Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production

Development of vaccines and management of viral diseases of crustaceans

Viruses from a wide variety of different virus families are capable of infecting shrimp. Many of these viruses, including white spot syndrome virus, have major impacts on the shrimp farming industry. As a consequence, during the past decade there has been increasing focus on the viruses infecting shrimp and on strategies to inhibit these viruses. This research has revealed two promising approaches for decreasing the impact of viruses on aquaculture: RNA interference and vaccination using viral proteins. Both strategies have recently proven successful in the laboratory, but a major challenge will be to adapt these for use in the field. In this chapter, we provide an overview of the viruses which cause problems, the progress on the understanding of the shrimp immune system and the development of strategies to inhibit viral infection

Pathogenicity of gill-associated virus and Mourilyan virus during mixed infections of black tiger shrimp (Penaeus monodon)

Gill-associated virus (GAV) and Mourilyan virus (MoV) can occur at very high prevalence in healthy black tiger shrimp (Penaeus monodon) in eastern Australia, and both have been detected in moribund shrimp collected from mid-crop mortality syndrome (MCMS) outbreaks. Experimental evidence presented here indicates that GAV, but not MoV, is the cause of MCMS. Firstly, in healthy P. monodon used for experimental infections, pre-existing MoV genetic loads were very high (mean.> 10 viral RNA copies μg total RNA) and did not increase significantly following lethal challenge with an inoculum containing both GAV and MoV. In contrast, GAV genetic loads prior to challenge were low (mean ~105 RNA copies μg total RNA) and increased > 10 -fold in moribund shrimp. Secondly, dsRNAs targeted to the GAV RNA-dependent RNA polymerase (RdRp) or helicase gene regions reduced GAV genetic loads, delayed the onset of mortalities and improved survival following challenge. In contrast, dsRNA targeted to the MoV RdRp gene (L RNA) was highly effective in reducing MoV genetic loads, but mortality rates were unaffected. Targeting of the MoV S2 RNA, encoding a small non-structural protein (NSs2), a putative supressor of RNA interference, did not reduce the MoV genetic loads or enhance knockdown of GAV when administered simultaneously with dsRNA targeted to the GAV helicase gene. Overall, the data show that P. monodon can tolerate a high-level MoV infection and that mortalities are associated with GAV infection

Gill-associated virus and recombinant protein vaccination in Penaeus monodon

Recent studies in penaeid shrimp and various crustaceans showing specific antiviral responses to the DNA based White spot syndrome virus (WSSV) induced by pre-exposure to viral proteins are of particular research interest as invertebrates lack the classical adaptive immune response characterised in vertebrate animals. However, this protein-induced defence response has not been reported for any other invertebrate virus, and the mechanisms remain unknown. Here we investigated the generality of this defence response by challenging Penaeus monodon (Black Tiger shrimp) with the RNA based Gill-associated virus (GAV) following pre-exposure to fragments of the GAV gp116 envelope glycoprotein or to the p20 nucleocapsid protein. Shrimp were injected twice with recombinant GAV protein fragments expressed and purified from bacteria before challenge with GAV. Protein vaccination did not protect the shrimp, with mortality rates in shrimp injected with any of the GAV proteins not differing from shrimp either not injected with protein or injected with a non-specific protein. In addition, there was no significant difference in GAV RNA loads amongst the virus challenged shrimp groups. The data suggest that the efficacy of protection following protein-based vaccination may vary amongst different shrimp viruses. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved

Temperature-dependent processing and secretion of SAV-E2.

<p>A) <i>Sf</i>9 cells were infected with Ac-SAV3 with an MOI of 10 at 27°C, 18°C, 15°C and 12°C. SAV-E2 expression in the cell-fraction was analyzed by WB using SAV α-E2 mab (17H23). B) Relative percentages of SAV-E3E2 and E2, indicating more efficient processing of E3E2, with decreasing temperatures. C) Secretion of E2 as a function of the temperature. The medium fraction of infected <i>Sf</i>9-cell cultures was PEG-precipitated and analyzed by WB using SAV α-E2 mab.</p

SAV3-E2 detection on the surface of <i>Sf</i>9 cells after recombinant baculovirus expression.

<p>Cells were infected with Ac-SAV3 at 12°C, 15°C, 18°C and 27°C. Cells were fixed with 4% paraformaldehyde and subjected to immunostaining with α-E2 mabs. Cells were analyzed by confocal microscopy and positive staining indicates the presence of E2 at the surface of infected cells.</p

SAV3 structural cassette expression and VLP formation by temperature-shift in <i>Sf</i>9 insect cells.

<p><i>Sf</i>9 cells were infected with Ac-SAV3, incubated for 2 days at 27°C and subsequently transferred to 12°C for 3 days. A) Cell cultures were treated with/without PNGase F and analyzed by WB using α-E2 mabs. B) Infected cells were treated with 4% paraformaldehyde and subjected to surface immunostaining with α-E2 mabs. C) The medium fraction the infected cell culture was analyzed by TEM for the presence of VLPs.</p

SAV-E2 antigenic mass determination and VLP production.

<p>A) SAV-E2 antigenic mass was determined using the SAV-neutralizing mab 17H23 on cell and medium fractions of infected <i>Sf</i>9-cells, incubated at 27°C, 18°C, 15°C and 12°C. B) The medium fraction of cells infected with Ac-SAV3 at 15°C was analyzed by TEM to analyze SAV VLP production. Medium fractions of Ac-GFP infected <i>Sf</i>9 cells and SAV3 infected Chinook salmon embryo cells were used as control samples.</p

Nuclear localization and assembly of SAV3-nucleocapsids.

<p><i>Sf</i>9 infection with Ac-SAV3 and Ac-GFP was analyzed by A) light-microscopy at 2 d.p.i. B) Ac-SAV3-infected cells were fixed and embedded in gelatin and ultrathin coupes were analyzed by TEM, with C, N, VS and NC indicating cytoplasm, nucleus, virogenic stroma and SAV nucleocapsids, respectively.</p

SAV3 structural cassette expression, using recombinant baculoviruses.

<p>A) Schematic representation of the SAV3 structural cassette as it is expressed by recombinant baculoviruses. The molecular mass of the proteins are indicated and shaded areas represent transmembrane domains or signal sequences (ss). Autocatalytic (A), furin (F) and signalase (S) cleavage sites are indicated, asterisks represent N-linked glycosylation sites. B) Protein expression in <i>Sf</i>9 cells was analyzed by CBB and WB using SAV α-E1 and SAV α-E2 mabs. C) Whole cell lysates were treated with/without PNGase F and analyzed with SAV α-E2 mabs. D) Cells infected with Ac-GFP and Ac-SAV3 and stained with Hoechst. CPE was evaluated by brightfield and fluorescence microscopy. Arrows indicate dense nuclear bodies in Ac-SAV3-infected insect cells.</p