Prokaryotic Production of Virus-Like Particle Vaccine of Betanodavirus
Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a serious disease of cultured marine fish worldwide. To control this disease, vaccines of subunit capsid proteins (recombinant proteins or peptides), inactivated viruses, and virus-
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Introduction Piscine nodaviruses, the causal agents of viral nervous necrosis or viral encephalopathy and retinopathy, are members of genus Betanodavirus under family Nodaviridae. Betanodaviruses are small, spherical, non-enveloped viruses with a bipartite singlestranded (+) RNA genome encapsulated by 180 molecules of a single self-assembly capsid protein (CP) [1]. They are important viruses in aquaculture because they can infect more than 39 marine fish species [2], especially high-value fish species, primarily at the larval and juvenile stages [3, 4], which result in mass mortality and serious economic losses. To efficiently control betanodavirus infections, vaccination is a pivotal strategy and several types of vaccines have been reported. Prokaryotic recombinant CP, synthetic peptides of neutralizing betanodavirus epitopes, DNA vaccine, inactivated virus, and viruslike particles (VLPs) were tested and VLPs are thought to be the most promising vaccine candidate because they can activate humoral immune response and induce cellular and innate immunities post-immunization with small quantities [5]. VLPs can be expressed by the baculovirus [6], yeast [7] or Escherichia coli (E. coli) [5, 8] system and provide relatively high protective immunity as efficient as inactivated betanodaviruses in several fish species. The structure of OGNNV VLP was revealed by cryo-electron microscopy (Fig. 1) and was found indistinguishable from the native virus on the outer surface [9]. VLPs can be produced eukaryotically and prokaryotically, of which the prokaryotic system is easier to manipulate, has higher yields and is faster and cheaper. VLPs should be purified from expression host by purification methods after expression. The laboratory purification protocols based on ultracentrifugation on sucrose or cesium chloride density
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_15, © Springer Science+Business Media New York 2016
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Fig. 1 Structural analysis of OGNNV. (a) 3D reconstruction of OGNNV VLP at 3.9 Å. The 3D structure of OGNNV VLP was resolved by cryo-electron microscopy and single particle reconstruction. VLP has a 38 nm in diameter with T = 3 icosahedral symmetry. There are three chemical identical monomers in each asymmetric unit. The capsid shells are shown in green and the protrusions are shown in blue. There is a high flexible loop to connect the protrusion to the capsid shell (not shown). (b) Structure prediction of CP monomer. We separate CP into three independent domains: the N-terminal domain (N-domain) (residues 1–50), the Shell domain (S-domain) (residues 51–220, the lower part of the structure contains N- and S-domain), and the Protrusion domain (P-domain) (residues 221–338, the upper part). N- and S-domains constitute the capsid shell while P-domains form the protrusion. Each protrusion is composed of three P-domains of three VPs in the asymmetry unit. Bet
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