RESUMO
Although prevention of shrimp mortality from yellow head virus (YHV) infection via dsRNA injection has been well demonstrated for many years, it has not yet been applied in a farm culture because of its impracticality. Hence, oral administration of dsRNA becomes an alternative and desirable approach. This study is the first to demonstrate that oral feeding of Escherichia coli expressing shrimp Rab7 gene (dsRab7) or YHV protease gene (dsYHV) could inhibit YHV replication and lowered shrimp mortality. E. coli HT115 expressing dsRab7 or dsYHV or a combination of these dsRNAs were embedded in agar and used to feed vannamei shrimp at early juvenile stage before YHV challenge. After 4 days of continuous feeding of dsRNAs, strong inhibitory effect on shrimp mortality was observed in which dsRab7 gave the highest effect (70% reduction from the control) whereas dsYHV showed a 40% reduction. Our results reveal the potential of anti-YHV strategy via orally delivered dsRNA for application in the shrimp farm industry.
Assuntos
Aquicultura , Penaeidae/virologia , RNA de Cadeia Dupla/farmacologia , RNA Viral/antagonistas & inibidores , Roniviridae/fisiologia , Proteínas Virais/antagonistas & inibidores , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/genética , Peptídeo Hidrolases/genética , RNA Viral/metabolismo , Roniviridae/enzimologia , Proteínas Virais/genética , Replicação Viral , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Yellow head virus (YHV) is one of the causative agents of shrimp viral disease. The prevention of YHV infection in shrimp has been developed by various methods, but it is still insufficient to protect the mass mortality in shrimp. New approaches for the antiviral drug development for viral infection have been focused on the inhibition of several potent viral enzymes, and thus the YHV protease is one of the interesting targets for developing antiviral drugs according to the pivotal roles of the enzyme in an early stage of viral propagation. In this study, a theoretical modeling of the YHV protease was constructed based on the folds of several known crystal structures of other viral proteases, and was subsequently used as a target for virtual screening-molecular docking against approximately 1364 NCI structurally diversity compounds. A complex between the protease and the hit compounds was investigated for intermolecular interactions by molecular dynamics simulations. Five best predicted compounds (NSC122819, NSC345647, NSC319990, NSC50650, and NSC5069) were tested against bacterial expressed YHV. The NSC122819 showed the best inhibitory characteristic among the candidates, while others showed more than 50 % of inhibition in the assay condition. These compounds could potentially be inhibitors for curing YHV infection.
Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Modelos Moleculares , Peptídeo Hidrolases/química , Roniviridae/enzimologia , Proteínas Virais/química , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Sítios de Ligação/genética , Inibidores Enzimáticos/farmacologia , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Molecular , Penaeidae/virologia , Peptídeo Hidrolases/genética , Ligação Proteica , Estrutura Terciária de Proteína , Roniviridae/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genéticaRESUMO
Large-scale production of long dsRNA is needed if antiviral applications of RNAi are to succeed in shrimp farm operations. A novel hairpin-RNA expression vector was developed based on the RNA-dependent RNA polymerase (RdRp) gene of yellow head virus (YHV), the cause of a lethal shrimp disease. Using transformed RNase-deficient Escherichia coli, large amounts (approximately 5 mg dsRNA from 130 ml bacterial culture) of long dsRNA (>300 nt) were produced. Large-scale in vivo dsRNA production was approximately one-fourth the cost of production of a commercial in vitro transcription kit. The hairpin-RNA consisted of the target RdRp sequence ("forward") and a 100-base shortened version of its inverted repeat ("reverse") to introduce a loop and bypass the difficulty of including a small "loop" connector into the "carrier" vector. A test group of whiteleg shrimp Penaeus (Litopenaeus) vannamei (approximately 10-15 g) was injected with 25 microg of this dsRNA 1-day prior to YHV challenge while control groups were injected with NaCl solution or similarly prepared dsGFP-RNA. The group injected with YHV-specific dsRNA did not develop yellow head disease during 14-day of observation after YHV challenge, whereas the control groups injected with NaCl and dsGFP-RNA developed gross signs of yellow head disease and died within 7-10 days after challenge. Quantitative RT-PCR and immunohistochemistry revealed that both viral mRNA and viral proteins were suppressed in the protected shrimp.
