Background Viruses cause significant economic losses to shrimp aquaculture worldwide. In severe cases, they can lead to 100% mortality within a matter of days, hence the aquaculture industry requires antiviral strategies to minimize economic impacts. Currently, a double-stranded RNA (dsRNA)-based platform has been proven effective at a laboratory scale. The bottleneck for its industrialization is the lack of low-cost, efficient and practical delivery approaches. In an effort to bridge the gap between laboratory and farm applications, virus-like particles (VLP) have been used as nanocarriers of dsRNA. However, the implementation of this approach still suffers from high costs and a lengthy procedure, co-expression of subunits of VLP or capsid proteins (CPs) and dsRNA can be the solution for the problem. CP and dsRNA are traditionally expressed in two different E. coli hosts: protease-deficient and RNase III-deficient strains. To condense the manufacturing of dsRNA-containing VLP, this study constructed a novel E. coli strain that is able to co-express viral capsid proteins and dsRNA in the same E. coli cell. Results A novel bacterial strain DualX-B15(DE3) was engineered to be both protease- and RNase III-deficiency via P1 phage transduction. The results revealed that it could simultaneously express recombinant proteins and dsRNA. Conclusion Co-expression of viral capsid proteins and dsRNA in the same cell has been shown to be feasible. Not only could this platform serve as a basis for future cost-effective and streamlined production of shrimp antiviral therapeutics, it may be applicable for other applications that requires co-expression of recombinant proteins and dsRNA.
The aim of this study was to examine the feasibility of employing a yeast functional complementation assay for shrimp genes by using the shrimp mitochondrial F1F0-ATP synthase enzyme complex as a model. Yeast mutants defective in this complex are typically respiratory-deficient and cannot grow on non-fermentable carbon sources such as glycerol, allowing easy verification of functional complementation by yeast growth on media with them as the only carbon source. We cloned the previous published sequence of ATP2 (coding for ATP synthase β subunit) from the Pacific white shrimp Penaeus vannamei (Pv) and also successfully amplified a novel PvATP3 (coding for the ATP synthase γ subunit). Analysis of the putative amino acid sequence of PvATP3 revealed a significant homology with the ATP synthase γ subunit of crustaceans and insects. Complementation assays were performed using full-length ATP2 and ATP3 as well as a chimeric form of ATP2 containing a leader peptide sequence from yeast and a mature sequence from shrimp. However, the shrimp genes were unable to complement the growth of respective yeast mutants on glycerol medium, even though transcriptional expression of the shrimp genes from plasmid-borne constructs in the transformed yeast cells was confirmed by RT-PCR. Interestingly, both PvATP2 and PvATP3 suppressed the lethality of the yeast F1 mutants after the elimination of mtDNA, which suggests the assembly of a functional F1 complex necessary for the maintenance of membrane potential in the ρ0 state. These data suggest an incompatibility of the shrimp/yeast chimeric F1-ATPase with the stalk and probably also the F0 sectors of the ATP synthase, which is essential for coupled energy transduction and ATP synthesis.
White spot syndrome virus (WSSV) is a highly infectious virus that can cause 100% mortality in farmed shrimp in the matter of days, leading to significant economic losses to shrimp aquaculture worldwide. To mitigate the adverse impact of WSSV, effective antiviral strategies are necessary. Currently, double‐stranded RNA (dsRNA), which can silence viral gene expression through an RNA interference (RNAi) pathway, is among the most promising therapeutic tools for WSSV, but its farm‐scale utilization is held back by high costs and the lack of delivery approach. Recently, virus‐like particles (VLPs) have been applied as nanocarriers to deliver dsRNA into shrimp tissues, resulting in prolonged half‐life of the encapsidated dsRNA and stimulation of shrimp innate immunity. However, recombinant VLPs and dsRNA are traditionally expressed in two different E. coli strains: a protease‐deficient BL21(DE3) strain and an RNase III‐deficient HT115(DE3) strain, respectively. The ability to produce both VLPs and dsRNA simultaneously in a cell will reduce the costs and steps associated with the production, but attempts at VLP‐dsRNA coexpression in each of these strains resulted in degradation of either VLP or dsRNA due to the presence of proteases or RNaseIII, respectively. Here, to streamline VLP‐dsRNA production and allow simultaneous expression of VLP and dsRNA in the same cell, P1 transduction was employed to generate a novel E. coli strain which is both protease‐ and RNase III‐deficient. The newly engineered E. coli strain is able to express both MrNV‐VLP and VP28 dsRNA efficiently. Therefore, this new expression platform will pave the way for development of effective and low‐cost production of VLP‐encapsulated dsRNA for protection against viral infection in farmed shrimp. Support or Funding Information The authors would like to thank the following institutions for supporting research and scholarship to graduate student: Thailand Graduate Institute of Science and Technology (TGIST), National Science and Technology Development Agency (NSTDA) and Mahidol University.
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