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All Journal Jurnal Ilmu Pertanian Indonesia Microbiology Indonesia The International Journal of Tropical Veterinary and Biomedical Research

Diah Iskandriati

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Author-ID : 2445936

Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Immunology & microbiology Medicine & Pharmacology Public Health Veterinary

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Ekspresi Enzim Rekombinan Reverse Transcriptase (RTRNase H) Simian Betaretrovirus Serotipe-2 Asal Macaca fascicularis Indonesia dalam Sistem Ekspresi Eschericia coli Uus Saepuloh; Diah Iskandriati; Joko Pamungkas; Dondin Sajuthi
Jurnal Ilmu Pertanian Indonesia Vol. 18 No. 1 (2013): Jurnal Ilmu Pertanian Indonesia
Publisher : Institut Pertanian Bogor

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Reverse transcriptase (RT) enzyme is a key tool in molecular biology for the synthesis of complementary DNA (cDNA) from messenger RNA (mRNA). Combining RT activity with PCR amplification has been a gold standard as the first step in cloning the coding region of any gene of interest. Evidently, RTs have been critical in advancing molecular biology, genetics and medicine to their current stage. In this study, we were developing the RTDRNase H recombinant enzyme isolated from serotype-2 simian betaretrovirus-2 (SRV-2) infected Indonesian Macaca fascicularis using Escherichia coli expression system. The study was conducted using RT SRV-2 gene expression using E. coli expression system, proteins purification, and application to RT PCR technique. The SDS PAGE expression analysis showed a specific band size of 32.7 kDa assumed as RT protein enzyme. Application of RT SRV-2 enzyme generated to the RT PCR technique of β-globin CDV and SRV-2 env gene target showed that the RT SRV-2 enzyme was capable to reverse transcribed mRNA into cDNA as indicated by the presence of specific DNA band compared with commercial RT enzymes. This RT SRV-2 enzyme showed its activity similar to that of commercial one, although the activity was lower.

Expression of Simian Retrovirus Type D Serotype 2 Envelope in Insect Cell Using Baculovirus Expression Vector System DIAH ISKANDRIATI; MOHAMAD SADIKIN; JOKO PAMUNGKAS
Microbiology Indonesia Vol. 3 No. 2 (2009): August 2009
Publisher : Indonesian Society for microbiology

Simian retrovirus type-D (SRV) is a causative agent of simian acquired immunodeficiency syndrome in Asian macaques, and can serve as a viral model in understanding of retrovirus infection because of some similarities to human AIDS pathogenesis. Study of infection and pathogenesis of SRV in macaques could be a strategy of vaccine and antiviral development for preventive and therapeutic purposes. We expressed the SRV-2 envelope gene using baculovirus expression vector system and transfected it to Spodoptera frugiferda insect cell line for SRV-2 recombinant protein production. Analysis using PCR and sequencing technique of recombinant in the passage-3 viral stock indicated the occurrence of recombination between SRV-2 envelope and baculovirus genome. Purification using immobilized metal ion affinity chromatography Ni2+-NTA to recombinant protein could minimize the presence non-specific proteins. The SDS-PAGE analysis showed a specific protein for SRV-2 gp70 envelope. Western blot analysis of this purified protein indicated a specific reaction with anti-SRV-2 antibody positive of Macaca fascicularis serum shown as SRV-2 gp70 envelope band.

Cloning and Expression of Serotype-2 Simian Betaretrovirus Reverse Transcriptase Gene Isolated from Indonesian Cynomolgus Monkey in Escherichia coli UUS SAEPULOH; DIAH ISKANDRIATI; FUNGKEY HOETAMA; SELA SEPTIMA MARIYA; DEDY DURYADI SOLIHIN; JOKO PAMUNGKAS; DONDIN SAJUTHI
Microbiology Indonesia Vol. 7 No. 2 (2013): June 2013
Publisher : Indonesian Society for microbiology

In this study, we isolated the simian betaretrovirus serotype-2 (SRV-2) reverse transcriptase (RT) gene from infected Indonesian cynomolgus monkey (Macaca fascicularis). The gene was then cloned in Escherichia coli expression system. The SRV-2 RT gene is located between nucleotides 3284-4925 in the polyprotein (Pol) region encodes 547 amino acids. Analysis of expression using SDS-PAGE and western blot techniques showed a specific band of 64.9 kDa, indicating SRV-2 RT recombinant enzyme. Purification of SRV-2 RT recombinant enzyme produced 312 μg mL-1 protein with 7.1 U μL-1 enzyme activities. Application of this recombinant enzyme in reverse transcription-PCR (RT-PCR) of β-globin and β-actin genes produced DNA fragments of 206 and 350 bp, indicating amplification of β-globin and β-actin genes, respectively. Therefore, the expressed SRV-2 RT enzyme was proven to be functional, although the activity was low.

Bat Coronavirus of Pteropus alecto from Gorontalo Province, Indonesia Wenty Dwi Febriani; Uus Saepuloh; Ellis Dwi Ayuningsih; R. Suryo Saputra; Azhari Purbatrapsila; Meis Jacinta Nangoy; Tiltje Andretha Ransaleh; Indyah Wahyuni; Safriyanto Dako; Rachmitasari Noviana; Diah Iskandriati; Ligaya ITA Tumbelaka; Joko Pamungkas
The International Journal of Tropical Veterinary and Biomedical Research Vol 3, No 2 (2018): Vol. 3 (2) November 2018
Publisher : The Faculty of Veterinary Medicine of Syiah Kuala University

Bats have been known as natural reservoirs for potential emerging infectious viruses, such as Lyssaviruses, Coronaviruses, Ebola viruses, Nipah virus, and many others. Because of their abudance in population, wide distribution and mobility, bats have a greater risk as source for zoonotic transmission than other animals. Despite the facts of their role as reservoirs for many pathogens, not until an epidemic of Severe Acute Respiratory Coronavirus (SARS-CoV) in 2003 and Middle-East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012, that people pay much attention about coronavirus in bats. SARS-like virus also found in bats with a higher prevalence rate. This study aims to detect the coronavirus of bats in Gorontalo province Indonesia, characterization at the molecular level of the coronavirus genome and determining the level of kinship (through trees filogenetic). This study was conducted as part of bigger PREDICT Indonesia project, in particular to examine coronavirus in bats from Gorontalo province, Indonesia. As many as 95 rectal swab samples collected from flying foxes (Pteropus alecto) were analyzed in the laboratory using Consensus Polymerase Chain Reaction (PCR) technique to amplify the target sequence from RNA-dependent RNA Polymerase (RdRp) gene with 434 basepair product, resulted 24 samples determined as presumptive positive. Eight out of 24 presumptive positive samples by PCR were analyzed further by nucleotide sequencing and confirmed coronavirus positive. Phylogenetic tree analyses to the eight coronavirus confirmed-sequences were constructed with MEGA-6.0 . The conclusion was 24 out of 95 samples suggested as presumptive positive to Bat CoV. Eight out of 24 samples were analyzed further by nucleotide sequencing and have similarities in the kinship. Three samples had the 98% nucleotide identity to BatCoV from Indonesia and five samples were 85-88% nucleotide identity to BatCoV from Thailand.

Primary Tupaia javanica Hepatocytes Cultures As In Vitro Replication System for Ape Hepatitis B Viruses MARYATI SURYA; DIAH ISKANDRIATI; SILMI MARIYA; UUS SAEPULOH; PERMANAWATI PERMANAWATI; DONDIN SAJUTHI; JOKO PAMUNGKAS
Microbiology Indonesia Vol. 10 No. 2 (2016): June 2016
Publisher : Indonesian Society for microbiology

Hepatitis B virus (HBV) is a DNA virus with liver as primary target organ.This virus caused chronic infection that can progress to cirrhosis, liver cancer and even death. In vitro model system of hepatocyte cultures is important and widely used to study a variety aspects of hepatitis B. Development of small animal Tupaia sp. for the in vitro model system is an alternative to the existinghepatocyte cultures. The specific purpose of the studyis to develop Tupaia javanica hepatocytes culture for HBV replication, and in a broader spectrum to answer the need for in vitro model of hepatocytes. Primary T. javanica hepatocytes (PTH) culture was successfully maintained for 14 days to reach 80% confluence, and infection of Javan gibbon HBV (GiHBV) and orangutan HBV (OuHBV) onto the culture on day 15 showed viral replication for up to eight days as measured by polymerase chain reaction (PCR). PCR quantification indicated that the highest copy number of DNA virus was detected onday two anddecreased until day 8 after infection. Cell receptor for HBV attachment, known as sodium taurocholate cotransporting polypeptide was expressed on the surface of PTH and shown as green luminenscent when observed by immunofluorescence assay. Sequence of partialS gene from the apes HBVs after the viruses have been infected to the PTH showed amino acid identity to their wildtype as high as 99.29% for GiHBV and 95.71% for OuHBV. This study suggested that the PTH can support the replication of GiHBV andÂ OuHBV.Â

Co-Authors Azhari Purbatrapsila Dondin Sajuthi Ellis Dwi Ayuningsih FUNGKEY HOETAMA Indyah Wahyuni MARYATI SURYA Mohamad Sadikin Nangoy, Meis Pamungkas, Joko PERMANAWATI PERMANAWATI R. Suryo Saputra Rachmitasari Noviana Safriyanto Dako SELA SEPTIMA MARIYA Silmi Mariya TARUNI SRI PRAWAST MIEN KAOMINI ANY ARYANI DEDY DURYADI SOLIHIN Tiltje Andretha Ransaleh UUS SAEPULOH Uus Saepuloh Wenty Dwi Febriani

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