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.

Application of dsRNA VP15-WSSV by Oral Vaccination to Increase Survival Rate and Response Immunes of Tiger Shrimp Penaeus monodon

“…A study of the dsRNA VP-24 vaccine at a dose of 0.2 μg showed a significant increase in survival rate in tiger shrimp by 65% compared to control (10%) (Mulyaningrum et al, 2018). Some studies also demonstrated that the intramuscular injection and oral delivery of dsRNA can provide better protection than control groups of tiger shrimp (Puneeth et al, 2017) and our studies indicated that the application of dsRNA by injection showed a better survival response than oral administration (Mulyaningrum et al, 2018;Parenrengi et al, 2018;2019). The shrimp survival by injected with dsRNA of VP24, VP19 and VP15 (Sarathi et al, 2010) and VP28 (Nilsen et al, 2017;Sarathi et al, 2008;Solis-Lucero et al, 2016) was significantly higher than that of control groups.…”

“…In contrast, Parenrengi et al (2019) reported that dsRNA vaccine had a significant effect on the proPO activity of tiger shrimp, where the highest proPO activity was obtained in the in-vivo VP15 dsRNA and subsequently invitro and the lowest in control (without dsRNA). This study also revealed that proPO activity increased the longer test animals were exposed to W SSV.…”

“…The successful production by invitro using MEGAscript RNAi kit and in-vivo (Mulyaningrum et al, 2018;Parenrengi et al, 2018) has been proven led to apply in aquaculture for preventing the WSD. Application of dsRNA produced by in-vivo showed better survival and immune response than control shrimp for both the injection and the immersion method (Parenrengi et al, 2018(Parenrengi et al, , 2019. The vaccination by oral administration is expected to be an efficient way in mass application.…”

Application of dsRNA VP24 vaccine by oral administration at different larval stages of Tiger Shrimp Penaeus monodon

“…As a preliminary step, isolation and characterization of the coding genes VP-24 and VP-15 [15] and [16]has been carried out as well as their application in the form of recombinant DNA dsRNA, which has been constructed and produced in-vitro using mega script RNAi kits [17,18]. The optimal injection dose can be obtained in fighting white spot disease in tiger shrimp [19]. In-vivo production of dsRNA is carried out by cloning of the VP-WSSV gene in the RNAi vector and transformation in the competent of E. coli bacteria that can produce plasmids.…”

Increased Resistance of Penaeus Monodon to Whispovirus Causes White Spot Syndrome Through Antivirus Gene Transfer

“…That issue made (Luo et al, 2003) conducted the first study with the transfer of the antiviral gene PmAV and the results showed that the transfer of antiviral genes could reduce the attack of WSSV pathogens on tiger shrimp. Parenrengi et al, (2019)…”