Analisis sekuens non-coding region dan coding region ujung-5' gen polimerase kompleks virus avianinfluenza subtipe H5N1 asal unggas dan babi di Indonesia
KENCANA, Gusti Ayu Yuniati, Prof. drh. Widya Asmara, S.U., Ph.D
2010 | Tesis |Penelitian tentang faktor genetik yang mempengaruhi spesifitas dan patogenitas hospes virus Avian Influenza (AI) subtipe H5N1 sangat penting dilakukan untuk mengetahui keganasan virus. Tujuan penelitian adalah untuk menemukan kemungkinan adanya variasi dan keterkaiatan pola sekuens non-coding region (NCR) dan coding region (CR) ujung -5’gen penyandi protein polimerase: PB2, PB1 dan PA dari isolat virus unggas (ayam, itik, puyuh) dan isolat babi. Sampel isolat virus AI subtipe H5N1 yang diteliti berasal dari ayam, itik, puyuh, dan babi asal Jawa dan Bali yang diisolasi tahun 2004 sampai tahun 2007. Isolat-isolat tersebut merupakan koleksi dari Fakultas Kedokteran Hewan Universitas Udayana, Universitas Gadjah Mada Yogyakarta, dan Institut Pertanian Bogor. Propagasi isolat virus dilakukan pada telur ayam bertunas spesific pathogen free (SPF) umur 9 hari. Fragmen gen target diperbanyak dengan reverse- transcriptasepolymerase chain reaction (RT-PCR) menggunakan primer depan universal baku dan primer belakang dirancang sendiri berdasarkan atas susunan gen dari genom virus AI yang diunduh dari GeneBank. Produk PCR disekuensing dengan DNA sequencer di Eijkman Institute for Molecular Biology, Jakarta. Sekuens nukleotida yang diperoleh disepadankan dan dianalisis dengan Clustal W dari program Mega 4. Kesimpulannya, variasi sekuens NCR gen PB2 dan PA antara isolat VAI asal unggas (ayam, itik, puyuh) dan mamalia (babi) tidak ditemukan. Variasi sekuens NCR gen PB1 antara isolat VAI asal berbagai spesies itu juga tidak ditemukan, kecuali satu varian NCR gen PB1 itik Duck/Badung/2006 sekuensnya 5’- AGCGAAAGCAGGCAAACTATTTGA-3’ (substitusi posisi C-7T ditulis miring dan digaris-bawahi). Spesies hospes asal isolat dan distribusi geografis tidak memberikan pola khusus pada sekuens ujung-5’CR gen PB2, PB1, PA dan tidak berpengaruh terhadap rataan jarak genetik antar spesies. Disamping itu, ujung -5’dari CR gen PB2, PB1, dan PA dari semua isolat virus yang dianalisis juga tidak menunjukkan pola geografis maupun pola spesies tertentu. Susunan asam amino yang khas isolat virus dari spesies tertentu hanya ditemukan pada gen PB2 isolat asal babi Sw/Tabanan/2006 berupa substitusi D26E, H27Q, M50I, dan H134Y.
Studies on the viral genetic factors that influence host specificity and pathogenecity of Avian Influenza Virus of H5N1 subtype (AIV H5N1) are invaluable to predict the pathogenicity of the isolates. This study was done to elucidate the possibility of nucleotide (nt) sequence variation of non-coding region (NCR) of 5’-ends of positive strand cRNA as well as nt and deduced amino acid (aa) sequences of 5- end coding region (CR) of PB2, PB1, and PA genes of isolates from poultry (chicken, duck, and quill) and swine. The object of the study was AIV H5N1 isolates that were isolated from chicken, duck, quail, and swine from Java and Bali in 2004-2007. The isolates belong to Faculty of Veterinary Medicine of Udayana University Bali,Gadjah Mada University Yogyakarta, and Institut Pertanian Bogor. The isolates were propagated in specific pathogenic free (SPF) chicken eggs of 9 days-old embryo. The targeted gene fragments were produced with reverse transcriptase-polymerase chain reaction (RTPCR) using published universal forward primers and specifically designed backward primers. Reverse Transcriptase Polymerase Chain Reaction products were sequenced using automated DNA sequencer in Eijkman Institute for Molecular Biology, Jakarta. All nt sequences and the corresponding aa sequences were aligned and deduced using Clustal W method of Mega 4. It is concluded that sequence variation of NCR of PB2 and PA genes between AIV isolates of poultry (chicken, duck, and quail) and mammal (pig) was not found. Sequence variation of NCR of PB1 gene between AIV isolated from those species was also not found, except that one variant sequence of 5’- AGCGAAAGCAGGCAAACTATTTGA-3’ (the substituton C-7T is written in italic and underlined) was observed in one isolate from duck (Duck/Badung/2006). Host diversity and geographical distribution did not influence the pattern of the 5-end CR sequences of PB2, PB1, PA genes and the genetic distance. Furthermore, the 5-end CR of PB2, PB1, and PA genes of all isolates under study do not show any specific geographical and species pattern. Isolate specific amino acid sequence was found in a swine isolate of Sw/Tabanan/2006 which had substitutions of D26E, H27Q, M50I, and H134Y.
Kata Kunci : Non coding-region, coding region, virus AI subtipe H5N1, gen polimerase kompleks (PB2, PB1, dan PA), analisis sekuens, 5’-End non-coding region, coding region, avian influenza virus of H5N1, polymerase genes (PB2, PB1 and PA), sequence analysis
