In order to achieve inhibition of CyHV-3 WT strains, the CyHV-3 ORF57 gene – which is partially absent in an existing attenuated vaccine strain (resulting in its attenuation), was chosen as a target for siRNAs processed from vaccine expressed shRNA precursors. Typically, U6 promoters such as the human U6 promoter (HU6) are used for this. Thus, we proposed to establish if this promoter could be used in carp cells, in addition to using the HU6 promoter to express shRNAs that act as anti-WT siRNA precursors, it could also be used to express separate structural variants (blunt-ended-shRNAs) designed to function as RIG-I agonists to provide stimulation of the immune system.
Thus, this project was successful in conducting the first initial characterization the HU6 promoter expression system in carp cells, confirming that it can drive the expression of both protein coding genes (Figure 1A and 1B) and non-coding genes (Figure 1C and 1D) but in both cases activity was lower than in human cells. While these expression levels may possibly be insufficient for RNAi, we reasoned that it may still be sufficient to achieve RIG-I mediated stimulation of the innate immune system.
To offset the potential risk that RNA expression levels from the HU6 promoter may be too low for RNAi (or even immune stimulation), we also conducted the first study aimed at identifying endogenous carp U6 expression systems which should naturally be more active in carp derived cells. Using existing genomic data and our own transcriptomic approach, we conducted the preliminary screening of a putative endogenous carp U6 promoter and uncovered strong evidence for the existence of a second much more active putative carp U6 promoter, which we are currently investigating further.
In parallel, in anticipation of the identification of a U6 promoter system that was sufficiently active to drive shRNA expression in carp cells, we proceeded with the development of methodology to routinely establish co-infected cell monolayers that would exhibit significant number of co-infected cells, which could be used to test inhibition of WT virus via vaccine-derived RNAi mechanism. Despite repeated attempts however, we were unable to observe any substantial amounts of co-infected cells in co-infected monolayers. Further investigations revealed that this was due to the fact that CyHV-3 exhibits rapid superinfection inhibition, thus blocking the main mechanism by which co-infected cells arise.
Despite this, our virus-host model could still be utilized to study the enhancement of live attenuated vaccine potency via expression of RNA-based agonists of the innate immune system. Prior to design of HU6 expression constructs we tested the immuno-stimulatory capacity of several dsRNA molecules in carp cells, revealing that heavily structured 5ppp-non-blunt-ended dsRNA (5ppp-NBE-dsRNA) molecules performed best (Figure 1E). We are currently moving towards testing expression constructs for these molecules (and our own custom molecules) in carp cells, with the aim of inserting functional IFN-I-stimulating expression constructs into the existing live attenuated CyHV-3 vaccine strain.
