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BackgroundRNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called “multi-WSSV dsRNA.” A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners.ResultsFor a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge.ConclusionThe present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

Tilapia lake virus (TiLV) is an emerging virus that is rapidly spreading across the world.Over the past 6 years (2014-2020), TiLV outbreaks had been reported in at least 16 countries, spanning three continents, including Asia, Africa, and America. Despite its enormous economic impact, its origin, evolution and epidemiology are still largely poorly characterized. Here, we report eight TiLV whole-genome sequences from Thailand sampled between 2014 and 2019. Together with publicly available sequences from various regions of the world, we estimated the origin of TiLV to be between 2003 and 2009, 5-10 years before the first report of the virus in Israel in 2014. Our analyses consistently showed that TiLV started to spread in 2000s, and reached its peak in 2014-2016, matching well with the timing of its first report. From 2016 onwards, the global TiLV population declined steadily. This could be a result of herd immunity building up in the fish population, and/or a reflection of a better awareness of the virus coupled with a better and more cautious protocol of Tilapia importation. Despite the fact that we included all publicly available sequences, our analyses revealed long unsampled histories of TiLVs in many countries, especially towards its basal diversification. This result highlights the lack and the need for systematic surveillance of TiLV in fish.