KARAKTERISASI MOLEKULER FRAGMEN GEN FUSI VIRUS NEWCASTLE DISEASE ISOLAT ASAL WILAYAH KERJA BALAI PENYIDIKAN DAN PENGUJIAN VETERINER REGIONAL III LAMPUNG TAHUN 2010-2013
ANGELIYA, Liza , Michael Haryadi Wibowo
2014 | Tesis |Newcastle Disease (ND) merupakan penyakit unggas yang bersifat infeksius disebabkan oleh virus ND yang merupakan keluarga Paramyxoviridae genus Avulavirus spesies Avian paramyxovirus. Penyakit ini menyebabkan penurunan produksi telur, mortalitas tinggi dan biaya pengendalian yang tinggi sehingga menyebabkan kerugian ekonomi yang besar bagi industri perunggasan baik di Indonesia maupun di dunia. Pengendalian penyakit ND di Indonesia dilakukan dengan vaksinasi namun demikian masih banyak ditemui kasus tersebut di lapangan. Data molekuler mengenai virus ND di wilayah kerja BPPV regional III Lampung belum dilaporkan. Penelitian ini bertujuan untuk melakukan karakterisasi fragmen gen fusi yang merupakan penentu patogenesitas molekuler virus ND. Sebanyak 12 sampel berasal dari wilayah kerja BPPV Regional III Lampung tahun 2010-2013 digunakan dalam penelitian ini. Sepuluh sampel berupa swab kloaka dan 2 sampel berupa organ (trakea, pulmo, esophagus dan intestinum). Suspensi organ dan swab kloaka diekstraksi untuk mendapatkan RNA. Amplifikasi DNA dengan tehnik Reverse Transcription Polymerase Chain Reaction (RT-PCR) untuk deteksi keberadaan virus ND menggunakan primer yang didesain oleh Australian Animal Health Laboratory (AAHL) Geelong, Australia. Primer dari BPPV Regional III Lampung digunakan untuk mengamplifikasi fragmen gen fusi untuk dilanjutkan proses sekuensing. Hasil penelitian menunjukkan bahwa seluruh sampel dapat diidentifikasi positif virus ND. Analisis hasil sekuensing dengan menggunakan perangkat lunak MEGA versi 5.01 didapatkan susunan asam amino penyusun cleavage site isolat tersebut digolongkan dalam virus Newcastle disease strain velogenik yang virulen dengan pola sekuen 112RRQKR↓F117 dan 112RRRKR↓F117. Berdasarkan analisis phylogenic tree isolat asal BPPV Regional III Lampung tahun 2010-2013 termasuk dalam genotip VII subtipe a (VIIa) dan d (VIId). Jarak genetik isolat asal BPPV Regional III Lampung dari tahun 2010-2013 dengan virus ND Indonesia yang pernah dilaporkan sebelumnya pada fragmen gen F posisi nukleotida 103-626 berkisar 0 - 8,6% dengan tingkat homologi mencapai 91,4%- 100%.
Newcastle Disease (ND) is an infectious poultry disease that caused by ND virus which is belongs to family Paramyxoviridae, genus Avulavirus, and Avian paramyxovirus species. This disease causes a drop in egg production and has high mortality and high cost of control, which cause economic losses in Indonesia and the world. Vaccination is a method for controlling Newcastle disease in Indonesia, however there were still a lot of cases encountered in the field. Molecular data on the ND virus in the working area of Disease Investigation Center Lampung had not been reported. The purpose of this study is to characterize the fragment of fusion gene, which acts as a molecular determinant of pathogenicity of ND viruses. A total of 12 samples from Disease Investigation Center Lampung from 2010 until 2013 were used in this study. The samples consist of 10 samples of cloacal swabs and two samples of internal organs (trachea, lung, esophagus and intestine). The suspension of organs and cloacal swabs were extracted to obtain RNA. The amplification of DNA was conducted with Reverse Transcription Polymerase Chain Reaction (RT-PCR) using primers designed by the Australian Animal Health Laboratory (AAHL) Geelong, Australia. Another primers from Disease Investigation Center Lampung were used to amplify a fragment from the fusion gene, and then followed by gene sequencing. The results showed that ND were positively identified in all of the samples. The result of sequence analysis with MEGA v5.01 software showed that the amino acids composition in the cleavage site showed that the isolates were classified as velogenic virulent strain of Newcastle Disease virus, with sequence patterns of 112RRQKR↓F117 and 112RRRKR↓F117. According to phylogenic tree analysis, the isolates from Disease Investigation Center Lampung were classified in the genotype VII subtype VIIa and VIId. Genetic distance between the isolates of Disease Investigation Center in 2010-2013 and Indonesian ND viruses that previously reported in nucleotide of fusion gene fragmen position 103-626 ranged from 0 to 8.6 % and homology between 91.4%-100%.
Kata Kunci : Newcastle disease virus, patogenesitas, amplifikasi dan sekuensing
