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"ABSTRACTDengue 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 Indonesiais needed to control dengue in Indonesia. In this research, cloning of Non Structural 1 (NS1) gene from dengue virustype 3 (Indonesian strain D3-1703) into pYES2/CT vector was performed. In the long run, NS1 recombinant proteinwill be expressed inSaccharomyces cerevisiaefor diagnostic materials. Polymerase Chain Reaction (PCR)amplification of NS1 gene fragments were done with optimal annealing temperature at 55 ºC. NS1 gene fragment andpYES2/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:insertratio 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 blastnanalysis showed that it had a 99% identity with dengue virus type 3 strain FW06. Finally, it was shown that NS1 clonewithin pYES2/CT was in the correct Open Reading Frame and ready to be expressed in S. cerevisiae."

"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.

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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."

"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.

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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."

"ABSTRAKPenelitian terkait karakteristik genetik sekuens virus dengue (DENV) diperlukan dalam menilai kekerabatan antara strain DENV yang tersebar di seluruh dunia. Tujuan dalam penelitian ini adalah membandingkan karakteristik genotype dan sekuens data DENV serotipe 2 (DENV-2) nukleotida envelope dibandingkan dengan sekuens data DENV-2 nukleotida Non-Struktural 1 (NS1). Data didapatkan dari Laboratorium Mikrobiologi Fakultas Kedokteran Universitas Indonesia untuk strain data yang berasal dari Indonesia dan GenBank untuk strain data yang berasal dari seluruh dunia sebagai data pembanding dengan jumlah sebanyak 42 data, yang kemudian dianalisis menggunakan Genetyx 5.1. Hasil penelitian didapatkan bahwa sekuens data NS1 strain DENV-2 yang berasal dari Indonesia termasuk ke dalam kelompok genotype Cosmopolitan, serupa dengan hasil analisis data dengan sekuens data envelope strain DENV-2. Sementara pada analisis epitope LX1 yang merupakan epitope khas dari NS1, terdapat berbagai peubahan komponen asam amino pada epitope tersebut dibandingkan dengan sekuens data strain Indonesia yang tergabung ke dalam kelompok genotype Cosmopolitan. Kesimpulan dari penelitian ini adalah bahwa filogenetik dengan menggunakan NS1 sebagai bahan analisis dapat digunakan untuk menentukan genotipe. Sehingga gen NS1 pada DENV-2 strain Indonesia masih dapat dipertimbangkan sebagai salah satu dasar metode pengembangan vaksin.

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ABSTRACTStudies about genetic characteristic between dengue virus serotype 2 (DENV-2) sequence data is needed to determine its relationship between DENV strain over the world. The main purpose of this study is to compare between characteristic of sequence data DENV-2 envelope nucleotide and sequence data DENV-2 Non- Structural 1 (NS1) nucleotide, and analyze between amino acid homology in this study and related previous studies. 42 data used for this study are obtained from Laboratory of Microbiology Faculty of Medicine University of Indonesia for data from Indonesia and form GenBank for data from other countries as a comparison, which is analyzed with software Genetyx 5.1. NS1 sequence data from DENV-2 strain from Indonesia is classified in Cosmpolitan genotype group, which are similar than data analysis from envelope sequence data from same data. Meanwhile in analysis of LX1 epitope, which is considered as typical epitope from NS1, there are any differences in amino acid component at it compared than strain Indonesia data sequences which are included in Cosmopolitan genotype group. As a conclusion, phylogenetic analysis of NS1 nucleotide is useful for determining the genotypes, which means NS1 DENV-2 gene strain Indonesia can be useful as the basis for vaccine development"

"ABSTRAKArbovirus (arthropode-borne virus) yang timbul dan timbul kembali telah memengaruhi berbagai aspek kehidupan manusia. Infeksi arbovirus terbanyak di Indonesia: dengue, Japanese encephalitis (JE) dan chikungunya (CHIK) menyebabkan kasus luar biasa tiap tahun. Ketersediaan metode deteksi JE dan CHIK sangat terbatas di Indonesia. Pengembangan in-house IgM antibody-capture Enzyme Linked Immunosorbent Assay (MAC ELISA) dengan antigen local terinaktivasi akan meningkatkan deteksi dan pemantauan dengan meningkatkan spesifisitas dan sensitivitas. Antigen diproduksi dalam kultur sel dengan sel BHK-21 dan sel Vero kemudian diinaktivasi dengan gamma-irradiasi dan 0,01% beta-propiolakton. Kinerja Antigen dievaluasi dengan uji MAC ELISA dan titer virus dihitung dengan uji plak. Virus Japanese encephalitis dan chikungunya terinaktivasi pada 20 kGy gamma- irradiasi dan 0,01% BPL. In-house MAC ELISA telah dioptimisasi dengan inkubasi 2 jam. Kit in-house MAC ELISA yang telah dikembangkan berguna untuk deteksi dan pemantauan JE dan Chik dengan fasilitas terbatas.

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ABSTRACTThe emerging and re-emerging arthropod-borne viruses (arboviruses) have effected many aspects of human existence. Three major arbovirus infection in Indonesia: dengue, Japanese encephalitis (JE) and chikungunya (CHIK) causes numerous outbreaks each year. However, availability of detection methods for JE and CHIK are very limited in Indonesia. Development of in-house IgM antibody-capture Enzyme Linked Immunosorbent Assay (MAC ELISA) with inactivated local antigen will improve detection and surveillance capability across Indonesia by increasing its specificity and sensitivity. Antigens were produced in cell culture using BHK-21 cells and Vero cells then inactivated using gamma-irradiation and 0.01% beta-propiolactone (BPL). Antigen performance was evaluated using MAC ELISA and virus titer were calculated using plaque assay. Japanese encephalitis virus and chikungunya virus was inactivated at 20 kGy with 0.01% BPL. Optimized in-house MAC ELISA protocol using these antigen has been developed. Developed in-house MAC ELISA kit will be beneficial for detection and surveillance of JE and CHIK with limited facility. "

"[Saat ini tengah banyak studi yang dilakukan untuk menemukan antivirus dengue(DENV) dari ekstrak bebagai tumbuhan sebagai alternatif pengobatan terhadapinfeksi DENV. Calophyllum flavoramulum (C. flavoramulum) merupakantanaman yang banyak hidup di daerah Asia Tenggara ini memiliki kandungan dariturunan xanthone dan flavonoid yang memiliki potensi sebagai antivirus. Padastudi ini dilakukan evaluasi efek inhibisi dari fraksi etanol ekstrak daun C.flavoramulum terhadap DENV-2 strain New Guinea C (NGC). Efek inhibisi(IC50) dievaluasi dengan menggunakan focus assay. Sedangkan efek toksisitas(CC50) terhadap sel dievaluasi pada sel Huh7it-1 menggunakan metode MTTassay. Hasil dari studi ini menunjukan CC50 = 473,50ug/ml, IC50 = 41,74ug/ml,dan SI=11,33. Hasil tersebut menunjukan bahwa C. flavoramulum tidak toksikterhadap sel hidup dan memiliki nilai SI yang cukup tinggi sehingga efektif untukdigunakan sebagai antivirus dengue. Akan tetapi, perlu diteliti lebih lanjutmengenai kandungan aktif dalam C. flavoramulum yang memiliki aktivitas untukmenghambat replikasi DENV.;In recent years, several studies have been conducted to find dengue (DENV)antiviral from natural extract as an alternative management for dengue infection.Calophyllum flavoramulum(C. flavoramulum), one of South East Asia plants,contains derivates of flavonoid and xanthone where both have been known aspotential dengue antivirals. This study evaluated inhibitory potential of ethanolextract from C. flavoramulum leaf to DENV-2 strain New Guinea C (NGC)through focus assay. Along with inhibitory effect (IC50), toxicity effect (CC50) toHuh7it-1 cell also evaluated through MTT assay. The result of this study shownIC50 = 41.74 μg/mL, CC50 = 473.50 μg/mL, and SI=11.33. In conclusion, extractof C. flavoramulum can be used selectively as a dengue antiviral, besides it is nottoxic for living cells. More studies are needed to find its active ingredients whichspecifically have the ability to inhibit DENV replication.;In recent years, several studies have been conducted to find dengue (DENV)antiviral from natural extract as an alternative management for dengue infection.Calophyllum flavoramulum(C. flavoramulum), one of South East Asia plants,contains derivates of flavonoid and xanthone where both have been known aspotential dengue antivirals. This study evaluated inhibitory potential of ethanolextract from C. flavoramulum leaf to DENV-2 strain New Guinea C (NGC)through focus assay. Along with inhibitory effect (IC50), toxicity effect (CC50) toHuh7it-1 cell also evaluated through MTT assay. The result of this study shownIC50 = 41.74 μg/mL, CC50 = 473.50 μg/mL, and SI=11.33. In conclusion, extractof C. flavoramulum can be used selectively as a dengue antiviral, besides it is nottoxic for living cells. More studies are needed to find its active ingredients whichspecifically have the ability to inhibit DENV replication., In recent years, several studies have been conducted to find dengue (DENV)antiviral from natural extract as an alternative management for dengue infection.Calophyllum flavoramulum(C. flavoramulum), one of South East Asia plants,contains derivates of flavonoid and xanthone where both have been known aspotential dengue antivirals. This study evaluated inhibitory potential of ethanolextract from C. flavoramulum leaf to DENV-2 strain New Guinea C (NGC)through focus assay. Along with inhibitory effect (IC50), toxicity effect (CC50) toHuh7it-1 cell also evaluated through MTT assay. The result of this study shownIC50 = 41.74 μg/mL, CC50 = 473.50 μg/mL, and SI=11.33. In conclusion, extractof C. flavoramulum can be used selectively as a dengue antiviral, besides it is nottoxic for living cells. More studies are needed to find its active ingredients whichspecifically have the ability to inhibit DENV replication.]"

"Indonesia merupakan negara dengan jumlah kasus dengue terbanyak dan terparah di Asia Tenggara Studi filogenetik virus dengue DENV diperlukan sebagai dasar pengembangan struktur vaksin yang cocok Meskipun demikian data sekuens DENV masih terbatas Penelitian ini bertujuan menganalisis sekuens dan filogenetik keseluruhan gen envelope DENV 1 dibandingkan dengan domain III saja Data didapatkan dari GenBank dan Laboratorium Mikrobiologi Fakultas Kedokteran Universitas Indonesia berupa whole genome DENV 1 sebanyak 30 sekuens yang diolah dengan Genetyx 5 1 Hasil analisis nukleotida gen envelope DENV 1 menunjukan strain Indonesia termasuk genotipe I dan IV Sedangkan analisis nukleotida dengan hanya domain III menunjukan adanya perbedaan cluster antar strain namun tetap dalam genotipe yang sama Dengan demikian studi filogenetik penentuan genotipe dapat dilakukan dengan hanya menggunakan domain III saja Analisis homologi asam amino domain III menunjukan epitope utama dilestarikan dan dapat menjadi landasan penting dalam pembuatan vaksin dengue berbasis domain III

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Phylogenetic study of dengue virus DENV is required as a basis to develop a suitable structure for vaccine development Nonetheless DENV sequence data is limited This study aims to analyze and compare the sequence and phylogenetic of DENV 1 envelope gene with domain III The data is obtained from GenBank and Laboratory of Microbiology Faculty of Medicine Universitas Indonesia Thirty sequences of DENV 1 whole genome were processed using Genetyx 5 1 Analysis using DENV 1 envelope nucleotide showed that strain Indonesia has genotype I and IV Analysis using only the nucleotide of domain III showed the same genotype with difference of clusters between strains Thus phylogenetic studies determining the genotype can be done using domain III alone Homology analysis of amino acid of domain III showed that the main epitope is well reserved This finding can be an important cornerstone in the development of domain III based dengue vaccine."