3 410 Shanti Ratnakomala.cdr Screening of Actinomycetes Producing an ATPase Inhibitor of RNA Helicase from Soil and Leaf Litter Samples Japanese Encephalitis Virus SHANTI RATNAKOMALA , RONI RIDWAN , PUSPITA LISDIYANTI , ABINAWANTO , ANDI UTAMA 1 1 1 2 1 * AND 1 2 Research Center for Biotechnology LIPI, Jalan Raya Bogor Km 46, Cibinong 16911, Indonesia; Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, UI Campus, Depok 16242, Indonesia Japanese encephalitis virus Actinoplanes philippinensis Streptomyces chartreusis Japanese encephalitis virus, Japanese ensefalitis virus Actinoplanes philippinensis Streptomyces chartreusis Japanese encephalitis virus Actinomycetes are commercially important microorganisms for the production of antibiotics, enzymes, inhibitors of enzymes, and other bioactive secondary metabolites. Some 853 isolates of actinomycetes were isolated from soil and leaf litter samples in Kupang NTT and Enrekang, South Sulawesi. Those isolates were then tested for inhibition of ATPase activity of RNA helicase from (JEV), in order to identify a drug candidate for the treatment of JEV infection. Results revealed that 14 isolates have relatively high inhibition-activity on JEV ATPase activity of the JEV-RNA- helicase, which range from approximately 40.0-50.0% inhibition. The highest inhibition-activity was identified in 5-849 with 49. 9% of inhibition-activity and 5-095 with 49.2% of inhibition-activity. Key words: actinomycetes, JEV, RNA helicase, inhibitor Aktinomiset merupakan mikroorganisme yang penting secara komersial untuk produksi antibiotik, enzim, enzim inhibitor, dan metabolit sekunder bioaktif. Sejumlah 853 isolat aktinomiset telah diisolasi dari sampel tanah dan serasah di Kupang (Nusa Tenggara Timur) dan Enrekang (Sulawesi Selatan). Isolat diuji kemampuannya untuk menghambat aktivitas ATP-ase dari RNA helikase (JEV), dalam rangka mencari kandidat obat bagi pengobatan infeksi JEV. Hasil penelitian menunjukkan ada 14 isolat yang memiliki aktivitas inhibisi 40-50% terhadap aktivitas ATPase dari RNA helikase-JEV, di antaranya ialah 5-849 dan 5-095. Kata kunci: aktinomiset, virus, , JEV, RNA helikase, inhibitor Japanese encephalitis virus C u l e x t r i t a e n i o r y n c h u s flavivirus Flaviviridae (JEV) is one of the most prevalent causative agents of viral encephalitis with high morbidity and mortality. Approximately 50 000 human cases occur annually in Asia. In nature, JEV is transmitted between vertebrates by the mosquito , a n d c a u s e s i n f e c t approximately 10% of the susceptible population in Southeast Asian countries each year. In Indonesia, particularly, JEV is endemic in Kalimantan, Bali, Nusa Tenggara, Sulawesi, Maluku, Papua and Lombok (Spicer 1997). This virus is recognized by the World Health Organization as a major threat to public health because of the high incidence of clinical infections, and approximately thirty percent of which are fatal and half result in neuropsychiatric sequelae. JEV belongs to genus in the family whose members include several pathogens of humans and animals. Although vaccines have been developed since 1960, unfortunately no effective drug is clinically available so far. Several efforts have been performed to find a drug as a candidate for treatment of JEV infection, including finding inhibitors of enzymes which are essential for JEV replication such as , , protease, RNA helicase and RNA polymerase (Borowski . 2002). Some studies recently discovered drug candidates for , particularly (HCV) and JEV, through RNA helicase inhibitor screening. Hatsu (2002) noted that secondary metabolites from broth cultures of sp. could be act as inhibitor to RNA helicase of JEV In general, the drug candidates were chemical substances such as ribavirin-5-triphosphate (Borowski . 2001) and SCH16 a derivatives o thiosemicarbazone (MIBT) (Sebastian 2008) which were able to completely inhibit JEV and (WNV) replication, however the studies on the inhibition by natural products is still limited. Actinomycetes are commonly found in natural substrates such as soils and litters, and play a significant role in the degradation of naturally organic substances (Williams . 1984). Therefore, actinomycetes have the ability to produce large amounts of secondary metabolites such as antibiotics, enzymes, enzyme inhibitors, and other bioactive compounds. About 70% of presently commercial antibiotics were found in secondary metabolites produced from actinomycetes (Miyadoh 1993). et al flavivirus Hepatitis C virus et al Streptomyces et al , et al in vitro West Nile virus et al . . . f N-methylisatin-β- *Corresponding author, Phone: +62-21-8754587, Fax. +62-21-8754588, E-mail: shanti_ratna01@yahoo.com ISSN 1978-3477, eISSN 2087-8575 Vol 5, No 1, March 2011, p 15-20 I N D O N E S I A Available online at: http://www.permi.or.id/journal/index.php/mionline DOI: 10.5454/mi.5.1.3 Furthermore, Indonesian actinomycetes have not been explored yet for screening of useful bioactive compounds such as inhibitors of JEV replication. Therefore, a study to find a novel strain with a novel secondary metabolite useful for pharmaceutical and industrial applications is important. In this study, we isolated actinomycetes from soil and leaf litter samples and screened the actinomycetes for inhibitor of ATPase activity of JEV RNA helicase. The 853 isolated actinomycetes used for testing for inhibition-of ATPase activity of JEV RNA helicase were obtained from soil and leaf litter samples in Kupang, Nusa Tenggara Timur, and Enrekang South Sulawesi, Indonesia under collaborative research between Indonesian Institute of Sciences (LIPI), Indonesia and National Institute of Technology & Evaluation (NITE), Japan in 2005. The project have been done from 2004-2009. (Widyastuti and Ando 2009). In the project in 2005 SDS-yeast extract (SDS-YE) method (Hayakawa & Nonomura 1987), rehydration-centrifugation (RC) method (Hayakawa 2000), and oil separation system (OSS) method were used in this study as referenced in Widyastuti and Ando (2009). All the isolates have been identified at the genus level by sequencing of their 16S rDNA at the project in 2005 as stated in Widyastuti and Ando (2009). In this study, the compiling 16S rDNA data of the isolates based on their isolation sources and isolation methods were done. Yeast extract-starch agar (YS agar) (Nonomura and Ohara 1969) was used as growth medium and contained 2 g yeast extract, 10 g soluble starch, 15 g agar added to 1 L distilled water and adjust pH to 7.3. International project 2 (ISP 2) medium was used as production medium for screening the JEV ATPase inhibitor from actinomycetes, and contained 4 g yeast extract, 10 g malt extract, 4 g dextrose, 20 g agar added to 1 L distilled water and adjust pH to 7.3. The expression plasmids containing JEV NS3 genes were independently transformed into BL21 (DE3) pLysS cells (Stratagene, La Jolla, CA, USA). After the IPTG induction at 37 C for 3 h, the cells were harvested by centrifugation at 8000 rpm. The harvested cells were resuspended in buffer B (10 mM Tris HCl buffer (pH 8.5), 100 mM NaCl, 0.25% v/v Tween 20), and disrupted by sonication for 5 min on ice. The soluble fraction of the cell lysate was mixed with TALON metal affinity resin (Clontech, Palo Alto, MATERIALS AND METHODS Microorganisms. , Growth Medium and Production Medium. Expression and Purification of JEV NS3 Proteins et al. Streptomyces E. coli . o CA, USA). After gentle mixing for 1 h at 4 C, the resin was collected by a brief centrifugation and washed with buffer B. Resin-bound protein was eluted with 2 volumes of buffer B containing 400 mM imidazole. Eluted protein fractions were dialyzed against dialysis buffer (10 mM Tris HCl (pH 8.5), 100 mM NaCl, 10% v/v glycerol) at 4 C. Purified JEV NS3 proteins from the transformant were then used as substrate helicase enzyme forATPase assay (Utama 2000a) The amount of phosphate moiety released from ATP was measured. A 50 µL per well of reaction mixture containing 10 mM MOPS buffer (pH 6.5), 2 mM ATP, 1 mM MgCl , purified NS3 protein (0.8 pmol) and 5 µL per well of culture supernatant as inhibitor substances was incubated in a 96 well microtiter plate at room temperature for 30 min. The reaction was stopped by adding 100 µL per well of dye solution (water:0.081% malachite green:5.7% ammonium molybdate in 6 N HCl:2.3% polyvinyl alcohol = 2:2:1:1, v/v). After the addition of 25 µL per well of 30% sodium citrate, the absorbance at 620 nm with a reference wavelength at 492 nm was measured. Percentage of inhibition (%) was measured using the equation (A-I) /A x 100, which A was absorbance without added inhibitor substances, and “I” was absorbance with added inhibitor substances (Hatsu . 2002). In this assay the amount of free phosphate moiety released from ATP was measured via absorbance of RNA helicase enzyme. We have measured the change of absorbance with and without added inhibitor substances. If there was no inhibitory effect from the supernatant of actinomycetes, the JEV RNA helicase enzyme would continue to hydrolyze ATP to ADP and inorganic phosphate released from this reaction will be bounded with malachite green and ammonium molibdate made a coloured complex compound of phosphomolybdate-malachite green. The absorbance of this coloured complex compound were detected using spectrophotometer. Of the 853 isolates of actinomycetes used in this study, 529 isolates were defined as a group of (Family ), and 324 isolates as a group of non- or so called rare actinomycetes (other family in the order ) Further, from the group of non- , 381 have zoospore (motile actinomycetes) and 153 do not have zoospore (non- motile actinomycetes). Based on results in origin, 716 were isolated from soil and 137 were isolated from leaf litter samples. Some 488 isolates from the soil sources o o E. coli et al . et al Streptomyces Streptomycetaceae Streptomyces Actinomycetales Streptomyces Colorimetric ATPase Assay. RESULTS Actinomycetes Isolates. . - . 2 16 RATNAKOMALA ET AL. Microbiol Indones were identified as belonging to the family and 228 isolates were identified as group of non- . A total of 137 isolates obtained from leaf litter samples were divided into 41 isolates of the family and 96 isolates of the group of non- group (Table 1). This result revealed that the genus was the dominant actinomycetes in the soils used. Based on results of the isolation methods, 468 were isolated by SDS-YE method resulted in as the major genus, 286 were isolated by RC method resulted as the major genus, and 99 were isolated by oil separation method which resulted as the major genus. Twenty eight genera were found in Kupang, Nusa Tenggara Timur and 40 genera were found in Enrekang. Furthermore, 33 genera were found in soils samples and 12 genera were found in litter samples. The genera of were only found in and (Table 2) The genus , , and were abundant in soil samples and the genus , , and were abundant in litter samples. All actinomycetes isolates obtained from soil and leaf litter samples in Kupang and Enrekang were then screened for their ability to inhibit ATPase activity of JEV RNA helicase. Of the 853 actinomycete isolates, 14 isolates produced metabolites having inhibitory activity as high as 40-50%. Thirty-five isolates demonstrated inhibition to 30-40% and 37 isolates showed 20-30% inhibitory activity ( Fig 1). Of the 14 isolates actinomycetes which were found to have the highest potency as antiviral compound were presented in Table 3, the bioactive compounds produced from secondary metabolites were released in broth cultures. The highest inhibition-activity was identified in 5-849 with Streptomycetaceae Streptomyces Streptomycetaceae Streptomyces Streptomyces Streptomyces Actinoplanes Streptomyces Dietzia, Kocuria, Planomonospora, and Sphaerosporangium Kupang, and the genera of Actinokineospora, Cellulomonas, Dermatophilus, Geodermatophilus, Agromyces, Virgosporangium, Planotetraspora were only found in Enrekang . Streptomyces Actinoplanes Nonomuraea Actinoplanes Streptomyces Kinesoporia Actinoplanes philippinensis Screening of Actinomycetes which Have Inhibition-Activity on ATPase of JEV RNA Helicase. Volume 5, 2011 Microbiol Indones 17 Sampling source Sampling site Σ isolate Streptomyces Non-Streptomyces Motile Non-motile Litter Kupang 59 31 23 5 Enrekang 78 10 54 14 Soil Kupang 318 210 52 56 Enrekang 398 278 42 78 Table 1 Actinomycetes isolated from leaf litter and soil samples Compiling data from Final Report of Collaborative Research Project in 2005 between LIPI-NITE 1 (Widyastuti and Ando 2009). Table 2 Genera of actinomycetes found in and samplessoil leaf litter Soil Actinokineospora - 2 2 Actinoplanes 47 25 72 Catenuloplanes 3 7 10 Couchioplanes 1 2 3 Dactylosporangium 1 2 3 Planotetraspora - 4 4 Actinomadura 1 4 5 Agromyces - 1 1 Cellulomonas - 1 1 Cryptosporangium 7 11 18 Dermatophilus - 1 1 Dietzia 1 - 1 Geodermatophilus - 1 1 Isoptericola 1 2 3 Kocuria 1 - 1 Kribbella 3 13 16 Microbispora - 1 1 Micromonospora 7 5 12 Mycobacterium 7 6 13 Nocardia 2 10 12 Nocardioides 1 4 5 Nonomuraea 16 11 27 Planomonospora - 4 4 Planobispora 1 - 1 Promicromonospora 2 2 4 Pseudonocardia 3 1 4 Saccharothrix 1 1 2 Sphaerosporangium 1 - 1 Streptacidiphilus - 2 2 Streptomyces 210 272 482 Streptosporangium 1 1 2 Virgosporangium - 1 1 Verrucosispora - 1 1 Leaf litter Actinoplanes 10 45 55 Catenuloplanes - 1 1 Couchioplanes - 2 2 Dactylosporangium 1 - 1 Kineosporia 12 6 18 Amycolatopsis - 1 1 Cryptosporangium - 11 11 Krasilnikovia 1 1 2 Micromonospora 2 1 3 Nocardia - 1 1 Promicromonospora - 1 1 Streptomyces 31 10 41 Total 853 Data source: Compiling data from Final Report of Collaborative Research Project in 2005 between LIPI-NITE 1 (Widyastuti and Ando 2009). 49. 9% of inhibition-activity and 5-095 with 49.2% of inhibition-activity. Actinomycetes are prokaryotic microorganisms belonging to a group of Gram positive bacteria that have high G+C contents, are saprophytic, produce large amount of sporangia and stable mycelium. Actinomycetes are widely distributed in nature and in man-made environments, and play an important role in the degradation of organic matter. They are also well known as a rich source of antibiotics and bioactive Streptomyces chartreusis. DISCUSSION 18 RATNAKOMALA ET AL. Microbiol Indones molecules, and are of considerable importance in industry (Seong 2001). Many are well known for their economic importance as producers of biologically active substances, such as antibiotics, vitamins and enzymes (Basilio 2003). This report revealed as stated also in Widyastuti and Ando (2009), that species diversity in the soil samples (33 genera) are higher when compared with the litter samples (12 genera), this is in line with the review of Hayakawa (2008) that states soil is the natural habitat of actinomycetes. The genus was widely distributed in Kupang, Nusa Tenggara Timur, and Enrekang both in sampling sources. As noted by Hayakawa (2000), rare actinomycetes were divided in two groups according to the type of zoospora produced, non-motile actinomycetes and motile actinomycetes. Non-motile actinomycetes formed non-flagellated zoospores, such as , etc. and motile actinomycetes formed flagellated zoospores. Examples of motile zoospora are and Actinomycetes isolated from et al et al Streptomyces et al. Nocardia Micromonospora, Actinokineospora, Actinoplanes, Catenuloplanes, Couchioplanes, Dactilosporangium, Planotetraspora. . , . Table 3 Actinomycetes have highest inhibitory activity to ATPase activity of JEV RNA helicase enzyme isolated from soils and leaf litter samples in Kupang and Enrekang Isolate Inhibition (%) Name of isolate 5-087 45.1 Streptomyces floridae 5-095 49.2 S. chartreusis 5-096 43.5 S. marokkonensis 5-119 47.0 Streptomyces Smarlab 330204 5-124 46.0 S. violens 5-217 40.9 S. albus 5-218 44.3 S. ginsengisoli 5-224 41.0 S. albus 5-264 43.6 S. cyanoalbus 5-304 44.2 S. durhamensis 5-320 41.4 S. pulveraceus 5-800 42.3 S. badius 5-849 49.9 Actinoplanes philippinensis 5-1036 40.4 Kribbella flavida Inhibition (%) Number of Isolates 0 594 0.01-10 96 10.01-20 77 20.01-30 37 30.01-40 35 40.01-50 14 Table 4 Actinomycetes having inhibitory activity to ATPase activity of JEV RNA helicase enzyme isolated from soils and leaf litter samples at Kupang and Enrekang Enrekang soils and leaf litter samples were abundant with rare actinomycetes from the genus . Based on the isolation method as stated in Widyastuti and Ando (2009) SDS YE methods were useful for isolation of the genus On the other hand RC methods gave an abundance of the genus Further, in this research it can be seen that the actinomycetes originating from litter sample was dominated by motile actinomycetes (the genus ), while from the soil was dominated by non-motile actinomycetes (the genus ) (Table 1). The RC method is a simple enrichment method incorporating differential centrifugation for the isolation of motile actinomycetes (Hayakawa 2000). The phosphate buffer-soil extract solution liberates motile zoospores and centrifugation eliminates and other non- motile actinomycetes from the supernatant, thereby facilitating selective growth of motile actinomycetes on the isolation plates subsequent to inoculation (Hayakawa 2008). The OSS method is a selective isolation methods expected to favour actinomycetes from the genus , but unfortunately in this study this genus is not obtained. Using OSS methods the genus This may be because the isolates were putatively closely related with the genus on the basis of their mycolic acid profiles. Our analysis of screening for inhibitory activities to JEV RNA helicase enzymes showed a high proportion of inhibitory activity was produced by species. Seeking an inhibitor to JEV RNA helicase was performed through RNA- stimulated-ATPase-activity (ATP hydrolysis). Inhibition of ATP hydrolysis, was derived from an indirect inhibition of RNA helicase-mediated hydrolytic processing so that the energy performed from hydrolysis ATP was not available to unwind dsRNA JEV. Colorimetric ATPase assay was chosen for detection of hydrolyzed ATP, because that was the easy and safe methods, without using radioactive compound which relatively unstable and caused radiation contamination (Utama 2000b; Boguszewska- Chachulska 2004). Robarts 2004 and Welbourn 2005 used colorimetric ATPase assay to detect ATP hydrolysis from RNA helicase JEV. Screening inhibitors of JEV RNA helicase from actinomycetes showed a varied percentage of inhi bition among the isolates, between 0% and 49.9% (Fig 1). The highest percentages of inhibition were 49.9% performed by isolate 5-849 identified as and 49.2% inhibition-activity obtained Actinoplanes Streptomyces Actinoplanes Actinoplanes Streptomyces et al Streptomyces Rhodococcus Mycobacterium was found Mycobacterium Rhodococcus Streptomyces et al et al et al et al - Actinoplanes philippinensis , . , . . , . . . . . Volume 5, 2011 Microbiol Indones 19 from isolate 5-095 identified as (Table 3). isolated from leaf litter samples at Enrekang was a rare actinomycetes. The role of rare actinomycetes as bioactive molecule sources became apparent as these organisms provided about 25% of the antibiotics of actinomycete origin reported during 1975 to 1980. Rare actinomycetes have usually been regarded as strains of actinomycetes whose isolation frequency by conventional methods is much lower than that of strains. Hatsu . (2002) noted that secondary metabolites from broth cultures of could act as inhibitor to RNA helicase enzyme of JEV. Helicase enzyme was a great potential target for the discovery new antiviral compounds because this enzyme is essential enzyme for replication of the virus. Helicase enzyme has a function as not only for helicase activity, but also RNA binding activity and RNA-stimulated- ATPase activity, both of these activities influence helicase activity. This function is interrupted or at least depressed by the existence of an ATPase inhibitor. Therefore this make the enzyme an attractive target for development new antiviral drugs, because an inhibitor of helicase enzyme could be due to the discovery of an inhibitor of RNA binding activity or an ATPase inhibitor. In general, the active isolates showed an inhibition of ATPase activity of JEV RNA helicase enzymes were mainly produced from genus , although some of the non isolates also had inhibitory activity. Whether the activities being detected in these cases were due to single inhibitor acting on multiple microbial species or were mixtures of compounds with different specificities is unclear without chemical fractionation of the active extracts. A diverse percentage of inhibition existed among the isolates, suggesting a varied ability of microorganisms to produce inhibitor compounds. are fast growing actinomycetes, will reach their stationary phase earlier at which stage the secondary metabolites are produced. Differences in incubation time to reach the stationary phase between and rare actinomycetes could act as a factor causing diverse inhibitory activity. Of some of the antiviral candidates discovered, the drug candidates were proven and , such as BAY 57-1293 a compound which suppresses (HSV) infection in the monkey (Betz . 2000). Some studies recently noted the discovery of drug candidates through helicase-inhibitor- screening, particularly for HCV and JEV. In general, the drug candidates were chemically substances such Streptomyces chartreusis Actinoplanes philippinensis Streptomycetes et al Streptomyces Streptomyces Streptomycetes Streptomyces Streptomyces in vitro in vivo Herpes simplex virus et al flavivirus , , - , , as ribavirin -5”-triphosphate (Borowski 2001), while the studies on naturally occuring inhibitors are rare or yet to be done. In the present study, we report on biologically active compound from actinomycetes which potentially have antiviral activity making them promising new drug candidates. The existence of candidate strains with high inhibition of ATPase activity of JEV RNA helicase enzymes, suggests that Indonesian actinomycetes have not been thoroughly investigated and therefore have potential as a source of novel bioactive compounds. These results not only suggested the usefulness of Indonesian actinomycetes as screening sources of natural product based drug discovery programs, but also may be useful as basic data such as distribution studies. et al. REFERENCES Basilio A, Gonza´ lez I, Vicente MF, Gorrochategui J, Cabello A, Gonza´lez A, Genilloud O. 2003. Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity. J Appl Microbiol 95(4):814 823 Betz UAK, Fischer R, Kleyman G, Hendrix M, Rubsamen-Waigmann H 2000. Potent antiviral activity of the herpes simplex virus primase-helicase inhibitor BAY 57-1293. Antimicrob Agents Chemother. 46(6):1766-1772. Boguszewska-Chachulska AM, Krawczyk M, Stankiewicz A, Gozdek A, Haenni AL, Strokovskaya L. 2004. Direct fluorometric measurement of hepatitis C virus helicase activity. Fed Eur Biochem Soc Lett. 567(2- 3):253-258. Borowski P, Lang M, Niebuhr A, Haag A, Schmitz H, Schulze zur Wiesch J, Choe J, Siwecka MA, Kulikowski T. 2001. Inhibition of the helicase activity of Acta Biochim Polon. 48(3):739-744. Borowski P, Lang M, Haag A, Schmitz H, Choe J, Chen HM, Ramachandra SH. 2002. Characterization of imidazo[4,5-d]pyridazine nucleosides as modulators of unwinding reaction mediated by West Nile virus nucleoside triphosphatase/helicase: evidence for activity on the level of substrate and/or enzyme. Antimicrob Agents Chemother. 46(5):1231- 39. Hatsu M, Tanaka M, Utama A, Shimizu H, Takamizawa K. 2002. A NS3 inhibitor produced by a sp. Actinomycetologica 16(1):6-8. Hayakawa M, Nonomura H. 1987. Efficacy of artificial humic acid as a selective nutrient in HV agar used for the isolation of soil actinomycetes. J Ferment Technol. 65(6):609-616. Hayakawa M, Otoguro M, Takeuchi T, Yamazaki T, Iimura Y. 2000. Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter. Antonie Leeuwenhoek. 78(2):171-185. Hayakawa M. 2008. Studies on the isolation and distribution of rare actinomycetes in soil. Actinomycetologica 22(1):12 19. Miyadoh S. 1993. Research on antibiotic screening in Japan over the last decade. A producing microorganism approach. Actinomycetologica. 9(2):100-106. Nonomura H, Ohara Y. 1969. Distribution of actinomycetes in soil. VII. A culture method effective for both of preferential isolatin and enumeration of and strains in soil (Part II). J Ferment Technol. 47:701-709. . - . - . . in vitro Japanese encephalitis virus Streptomyces Microbispora Streptosporangium HCV NTPase/helicase by 1-β-D-ribofuranosyl-1,2,4- triazole-3-carboxamide-5'-triphosphate (ribavirin-TP). . doi:10.1046/j.1365-2672.2003.02049.x doi:10.1128/AAC.46.6.1766-1772.2002. doi:10.1128/AAC.46.5.1231-1239.2002. doi:10.1128/AAC.46.5.1231-1239.2002. doi:10.3209/saj.16_6 doi:10.1016/0385- 6380(87)90001-X. doi:10.1023/A:1026579426265. doi:10.3209/saj.SAJ220103 doi:10.3209/saj.7_100. . . . Robarts MY, Blouin AG, Bleker S, Kleinschmidt JA, Aggarwal AK, Escalante CR, Linden RM. 2004. Residues within the B' motif is critical for DNA binding by the superfamily 3 helicase Rep40 of adenoassociated virus type 2. J Biol Chem. 279(48):50472-50481. Sebastian L, Desai A, Shampur MN, Perumal Y, Sriram D, Vasanthapuram R. 2008. N-methylisatin-beta-thiosemicarbazone derivative (SCH 16) is an inhibitor of Japanese encephalitis virus infection and . Virol J 5(1)64:1-12 Seong CN, Choi JH, Baik KS. 2001. An improved selective isolation of rare actinomycetes from forest soil. J Microbiol. 39(1):17-23. Spicer PE. 1997. Japanese encephalitis in Western Irian Jaya. J Travel Med. 4:146-147. Utama A, Shimizu H, Hasebe F, Morita K, Igarashi A, Shoji I, Matsuura Y, Hatsu M, Takamizawa K, Hagiwara A, Miyamura T. 2000a. Role of the DExH motif of the and NS3 proteins in the ATPase and RNA helicase activities. Virology. 273(2):316-324. in vitro in vivo . Japanese encephalitis virus Hepatitis C virus . doi:10.1074/jbc.M403900200. doi:10.1186/1743-422X-5-64 doi:10.1111/j.1708-8305.1997.tb00803.x. doi:10.1006/viro.2000.0417. . Utama A, Shimizu H, Morikawa S, Hasebe F, Morita K, Igarashi A, Hatsu M, Takamizawa K, Miyamura T. 2000b. Identification and characterization of the RNA helicase activity of NS3 protein. FEBS Lett. 465(1):74- 8. Welbourn S, Green R, Gamache I, Dandache S, Lohmann V, Bartenschlager R, Meerovitch K, Pause R. 2005. NS2/3 processing is required for NS3 stability and viral RNA replication. J Biol Chem. 280(33):29604-29611. Widyastuti Y Ando K 2009. Taxonomic and ecological studies of actinomycetes in Indonesia. [Final Report Joint Research Project between Indonesian Institute of Sciences (LIPI), representing the Government Research Centers (GRC) of the Republic of Indonesia and National Institute of Technology and Evaluation (NITE) of Japan]. Jakarta (ID): LIPI Pr. p 709-711. Williams ST, Lanning S, Wellington EMH. 1984. Ecology of actinomycetes. In: Goodfellow M, Mordarski M, Williams ST, editors. The biology of the actinomycetes. London (GB):Academic Pr. p 481-528. Japanese encephalitis virus 7 Hepatitis C virus , . doi:10.1016/S0014- 5793(99)01705-6. doi:10.1074/jbc.M505019200. 20 RATNAKOMALA ET AL. Microbiol Indones