Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes

Abstract

The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs) targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNAval Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE) Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR), comparative TCID50-IFA analysis, in situ Immuno-fluorescent Assays (IFA) and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies

Engineered resistance to Zika virus in transgenic Aedes aegypti expressing a polycistronic cluster of synthetic small RNAs

“…Previously in Ae. aegypti , resistance to DENV has been engineered by transgenic activation of antiviral pathways (26), transgene-based RNAi in either the midgut (27, 46) or salivary glands (28), and antiviral hammerhead enzymes (47), and expression of synthetic miRNAs has also been demonstrated to induce partial resistance to DENV-3 and CHIKV (32). However, similar approaches have not been successfully demonstrated for ZIKV, and the currently described system is potentially especially advantageous since targeting six of 10 conserved protein-coding genes from the ZIKV genome with eight separate synthetic small RNAs may reduce the possibility of escapee mutants, and thus ensure evolutionary stability.…”

Engineered resistance to Zika virus in transgenicAe. aegyptiexpressing a polycistronic cluster of synthetic miRNAs

“…Previously in Ae. aegypti , resistance to DENV has been engineered by transgenic activation of antiviral pathways 25 , transgene-based RNAi in either the midgut 26,43 or salivary glands 27 , and antiviral hammerhead enzymes 44 , and expression of synthetic miRNAs has also been demonstrated to induce partial resistance to DENV-3 and CHIKV 31 . However, similar approaches have not been successfully demonstrated for ZIKV; and, the currently described system is potentially especially advantageous, since targeting 6/10 conserved protein-coding genes from the ZIKV genome with 8 separate synthetic miRNAs may reduce the possibility of escapee mutants and thus ensure evolutionary stability.…”

Winning the Tug-of-War Between Effector Gene Design and Pathogen Evolution in Vector Population Replacement Strategies

“…aegypti -transmitted arboviruses are of particular concern at present, as the mosquito vector has rapidly expanded its range in recent years (Huang et al, 2017; Lee et al, 2019; Ryan et al, 2019). In response, a variety of approaches have recently been taken to engineer mosquitoes refractory to these diseases, including: i) transgene-based RNA interference (RNAi) in the midgut (Franz et al, 2006) and salivary glands (Mathur et al, 2010), ii) expression of synthetic miRNAs (Buchman et al, 2019b), iii) use of broadly neutralizing, single-chain variable fragments (scFv) (Buchman et al, 2019a), iv) use of antiviral hammerhead enzymes (Mishra et al, 2016), and v) transgenic activation of antiviral pathways (Jupatanakul et al, 2017). Nonetheless, these arboviruses are prone to rapidly evolving resistance to antiviral strategies.…”

“…Another approach to minimize the emergence of escape mutants is to use small antiviral hammerhead ribozymes to increase the number of sites being targeted in the viral genome. This was used by Mishra et al (2016) to target the CHIKV genome (Mishra et al, 2016). Error-prone activities of RNA polymerase provide opportunities for viruses to escape from ribozyme catalysis (Scherer and Rossi, 2003); however this can be overcome by using antiviral group-I introns, and by targeting conserved sequences in arboviruses (Carter et al, 2015).…”

“…Most of the discussion thus far has focused on the former. Strategies to increase the evolutionary hurdle include: i) using multiple effectors that attack the pathogen at multiple sites on its genome and/or multiple stages of development ( Figure 3 ) (Mishra et al, 2016; Buchman et al, 2019b), and ii) ensuring that the effectors used are effective at substantially reducing pathogen transmission, and in a wide range of individuals representing the genetic diversity of the species. As mentioned earlier, a crucial goal is to prevent selection of escape mutants with a higher viral or parasitic load.…”