Kandidat Vaksin Flu Burung H5N1 Bagi Ternak Ayam Isolat Asal Jawa Timur Avian Influenza H5N1 Vaccine Candidate for Chicken from East Java Isolate virus 1 2 3 Rahayu Ernawati , E. Djoko Putranto , Maulana Hanief R 1 Fakultas Kedokteran Hewan Unair 2 Mahasiswa Program DoktorPascasarjana Unair 3 Mahasiswa Program MagisterPascasarjana Unair Kampus C Unair, Jl. Mulyorejo Surabaya-60115 Telp. 031-5992785, Fax. 031-5993015 Email : [email protected]Abstract Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is an ongoing public health and socio-economic challenge, particularly in Indonesia. Avian Influenza (H5N1) is now endemic in poultry in many countries, and represents a major pandemic threat. Now, the evolution of H5N1 virus in Indonesia has evolved with genetic variations affecting virulence, drug-resistance, and adaptation to new host species. The reassortment events leading to high genetic diversity in the region, and factors responsible for virus spread. Development of H5N1-specific vaccine may be become a good strategy for the prevention or controlling the spread of AI. This study was aimed to find a vaccine candidate for Avian Influenza subtype H5N1 which has a good quality, safe, homolog genetically and antigenically with the virus in East Java. The sample viruses were isolated from village chicken in traditional market at Surabaya, Jombang, Pare, and Kediri. Suspensions in antibiotic solution of cloacal swabs (or organs), taken from village chicken, inoculated into the allantoic cavity of 9 to 11-day-old embryonating chicken eggs. The eggs are incubated at 37°C (range 35–39°C) for 4–7 days. The allantoic fluid of any eggs containing dead or dying embryos during the incubation and all eggs at the end of the incubation period are tested for the presence of haemagglutinating activity by HA and HI test. The presence of Hemagglutinin gene which are common to all influenza A viruses can be confirmed by One Step PCR using 4 pairs of specific primers. Then, the product being purificated and sequenced to get the hemagglutinin nucleotide data. The data were analysed for homology relationship. The antigenic test against confirmed antisera panel was conducted to observed the antigenic potential from the isolate. The study results two isolates, Jombang and Pare isolate. The result of this study show that the isolate have a high homology of hemagglutinin gene with another virus isolate (A/chicken/WestJava/Tasiksol/2006) which have wide covered spatial area. The antigenic test showed the same result, but Pare isolate have much wider covered area because it can react with all of antigenic test panel antisera. Keywords : Vaccine, Avian Influenza (H5N1), Chicken, Hemagglutinin, Homology Jurnal Ilmiah Kedokteran Hewan Vol. 4, No. 1, Februari 2011 Pendahuluan Kejadian wabah Flu Burung ( Avian Influenza) selain mengakibatkan kerugian ekonomi yang sangat tinggi karena kematian pada ternak unggas juga menimbulkan kepanikan yang luar biasa karena dapat menular dan mematikan pada manusia. Di Indonesia sejak Agustus 2003 hingga 10 September 2008 telah menyebabkan kematian lebih dari 10 juta ayam dengan menimbulkan kerugian lebih dari 4 trilyun rupiah dan hingga bulan Januari 2010 tercatat 163 kasus pada manusia dengan kematian 135 orang (Hasan, 2009; Dharmawan dan Wahyuningsih, 2010). Virus Avian Influenza (AI) mempunyai struktur antigen permukaan antara lain Hemaglutinin (HA) dan Neuroamidase (NA). Penggolongan subtipe AI adalah atas dasar kandungan Hemaglutinin yang terdiri atas 16 subtipe dan atas dasar Neuroamidase yang terdiri atas 9 subtipe. Jenis yang menyerang Indonesia, menurut pemerintah yang diumumkan oleh Ditjenak adalah subtype H5N1 (Nidom, 2009; Kawaoka, 2009). Virus AI mempunyai 8 segmen gen yang menyandi 10 macam protein, yakni PB-2 (Polymerase Basic-2), PB-1(Polymerase Basic-1), PA (Polymerase acidic), HA (Haemaglutinin), NP (Nucleo Protein) , NA (Neuroamidase) , M1(Matrix-1), M2 (Matrix-2) , NS1 (Non Struktural- 1) dan NS2 (Non Struktural-2). Protein HA merupakan target awal dalam pembentukan antibody inang. Pada awal infeksi protein ini akan berikatan dengan reseptor sel inang dan melepaskan ribonukleoprotein. Akibat aktivasi precursor HA (HAO) oleh protease inang, protein akan terbelah menjadi HA1 dan HA2. Protein HA1 akan berikatan dengan reseptor dan merupakan target utama untuk timbulnya respons imun, sedangkan protein HA2 akan memfasilitasi fusi antara amplop virus dengan membrane endosomal inang. Oleh karenanya Protein HA merupakan pertimbangan utama dalam 19
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Kandidat Vaksin Flu Burung H5N1 Bagi Ternak Ayam Isolat Asal Jawa Timur
Avian Influenza H5N1 Vaccine Candidate for Chicken from East Java Isolate virus
1 2 3Rahayu Ernawati , E. Djoko Putranto , Maulana Hanief R
1Fakultas Kedokteran Hewan Unair 2Mahasiswa Program DoktorPascasarjana Unair
3Mahasiswa Program MagisterPascasarjana Unair
Kampus C Unair, Jl. Mulyorejo Surabaya-60115 Telp. 031-5992785, Fax. 031-5993015
Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is an ongoing public health and socio-economic challenge, particularly in Indonesia. Avian Influenza (H5N1) is now endemic in poultry in many countries, and represents a major pandemic threat. Now, the evolution of H5N1 virus in Indonesia has evolved with genetic variations affecting virulence, drug-resistance, and adaptation to new host species. The reassortment events leading to high genetic diversity in the region, and factors responsible for virus spread. Development of H5N1-specific vaccine may be become a good strategy for the prevention or controlling the spread of AI. This study was aimed to find a vaccine candidate for Avian Influenza subtype H5N1 which has a good quality, safe, homolog genetically and antigenically with the virus in East Java. The sample viruses were isolated from village chicken in traditional market at Surabaya, Jombang, Pare, and Kediri. Suspensions in antibiotic solution of cloacal swabs (or organs), taken from village chicken, inoculated into the allantoic cavity of 9 to 11-day-old embryonating chicken eggs. The eggs are incubated at 37°C (range 35–39°C) for 4–7 days. The allantoic fluid of any eggs containing dead or dying embryos during the incubation and all eggs at the end of the incubation period are tested for the presence of haemagglutinating activity by HA and HI test. The presence of Hemagglutinin gene which are common to all influenza A viruses can be confirmed by One Step PCR using 4 pairs of specific primers. Then, the product being purificated and sequenced to get the hemagglutinin nucleotide data. The data were analysed for homology relationship. The antigenic test against confirmed antisera panel was conducted to observed the antigenic potential from the isolate. The study results two isolates, Jombang and Pare isolate. The result of this study show that the isolate have a high homology of hemagglutinin gene with another virus isolate (A/chicken/WestJava/Tasiksol/2006) which have wide covered spatial area. The antigenic test showed the same result, but Pare isolate have much wider covered area because it can react with all of antigenic test panel antisera.