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 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.

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 River's 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.

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.

TaqMan real-time PCR for detection of hepatopancreatic parvovirus from Australia.

Hepatopancreatic parvovirus is an emerging disease in crustacean aquaculture. Consequently, methods of detection are needed that enable the sensitive detection and confirmation of the virus better than currently used methods such as histology and conventional polymerase chain reaction (PCR). A TaqMan based real-time PCR assay was developed for the detection of the Australian isolate of hepatopancreatic parvovirus which is only 85% similar to its nearest known relative. The TaqMan assay was developed within the capsid protein region of the genome and is optimised to detect as little as 10 copies of the targeted sequence per PCR vial. The hepatopancreatic parvovirus primers and probe were HPV140F 5'-CTA CTC CAA TGG AAA CTT CTG AGC-3', HPV140R 5'-GTG GCG TTG GAA GGC ACT TC-3' and HPV140probe 5'-FAM TAC CGC CGC ACC GCA GCA GC TAMRA-3', respectively. The assay was specific for the hepatopancreatic parvovirus strain from Australian Penaeus merguiensis as it did not detect related crustacean and canine parvoviruses from Australia. In addition, the very low homology of the target sequence with published sequences from the Thai and Korean strains of hepatopancreatic parvovirus and other prawn viruses such as WSSV, suggested this assay would be specific for the Australian hepatopancreatic parvovirus isolate. Furthermore, it detected hepatopancreatic parvovirus in 22/22 wild-caught P. merguiensis clinical samples and 473/545 (87%) farmed P. merguiensis. This assay has the potential to be used for diagnostic purposes and in robotic applications, particularly for the detection and quantitation of low-grade infections.

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.

Genomics, Molecular Epidemiology and Diagnostics of Infectious hypodermal and hematopoietic necrosis virus.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimps worldwide, which has resulted in severe mortalities of up to 90 % in cultured Penaeus (Litopenaeus) stylirostris from Hawaii and hence designated Penaeus stylirostris densovirus (PstDNV). IHHNV is distributed in shrimp culture facilities worldwide. It causes large economic loss to the shrimp farming industry. Our knowledge about the natural reservoirs of IHHNV is still scarce. Recent studies suggest that there is sufficient sequence variation among the isolates from different locations in Asia, suggesting multiple geographical strains of the virus. Four complete genomes and several partial sequences of the virus are available in the GenBank. Complete genome information would be useful for assessing the specificity of diagnostics for viruses from different geographical areas. Comparisons of complete genome sequences will help us gain insights into point mutations that can affect virulence of the virus. In addition, because of unavailability of shrimp cell lines for culturing IHHNV in vitro, quantification of virus is difficult. The recent progress in research regarding clinical signs, geographical distribution, complete genome sequence and genetic variation, transmission has made it possible to obtain information on IHHNV. A comprehensive understanding of IHHNV infection process, pathogenesis, structural proteins and replication is essential for developing prevention measures. To date, no effective prophylactic measure for IHHNV infection is available for shrimp to reduce its impact. This review provides an overview of key issues regarding IHHNV infection and disease in commercially important shrimp species.

Molecular characterisation of hepatopancreatic parvovirus (PmergDNV) from Australian Penaeus merguiensis.

Hepatopancreatic parvovirus infection is associated with reduced growth rates of prawns during the juvenile stages and overt mortalities. Hepatopancreatic parvovirus was purified from Penaeus merguiensis from northern Queensland and a partial consensus sequence of 5.9 kb was obtained. Nucleotide comparisons revealed that the Australian isolate of HPV has a nucleotide similarity (87%) closer to HPVchin and the full sequence of HPV Penaeus monodon (PmDNV) (6321 bp) than to HPVsemi (83%). Three putative open reading frames were identified. The first open reading frame encoded a nonstructural protein (NS2) and shared an amino acid similarity of 86% with PmDNV. The second ORF overlapped the first open reading frame and shared 93% and 26% amino acid similarity with PmDNV and PstDNV, respectively, and encoded NS1. The third ORF encoded the viral structural protein and shared an amino acid similarity of 73% with the capsid protein of PmDNV and HPVchin. The phylogeny suggests that the Australian HPV isolate is closely related to the Korean HPVchin isolate than to the Indian HPVsemi and Thai PmDNV isolates. HPV strains may be following the phylogenetic relationship of penaeid prawn hosts rather than their geography.