Andi Tenriulo
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PENGARUH APLIKASI dsRNA VP-15 IN VITRO DAN IN VIVO TERHADAP SINTASAN DAN RESPONS IMUN UDANG WINDU Penaeus monodon Andi Parenrengi; Andi Tenriulo; Sri Redjeki Hesti Mulyaningrum; Samuel Lante; Agus Nawang
Jurnal Riset Akuakultur Vol 14, No 4 (2019): (Desember, 2019)
Publisher : Pusat Riset Perikanan, Badan Riset dan Sumber Daya Manusia Kelautan dan Perikanan

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (277.291 KB) | DOI: 10.15578/jra.14.4.2019.213-223

Abstract

Teknologi RNA interference (RNAi) merupakan salah satu pendekatan yang digunakan untuk meningkatkan resistensi udang windu terhadap infeksi patogen termasuk WSSV. Pengembangan teknologi RNAi melalui aplikasi untai ganda RNA (dsRNA) yang berasal dari gen pengkode viral protein (VP) dari WSSV telah mulai dikembangkan pada udang. Penelitian ini bertujuan untuk mengkaji sintasan dan respons imun udang windu yang diberi VP-15 pasca uji tantang dengan WSSV. Udang windu (panjang 15,21 ± 1,19 cm dan bobot 32,5 ± 1,83 g) diinjeksi dengan 0,2 µg/ekor dsRNA in vitro (A), dsRNA in vivo (B), dan larutan garam/kontrol (C). Setelah tiga hari vaksinasi, udang windu ditantang dengan WSSV dengan dosis 50 µL/ekor. Pengamatan sintasan dilakukan setiap hari, sedangkan respons imun (THC dan aktivitas proPO) dilakukan pada awal dan hari ke-1, ke-3, dan ke-5 pasca uji tantang, serta analisis ekspresi gen antivirus dan histopatologi hepatopankreas dilakukan pada akhir penelitian. Hasil penelitian menunjukkan bahwa aplikasi dsRNA berpengaruh nyata (P<0,05) terhadap sintasan, THC, dan proPO. Sintasan udang windu yang diberi dsRNA VP-15 in vitro dan in vivo memberikan sintasan yang lebih tinggi 75% dibandingkan dengan kontrol. Nilai proPO tertinggi didapatkan pada dsRNA in vivo (0,138); kemudian dsRNA in vitro (0,093); dan terendah kontrol (0,061); sedangkan THC tertinggi (5.704 x 104 sel/mL) pada dsRNA in vivo, kemudian dsRNA in vitro (3.516 x 104 sel/mL) dan terendah pada perlakuan kontrol (3.322 x 104 sel/mL). Ekspresi gen antivirus semakin meningkat dengan semakin lamanya udang windu terpapar dengan WSSV. Jaringan hepatopankreas udang windu pada perlakuan kontrol (tanpa dsRNA) menunjukkan adanya kerusakan sel akibat infeksi virus.RNA interference (RNAi) technology is one of the approaches used to improve tiger shrimp Penaeus monodon resistance against WSSV infection. The development of RNAi technology through double-stranded RNA (dsRNA) isolated from gene encoding viral protein (VP) of WSSV has been applied to shrimp. This study was aimed to assess the survival rate and immune response of injected-VP-15 WSSV tiger shrimp after a challenge with WSSV. The tiger shrimp (15.21 ± 1.19 cm in length and 32.5 ± 1.83 g in weight) were injected with 0.02 µg/shrimp of in vitro dsRNA (A), in vivo dsRNA (b) and saline solution (C). After three days of vaccination, the tiger shrimp were challenged with WSSV using a dosage of 50 µL/shrimp. The survival rate was observed daily. Analyses of immune responses (hemocyte total and PO activity) were performed in several stages: before the challenge test and day-1, day-3, and day-5 post-challenge test. The expression of the antivirus gene and hepatopancreas histophatology were was observed at the end of the experiment. The results showed that the application of dsRNA significantly influenced the shrimp survival rate, THC, and proPO. Tiger shrimp injected with dsRNA VP-15 of in vitro and in vivo exhibited a higher 75% survival rate than the control (P<0.05). The highest proPO activity (0.138) was obtained at dsRNA in vivo, followed by dsRNA in vitro (0.093) and the lowest (0.061) in the control. The highest THC (5,704 x 104 cell/mL) was in vivo dsRNA, then in vitro dsRNA (3,516 x 104 cell/mL), and the lowest in the control (3,322 x 104 cell/mL). The longer the exposure with WSSV, the higher the antivirus gene expression. Histopathology analysis showed some damages to the hepatopancreas cells in the control shrimp (without dsRNA) caused by the virus infection.
PENGKLONAN GEN PENYANDI VIRAL PROTEIN 15 (VP-15) WSSV DAN APLIKASINYA SEBAGAI VAKSIN REKOMBINAN PADA UDANG WINDU Andi Parenrengi; Sri Redjeki Hesti Mulyaningrum; Andi Tenriulo; Agus Nawang
Jurnal Riset Akuakultur Vol 13, No 1 (2018): (Maret 2018)
Publisher : Pusat Riset Perikanan, Badan Riset dan Sumber Daya Manusia Kelautan dan Perikanan

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (368.272 KB) | DOI: 10.15578/jra.13.1.2018.57-65

Abstract

Infeksi white spot syndrome virus (WSSV) dapat menyebabkan kematian massal pada budidaya udang windu Penaeus monodon di Indonesia. Infeksi yang terjadi secara sistematis tersebut disebabkan oleh peran gen nucleocapsid viral protein (VP-15). Upaya pengembangan gen VP-15 WSSV untuk menginduksi respons imun dan menetralisasi terhadap infeksi WSSV pada udang windu perlu dilakukan. Penelitian ini bertujuan untuk mengisolasi dan merekombinasikan gen penyandi VP-15 WSSV sebagai vaksin dsRNA, serta menganalisis aplikasinya pada udang windu. Gen VP-15 diisolasi dari udang windu yang terinfeksi WSSV, dikloning ke dalam suatu vektor dan ditransformasikan ke sel kompeten (bakteri Escheria coli DH5a). Plasmid diisolasi untuk mengonfirmasi insert region gen VP-15 melalui sekuensing nukleotida. Pembuatan vaksin rekombinan dilakukan secara in-vitro menggunakan kit MEGAscript RNAi dan diaplikasikan ke udang windu melalui metode injeksi dengan dosis tunggal 0,2 µg dan kontrol (tanpa injeksi vaksin). Hewan uji yang digunakan berukuran panjang 14,75±3,17 g dan bobot 11,64±0,76 cm; serta dipelihara pada wadah bak fiber volume 250 L dengan kepadatan 10 ekor/bak. Hasil penelitian menunjukkan bahwa gen penyandi VP-15 telah diisolasi dari udang windu dan vaksin rekombinan telah dihasilkan secara in-vitro. Analisis sekuens nukleotida memperlihatkan bahwa sisipan gen DNA VP-15 sebesar 253 bp dan menunjukkan kemiripan yang tinggi (99%) pada GenBank. Penggunaan vaksin rekombinan dsRNA dengan dosis 0,2 µg memperlihatkan sintasan udang windu yang dapat mencapai 40,0% dibandingkan dengan kontrol hanya 3,3% (peningkatan 36,7%). Gambaran histopatologi pada jaringan hepatopankreas udang windu pada perlakuan kontrol menunjukkan adanya kerusakan inti sel, akibat infeksi WSSV. Gene VP-15 berpotensi sebagai bahan vaksin rekombinan dsRNA dalam mencegah infeksi WSSV.Infection of white spot syndrome virus (WSSV) causes bulk mortalities of tiger shrimp Penaeus monodon cultured in Indonesia. The nucleocapsid viral protein-15 (VP-15) is strongly suspected to be responsible for the systemic infection of WSSV. The development of VP-15 WSSV gene for inducing the immune response to and neutralize WSSV infection of tiger shrimp is vitally needed. The aim of this study was to isolate and clone the gene encoding VP-15 WSSV as dsRNA vaccine and assess the vaccine application to tiger shrimp. VP-15 gene was isolated from the genomic DNA of infected tiger shrimps, cloned into the vector, and transformed into competent cells (Escheria coli DH5a). The plasmid was isolated to confirm the insert region gene of VP-15 by the nucleotide sequence. Production of dsRNA vaccine was performed by in-vitro using MEGAscript RNAi kit and applied to tiger shrimp through muscular injection at a single dosage of 0.2 µg and without dsRNA as a control treatment. The average size of tiger shrimps used was 14.75±3.17 g in weight and 11.64±0.76 cm in length and stocked in 250 L fiber tank at 10 ind./tank. The results of the study showed the VP-15 gene was successfully isolated from the tiger shrimps and the recombinant vaccine was produced by in-vitro. The analysis of nucleotide sequence showed that the inserted DNA was 253 bp and showed a high similarity (99%) with VP-15 gene deposited in the GenBank. The application of dsRNA vaccine showed that the dosage of 0.2 ¼g resulted in the survival rate of 40.0% compared with without dsRNA (control) of 3.3% (36.7% increment). Hepatopancreas histology indicated obvious damages to cell nucleus in the un-vaccinated tiger shrimp caused by the virus infection. We suggest that the VP-15 gene is a very promising dsRNA recombinant vaccine against WSSV infection.