Newcastle disease (ND) merupakan salah satu penyakit infeksius paling penting dalam industri perunggasan. Virus ND termasuk dalam ordo Mononegavirales, famili Paramyxoviridae, dan genus Avulavirus. Virus ND terdiri dari satu serotipe dan disebut juga sebagai avian paramyxovirus serotipe-1 (APMV-1). Kerugian ekonomi yang tinggi akibat mortalitas dan morbiditas yang tinggi, penurunan produksi telur serta tingginya biaya pengendalian menyebabkan penentuan patotipe virus ND secara cepat dan spesifik penting untuk dilakukan. Penelitian ini bertujuan untuk menentukan patotipe virus ND secara cepat dan spesifik dengan RT-PCR dan REA serta menentukan hubungan genetik antara virus yang ditemukan pada ayam broiler dengan virus yang telah ditemukan sebelumnya. Sebanyak 4 isolat virus ND ayam broiler yang berasal dari BBVet Wates diamplifikasi dengan sepasang primer A (5'-TTGATGGCAGGCCTCTTGC-3') dan primer B (5'-GGAGGATGTTGGCAGCATT-3'). RT-PCR diawali dengan satu siklus reverse transcription dilanjutkan dengan 40 siklus proses amplifikasi. Produk amplifikasi protein fusion (F) divisualisasikan dengan elektroforesis gel agarose 1,5 % dengan pewarnaan florosafe DNA stain. REA dengan enzim Hin1l digunakan untuk membedakan virus ND strain virulen dan avirulen berdasarkan pola pemotongan yang terbentuk. Enzim Hin1l dapat memotong produk amplifikasi protein F virus ND avirulen melalui inkubasi pada suhu 37°C selama 3 jam. Visualisasi pemotongan DNA dilakukan dengan elektroforesis gel agarose konsentrasi 2,5 % dengan pewarnaan florosafe DNA stain. Produk amplifikasi disekuensing untuk mengetahui urutan nukleotida dan asam amino. Hasil sekuensing DNA dilakukan penjajaran berganda dengan data sekuen protein F virus ND yang berasal dari Genbank, dilanjutkan dengan analisis filogram menggunakan metode neighbor joining. Patotipe virus ND dapat diketahui dengan melihat keberadaan asam amino basa pada cleavage site. Visualisasi produk amplifikasi dengan elektroforesis gel agarose menghasilkan fragmen DNA 363 bp pada semua isolat. Sebanyak 2 dari 4 isolat menunjukkan adanya sekuen pengenalan Hin1l sehingga terlihat pola pemotongan. Berdasarkan urutan asam amino dan keberadaan asam amino basa 2 isolat digolongkan sebagai strain lentogenik/avirulen dan 2 isolat sebagai strain mesogenik/virulen. Analisis filogram dengan menggunakan metode neighbor joining dapat menunjukkan hubungan genetik virus yang ditemukan pada ayam broiler dengan virus yang telah ditemukan sebelumnya.

Newcastle disease (ND) is one of the most important infectious diseases of poultry. Newcastle disease virus (NDV) belongs to the order Mononegavirales, family Paramyxoviridae, and genus Avulavirus. NDV contain one serotype and also known as avian paramyxovirus serotype-1 (APMV-1). The economic losses due to high mortality and morbidity, decreased egg production, and the high cost of control causing rapidly and specifically patotyping of NDV important to perform. This research was conducted in order to patotyping NDV rapidly and specifically by RT-PCR and REA also to determine the genetic relationship between broiler chicken viruses and viruses that have been found previously. Newcastle disease isolates of 4 broiler chickens collected by BBVet Wates were amplified with a pair of primer A (5'-TTGATGGCAGGCCTCTTGC-3') and primer B (5'-GGAGGATGTTGGCAGCATT-3'). RT-PCR began with a single cycle of reverse transcription followed by 40 cycles of PCR amplification process. The RT-PCR products of fusion protein were visualized using 1,5% agarose gel electrophoresis with florosafe DNA staining. REA by Hin1l enzyme used to distinguish avirulent and virulent NDV strains based on the restriction pattern. Hin1l can digest the RT-PCR products of avirulent NDV strains into several fragments amplification products through incubation at 37° C for 3 hours. Restriction products from the REA process were visualized using 2,5% agarose gel electrophoresis with florosafe DNA staining. Amplification products were sequenced to determine the sequence of nucleotides and amino acids. Multiple alignments were performed to compare our sequences with Genbank sequences, followed by neighbor joining phylogram analysis. NDV patotipe can be determined based on the presence of basic amino acids at the cleavage site. Agarose gel electrophoresis of the amplification products showed 363 bp fragments in all isolates. Restriction pattern can be seen in 2 from 4 isolates because of the presence of recognition sequence Hin1l. Based on the amino acid sequences and the presence of basic amino acids 2 isolates were classified as lentogenic/avirulent strains and 2 isolates as mesogenic/virulent strains. Phylogram analysis using neighbor joining method can show genetic relationship between broiler chicken viruses and viruses that have been found previously.

Kata Kunci : Newcastle Disease, Patotipe, RT-PCR, Protein Fusion, REA, Hin1l