Kathy La Fauce

Macrobrachium rosenbergii nodavirus disease (white tail disease) in Australia

The index case of white tail disease (WTD) is presented in adult broodstock prawns Macrobrachium rosenbergii from the Flinders River in western Queensland, Australia, in mid-2007. Histological examination revealed extensive myonecrosis with massive infiltration of myonuclei and some haemocytes. Juveniles from the same broodstock but not from 3 other families displayed white muscle lesions. Low-grade chronic mortalities approaching 100% over 1 yr occurred. Reverse transcriptase polymerase chain reactions (RT-PCR) were attempted for both M. rosenbergii nodavirus (MrNV) with 2 sets of primers and for the satellite virus, extrasmall virus (XSV). All 3 PCRs generated amplicons of the expected sizes. Basic local alignment search tool (BLAST) analyses of the 3 consensus sequences identified a 91% match with MrNV viral capsid protein gene, 96% match with MrNV RNA-directed RNA polymerase gene, and a 99% match with M. rosenbergii XSV capsid protein gene. The clinical signs, histopathological lesions and RT-PCR amplicons could be reproduced in M. rosenbergii inoculated with cell-free extracts fulfilling Rivers postulates. We conclude that this is an endemic strain of MrNV as the sequences are dissimilar to strains of MrNV circulating around Asia and the Americas. This case only poorly meets the Office International des Epizooties (OIE) case definition for WTD due to the age of the prawns involved and the nature of the inclusion bodies. Perhaps the OIE case definition needs broadening.

RNA interference reduces PmergDNV expression and replication in an in vivo cricket model.

RNA interference (RNAi) is an attractive anti-viral preventative because it allows interference with the expression of a viral gene in a highly sequence-specific manner. Thus, essential viral genes can be targeted by design, with little or no risk of undesired off-target effects. To investigate if stealth RNAis can mediate a sequence-specific anti-viral effect against PmergDNV, adult Acheta domesticus were injected with 5 microg of stealth RNAi or control stealth RNAi, targeting the capsid protein. Twenty-four hours post-injection, crickets were challenged with PmergDNV. Mortality was monitored for 14 days and real-time reverse transcriptase PCR was used to enumerate the number of copies of PmergDNV in cricket tissues. Whilst statistically not significant, trends in mortality suggest crickets injected with RNAi targeting PmergDNV had the lowest mortality rate (11.5%) compared to crickets injected with control dsRNAi (33%) and PmergDNV alone (25%). Crickets challenged with specific dsRNAi had statistically significantly reduced PmergDNV titres by one log (3.58 x 10(2)) compared to crickets challenged with PmergDNV alone (3.42 x 10(3)). Interestingly, even the control dsRNAi was capable of reducing PmergDNV titres by one log (3.95 x 10(2)), but did not produce an inhibitory effect quite as strong as the targeted dsRNAi for the capsid protein of PmergDNV. The introduction of dsRNAi corresponding to the capsid protein of PmergDNV, was effective in reducing viral replication in Acheta domesticus. Administration of PmergDNV-specific dsRNAis may provide an efficient counter measure against PmergDNV in prawns.

Suppression of Penaeus merguiensis densovirus following oral delivery of live bacteria expressing dsRNA in the house cricket (Acheta domesticus) model.

Penaeus merguiensis densovirus (PmergDNV) is a serious pathogen of the banana prawn, Penaeus merguiensis leading to at least 28% production loss due to reduced growth rates and mortality of juveniles. In the present study, we reduced PmergDNV titres and subsequent mortality by feeding Acheta domesticus (previously determined as an appropriate animal model for P. merguiensis) with dsRNA specific to the capsid protein by mixing it into their food. Feeding A. domesticus with PmergDNV-specific dsRNA in advance of viral challenge increased their longevity, decreased mortality by 84.4% and reduced viral loads 24-fold below the threshold level required for mortality. Mortalities and viral loads were significantly (both P < 0.001) lower in treatments challenged with PmergDNV following exposure to bacterially expressed PmergDNV-dsRNA. This is the first study to demonstrate gene silencing via RNAi against PmergDNV in vivo through oral administration of live bacteria expressing dsRNA in a model system.

RNA Interference with Special Reference to Combating Viruses of Crustacea

RNA interference has evolved from being a nuisance biological phenomenon to a valuable research tool to determine gene function and as a therapeutic agent. Since pioneering observations regarding RNA interference were first reported in the 1990s from the nematode worm, plants and Drosophila, the RNAi phenomenon has since been reported in all eukaryotic organisms investigated from protozoans, plants, arthropods, fish and mammals. The design of RNAi therapeutics has progressed rapidly to designing dsRNA that can specifically and effectively silence disease related genes. Such technology has demonstrated the effective use of short interfering as therapeutics. In the absence of a B cell lineage in arthropods, and hence no long term vaccination strategy being available, the introduction of using RNA interference in crustacea may serve as an effective control and preventative measure for viral diseases for application in aquaculture.

The use of insects as a bioassay for Penaeus merguiensis densovirus (PmergDNV)

The lack of available cell lines has hampered the study of viral diseases in crustaceans. This is particularly important for aquaculture which has been plagued by viral diseases since its rapid expansion to meet with the growing demand for seafood products. This study was designed to find an alternative bioassay to cell lines by investigating the use of insects as potential animal models for Penaeus merguiensis densovirus (PmergDNV). Acheta domesticus (house cricket) and Tenebrio molitor (mealworms) were challenged with approximately 1x10(6) virions of PmergDNV by inoculation. PmergDNV was detected in 20% of Tenebrio molitor and 86.6% of Acheta domesticus challenged with PmergDNV. During a subsequent time course experiment, there was a non significant increase in PmergDNV titres (10(4-5) virions), reaching a maximum peak at day 5 (10(6) copies). A threshold of PmergDNV DNA level equal to or greater than 10(3) virions was necessary for mortality in Acheta domesticus. As the inoculum increased from 10(3) DNA copies to 10(4), 10(5), 10(6), mortality increased from 20% to 60%, 80% and 100%, respectively. This is the first evidence that insects may be directly used to study viruses from crustaceans and concludes Acheta domesticus may be used as a potential model to study Penaeus merguiensis densovirus.

Dietary uptake of green fluorescent protein for delivery of dsRNA to induce RNA interference

Double stranded RNA (dsRNA) is a potent initiator of the RNA interference (RNAi) pathways in a diverse range of organisms including plants, invertebrates and vertebrates. RNAi is increasingly being investigated as an antiviral mechanism, but often the research is hampered by the safe and cost effective delivery of the dsRNA to the target cells. This study uses specifically designed E. coli-expressed plasmids containing green fluorescent protein (gfp), to determine if the plasmids still inside the bacterial cells can be successfully incorporated into host cells of the house cricket (Acheta domesticus), following ingestion of feed or water. This approach was trialled to determine, if using similar designed plasmids containing siRNAs can be effectively administered and provide protection against a target pathogen. Daily exposure through feed is the most effective for gfp expression (78%-91.8%) in A. domesticus, although weekly exposure was statistically equivalent and almost as good (75%-99.9%). Exposure via water gave reasonable uptake either daily or weekly (60%-84%, 14%-56%, respectively), but intensity was reduced and muscles did not demonstrate uptake (P<0.002). Continual or semi-continual exposure to gfp expressing bacteria resulted in increased uptake within cricket tissues, and increasing fluorescence (+++) with increasing exposure. Plasmid-based delivery via bacterial cells is therefore, an effective delivery tool and incorporation into feed allows a cost-effective method of providing continuous exposure for therapeutic purposes.