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Abstrak :
ABSTRACT
Dengue is an infectious disease caused by dengue virus. Dengue endemic region includes America, Western Pacific, Africa, East Mediterranian, and South East Asia including Indonesia. An early diagnostic system specific for Indonesia is needed to control dengue in Indonesia. In this research, cloning of Non Structural 1 (NS1) gene from dengue virus type 3 (Indonesian strain D3-1703) into pYES2/CT vector was performed. In the long run, NS1 recombinant protein will be expressed in Saccharomyces cerevisiae for diagnostic materials. Polymerase Chain Reaction (PCR) amplification of NS1 gene fragments were done with optimal annealing temperature at 55 ºC. NS1 gene fragment and pYES2/CT were cut by Bam H I and Not I enzymes. The digested pYES2/CT was dephosphosrylated using Calf Intestine Alkaline Phospatase enzyme. Ligation with the vector:insert ratio of 1:12 and 1:20 resulted in 6 and 5 recombinant colony candidates respectively. Restriction enzyme and PCR verifications showed that 5 recombinant plasmids contained NS1 gene. Sequencing of the first 600 bp of one recombinant plasmid was performed. The blastn analysis showed that it had a 99% identity with dengue virus type 3 strain FW06. Finally, it was shown that NS1 clone within pYES2/CT was in the correct Open Reading Frame and ready to be expressed in S. cerevisiae.

Abstrak :
Virus dengue ialah penyebab demam dengue yang ditransmisikan melalui nyamuk betina Aedes aegypti. Manifestasi klinis dari infeksi virus ini sangat beragam, mulai dari tidak bergejala hingga dapat menyebabkan sindrom renjatan dengue. Kejadian infeksi dengue diperkirakan mencapai 390 juta kasus per tahun. Belum terdapat pengobatan antivirus spesifik yang bisa digunakan dalam menangani kasus infeksi dengue. Tanaman Moringa oleifera merupakan salah satu tanaman herbal yang diketahui memiliki banyak manfaat akibat zat-zat metabolit yang terkandung di dalamnya. Ekstrak daun ini berpotensi sebagai antivirus DENV, namun belum diketahui bagaimana mekanisme penghambatan yang terjadi. Penelitian ini dilakukan untuk mengevaluasi mekanisme penghambatan replikasi DENV secara in vitro oleh fraksi heksana dari ekstrak daun Moringa oleifera. Sel vero diinfeksi dengan DENV-2 dan diberi fraksi heksan ekstrak daun Moringa oleifera pada tahapan entry-step (awal) dan post-step (akhir). Pada penelitian ini, dilakukan focus assay untuk menghitung persentase penghambatan dan MTT assay untuk menghitung persentase viabilitas. Persentase penghambatan pemberian fraksi heksana ekstrak daun M. oleifera pada perlakuan entry step dan post adalah sebesar 98,08% dan 99,51 dengan persentase viabilitas masing-masing sebesar 104,04% dan 96,4%. Pemberian fraksi heksana ekstrak daun M. oleifera pada perlakuan entry step maupun post infeksi menunjukkan aktivitas antivirus DENV yang baik dan aman bagi sel. ......Dengue virus is the cause of dengue fever that can be transmitted by female mosquitoes from Aedes aegypti Sp. The clinical manifestation of dengue infection varies, from asymptomatic to dengue shock syndrome. The incidence of dengue infection is estimated to reach 390 million cases each year. There is no specific antivirus yet to treat DENV. Moringa oleifera is a herbal plant that is known to have many benefits as it has lots of important nutrients and essential phytochemicals. The objective of this study is to evaluate the cytotoxic effect and inhibition mechanism of dengue virus replication by hexane fraction of Moringa oleifera leaf extract using vero cells. Inhibitory mechanism was done by entry-step infection method and post infection method. Inhibitory percentage was determined by focus assay meanwhile viability percentage was determined by MTT assay. The viability percentage of vero cells from the entry step infection and post infection were 104,04% and 96,4%. While inhibitory percentage of DENV-2 were 98,08% and 99,51%, respectively. The entry-step infection and post infection mechanism of hexane fraction of Moringa oleifera leaf extract towards dengue virus shows a good antiviral potential with high inhibitory percentage and low cytotoxic effect.

Abstrak :
Latar belakang: Demam Berdarah Dengue (DBD) merupakan penyakit yang disebabkan virus dengue (DENV) dan ditularkan oleh nyamuk. Peningkatan kasus DBD masih menjadi masalah kesehatan utama di berbagai negara. Belum terdapat obat antivirus spesifik untuk mengobati atau mencegah infeksi DENV. Moringa oleifera, atau dikenal dengan tanaman kelor, memiliki kandungan bioaktif sebagai antivirus. Pada penelitian sebelumnya, Moringa oleifera memiliki potensi sebagai antivirus DENV dengan nilai IC50 31.51μg/ml,CC50>320μg/ml,danSI>10,2.Namun,mekanismepenghambatannya masih belum diketahui. Penelitian ini bertujuan untuk mengetahui mekanisme penghambatan replikasi DENV-2 oleh fraksi butanol dari ekstrak etanolik daun Moringa oleifera. Metode: DENV-2 secara in vitro diinfeksikan terhadap sel Vero serta diberi perlakuan ekstrak melalui mekanisme penghambatan yang berbeda yaitu: pre, prepost, post, dan entry step. Metode focus assay dan MTT assay digunakan untuk mengetahui persentase penghambatan dan persentase viabilitas. Hasil: Hasil persentase viabilitas pada perlakuan entry step, pre, pre-post dan post adalah 106,2 %, 125,3%, 112,6% dan 111,8%. Sementara itu, nilai persentase penghambatan tiap perlakuan secara berurutan adalah sebesar 7,42%, 100%, 95,91% dan 93,76% Kesimpulan: Fraksi butanol dari ekstrak etanolik daun Moringa oleifera secara aman memiliki efek antivirus baik pada awal maupun akhir infeksi. Mekanisme penghambatan dengan hasil yang lebih baik ditunjukkan oleh perlakuan pre diikuti perlakuan prepost, post, dan entry step. Namun, penelitian lebih lanjut diperlukan untuk menjawab mengenai kandungan zat bioaktif spesifik yang berperan dalam inhibisi ekstrak daun M. oleifera terhadap DENV. ......Introduction: Dengue Hemorrhagic Fever (DHF) is a disease caused by the dengue virus (DENV) and is transmitted by mosquitoes. The increase in dengue cases is still a major health problem in many countries. There are no specific antiviral drugs to treat or prevent DENV infection. Moringa oleifera, otherwise known as the Moringa plant, has bioactive properties as an antiviral. In previous studies, Moringa oleifera has potential as a DENV antiviral with IC50 values of IC50 31.51 μg/ml, CC50 >320 μg/ml, and SI >10,2. However, the mechanism of inhibition is still unknown. This study aimed to determine the mechanism of inhibition of DENV-2 replication by the butanol fraction of the ethanolic extract of Moringa oleifera leaves. Method: DENV-2 was infected with Vero cells in vitro and treated with extracts through different inhibition mechanisms, namely: pre, prepost, post, and entry step. Focus assay and MTT assay methods were used to determine the percentage of inhibition and the percentage of viability. Result: The results of the percentage of viability in the entry step, pre, pre-post and post treatments were 106.2%, 125.3%, 112.6% and 111.8%. Meanwhile, the percentage inhibition values for each treatment were respectively 7.42%, 100%, 95.91% and 93.76%. Conclusion: The butanol fraction of the ethanolic extract of Moringa oleifera leaves safely has an antiviral effect both at the beginning and at the end of the infection. The inhibition mechanism with better results was shown by pre treatment followed by prepost, post, and entry steps. However, further research is needed to answer the specific content of bioactive substances that play a role in the inhibition of M. oleifera leaf extract against DENV.