Antiviral therapy in shrimp through plant virus VLP containing VP28 dsRNA against WSSV

The white spot syndrome virus (WSSV), currently affecting cultured shrimp, causes substantial economic losses to the worldwide shrimp industry. An antiviral therapy using double-stranded RNA interference (dsRNAi) by intramuscular injection (IM) has proven the most effective shrimp protection against WSSV. However, IM treatment is still not viable for shrimp farms. The challenge is to develop an efficient oral delivery system that manages to avoid the degradation of antiviral RNA molecules. The present work demonstrates that VLPs (virus-like particles) allow efficient delivery of dsRNAi as antiviral therapy in shrimp. In particular, VLPs derived from a virus that infects plants, such as cowpea chlorotic mottle virus (CCMV), in which the capsid protein (CP) encapsidates the dsRNA of 563 bp, are shown to silence the WSSV glycoprotein VP28 (dsRNAvp28). In experimental challenges in vivo, the VLPs- dsRNAvp28 protect shrimp against WSSV up to 40% by oral administration and 100% by IM. The novel research demonstrates that plant VLPs, which avoid zoonosis, can be applied to pathogen control in shrimp and also other organisms, widening the application window in nanomedicine.

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The impact of size on particle drainage dynamics and antibody response

10.1101/2020.09.28.316612 ◽

2020 ◽

Author(s):

Simon Zinkhan ◽

Anete Ogrina ◽

Ina Balke ◽

Gunta Reseviča ◽

Andris Zeltins ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

Lymphoid Organs ◽

Cowpea Chlorotic Mottle Virus ◽

Chlorotic Mottle Virus ◽

Chlorotic Mottle ◽

Secondary Lymphoid Organs ◽

The Impact

AbstractVaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B-cells and other innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T=3 icosahedron particles. They can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxoid (TT) epitope at the N-terminus. The modified CCMVTT-VLPs successfully form icosahedral particles T=3, with a diameter of ∼30nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMVTT-VLPs results in the formation of Rod-shaped VLPs, ∼1µm in length and ∼30nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMVTT-VLPs and Rod-shaped CCMVTT-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMVTT-VLPs are more efficient in draining to secondary lymphoid organs to charge antigen-presenting cells as well as B-cells. Furthermore, compared to Rod-shaped CCMVTT-VLPs, Round-shaped CCMVTT-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMVTT-VLPs could also polarize the induced immune response towards TH1. Up to our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedrons or rods in the micrometer size.

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Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints

Journal of Virology ◽

10.1128/jvi.79.6.3277-3288.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3277-3288 ◽

Cited By ~ 28

Author(s):

Padmanaban Annamalai ◽

Swapna Apte ◽

Stephan Wilkens ◽

A. L. N. Rao

Keyword(s):

Northern Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Rna Binding ◽

Binding Motif ◽

Cowpea Chlorotic Mottle Virus ◽

Viral Rnas ◽

Chlorotic Mottle Virus ◽

Chlorotic Mottle

ABSTRACT The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Δ919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Δ919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Δ919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.

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Assembly of a spherical plant virus

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1976.0102 ◽

1976 ◽

Vol 276 (943) ◽

pp. 113-122 ◽

Cited By ~ 35

Keyword(s):

Buoyant Density ◽

Sedimentation Coefficient ◽

Protein Subunit ◽

Cowpea Chlorotic Mottle Virus ◽

Average Mass ◽

Chlorotic Mottle Virus ◽

Chlorotic Mottle ◽

Average Molecular Masses

The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 °C and did not require the presence of Mg 2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.

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