RNA interference (RNAi) as an antiviral mechanism against Penaeus merguiensis densovirus (PmergDNV)

La Fauce, Kathy Ann (2008) RNA interference (RNAi) as an antiviral mechanism against Penaeus merguiensis densovirus (PmergDNV). PhD thesis, James Cook University.

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Abstract

The Australian hepatopancreatic parvovirus (HPV) isolate is currently known to have a high prevalence in wild and cultured penaeid species throughout Queensland, Australia. Despite penaeid prawns being the main crustacean cultured in Queensland, there has been no investigation into the production losses resulting from HPV. The aim of this project was to characterise the virus from, estimate production losses on the culture of P. merguiensis due to HPV disease and determine if RNA interference (RNAi) can mediate a sequence specific anti-viral effect against the Australian HPV isolate.

The Australian HPV isolate from P. merguiensis is the fourth strain of penaeid prawn HPV identified. Following the convention of the International Committee for the Taxonomy of Viruses, this virus has been named Penaeus merguiensis densovirus (PmergDNV). Complete genome sequence (6299 bp) analysis revealed a nucleotide similarity (86%) closer to the complete genome sequence of the Korean HPV isolate from P. chinensis than to the complete genome sequence of the Thai HPV isolate from P. monodon (83%) and the Indian HPV isolate from P. semisulcatus (83%). Hence, HPV strains may be following the phylogenetic relationship of the penaeid prawn hosts rather than their geography.

A TaqMan based real-time PCR assay was developed for the detection of PmergDNV. The TaqMan assay was developed within the capsid protein region of the genome and was optimised to detect as little as 10 copies of the targeted sequence per reaction. The assay was specific for PmergDNV as it did not detect related crustacean and canine parvoviruses from Australia. 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, permitting exclusion of potential carriers of the virus from culture facilities and subsequently preventing disease outbreaks.

A PCR analysis of 10-day old postlarvae of P. merguiensis from three northern Queensland farms from 190 ponds over two years revealed that the odds were approximately 2:1 that ponds with moderate to heavy loads of PmergDNV will have below mean survival. Of particular interest is that production will increase by at least 14% across the farms if survival is improved to match the current mean loss by removing PmergDNV.

Since natural infections of PmergDNV in P. merguiensis can interfere with results of infection studies, infection experiments were conducted on the house cricket (Acheta domesticus) and mealworm beetle larvae (Tenebrio molitor) to find an alternate bioassay species. Acheta domesticus and T. molitor were challenged with approximately 1 x 106 virions of PmergDNV by inoculation. PmergDNV was detected in 20% of T. molitor and 86.6% of A. domesticus challenged with PmergDNV. During a subsequent time course experiment, there was a slight increase in PmergDNV titres (104-5 virions), reaching a maximum peak at day 5 (106 copies). A threshold of PmergDNV DNA level equal to or greater than 103 virions was necessary for mortality in A. domesticus. As the inoculum increased from 103 DNA copies to 104, 105, 106, mortality increased from 20% to 60%, 80% and 100% respectively.

Since an alternate bioassay species (A. domesticus) had been established, investigations continued to determine if RNAi can be used to provide protection against PmergDNV in A. domesticus. Adult A. domesticus were injected with 5 μg of stealth RNAi or control stealth RNAi, targeting the capsid protein and challenged with PmergDNV twenty four hours post-injection. 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%). The introduction of dsRNAi corresponding to the capsid protein of PmergDNV, was effective in reducing viral replication in A. domesticus. Crickets challenged with specific dsRNA significantly reduced PmergDNV production by one log (3.58 x 102) compared to crickets challenged with PmergDNV alone (3.42 x 103). Control dsRNA also resulted in a one log reduction of PmergDNV (3.95 x 102), but did not produce an inhibitory effect quite as strong as the targeted dsRNAi for the capsid protein of PmergDNV.

The RNAi assay targeting the NS2 gene was repeated jointly with a post-doctoral scientist from India as part of the Australia-India strategic research fund. The greatest cumulative percentage mortality (70%) was recorded in the target RNAi + PmergDNV treatment, followed by in the control RNAi + PmergDNV (50%) and PmergDNV only treatment (46.5%) and the target RNAi only treatment (43%). Similarly, the introduction of dsRNA corresponding to the NS2 protein of PmergDNV was effective in reducing viral replication in A. domesticus. However, a 10-fold reduction in PmergDNV titres was only recorded in the target RNAi + PmergDNV treatment. Crickets challenged with specific dsRNA significantly reduced PmergDNV production by one log (8.1 x 103) compared to crickets challenged with PmergDNV alone (9.85 x 104). Control dsRNA also resulted in a reduction of PmergDNV (2.2 x 104), but did not produce an inhibitory effect as the targeted stealth interfering RNAs.

As mud crabs Scylla serrata cohabit the environment around prawn farms and have been used as a maturation diet for prawn broodstock, they were examined in conjunction with other students for PmergDNV. Approximately 74% of haemolymph from adults and 100% of inclusion body positive batches of larvae were positive for PmergDNV by quantitative real-time polymerase chain reaction (PCR), with PmergDNV titres ranging from 6 x 102 to 1.5 x 105. Sequencing of 2,475 base pairs of viral genome confirmed the virus shared 99% similarity to PmergDNV. This is the first record of an isolate of hepatopancreatic virus to be found outside penaeid prawns and further complicates the epidemiology of PmergDNV since control of the virus must involve excluding both wild host species.

This project establishes an A. domesticus model for genetic studies of the virus-host interactions and demonstrates the RNAi pathway is a potent antiviral mechanism against PmergDNV. In this context, administration of PmergDNV-specific dsRNAi may provide an efficient counter measure against PmergDNV in prawns and therefore holds considerable promise as a preventative of viral diseases in aquaculture.

Item ID: 34444
Item Type: Thesis (PhD)
Keywords: aquaculture; banana prawn; Fenneropenaeus merguiensis; hepatopancreas; hepatopancreatic parvovirus; HPV; infections; penaeid prawns; penaeid shrimp; Penaeidae; Penaeus merguiensis densovirus; Penaeus merguiensis; PmergDNV; RNA interference; RNAi; viral diseases; viruses
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Additional Information:

Publications arising from this thesis are available from the Related URLs field. The publications are:

La Fauce, Kathy, and Owens, Leigh (2007) Investigation into the pathogenicity of Penaeus merguiensis densovirus (PmergDNV) to juvenile Cherax quadricarinatus. Aquaculture, 271 (1-4). pp. 31-38.

La Fauce, Kathy A., Elliman, Jennifer, and Owens, Leigh (2007) Molecular characterisation of hepatopancreatic parvovirus (PmergDNV) from Australian Penaeus merguiensis. Virology, 362 (2). pp. 397-403.

La Fauce, Kathy A., Layton, Ramon, and Owens, Leigh (2007) TaqMan real-time PCR for detection of hepatopancreatic parvovirus from Australia. Journal of Virological Methods, 140 (1-2). pp. 10-16.

La Fauce, Kathy A., and Owens, Leigh (2008) The use of insects as a bioassay for Penaeus merguiensis densovirus (PmergDNV). Journal of Invertebrate Pathology, 98 (1). pp. 1-6.

La Fauce, Kathy A., and Owens, Leigh (2009) RNA interference reduces PmergDNV expression and replication in an in vivo cricket model. Journal of Invertebrate Pathology, 100 (2). pp. 111-115.

Owens, L., Liessmann, L., La Fauce, K., Nyguyen, T., and Zeng, C. (2010) Intranuclear bacilliform virus and hepatopancreatic parvovirus (PmergDNV) in the mud crab Scylla serrata (Forskal) of Australia. Aquaculture, 310 (1-2). pp. 47-51.

Owens, Leigh, La Fauce, Kathy , Juntunen, Karen, Hayakijkosol, Orachun, and Zeng, Chaoshu (2009) Macrobrachium rosenbergii nodavirus disease (white tail disease) in Australia. Diseases of Aquatic Organisms, 85 (3). pp. 175-180.

Date Deposited: 20 Nov 2014 02:01
FoR Codes: 07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070404 Fish Pests and Diseases @ 50%
06 BIOLOGICAL SCIENCES > 0604 Genetics > 060405 Gene Expression (incl Microarray and other genome-wide approaches) @ 25%
06 BIOLOGICAL SCIENCES > 0604 Genetics > 060412 Quantitative Genetics (incl Disease and Trait Mapping Genetics) @ 25%
SEO Codes: 83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830105 Aquaculture Prawns @ 100%
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