Therapeutic trials against pre-existing Chequa iflavirus in redclaw crayfish (Cherax quadricarinatus)

Chequa iflavirus is associated with mortalites reaching 20-40% after about three weeks following a stress event in farmed redclaw crayfish (Cherax quadricarinatus). Farmers need cost-effective interventions that can reduce the impact of this virus. This study was to identify strategies that would reduce the viral copy number as measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). With increasing viral copy number, there was significantly (P < 0.002) less weight gain at ~16% reduction in growth. Chequa iflavirus (>104 copies) was distributed widely in all crayfish tissues sampled suggesting dispersion throughout the body via the haemolymph. In two experiments each of 70 crayfish in 2 × 5 treatments, the source of the crayfish, the polyphenol flavonoid quercetin, inhabiting water temperature at 32 °C all had statistically significant effects (P < 0.001; P < 0.018; P < 0.05 respectively). Viral copy numbers dropped 10-fold to 104.1 with quercetin and 82% to 102.75 with 32 °C water temperature. Unexpectedly, delivery of specific RNA interference (RNAi) within a plasmid allowed viral copy number to initially increase due to presumed activation of the inflammasome via the dsDNA plasmid (unmethylated CpG) or endotoxin (lipopolysaccharide) inclusion and immunosuppression. Nevertheless, RNAi followed its predicted half-life and by the end of the experiment, a 5-fold reduction of viral copy numbers had occurred. A one minute, 5000 ppm formalin dip of eggs dropped viral copy numbers by 99.997% to 2.4 × 103. Five crayfish out of 140 that were repetitively bled and tested 5 or 6 times were test-negative with RT-qPCR that had a sensitivity of 100 copies. They were likely to be virus-free and could be used to start a specific pathogen free breeding program. This study demonstrated a number of ways forward for the crayfish farmers with the inflammasome suppressant, quercetin being the most favoured as it is abundant, cheap and readily incorporated into the diet and should ameliorate the original, transport-stress mortality which started this study.