkinan peningkatan keganasan virus serta memperbaharui seed vaksin yang sesuai dengan virus yang bersirkulasi di lapangan. UCAPAN TERIMA KASIH Terima kasih kepada Bapak Nana Suryana, SE dan Teguh Suyatno, Amd atas bantuan teknisnya. Terima kasih juga diucapkan kepada Drh Endri Baharianto atas kontribusinya. Penelitian ini didanai oleh APBN-2009, Badan LitBang Pertanian, Kementrian Pertanian. DAFTAR PUSTAKA COLMAN, P.M. 1989. Neuraminidase: Enzyme and antigen. In: The Influenza Viruses. R.M. Krug (ed). Plenum Press. New York. pp. 175-218. DESSELBERGER, U., V.R. RACAINELLO, J.J. ZAZRA and P. PALASE. 1980. The 3’ and 5’ terminal sequences of influenza A,B and C virus RNA segments are highly concerved and show partial inverted complementary. Gene. 8: 315-328. DHARMAYANTI, N.L.P.I. 2009. 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