Analisis molekuler regio Cleavage Site hemaglutinin virus Avian Influenza H5N1 isolat asal demak dan Purworejo Jawa Tengah

WULANSIH, Dwi, Prof.drh. Widya Asmara, SU.,Ph.D

2007 | Tesis | S2 Ilmu Kedokteran Tropis (Imunologi dan Biologi M

Virus avian influenza sub tipe H5N1 dapat menginfeksi unggas maupun mamalia termasuk manusia. Infeksi virus H5N1 pertama kali menginfeksi manusia di Hongkong tahun 1997 yang kemudian meluas ke negara-negara lain di dunia termasuk Indonesia dengan angka kematian tinggi. Virus H5N1 penyebab infeksi pada manusia dan hewan peternakan dengan angka kematian yang tinggi termasuk dalam kelompok virus avian influenza dengan patogenisitas tinggi. Keberadaan asam amino polybasic di cleavage site HA dan terdapatnya mutasi regio tapak glikosilasi dihubungkan dengan virulensi virus. Penelitian ini untuk menganalisis susunan asam amino regio cleavage site HA H5N1 untuk mengetahui keberadaan asam amino polybasic di region CS HA, urutan dan posisi asam amino tapak glikosilasi di sekitar regio cleavage site HA serta mengetahui urutan asam amino regio fusion peptide HA yang berhubungan dengan proses fusi ke dalam membran sel hospes. Kekerabatan kedua sampel dengan virus H5N1 lain yang dipilih dari bank data menggunakan analisis filogenetik. Penelitian ini menggunakan metode analisis deskriptif. Sampel diperoleh dari isolasi virus H5N1 pada burung puyuh asal Demak (A/Quail/Demak- Mg/3/2005 ) dan ayam kampung asal Purworejo (A/Chicken/Purworejo/6/2005 ). Metode penelitian yang dirancang meliputi isolasi RNA, amplifikasi tempat perlekatan reseptor menggunakan primer spesifik dilakukan dengan one step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), elektroforesis dan proses sekuensing dengan mesin sekuenser otomatis. Analisa hasil sekuensing nukleotida regio cleavage site HA sampel dan 42 isolat H5N1 lain yang telah dipilih dari bank data menggunakan program ClustalX (1.81), BioEdit 5.0.6., Genedoc 2.6.002. dan Accelrys DS Visualizer 1.7. Pohon filogenetik menggunakan program Clustal X 1.81 yang diplotkan di TreeView 1.6.6. Kedua isolat virus H5N1 A/Quail/Demak-Mg/3/2005 dan A/Chicken/Purworejo/C/05 memiliki asam amino polybasic pada cleavage site HA yang menunjukkan virus dengan patogenisitas tinggi. Asam amino polybasic tersebut terletak di antara posisi Proline 321 dan Glisine 331 dengan urutan 321PQ- K-E-R-R-R-K-K-R/G331. Isolat sampel tidak menunjukkan perubahan pada tapak glikosilasi sekitar CS HA 286N-S-S288 dan fusion peptide HA 331GLFGAIAGFIEGGWQGMVDGWYG353. Analisis filogenetik berdasar sekuen nukleotida HA menunjukkan kekerabatan kedua sampel dengan homologi 99,0 % serta adanya indikasi diversitas VAI H5N1 di Indonesia dari tahun 2003- 2007.

The avian influenza virus subtype of H5N1 can infect birds and mammals and also human being. It infected human being for the first time in Hong Kong in 1997 and then it spread out to other countries, including Indonesia with a high mortality rate. Infection in human and poultry with highly case fatality rate have caused highly pathogenic avian influenza H5N1. The presence of amino acid in the cleavage site hemagglutinin the mutation in the glycosylation site are related with virus of virulence. This study are to investigate amino acid of sequence in the cleavage site of region of hemagglutinin to investigate the presence of the polybasic amino acid in this region, the sequence and the position of amino acid in the glycosylation site and to investigate the sequence of amino acid in the fusion peptide of HA that related with fusion activity to cell membrane of host. Phylogenetic analysis of both the isolate sample compared to another of the isolate H5N1 on the basis of the data of HA sequence data from the gene bank. The study uses a descriptive analysis method. The sample drawn from the isolation of the virus through the organs of the quail from Demak (A/Quail/Demak-Mg/3/2005 ) and the cloacae swab of free-range chicken from Purworejo (A/Chicken/Purworejo/6/2005 ). The methods were constructed in this research consists of RNA isolation, amplification of receptor binding site using specific primers by one step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), electrophoresis and sequencing process with automated sequencer. The results of the sequencing of the nucleotide in the cleavage site of hemagglutinin from both the isolate sampel and 42 isolated selected from the GenBank using the program Clustal X 1.81, BioEdit 5.0.6., Genedoc 2.6.002 and Accelrys DS Visualizer 1.7. The phylogenetic tree is drawn using the program Clustal X 1.81 that is subsequently plotted using TreeView 1.6.6. Both of the sample isolate H5N1 viruses A/Quail/Demak-Mg/3/2005 and A/Chicken/Purworejo/6/2005 have the polybasic amino acid residues in the HA cleavage site region of hemagglutinin of H5N1 indicates the highly pathogenic avian influenza virus. The polybasic amino acid lies between the position of 321 proline and 331 glycine with the amino acid sequencing 321P-Q-E-R-R-R-K-KR/ G331. The sample isolate did not indicate the change in the glycosylation site in the near CS region of HA with 286N-S-S288 of sequence and the fusion peptide HA with 331GLFGAIAGFIEGGWQGMVDGWYG353 of sequence. The phylogenetic analysis based on the HA nucleotide sequence revealed that both the sample have homology 99% and indicate the presence of the diversity of avian influenza virus H5N1 subtype between the isolates of virus from birds, mammals and human being in Indonesia from 2003-2007.

Kata Kunci : Virus Avian Influenza,Cleavage Site,Hemaglutinin,hemagglutinin, cleavage site, glycosylation site, avian influenza virus of H5N1 subtype, phylogenetic analysis