RNA interference offers promising antiviral methods to minimise nodovirus outbreaks in aquaculture
Following on from the talk by Leigh Owens and the recent complementary lecture by Colin, this research used interfering RNA (RNAi) to assess its effectiveness of reducing mortality from white tail disease (WTD) in giant freshwater prawns, Macrobrachium rosenbergii, using redclaw crayfish, Cherax quadricarinatus as a model organism.
Global demand for seafood is increasing and with intensive farming comes disease. WTD has lead to mass mortalities in prawn hatcheries the world over, causing economic problems and loss in production. M. rosenbergii nodavirus (MrNV) has been identified as the causative agent of WTD and has caused 100% mortality in hatcheries within one week of the first clinical signs in the hosts.
RNAi is the sequence-specific degradation of complementary mRNA using small, functional pieces of double stranded RNA (dsRNA) to target specific genes. It has been used as an antiviral mechanism, making it possible to knock-down specific viral genes and limiting any off-target effects. This experiment targeted protein B2 of MrNV, which inhibits the degradation of viral RNA that would normally occur in the host cells. Sequence specific RNAi was therefore used to silence the production of protein B2 by degrading viral mRNA – basically an arms race between virus and host cell.
MrNV from infected M. rosenbergii was homogenised with phosphate buffered saline (PBS) and injected into select C. quadricarinatus. Sequence specific RNAi (targeting protein B2), along with control RNAi (non-specific), was designed and injected into select animals in a cross-over experimental design, also controlling for exposure to MrNV and RNAi by using placebo treatments and PBS. All experimental animals, unless mortality occurred during the trial, were sacrificed at the end of 60 days. RNA was extracted from C. quadricarinatus muscle for qPCR analyses and tissue sections prepared for histopathological analysis.
Invertebrates have no acquired immunity and RNAi is important for antiviral innate immunity. Only specific dsRNA to protein B2 was effective in affording increased antiviral defence against MrNV infection in focal C. quadricarinatus (10% mortality) compared with high mortality (60%) and clinical signs consistent with MrNV infection reported in the control RNAi group (see Fig. 1).
The authors note that injection of RNAi + MrNV did not reduce the average viral titre in comparison to the control RNAi + MrNV group, contrasting their results to a similar study in crickets that reported a 10-fold reduction in viral titres (La Fauce & Owens, 2009). An alternative method of delivering sequence specific dsRNA may yield lower viral counts, for example including it in feeds via a bacterial plasmid. This may also allow viable scale up for commercial use and offer an alternative to phage therapy (see Sanket’s recent posts). In Leigh’s talk, he stated that his group has had difficulty in scaling up quantities for phage therapy to be effective outside of laboratory conditions and considers RNAi a more promising viral control technique. Inclusion of plasmid contained sequence specific dsRNA in commercial feeds may still offer antiviral protection to the prawns, whilst maintaining its select target (eg. the virus) and minimising other undesirable, off target effects. It will be interesting to see where this promising technique leads, although further long-term, transgenerational research would benefit here, to gain a better understanding of potential long-term impacts this may have.
Hayakijkosol, O., & Owens, L. (2012). B2 or not B2: RNA interference reduces (Macrobrachium rosenbergii) nodavirus replication in redclaw crayfish (Cherax quadricarinatus). Aquaculture, 326, 40-45.