Journal of Biotechnology 228 (2016) 95–102 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei Poohrawind Sanitt a , Nuttapon Apiratikul b , Nattisa Niyomtham c , Boon-ek Yingyongnarongkul c , Wanchai Assavalapsakul d , Sakol Panyim a,e , Apinunt Udomkit a,∗ a Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand b Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110, Thailand c Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok, 10240, Thailand d Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand e Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand a r t i c l e i n f o Article history: Received 3 February 2016 Received in revised form 13 April 2016 Accepted 28 April 2016 Available online 29 April 2016 Keywords: RNA interference Shrimp Cationic liposome Yellow head virus Double-stranded RNA delivery a b s t r a c t Protection of shrimp from yellow head virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA deliv- ery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05 g dsYHV/g shrimp could give compara- ble level of YHV protection to the injection with 1.25 g naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25 g dsRNA/g shrimp showed only 50% mortality at 60 days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encap- sulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4 days in the total of 960 g dsRab7/g shrimp could enhance YHV protection efficiency com- pared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Yellow head virus (YHV) is a rod shape, positive-sense, single- stranded RNA virus with the genome size of 26,622 nucleotides (Sittidilokratna et al., 2008). The virus was found in Thailand in 1992 and is named for the yellowish cephalothorax and very sallow overall coloration (Flegel, 1997). The virus is a virulent pathogen capable of infecting several penaeid shrimps resulting in huge economic loss in shrimp farm industry (Flegel, 1997; Senapin et al., 2010). Upon YHV infection, severe damage in shrimp lym- phoid organ, gill, connective tissues, hemocytes and hematopoietic organ was observed (Wang et al., 1996). YHV binds to a specific ∗ Corresponding author. E-mail address: apinunt.udo@mahidol.ac.th (A. Udomkit). receptor (YRP65) at the cell membrane of primary lymphoid cell, which was thought to be the primary target of the virus (Assavalapsakul et al., 2006). YHV was shown to internalize into a target cell via the clathrin mediated endocytosis, in which at least clathrin heavy chain and clathrin coat assembly protein 17 were demonstrated to be involved (Jatuyosporn et al., 2014; Posiri et al., 2015). The binding of the virus to its specific receptor YRP65 leads to endocytosis via the assembly of clathrin and the forma- tion of a coated pit on the plasma membrane. The coated pit later invaginates and pinches off to form an intracellular clathrin-coated vesicle. Depolymerization of clathrin-coated vesicles results in the formation of early endosome and subsequently late endosome, which is then transported to lysosome. Viruses somehow escaped from the late endosome prior to acidification in the lysosome to cytosol by either membrane fusion or membrane disruption mech- anisms (Mousavi et al., 2004). The endosomal trafficking of viruses http://dx.doi.org/10.1016/j.jbiotec.2016.04.049 0168-1656/© 2016 Elsevier B.V. All rights reserved.