Artículos de revistas: "Empty virus like particles"

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Wu, Hui-Lin, Pei-Jer Chen, Jung-Jung Mu, Wei-Kuang Chi, Tso-Liang Kao, Lih-Hwa Hwang y Ding-Shinn Chen. "Assembly of Hepatitis Delta Virus-like Empty Particles in Yeast". Virology 236, n.º 2 (septiembre de 1997): 374–81. http://dx.doi.org/10.1006/viro.1997.8743.

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Li, T. C., Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda y T. Miyamura. "Expression and self-assembly of empty virus-like particles of hepatitis E virus." Journal of virology 71, n.º 10 (1997): 7207–13. http://dx.doi.org/10.1128/jvi.71.10.7207-7213.1997.

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Hainisch, Edmund K., Christoph Jindra, Reinhard Kirnbauer y Sabine Brandt. "Papillomavirus-Like Particles in Equine Medicine". Viruses 15, n.º 2 (25 de enero de 2023): 345. http://dx.doi.org/10.3390/v15020345.

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Papillomaviruses (PVs) are a family of small DNA tumor viruses that can induce benign lesions or cancer in vertebrates. The observation that animal PV capsid-proteins spontaneously self-assemble to empty, highly immunogenic virus-like particles (VLPs) has led to the establishment of vaccines that efficiently protect humans from specific PV infections and associated diseases. We provide an overview of PV-induced tumors in horses and other equids, discuss possible routes of PV transmission in equid species, and present recent developments aiming at introducing the PV VLP-based vaccine technology into equine medicine.

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Huynh, Nhung T., Emma L. Hesketh, Pooja Saxena, Yulia Meshcheriakova, You-Chan Ku, Linh T. Hoang, John E. Johnson, Neil A. Ranson, George P. Lomonossoff y Vijay S. Reddy. "Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus". Structure 24, n.º 4 (abril de 2016): 567–75. http://dx.doi.org/10.1016/j.str.2016.02.011.

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Richterová, Zuzana, David Liebl, Martin Horák, Zdena Palková, Jitka Štokrová, Pavel Hozák, Jan Korb y Jitka Forstová. "Caveolae Are Involved in the Trafficking of Mouse Polyomavirus Virions and Artificial VP1 Pseudocapsids toward Cell Nuclei". Journal of Virology 75, n.º 22 (15 de noviembre de 2001): 10880–91. http://dx.doi.org/10.1128/jvi.75.22.10880-10891.2001.

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ABSTRACT Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids (“empty” or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both “empty” vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

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Hord, M., W. Villalobos, A. V. Macaya-Lizano y C. Rivera. "Chayote Mosaic, a New Disease in Sechium edule Caused by a Tymovirus". Plant Disease 81, n.º 4 (abril de 1997): 374–78. http://dx.doi.org/10.1094/pdis.1997.81.4.374.

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A sap-transmissible virus was isolated from chayote (Sechium edule) in Costa Rica. Infected plants showed chlorotic spots and rings, and blotchy mosaics, which often coalesced to give a complete mosaic and leaf deformation. By electron microscopy, spherical virus-like particles of approximately 29 nm in diameter were visible, and cytological changes associated with the chloroplasts were observed. The virus particles sedimented in sucrose density gradients as two components, a top component of empty protein shells and a bottom component of electron-dense particles. Electrophoretic analysis showed a single-stranded RNA of approximately 5.7 kb and capsid protein (CP) subunits of ∼22 kDa. The virus was identified as a member of the tymovirus group on the basis of particle morphology, size, sedimentation in sucrose gradients, cytopathological effects, and capsid protein and genome properties, and it was tentatively named chayote mosaic virus (ChMV).

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Ammar, E. D., R. E. Gingery y L. R. Nault. "Cytopathology and ultrastructure of mild and severe strains of maize chlorotic dwarf virus in maize and johnsongrass". Canadian Journal of Botany 71, n.º 5 (1 de mayo de 1993): 718–24. http://dx.doi.org/10.1139/b93-083.

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In maize leaves experimentally infected with various isolates or strains of maize chlorotic dwarf virus, including a newly characterized strain (M1), and in naturally infected johnsongrass, only two types of cytoplasmic inclusions were consistently observed: (i) quasi-spherical electron-dense granular inclusions, and (ii) curved or straight bundles of fibrous inclusions. Both types were detected by light and (or) electron microscopy in vascular parenchyma and phloem cells, and less frequently in bundle-sheath and adjacent mesophyll cells. The dense granular inclusions usually contained numerous isometric virus-like particles, some of which may have been released into the surrounding cytoplasm. However, a high proportion of these inclusions in cells infected with the mild type strain and a type-like isolate (M8) were either devoid of or contained very few viruslike particles. In maize leaves infected with the white stripe (WS) isolate, the chloroplasts were markedly deformed; in leaves of stunted plants doubly infected with M8 and the serologically distinct M1 strain, some phloem cells appeared degenerated. Electron microscopy of preparations of purified M1 stained with uranyl acetate revealed both stain-impenetrable full particles and stain-penetrable empty or partially empty particles. Both full and apparently empty particles were also found in cells of maize leaves infected with M1, whereas with other strains and isolates, mainly full particles were found both in situ and in vitro. Key words: maize chlorotic dwarf virus, cytopathology, ultrastructure, maize, johnsongrass.

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Bardi, Giuseppe. "Nanometric Virus-Like Particles: Key Tools for Vaccine and Adjuvant Technology". Vaccines 8, n.º 3 (31 de julio de 2020): 430. http://dx.doi.org/10.3390/vaccines8030430.

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The ideal vaccine should trigger a specific response against pathogens and induce the immune system memory to be prepared for eventual following infections. Although different approaches to develop new vaccines are currently taken, several of the features of natural pathogens that allow a tailored immune reaction are difficult to mimic. The viral capsids are the physical interface between a virus and the host defense machinery which recognizes specific patterns of the viral supramolecular complexes. Therefore, empty viral particles deprived of their genomes represent optimal targets to induce immune reactions with several advantages for vaccination and adjuvant realization.

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Suárez, Cristina, María L. Salas y Javier M. Rodríguez. "African Swine Fever Virus Polyprotein pp62 Is Essential for Viral Core Development". Journal of Virology 84, n.º 1 (21 de octubre de 2009): 176–87. http://dx.doi.org/10.1128/jvi.01858-09.

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ABSTRACT One of the most characteristic features of African swine fever virus gene expression is its use of two polyproteins, pp220 and pp62, to produce several structural proteins that account for approximately 32% of the total protein virion mass. Equimolecular amounts of these proteins are the major components of the core shell, a thick protein layer that lies beneath the inner envelope, surrounding the viral nucleoid. Polyprotein pp220, which is located immediately underneath the internal envelope, is essential for the encapsidation of the core of the viral particle. In its absence, the infection produces essentially coreless particles. In this study we analyzed, by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible virus, the role of polyprotein pp62 in virus assembly. Polyprotein pp62 is indispensable for viral replication. The repression of polyprotein pp62 expression does not alter late gene expression or the proteolytic processing of the polyprotein pp220. However, it has a profound impact on the subcellular localization of polyprotein pp220. Electron microscopy studies revealed that polyprotein pp62 is necessary for the correct assembly and maturation of the core of the viral particle. Its repression leads to the appearance of a significant fraction of empty particles, to an increase in the number of immature-like particles, and to the accumulation of defective particles. Immunoelectron microscopy analysis showed a clear correlation between the amount of polyprotein pp62, the quantity of polyprotein pp220, and the state of development of the core, suggesting that the complete absence of polyprotein pp62 during morphogenesis would produce a homogenous population of empty particles.

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Ren, Jingshan, Xiangxi Wang, Ling Zhu, Zhongyu Hu, Qiang Gao, Pan Yang, Xuemei Li et al. "Structures of Coxsackievirus A16 Capsids with Native Antigenicity: Implications for Particle Expansion, Receptor Binding, and Immunogenicity". Journal of Virology 89, n.º 20 (12 de agosto de 2015): 10500–10511. http://dx.doi.org/10.1128/jvi.01102-15.

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ABSTRACTEnterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production.IMPORTANCEHand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles, produced in insect cells (a system suitable for making vaccine antigen), are stabilized by recruiting from the insect cells a small molecule that is different from that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug design and vaccine development.

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Shanks, Michael y George P. Lomonossoff. "Co-expression of the capsid proteins of Cowpea mosaic virus in insect cells leads to the formation of virus-like particles". Journal of General Virology 81, n.º 12 (1 de diciembre de 2000): 3093–97. http://dx.doi.org/10.1099/0022-1317-81-12-3093.

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The regions of RNA-2 of Cowpea mosaic virus (CPMV) that encode the Large (L) and Small (S) coat proteins were expressed either individually or together in Spodoptera frugiperda (sf21) cells using baculovirus vectors. Co-expression of the two coat proteins from separate promoters in the same construct resulted in the formation of virus-like particles whose morphology closely resembled that of native CPMV virions. No such particles were formed when the individual L and S proteins were expressed. Sucrose gradient centrifugation of the virus-like particles showed that they had the sedimentation characteristics of empty (protein-only) shells. The results confirm that the 60 kDa L–S fusion is not an obligate intermediate in the virion assembly pathway and indicate that expression of the coat proteins in insect cells will provide a fruitful route for the study of CPMV morphogenesis.

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Meshcheriakova, Yulia, Alex Durrant, Emma L. Hesketh, Neil A. Ranson y George P. Lomonossoff. "Combining high-resolution cryo-electron microscopy and mutagenesis to develop cowpea mosaic virus for bionanotechnology". Biochemical Society Transactions 45, n.º 6 (3 de noviembre de 2017): 1263–69. http://dx.doi.org/10.1042/bst20160312.

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Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as nanoparticles. The development of a system for producing empty virus-like particles (eVLPs) of the virus, which are non-infectious and have the potential to be loaded with heterologous material, has increased the number of possible applications for CPMV-based particles. However, for this potential to be realised, it was essential to demonstrate that eVLPs were accurate surrogates for natural virus particles, and this information was provided by high-resolution cryo-EM studies of eVLPs. This demonstration has enabled the approaches developed for the production of modified particles developed with natural CPMV particles to be applied to eVLPs. Furthermore, a combination of cryo-EM and mutagenic studies allowed the development of particles which are permeable but which could still assemble efficiently. These particles were shown to be loadable with cobalt, indicating that they can, indeed, be used as nano-containers.

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Li, Tian-Cheng, Naokazu Takeda, Tatsuo Miyamura, Yoshiharu Matsuura, Joseph C. Y. Wang, Helena Engvall, Lena Hammar, Li Xing y R. Holland Cheng. "Essential Elements of the Capsid Protein for Self-Assembly into Empty Virus-Like Particles of Hepatitis E Virus". Journal of Virology 79, n.º 20 (15 de octubre de 2005): 12999–3006. http://dx.doi.org/10.1128/jvi.79.20.12999-13006.2005.

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ABSTRACT Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.

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Kimura, Tatsuji, Nobuhiko Ohno, Nobuo Terada, Akinori Rokuhara, Akihiro Matsumoto, Shintaro Yagi, Eiji Tanaka, Kendo Kiyosawa, Shinichi Ohno y Noboru Maki. "Hepatitis B Virus DNA-negative Dane Particles Lack Core Protein but Contain a 22-kDa Precore Protein without C-terminal Arginine-rich Domain". Journal of Biological Chemistry 280, n.º 23 (4 de abril de 2005): 21713–19. http://dx.doi.org/10.1074/jbc.m501564200.

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DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.

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Ericson, Brad L., Darby J. Carlson y Kimberly A. Carlson. "Characterization of Nora Virus Structural Proteins via Western Blot Analysis". Scientifica 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/9067848.

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Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infectedDrosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera.ORF3,ORF4a,ORF4b, andORF4cwere individually cloned and expressed inE. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infectedD. melanogasterlysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

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Bertioli, D. J., R. D. Harris, M. L. Edwards, J. I. Cooper y W. S. Hawes. "Transgenic Plants and Insect Cells Expressing the Coat Protein of Arabis Mosaic Virus Produce Empty Virus-like Particles". Journal of General Virology 72, n.º 8 (1 de agosto de 1991): 1801–9. http://dx.doi.org/10.1099/0022-1317-72-8-1801.

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Yuste-Calvo, Carmen, Pablo Ibort, Flora Sánchez y Fernando Ponz. "Turnip Mosaic Virus Coat Protein Deletion Mutants Allow Defining Dispensable Protein Domains for ‘in Planta’ eVLP Formation". Viruses 12, n.º 6 (19 de junio de 2020): 661. http://dx.doi.org/10.3390/v12060661.

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The involvement of different structural domains of the coat protein (CP) of turnip mosaic virus, a potyvirus, in establishing and/or maintaining particle assembly was analyzed through deletion mutants of the protein. In order to identify exclusively those domains involved in protein–protein interactions within the particle, the analysis was performed by agroinfiltration “in planta”, followed by the assessment of CP accumulation in leaves and the assembly of virus-like particles lacking nucleic acids, also known as empty virus-like particles (eVLP). Thus, the interactions involving viral RNA could be excluded. It was found that deletions precluding eVLP assembly did not allow for protein accumulation either, probably indicating that non-assembled CP protein was degraded in the plant leaves. Deletions involving the CP structural core were incompatible with particle assembly. On the N-terminal domain, only the deletion avoiding the subdomain involved in interactions with other CP subunits was incorporated into eVLPs. The C-terminal domain was shown to be more permissive to deletions. Assembled eVLPs were found for mutants, eliminating the whole domain. The C-terminal domain mutants were unusually long, suggesting some role of the domain in the regulation of particle length. The identification of the CP domains responsible for eVLP formation will allow for new approaches to protein stretch replacement with peptides or proteins of nanobiotechnological interest. Finally, specific cases of application are considered.

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Klut, M. Emilia y John G. Stockner. "Virus-Like Particles in an Ultra-Oligotrophic Lake on Vancouver Island, British Columbia". Canadian Journal of Fisheries and Aquatic Sciences 47, n.º 4 (1 de abril de 1990): 725–30. http://dx.doi.org/10.1139/f90-082.

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Transmission electron microscopy (TEM) seasonal studies of concentrated water samples from Sproat Lake, Vancouver Island, British Columbia, revealed numerous polygonal virus-like particles of variable size (60–200 nm). These particles (ca. 107/mL) were either free-living or associated with host picoplankters. Negative staining of living samples provides clear evidence of early stages of phage–picoplankton interactions. These phages display a six-sided head (ca. 90 nm dia.) with a distinct appendage (ca. 200 nm) or striated tail (ca. 130 nm). Viruses with dense matrices, deprived of envelopes or occurring as empty shells were found in the marginal area of invaded cells. Morphological changes such as invagination of the photosynthetic lamellae with the appearance of 'virogenic stroma' or with disruption of the cell membrane and the cell wall are described. Comments on the possible functional significance of viral agents in the biology and ecology of host cells are presented.

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Kim, Hyun-Soon, Jae-Heung Jeon, Kyung Jin Lee y Kisung Ko. "N-Glycosylation Modification of Plant-Derived Virus-Like Particles: An Application in Vaccines". BioMed Research International 2014 (2014): 1–8. http://dx.doi.org/10.1155/2014/249519.

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Plants have been developed as an alternative system to mammalian cells for production of recombinant prophylactic or therapeutic proteins for human and animal use. Effective plant expression systems for recombinant proteins have been established with the optimal combination of gene expression regulatory elements and control of posttranslational processing of recombinant glycoproteins. In plant, virus-like particles (VLPs), viral “empty shells” which maintain the same structural characteristics of virions but are genome-free, are considered extremely promising as vaccine platforms and therapeutic delivery systems. Unlike microbial fermentation, plants are capable of carrying outN-glycosylation as a posttranslational modification of glycoproteins. Recent advances in the glycoengineering in plant allow human-like glycomodification and optimization of desired glycan structures for enhancing safety and functionality of recombinant pharmaceutical glycoproteins. In this review, the current plant-derived VLP approaches are focused, andN-glycosylation and its in planta modifications are discussed.

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Granato, Marisa, Regina Feederle, Antonella Farina, Roberta Gonnella, Roberta Santarelli, Birgit Hub, Alberto Faggioni y Henri-Jacques Delecluse. "Deletion of Epstein-Barr Virus BFLF2 Leads to Impaired Viral DNA Packaging and Primary Egress as Well as to the Production of Defective Viral Particles". Journal of Virology 82, n.º 8 (20 de febrero de 2008): 4042–51. http://dx.doi.org/10.1128/jvi.02436-07.

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ABSTRACT Previous genetic and biochemical studies performed with several members of the Alphaherpesvirus subfamily have shown that the UL31 and UL34 proteins are essential components of the molecular machinery that mediates the primary egress of newly assembled capsids across the nuclear membrane. Further, there is substantial evidence that BFLF2 and BFRF1, the respective positional homologs of UL31 and UL34 in the Epstein-Barr virus (EBV) genome, are also their functional homologs, i.e., that the UL31/UL34 pathway is common to distant herpesviruses. However, the low degree of protein sequence identity between UL31 and BFLF2 would argue against such a hypothesis. To further clarify this issue, we have constructed a recombinant EBV strain devoid of BFLF2 (ΔBFLF2) and show that BFLF2 is crucial for efficient virus production but not for lytic DNA replication or B-cell transformation. This defective phenotype could be efficiently restored by trans complementation with a BFLF2 expression plasmid. Detailed analysis of replicating cells by electron microscopy revealed that, as expected, ΔBFLF2 viruses not only failed to egress from the nucleus but also showed defective DNA packaging. Nonfunctional primary egress did not, however, impair the production and extracellular release of enveloped but empty viral particles that comprised L particles containing tegument-like structures and a few virus-like particles carrying empty capsids. The ΔBFLF2 and ΔUL31 phenotypes therefore only partly overlap, from which we infer that BFLF2 and UL31 have substantially diverged during evolution to fulfil related but distinct functions.

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Ren, Yupeng, Sek-Man Wong y Lee-Yong Lim. "In vitro-reassembled plant virus-like particles for loading of polyacids". Journal of General Virology 87, n.º 9 (1 de septiembre de 2006): 2749–54. http://dx.doi.org/10.1099/vir.0.81944-0.

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The coat protein (CP) of certain plant viruses may reassemble into empty virus-like particles (VLPs) and these protein cages may serve as potential drug delivery platforms. In this paper, the production of novel VLPs from the Hibiscus chlorotic ringspot virus (HCRSV) is reported and the capacity to load foreign materials was characterized. VLPs were readily produced by destabilizing the HCRSV in 8 M urea or Tris buffer pH 8, in the absence of calcium ions, followed by removal of viral RNA by ultrahigh-speed centrifugation and the reassembly of the CP in sodium acetate buffer pH 5. The loading of foreign materials into the VLPs was dependent on electrostatic interactions. Anionic polyacids, such as polystyrenesulfonic acid and polyacrylic acid, were successfully loaded but neutrally charged dextran molecules were not. The molecular-mass threshold for the polyacid cargo was about 13 kDa, due to the poor retention of smaller molecules, which readily diffused through the holes between the S domains present on the surface of the VLPs. These holes precluded the entry of large molecules, but allowed smaller molecules to enter or exit. The polyacid-loaded VLPs had comparable size, morphology and surface-charge density to the native HCRSV, and the amount of polyacids loaded was comparable to the weight of the native genomic materials. The conditions applied to disassembly–reassembly of the virions did not change the structural conformation of the CP. HCRSV-derived VLPs may provide a promising nano-sized protein cage for delivery of anionic drug molecules.

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Strugała, Aleksander, Jakub Jagielski, Karol Kamel, Grzegorz Nowaczyk, Marcin Radom, Marek Figlerowicz y Anna Urbanowicz. "Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System". International Journal of Molecular Sciences 22, n.º 6 (18 de marzo de 2021): 3098. http://dx.doi.org/10.3390/ijms22063098.

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Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

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Sakaguchi, Takemasa, Atsushi Kato, Fumihiro Sugahara, Yukie Shimazu, Makoto Inoue, Katsuhiro Kiyotani, Yoshiyuki Nagai y Tetsuya Yoshida. "AIP1/Alix Is a Binding Partner of Sendai Virus C Protein and Facilitates Virus Budding". Journal of Virology 79, n.º 14 (julio de 2005): 8933–41. http://dx.doi.org/10.1128/jvi.79.14.8933-8941.2005.

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ABSTRACT The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix.

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Li, Tian-Cheng, Kumiko Yoshimatsu, Shumpei P. Yasuda, Jiro Arikawa, Takaaki Koma, Michiyo Kataoka, Yasushi Ami et al. "Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses". Journal of General Virology 92, n.º 12 (1 de diciembre de 2011): 2830–37. http://dx.doi.org/10.1099/vir.0.034835-0.

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Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.

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Patient, Romuald, Christophe Hourioux, Pierre-Yves Sizaret, Sylvie Trassard, Camille Sureau y Philippe Roingeard. "Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking". Journal of Virology 81, n.º 8 (31 de enero de 2007): 3842–51. http://dx.doi.org/10.1128/jvi.02741-06.

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ABSTRACT Hepatitis B virus (HBV) is unusual in that its surface proteins (small [S], medium, and large [L]) are not only incorporated into the virion envelope but they also bud into empty subviral particles in great excess over virions. The morphogenesis of these subviral envelope particles remains unclear, but the S protein is essential and sufficient for budding. We show here that, in contrast to the presumed model, the HBV subviral particle formed by the S protein self-assembles into branched filaments in the lumen of the endoplasmic reticulum (ER). These long filaments are then folded and bridged for packing into crystal-like structures, which are then transported by ER-derived vesicles to the ER-Golgi intermediate compartment (ERGIC). Within the ERGIC, they are unpacked and relaxed, and their size and shape probably limits further progression through the secretory pathway. Such progression requires their conversion into spherical particles, which occurred spontaneously during the purification of these filaments by affinity chromatography. Small branched filaments are also formed by the L protein in the ER lumen, but these filaments are not packed into transport vesicles. They are transported less efficiently to the ERGIC, potentially accounting for the retention of the L protein within cells. These findings shed light on an important step in the HBV infectious cycle, as the intracellular accumulation of HBV subviral filaments may be directly linked to viral pathogenesis.

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Li, Tian-Cheng, Jing Zhang, Haruhide Shinzawa, Masamichi Ishibashi, Michio Sata, Eric E. Mast, Kisoon Kim, Tatsuo Miyamura y Naokazu Takeda. "Empty virus-like particle-based enzyme-linked immunosorbent assay for antibodies to hepatitis E virus". Journal of Medical Virology 62, n.º 3 (2000): 327–33. http://dx.doi.org/10.1002/1096-9071(200011)62:3<327::aid-jmv4>3.0.co;2-1.

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Srivastava, Vartika, Kripa N. Nand, Aijaz Ahmad y Ravinder Kumar. "Yeast-Based Virus-like Particles as an Emerging Platform for Vaccine Development and Delivery". Vaccines 11, n.º 2 (18 de febrero de 2023): 479. http://dx.doi.org/10.3390/vaccines11020479.

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Virus-like particles (VLPs) are empty, nanoscale structures morphologically resembling viruses. Internal cavity, noninfectious, and particulate nature with a high density of repeating epitopes, make them an ideal platform for vaccine development and drug delivery. Commercial use of Gardasil-9 and Cervarix showed the usefulness of VLPs in vaccine formulation. Further, chimeric VLPs allow the raising of an immune response against different immunogens and thereby can help reduce the generation of medical or clinical waste. The economically viable production of VLPs significantly impacts their usage, application, and availability. To this end, several hosts have been used and tested. The present review will discuss VLPs produced using different yeasts as fermentation hosts. We also compile a list of studies highlighting the expression and purification of VLPs using a yeast-based platform. We also discuss the advantages of using yeast to generate VLPs over other available systems. Further, the issues or limitations of yeasts for producing VLPs are also summarized. The review also compiles a list of yeast-derived VLP-based vaccines that are presently in public use or in different phases of clinical trials.

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Kerstetter-Fogle, Shukla, Wang, Beiss, Harris, Sloan y Steinmetz. "Plant Virus-Like Particle In Situ Vaccine for Intracranial Glioma Immunotherapy". Cancers 11, n.º 4 (10 de abril de 2019): 515. http://dx.doi.org/10.3390/cancers11040515.

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Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.

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Zamani-Babgohari, Mahbobeh, Kathleen L. Hefferon, Tsu Huang y Mounir G. AbouHaidar. "How Computational Epitope Mapping Identifies the Interactions between Nanoparticles Derived from Papaya Mosaic Virus Capsid Proteins and Immune System". Current Genomics 20, n.º 3 (22 de julio de 2019): 214–25. http://dx.doi.org/10.2174/1389202920666190527080230.

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Background: Nanoparticles derived from plant viruses possess fascinating structures, versatile functions and safe properties, rendering them valuable for a variety of applications. Papaya mosaic Virus-Like Particles (VLPs) are nanoparticles that contain a repetitive number of virus capsid proteins (PMV-CP) and are considered to be promising platforms for vaccine design. Previous studies have reported the antigenicity of PMV nanoparticles in mammalian systems. Materials and Methods: As experiments that concern vaccine development require careful design and can be time consuming, computational experiments are of particular importance. Therefore, prior to expressing PMV-CP in E. coli and producing nanoparticles, we performed an in silico analysis of the virus particles using software programs based on a series of sophisticated algorithms and modeling networks as useful tools for vaccine design. A computational study of PMV-CP in the context of the immune system reaction allowed us to clarify particle structure and other unknown features prior to their introduction in vitro. Results: The results illustrated that the produced nanoparticles can trigger an immune response in the absence of fusion with any foreign antigen. Conclusion: Based on the in silico analyses, the empty capsid protein was determined to be recognised by different B and T cells, as well as cells which carry MHC epitopes.

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Dalba, Charlotte, Bertrand Bellier, Noriyuki Kasahara y David Klatzmann. "Replication-competent Vectors and Empty Virus-like Particles: New Retroviral Vector Designs for Cancer Gene Therapy or Vaccines". Molecular Therapy 15, n.º 3 (marzo de 2007): 457–66. http://dx.doi.org/10.1038/sj.mt.6300054.

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Chen, Chao, Joseph Che-Yen Wang, Elizabeth E. Pierson, David Z. Keifer, Mildred Delaleau, Lara Gallucci, Christian Cazenave, Michael Kann, Martin F. Jarrold y Adam Zlotnick. "Importin β Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes". PLOS Pathogens 12, n.º 8 (12 de agosto de 2016): e1005802. http://dx.doi.org/10.1371/journal.ppat.1005802.

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Goldmann, Claudia, Harald Petry, Stephan Frye, Oliver Ast, Susanne Ebitsch, Klaus-Dieter Jentsch, Franz-Josef Kaup et al. "Molecular Cloning and Expression of Major Structural Protein VP1 of the Human Polyomavirus JC Virus: Formation of Virus-Like Particles Useful for Immunological and Therapeutic Studies". Journal of Virology 73, n.º 5 (1 de mayo de 1999): 4465–69. http://dx.doi.org/10.1128/jvi.73.5.4465-4469.1999.

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ABSTRACT The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.

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Rieser, Ruth, Johanna Koch, Greta Faccioli, Klaus Richter, Tim Menzen, Martin Biel, Gerhard Winter y Stylianos Michalakis. "Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors". Pharmaceutics 13, n.º 5 (18 de mayo de 2021): 748. http://dx.doi.org/10.3390/pharmaceutics13050748.

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Recombinant adeno-associated virus (rAAV) vectors have evolved as one of the most promising technologies for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in nondividing cells. rAAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. There is a high demand for efficient and scalable production and purification methods for rAAVs. This is particularly true for the downstream purification methods. The current standard methods are based on multiple steps of gradient ultracentrifugation, which allow for the purification and enrichment of full rAAV particles, but the scale up of this method is challenging. Here, we explored fast, scalable, and universal liquid chromatography-based strategies for the purification of rAAVs. In contrast to the hydrophobic interaction chromatography (HIC), where a substantial amount of AAV was lost, the cation exchange chromatography (CEX) was performed robustly for multiple tested serotypes and resulted in a mixture of full and empty rAAVs with a good purity profile. For the used affinity chromatography (AC), a serotype dependence was observed. Anion exchange chromatography (AEX) worked well for the AAV8 serotype and achieved high levels of purification and a baseline separation of full and empty rAAVs. Depending on the AAV serotype, a combination of CEX and AEX or AC and AEX is recommended and holds promise for future translational projects that require highly pure and full particle-enriched rAAVs.

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Weerachatyanukul, Wattana, Pauline Kiatmetha, Ponlawoot Raksat, Supawich Boonkua, Orawan Thongsum, Pitchanee Jariyapong, Charoonroj Chotwiwatthanakun, Puey Ounjai y Zoltan Metlagel. "Viral Capsid Change upon Encapsulation of Double-Stranded DNA into an Infectious Hypodermal and Hematopoietic Necrosis Virus-like Particle". Viruses 15, n.º 1 (30 de diciembre de 2022): 110. http://dx.doi.org/10.3390/v15010110.

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In this study, we aimed to encapsulate the sizable double-stranded DNA (dsDNA, 3.9 kbp) into a small-sized infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP; T = 1) and compared the changes in capsid structure between dsDNA-filled VLP and empty VLP. Based on our encapsulation protocol, IHHNV-VLP was able to load dsDNA at an efficiency of 30–40% (w/w) into its cavity. Structural analysis revealed two subclasses of IHHNV-VLP, so-called empty and dsDNA-filled VLPs. The three-dimensional (3D) structure of the empty VLP produced in E. coli was similar to that of the empty IHHNV-VLP produced in Sf9 insect cells. The size of the dsDNA-filled VLP was slightly bigger (50 Å) than its empty VLP counterpart; however, the capsid structure was drastically altered. The capsid was about 1.5-fold thicker due to the thickening of the capsid interior, presumably from DNA–capsid interaction evident from capsid protrusions or nodules on the interior surface. In addition, the morphological changes of the capsid exterior were particularly observed in the vicinity of the five-fold axes, where the counter-clockwise twisting of the “tripod” structure at the vertex of the five-fold channel was evident, resulting in a widening of the channel’s opening. Whether these capsid changes are similar to virion capsid maturation in the host cells remains to be investigated. Nevertheless, the ability of IHHNV-VLP to encapsulate the sizable dsDNA has opened up the opportunity to package a dsDNA vector that can insert exogenous genes and target susceptible shrimp cells in order to halt viral infection.

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Depta, Philipp Nicolas, Maksym Dosta, Wolfgang Wenzel, Mariana Kozlowska y Stefan Heinrich. "Hierarchical Coarse-Grained Strategy for Macromolecular Self-Assembly: Application to Hepatitis B Virus-Like Particles". International Journal of Molecular Sciences 23, n.º 23 (24 de noviembre de 2022): 14699. http://dx.doi.org/10.3390/ijms232314699.

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Macromolecular self-assembly is at the basis of many phenomena in material and life sciences that find diverse applications in technology. One example is the formation of virus-like particles (VLPs) that act as stable empty capsids used for drug delivery or vaccine fabrication. Similarly to the capsid of a virus, VLPs are protein assemblies, but their structural formation, stability, and properties are not fully understood, especially as a function of the protein modifications. In this work, we present a data-driven modeling approach for capturing macromolecular self-assembly on scales beyond traditional molecular dynamics (MD), while preserving the chemical specificity. Each macromolecule is abstracted as an anisotropic object and high-dimensional models are formulated to describe interactions between molecules and with the solvent. For this, data-driven protein–protein interaction potentials are derived using a Kriging-based strategy, built on high-throughput MD simulations. Semi-automatic supervised learning is employed in a high performance computing environment and the resulting specialized force-fields enable a significant speed-up to the micrometer and millisecond scale, while maintaining high intermolecular detail. The reported generic framework is applied for the first time to capture the formation of hepatitis B VLPs from the smallest building unit, i.e., the dimer of the core protein HBcAg. Assembly pathways and kinetics are analyzed and compared to the available experimental observations. We demonstrate that VLP self-assembly phenomena and dependencies are now possible to be simulated. The method developed can be used for the parameterization of other macromolecules, enabling a molecular understanding of processes impossible to be attained with other theoretical models.

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Saunders, Keith, Frank Sainsbury y George P. Lomonossoff. "Efficient generation of cowpea mosaicvirus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants". Virology 393, n.º 2 (octubre de 2009): 329–37. http://dx.doi.org/10.1016/j.virol.2009.08.023.

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Duran-Meza, A. L., M. V. Villagrana-Escareño, J. Ruiz-García, C. M. Knobler y W. M. Gelbart. "Controlling the surface charge of simple viruses". PLOS ONE 16, n.º 9 (10 de septiembre de 2021): e0255820. http://dx.doi.org/10.1371/journal.pone.0255820.

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The vast majority of plant viruses are unenveloped, i.e., they lack a lipid bilayer that is characteristic of most animal viruses. The interactions between plant viruses, and between viruses and surfaces, properties that are essential for understanding their infectivity and to their use as bionanomaterials, are largely controlled by their surface charge, which depends on pH and ionic strength. They may also depend on the charge of their contents, i.e., of their genes or–in the instance of virus-like particles–encapsidated cargo such as nucleic acid molecules, nanoparticles or drugs. In the case of enveloped viruses, the surface charge of the capsid is equally important for controlling its interaction with the lipid bilayer that it acquires and loses upon leaving and entering host cells. We have previously investigated the charge on the unenveloped plant virus Cowpea Chlorotic Mottle Virus (CCMV) by measurements of its electrophoretic mobility. Here we examine the electrophoretic properties of a structurally and genetically closely related bromovirus, Brome Mosaic Virus (BMV), of its capsid protein, and of its empty viral shells, as functions of pH and ionic strength, and compare them with those of CCMV. From measurements of both solution and gel electrophoretic mobilities (EMs) we find that the isoelectric point (pI) of BMV (5.2) is significantly higher than that of CCMV (3.7), that virion EMs are essentially the same as those of the corresponding empty capsids, and that the same is true for the pIs of the virions and of their cleaved protein subunits. We discuss these results in terms of current theories of charged colloidal particles and relate them to biological processes and the role of surface charge in the design of new classes of drug and gene delivery systems.

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Li, Haozhou, Aldo Dekker, Shiqi Sun, Alison Burman, Jeroen Kortekaas y Michiel M. Harmsen. "Novel Capsid-Specific Single-Domain Antibodies with Broad Foot-and-Mouth Disease Strain Recognition Reveal Differences in Antigenicity of Virions, Empty Capsids, and Virus-Like Particles". Vaccines 9, n.º 6 (8 de junio de 2021): 620. http://dx.doi.org/10.3390/vaccines9060620.

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Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs.

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Sokullu, Esen, Hoda Soleymani Abyaneh y Marc A. Gauthier. "Plant/Bacterial Virus-Based Drug Discovery, Drug Delivery, and Therapeutics". Pharmaceutics 11, n.º 5 (3 de mayo de 2019): 211. http://dx.doi.org/10.3390/pharmaceutics11050211.

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Viruses have recently emerged as promising nanomaterials for biotechnological applications. One of the most important applications of viruses is phage display, which has already been employed to identify a broad range of potential therapeutic peptides and antibodies, as well as other biotechnologically relevant polypeptides (including protease inhibitors, minimizing proteins, and cell/organ targeting peptides). Additionally, their high stability, easily modifiable surface, and enormous diversity in shape and size, distinguish viruses from synthetic nanocarriers used for drug delivery. Indeed, several plant and bacterial viruses (e.g., phages) have been investigated and applied as drug carriers. The ability to remove the genetic material within the capsids of some plant viruses and phages produces empty viral-like particles that are replication-deficient and can be loaded with therapeutic agents. This review summarizes the current applications of plant viruses and phages in drug discovery and as drug delivery systems and includes a discussion of the present status of virus-based materials in clinical research, alongside the observed challenges and opportunities.

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Mani, Bernhard, Claudia Baltzer, Noelia Valle, José M. Almendral, Christoph Kempf y Carlos Ros. "Low pH-Dependent Endosomal Processing of the Incoming Parvovirus Minute Virus of Mice Virion Leads to Externalization of the VP1 N-Terminal Sequence (N-VP1), N-VP2 Cleavage, and Uncoating of the Full-Length Genome". Journal of Virology 80, n.º 2 (15 de enero de 2006): 1015–24. http://dx.doi.org/10.1128/jvi.80.2.1015-1024.2006.

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ABSTRACT Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.

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Silvestri, Lynn S., M. Alejandra Tortorici, Rodrigo Vasquez-Del Carpio y John T. Patton. "Rotavirus Glycoprotein NSP4 Is a Modulator of Viral Transcription in the Infected Cell". Journal of Virology 79, n.º 24 (15 de diciembre de 2005): 15165–74. http://dx.doi.org/10.1128/jvi.79.24.15165-15174.2005.

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ABSTRACT The outer shell of the rotavirus triple-layered virion is lost during cell entry, yielding a double-layered particle (DLP) that directs synthesis of viral plus-strand RNAs. The plus-strand RNAs act as templates for synthesis of the segmented double-stranded RNA (dsRNA) genome in viral inclusion bodies (viroplasms). The viral endoplasmic reticulum (ER)-resident glycoprotein NSP4 recruits progeny DLPs formed in viroplasms to the ER, where the particles are converted to triple-layered particles (TLPs) via budding. In this study, we have used short interfering RNAs to probe the role of NSP4 in the viral life cycle. Our analysis showed that knockdown of NSP4 expression had no marked effect on the expression of other viral proteins or on the replication of the dsRNA genome segments. However, NSP4 loss of function suppressed viroplasm maturation and caused a maldistribution of nonstructural and structural proteins that normally accumulate in viroplasms. NSP4 loss of function also inhibited formation of packaged virus particles, instead inducing the accumulation of empty particles. Most significant was the observation that NSP4 knockdown led to dramatically increased levels of viral transcription late in the infection cycle. These findings point to a multifaceted role for NSP4 in virus replication, including influencing the development of viroplasms, linking genome packaging with particle assembly, and acting as a modulator of viral transcription. By recruiting transcriptionally active or potentially active DLPs to the ER for conversion to quiescent TLPs, NSP4 acts as a feedback inhibitor down-regulating viral transcription when adequate levels of plus-strand RNAs are available to allow for productive infection.

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Sakaguchi, Takemasa, Tsuneo Uchiyama, Cheng Huang, Noriko Fukuhara, Katsuhiro Kiyotani, Yoshiyuki Nagai y Tetsuya Yoshida. "Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the Matrix Protein". Journal of Virology 76, n.º 4 (15 de febrero de 2002): 1682–90. http://dx.doi.org/10.1128/jvi.76.4.1682-1690.2002.

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ABSTRACT The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate virus from cDNA, SeV M-C83S, SeV M-C106S, and SeV M-C295S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C83S and SeV M-C106S had smaller virus particles than did the wild-type SeV, whereas SeV M-C295S had larger and heterogeneously sized particles. Furthermore, SeV M-C106S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle.

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de Souza, Theo Luiz Ferraz, Sheila Maria Barbosa de Lima, Vanessa L. de Azevedo Braga, David S. Peabody, Davis Fernandes Ferreira, M. Lucia Bianconi, Andre Marco de Oliveira Gomes, Jerson Lima Silva y Andréa Cheble de Oliveira. "Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein". PeerJ 4 (9 de noviembre de 2016): e2670. http://dx.doi.org/10.7717/peerj.2670.

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BackgroundHepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs)in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood.MethodsSpectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs.ResultsThe structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs.DiscussionTogether, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

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Cao, Yimei, Zengjun Lu, Jiachuan Sun, Xingwen Bai, Pu Sun, Huifang Bao, Yingli Chen et al. "Synthesis of empty capsid-like particles of Asia I foot-and-mouth disease virus in insect cells and their immunogenicity in guinea pigs". Veterinary Microbiology 137, n.º 1-2 (mayo de 2009): 10–17. http://dx.doi.org/10.1016/j.vetmic.2008.12.007.

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45

Werr, Margaret y Reinhild Prange. "Role for Calnexin and N-Linked Glycosylation in the Assembly and Secretion of Hepatitis B Virus Middle Envelope Protein Particles". Journal of Virology 72, n.º 1 (1 de enero de 1998): 778–82. http://dx.doi.org/10.1128/jvi.72.1.778-782.1998.

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ABSTRACT Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is required for efficient secretion of M empty envelope particles. Moreover, this glycan mediates specific association of M with the chaperone calnexin. Conversely, the N glycan, common to all three envelope proteins, is involved neither in calnexin binding nor in subviral particle release. As proper folding and trafficking of M need the assistance of the chaperone, the glycan-dependent association of M with calnexin may thus play a crucial role in the assembly of HBV. Beyond being modified by N glycosylation, M is modified by O glycosylation occurring within its amino acid sequence at positions 27 to 47. The O glycans, however, were found to be dispensable for secretion of M but may rather support viral infectivity. Surprisingly, nonglycosylated M localizes exclusively to the cytosol, either for degradation or for a yet-unknown function.

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46

Castón, José R., Jorge L. Martı́nez-Torrecuadrada, Antonio Maraver, Eleuterio Lombardo, José F. Rodrı́guez, J. Ignacio Casal y José L. Carrascosa. "C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly". Journal of Virology 75, n.º 22 (15 de noviembre de 2001): 10815–28. http://dx.doi.org/10.1128/jvi.75.22.10815-10828.2001.

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ABSTRACT Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.

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47

Kontou, Maria, Lakshmanan Govindasamy, Hyun-Joo Nam, Nathan Bryant, Antonio L. Llamas-Saiz, Concepción Foces-Foces, Eva Hernando et al. "Structural Determinants of Tissue Tropism and In Vivo Pathogenicity for the Parvovirus Minute Virus of Mice". Journal of Virology 79, n.º 17 (1 de septiembre de 2005): 10931–43. http://dx.doi.org/10.1128/jvi.79.17.10931-10943.2005.

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ABSTRACT Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMpb) and native wild-type (wt) empty capsids (MVMpe), determined and refined to 3.25 and 3.75 Å resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 Å resolution in this study. A comparison of the MVMpb and MVMpe capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the “shoulder” of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.

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48

Ahi, Yadvinder S., Sai V. Vemula y Suresh K. Mittal. "Adenoviral E2 IVa2 protein interacts with L4 33K protein and E2 DNA-binding protein". Journal of General Virology 94, n.º 6 (1 de junio de 2013): 1325–34. http://dx.doi.org/10.1099/vir.0.049346-0.

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Adenovirus (AdV) is thought to follow a sequential assembly pathway similar to that observed in dsDNA bacteriophages and herpesviruses. First, empty capsids are assembled, and then the genome is packaged through a ring-like structure, referred to as a portal, located at a unique vertex. In human AdV serotype 5 (HAdV5), the IVa2 protein initiates specific recognition of viral genome by associating with the viral packaging domain located between nucleotides 220 and 400 of the genome. IVa2 is located at a unique vertex on mature capsids and plays an essential role during genome packaging, most likely by acting as a DNA packaging ATPase. In this study, we demonstrated interactions among IVa2, 33K and DNA-binding protein (DBP) in virus-infected cells by in vivo cross-linking of HAdV5-infected cells followed by Western blot, and co-immunoprecipitation of IVa2, 33K and DBP from nuclear extracts of HAdV5-infected cells. Confocal microscopy demonstrated co-localization of IVa2, 33K and DBP in virus-infected cells and also in cells transfected with IVa2, 33K and DBP genes. Immunogold electron microscopy of purified HAdV5 showed the presence of IVa2, 33K or DBP at a single site on the virus particles. Our results provide indirect evidence that IVa2, 33K and DBP may form a complex at a unique vertex on viral capsids and cooperate in genome packaging.

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49

Lockhart, B. E. L. "Occurrence of Arabis mosaic virus in Hostas in the United States". Plant Disease 90, n.º 6 (junio de 2006): 834. http://dx.doi.org/10.1094/pd-90-0834c.

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Hostas (Hosta spp.) are one of the most widely grown and economically important landscape perennials in the nursery industry in North America. Several viruses including Hosta virus X (HVX), Tobacco rattle virus (TRV), Tobacco ringspot virus (ToRSV), Tomato ringspot virus (TomRSV), Impatiens necrotic spot virus (INSV), and Tomato spotted wilt virus (TSWV) are known to occur in hostas (4). This report confirms the occurrence of an additional virus, Arabis mosaic virus (ArMV), in hostas in North America. This virus was first identified during the summer of 2004 in Hosta fortunei ‘Sharmon’ in several garden centers in Minneapolis and St. Paul, MN. Entire lots of this variety, numbering several dozen plants, showed symptoms consisting of blanching of the foliage similar to those caused by ToRSV and TomRSV infection (4). Symptoms persisted throughout the growing season. Virus-like particles, 28 to 30 nm in diameter, were observed by electron microscopy in partially purified extracts of symptomatic leaf tissue following fixation with 5% glutaraldehyde and negative staining with 2% sodium phosphotungstate, pH 7.0. Particles had an angular outline and some were penetrated by stain. No other virus-like particles were observed in these extracts. The particles were identified as those of ArMV. Identification was made using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) with antiserum to ArMV (PVAS-587) obtained from the American Type Culture Collection, Manassas, VA. In the spring and summer of 2005, ArMV was again identified as described above in ‘Sharmon’, H. undulata ‘Albomarginata’ samples from Minnesota, Michigan, and Nebraska, and H. ‘Marion Bachman’ and H. ‘Touch of Class’ from two wholesale nurseries in Minnesota. Symptoms in these hosta cultivars were similar to those observed in ‘Sharmon’ and were accompanied by stunting and leaf deformation. A portion of the coat protein (CP) gene of the ArMV isolate from ‘Sharmon’, designated ArMV-H, was amplified using reverse transcription-polymerase chain reaction (RT-PCR) with ArMV-specific CP primers (3) and total RNA extracted with a RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Amplicons of the expected size (220 bp) were cloned and five clones were sequenced. Nucleotide sequence identities of the ArMV-H CP sequence to corresponding ArMV databank entries varied from 94 to 88% (Genbank Accession Nos. AY017339 and D10086 and X55460 and X81815, respectively). Interestingly, the hosta ArMV isolate was not transmitted by mechanical inoculation to diagnostically susceptible indicator plants (cucumber, tobacco, and petunia) (2) or to hosta (H. undulata ‘Albormarginata’, H. ‘Honeybells’, and H. ‘Royal Standard’). Testing by using ELISA and ISEM showed that ‘Sharmon’ source plants contained high levels of ArMV antigen and virions, and a high percentage of virions were not penetrated by negative stain, indicating that they were not empty (i.e., devoid of RNA). It appears that ArMV-H may be transmitted only vertically, (i.e., clonal propagation) and this raises some interesting questions about the molecular basis of this anomaly. An isolate of ArMV from hops was similarly reported to have a very restricted host range (1) suggesting a possibility of a common mechanism of host range restriction. References: (1) K. R. Bock. Ann. Appl. Biol. 57:431, 1966. (2) A. A. Brunt et al. Viruses of Plants. CAB Internacional Mycological Institute, Wallingford, UK, 1995. (3) P. Kominek et al. Acta Virol. 47:199, 2003. (4) B. E. L. Lockhart and S. Currier. Acta Hortic. 432:62, 1996.

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Xie, Yinli, Haitao Li, Xingcai Qi, Youji Ma, Bo Yang, Shumin Zhang, Huiyun Chang, Xiangping Yin y Zhiyong Li. "Immunogenicity and protective efficacy of a novel foot-and-mouth disease virus empty-capsid-like particle with improved acid stability". Vaccine 37, n.º 14 (marzo de 2019): 2016–25. http://dx.doi.org/10.1016/j.vaccine.2019.02.032.

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