ANALISIS GENETIK GEN HEMAGLUTININ VIRUS AVIAN INFLUENZA SUBTIPE H5N1 ISOLAT LAMPUNG TAHUN 2008-2013

EKO AGUS SRIHANTO, Prof. drh. Widya Asmara, SU, Ph.D

2013 | Tesis | S2 Sain Veteriner

Penyakit Avian Influenza merupakan penyakit unggas yang bersifat infeksius disebabkan oleh virus Avian influenza tipe A yang merupakan keluarga Orthomyxoviridae. Wabah AI di Lampung dilaporkan pada tahun 2003 di Kabupaten Lampung Selatan yang akhirnya menyebar di beberapa kabupaten/kota di wilayah kerja BPPV Regional III. Data molekuler isolat Lampung belum banyak diungkap. Penelitian ini bertujuan untuk melakukan karakterisasi gen HA secara utuh. Isolat virus AI berasal dari wilayah kerja BPPV Regional III Lampung yang diisolasi pada spesific antibody negative (SAN) terhadap AI telur ayam bertunas (TAB) umur 10 hari. Cairan allantois diekstraksi untuk mendapatkan RNA dan dilakukan identifikasi subtipe H5N1 dengan tehnik Reverse Transcription Polymerase Chain Reaction (RT-PCR). Amplifikasi gen HA menggunakan 4 pasang primer referens dari Australian Animal Health Laboratory (AAHL) Geelong Australia (HA10, HA20, HA30 dan HA40) dan dilanjutkan dengan proses sekuensing. Analisis hasil sekuensing denganm menggunakan perangkat lunak MEGA versi 5.05 didapatkan hasil perbedaan genetik antar isolat Lampung dari tahun 2003-2013 berkisar 1,1 – 9,1 % dengan tingkat homologi mencapai 90,9 – 98,9%. Variasi genetik ditemukan di antigenic site dan receptor binding site dengan adanya substitusi pada posisi 53 (R53K), 126 (D126E), 136 (P136S), 138 (H138Q dan H138L), 140 (R140K, R140S dan R140N), 141 (S141P), 189 (K189R), 129 (S129L) dan 189 (K189R). Analisis terhadap susunan asam amino penyusun cleavage site isolat Lampung digolongkan HPVAI dengan pola sekuens QRERRRKKRG, QRESRRKKRG dan QRERRRKG. Berdasarkan analisis filogenic tree isolat Lampung tahun 2008- 2011 termasuk ke dalam clade 2.1.3. Analisis filogenik isolat AI tahun 2012-2013 yang menginfeksi unggas air mempunyai homologi sekitar 98,5-99,1% dibandingkan dengan isolat AI yang menginfeksi unggas air asal Jawa dan termasuk ke dalam clade 2.3.2.2.

Avian influenza is an infectious poultry disease which be caused by type A Avian Influenza virus in the family of Orthomyxoviridae. AI outbreaks in Lampung was reported in 2003 in South Lampung district which continued to spread in several districts/cities in the region of DIC Region III. Molecular data of the Lampung isolates has not been reported. This study was aimed to characterize the full HA gene. AI virus isolates derived from the work area of DIC Region III were isolated on chicken embrionated eggs negative antibody spesific (SAN) to AI at 10 days of age. Allantois fluid was extracted to obtain RNA and the subtype identification of H5N1 was done by Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique. Amplification of the HA gene using 4 pairs references primer from the Australian Animal Health Laboratory (AAHL) Geelong Australia (HA10, HA20, HA30 and HA40) and was continued with the sequencing process. Sequences were analysed by the MEGA software version 5.05. Analysis of genetic differences between isolat Lampung from 2003-2008 compare with isolat from 2008-2013 indicated genetic differences among 1.1- 9.1% and homology among this isolat were 90.9-98.9%. Genetic variation found in the antigenic site and the receptor binding site with a substitution at position 53 (R53K), 126 (D126E), 136 (P136S), 138 (H138Q and H138L), 140 (R140K, R140S and R140N), 141 (S141P), 189 (K189R), 129 (S129L) and 189 (K189R). Analysis of the composition of amino acids at cleavage site of Lampung isolates was classified to HPVAI. The feature of sequences were QRERRRKKRG, QRESRRKKRG and QRERRRKG. Phylogenic tree analysis of tree Lampung in 2008-2011 isolates belong to the clade 2.1.3. Analysis of the 2012-2013 AI isolates that infect waterfowl have approximately 98.5-99.1% homology compared with AI isolates that infect waterfowl Javanese origin and belong to the clade 2.3.2.2.

Kata Kunci : Avian Influenza virus, sekuensing, analisis genetik, substitusi, clade