PENGARUH APLIKASI dsRNA VP-15 IN VITRO DAN IN VIVO TERHADAP SINTASAN DAN RESPONS IMUN UDANG WINDU Penaeus monodon

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.