1,721,275 research outputs found
Nonspreading Rift Valley fever virus: A potent and flexible vaccine platform
No full textRift Valley fever virus (RVFV) is a serious pathogen for both ruminants and humans. RVF outbreaks have a major impact on the agricultural and related sectors in affected areas with severe socio-economic consequences. Although the virus is confined to the African continent and the Arabian Peninsula, globalization, climate change and global prevalence of potential mosquito vectors constitute a significant risk for further spread to new areas. All these features explain the need for safe, effective and affordable vaccines for both endemic and yet unaffected areas. The aim of the work described in this thesis was to develop a RVF vaccine that optimally combines safety and efficacy. We report the first successful creation of RVFV replicon particles, referred to as nonspreading RVFV (NSR). NSR particles are able to infect new cells, resulting in autonomous genome replication. However, these particles lack the genes encoding surface glycoproteins, therefore no progeny particles can be produced. We demonstrate that a single vaccination with NSR provides full protection in mice and prevents clinical sings and significantly reduces viremia in lambs. Further improvement of NSR resulted in the development of NSR-Gn, containing the gene of one of the viral glycoproteins, Gn. Gn is the major target for neutralizing antibodies and its expression in NSR-Gn infected cells significantly improved immune responses, eliciting sterile protection in lambs. In addition to RVFV vaccine, we aimed to develop a vaccine vector platform suitable for mucosal administration. Many pathogens invade the body via mucosal surfaces. Therefore, vaccines that elicit protective immunity on mucosal surfaces are particularly valuable. As RVFV is very infectious via the respiratory route, we were especially interested in using NSR for respiratory application. As a model for respiratory infection, influenza was used. We developed NSR-HA, which contains the gene of influenza A virus hemagglutinin (HA), one of the major influenza A virus immunogens. A single intranasal vaccination of mice with NSR-HA completely prevented death and clinical signs induced by an influenza virus infection, revealing the potency of NSR as a vector platform suitable for administration via the respiratory mucosa. The efficacy of NSR as a viral vector vaccine for cancer immunotherapy was also studies. Our results in a mouse model show that NSR can reduce tumor growth and even eradicate tumors, suggesting that it is a promising vector for cancer immunotherapy. In our vaccination experiments we consistently observed a high efficaciousness of NSR-based vaccines. We were interested to unravel the molecular basis of the NSR-induced immune response. To that end we investigated the interaction between NSR and dendritic cells (DC). We observed that NSR-infected DCs undergo incomplete maturation, associated with a decrease in CD83, the most prominent marker for DC maturation. However, bystander DCs undergo complete maturation and upregulate CD83. Our results suggest that not only infected, but also bystander DCs play an important role in NSR-mediated immunity. Although we were unable to unravel the exact mechanism of the observed decrease of CD83 in NSR-infected cells, we established that it occurs at the level of protein translation
Tuning of influenza A virus neuraminidase activity
No full textInfluenza A viruses (IAVs) are zoonotic pathogens that constantly circulate in a wide variety of species, including birds, pigs and humans. In humans, IAVs cause seasonal epidemics and occasional influenza pandemics. Annual epidemics caused by seasonal IAVs usually lead to millions of human infections, posing great threats to public health and cause large economic burdens. Influenza pandemic occurs when animal viruses managed to cross the host species barrier and became transmissible among humans. IAV pandemics have occurred four times in the 100 years, causing millions of deaths and global devastating effects. IAVs are enveloped, segmented negative-strand RNA viruses belonging to the Orthomyxoviridae family. There are two main important glycoproteins in the IAV virus membrane, hemagglutinin (HA) and neuraminidase (NA), both of which recognize sialic acids (SIAs). The HA protein is responsible for virus-cell attachment via binding to sialylated receptors at the cell surface. The NA protein is the receptor-destroying enzyme and responsible for removing SIA from host glycoproteins as well as glycolipids, thereby allowing release of progeny virions from cells and decoy receptors and preventing virus self-aggregation. A functional balance between the HA and NA proteins is of importance for maintaining optimal virus replication as well as transmission across different host speciesADDIN RW.CITE{{36 Xu,R. 2012; 37 Yen,H.L. 2011; 38 de Wit,E. 2010}}. While HA receptor-binding avidity and specificity have been studied in detail, much less is known about the molecular determinants that mediate (changes in) the specificity and activity of IAV NA proteins. The overall aim of this thesis was to unravel to what extent and how IAVs modulate the activity of their NA proteins during virus evolution. To this end, NA functionality, mainly enzymatic activity has been extensively investigated by using an optimized recombinant soluble protein approach. By doing so, we not only identified an optimal recombinant soluble NA expression approach, but also identified residues that affected NA folding and/or enzymatic activity. With the established recombinant soluble approach, we further found an important role of the 2nd SIA-binding site in NA enzymatic activity. Mutation of the 2nd SIA binding site provides viruses with an additional mechanism to manipulate the enzymatic activity of their NA proteins without having to mutate their active site residues directly. The studies presented in this thesis also highlight some complexities of HA/NA balance during virus evolution. Different NA phenotypic properties of H1N1pdm09 virus were found to be intertwined, with several NA substitutions affecting more than one phenotypic characteristic. The phenotypic changes of NA are probably also linked to the properties of the HA protein and corresponding HA/NA balance, which makes evolution of HA and NA more difficult to understand
Sweet attachment: Structural and functional studies on nidovirus hemagglutinin-esterases
No full textThe thesis described our investigations in the fields of O-acetylsialoglycobiology and the structure and function of corona- and torovirus hemagglutininesterase (HE) proteins. There are three topics that cover the experimental contents of this thesis: i) the distribution and synthesis of O-Ac-sialoglycans ii) common principles underlying the structural recognition of O-Ac-Sia by viral proteins iii) the dynamic balance of virionglycan interactions by the opposing activities of attachment and release
Novel bluetongue vaccine platform: NS3/NS3a knockout virus as Disabled Infectious Single Animal (DISA) vaccine
No full textBluetongue (BT) is a disease of ruminants caused by the bluetongue virus (BTV) transmitted by bites of Culicoides midges. Bluetongue has a worldwide prevalence and mortality in sheep varies from 0 to 30%. There are at least 27 BTV serotypes showing no or little cross protection. In 2006, BTV has been reported in north-western Europe for the first time, and has caused a huge outbreak with large economic losses. BTV (family Reoviridae, genus Orbivirus) is a non-enveloped virus with a ten-segmented dsRNA genome encoding seven viral proteins (VP1-7) and four non-structural (NS1-4) proteins. VP2 is the outer capsid protein, and is the serotype specific target for neutralizing antibodies. NS3 and its N-terminal truncated form NS3a (Seg-10) enable virus release from infected cells. Currently, live-attenuated and inactivated BT vaccines have been marketed, but these have several shortcomings. Therefore there is a need for next-generation types of BT vaccines with improved vaccine profile. Here, such a novel vaccine platform based on BTV without NS3/NS3a expression is described. BTV based on lab adapted strain BTV1, vaccine related BTV6, and virulent BTV8 were equipped with a deletion in Seg-10 abolishing NS3/NS3a expression and with Seg-2 encoding serotype determining VP2 from serotype 8. All three vaccine candidates were not virulent in sheep and did not show viremia, whereas seroconversion was dependent on replication of vaccine virus. This suggests that BTV without NS3/NS3a replicates only locally. Sheep were completely protected against infection of virulent BTV8. In a second efficacy experiment in sheep, complete serotype specific protection was demonstrated at nine weeks after booster vaccination. A competitive ELISA based on NS3 antibodies enables the differentiation of infected from vaccinated animals (DIVA). Uptake of the NS3/NS3a knockout vaccine candidate by midges is highly unlikely, due to the absence of viremia after vaccination. This vaccine candidate has therefore been named the Disabled Infectious, Single Animal (DISA) vaccine. Additionally, replication of BT DISA vaccine in midges is abolished as shown after injection of Culicoides sonorensis midges. The BT DISA vaccine platform was also adapted for other BTV serotypes by exchange of Seg-2 (VP2) or by use of chimeric Seg-2 originating from two different serotypes. The latter showed a broader application of the vaccine platform and neutralizing responses against both ancestor serotypes. In conclusion, BT DISA vaccine is effective, safe, enables DIVA, and can be used for many serotypes by exchange of VP2
Exploring picornavirus receptors and uncoating mechanisms
No full textThe Picornaviridae are a large family of non-enveloped RNA viruses that cause a wide range of human and veterinary diseases. The life cycle of a picornavirus begins with binding to its cognate receptor(s), which mediates attachment to a target cell, promotes endocytic uptake and in many cases destabilizes the capsid to stimulate uncoating; that is the delivery of the viral genome from the virus particle into the cytoplasm of the host cell. In this thesis, we aimed to identify novel picornavirus receptors and to investigate the impact of receptor binding on virion structure, focusing on the emerging human pathogens enterovirus D68 (EV-D68) and coxsackievirus A24 variant (CV-A24v), and the animal pathogen encephalomyocarditis virus (EMCV). EV-D68 is an emerging virus that causes outbreaks of respiratory disease and is associated with cases of acute flaccid paralysis in children. We explored EV-D68 receptor requirements via a genome-wide haploid genetic screen, which identified sialic acid (Sia) as an essential receptor and showed that EV-D68 can employ both α2,3- and α2,6-linked Sia as receptors. By determining a crystal structure of the EV-D68 prototype Fermon in complex with sialylated trisaccharides, we identified the Sia-binding site on the virion and revealed that Sia binding induces structural rearrangements in the viral capsid that result in ejection of the pocket factor, a fatty acid that regulates virus stability. These findings first showed that a glycan receptor can initiate enterovirus uncoating, via a similar mechanism as proteinaceous receptors. Although several EV-D68 strains strictly required Sia for infection, other strains could infect cells via a non-sialylated receptor. Using a haploid screen in Sia-deficient cells we showed that a Sia-independent strain could employ both Sia and sulfated glycosaminoglycans (sGAGs) as functional receptors. Remarkably, this screen did not identify the phospholipase PLA2G16, a recently discovered picornavirus host factor that promotes viral RNA translocation into the cytoplasm. We showed that binding of sGAGs, but not Sia, allowed the virus to circumvent PLA2G16. Cryo-EM analysis revealed that sGAG binding promotes virus uncoating, via extensive capsid rearrangements that enlarge the putative openings for genome release. These findings suggested that the need for PLA2G16 can be overcome by excessive receptor-mediated virion destabilization. We explored the receptor usage of EMCV, a commonly used model virus that causes fatal disease in domestic animals. Using a haploid genetic screen, we identified genes involved in FGF signaling as novel host factors and showed that the potential receptor ADAM9 plays a role in EMCV entry. We also investigated CV-A24v, the main cause of acute hemorrhagic conjunctivitis (AHC). Originally, CV-A24 was not associated with a human disease, but in 1970 a pathogenic “variant” emerged that caused AHC outbreaks and two pandemics. We showed that ICAM-1 is an essential CV-A24 receptor and, via a high-resolution cryo-EM structure, identified residues mediating contact between ICAM-1 and the virion. Moreover, we identified a capsid substitution that occurs in all pandemic CV-A24v strains and enhances the capacity of CV-A24v to bind Sia, revealing a possible link between Sia and viral adaptation to the eye
Broad activity against porcine bacterial pathogens displayed by two insect antimicrobial peptides moricin and cecropin B
No full textIn response to infection, insects produce a variety of antimicrobial peptides (AMPs) to kill the invading pathogens. To study their physicochemical properties and bioactivities for clinical and commercial use in the porcine industry, we chemically synthesized the mature peptides Bombyx mori moricin and Hyalophora cecropia cecropin B. In this paper, we described the antimicrobial activity of the two AMPs. Moricin exhibited antimicrobial activity on eight strains tested with minimal inhibitory concentration values (MICs) ranging between 8 and 128 mu g/ml, while cecropin B mainly showed antimicrobial activity against the Gramnegative strains with MICs ranging from 0.5 to 16 mu g/ml. Compared to the potent antimicrobial activity these two AMPs displayed against most of the bacterial pathogens tested, they exhibited limited hemolytic activity against porcine red blood cells. The activities of moricin and cecropin B against Haemophilus parasuis SH 0165 were studied in further detail. Transmission electron microscopy (TEM) of moricin and cecropin B treated H. parasuis SH 0165 indicated extensive damage to the membranes of the bacteria. Insights into the probable mechanism utilized by moricin and cecropin B to eliminate pathogens are also presented. The observations from this study are important for the future application of AMPs in the porcine industry
Coronavirus particle assembly: primary structure requirements of the membrane protein
No full textCoronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly
Mapping of the coronavirus membrane protein domains involved in interaction with the spike protein
No full textThe coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes
9-Norbornyl-6-chloropurine (NCP) induces cell death through GSH depletion-associated ER stress and mitochondrial dysfunction
No full textAbstract9-Norbornyl-6-chloropurine (NCP) is a representative of a series of antienteroviral bicycle derivatives with selective cytotoxicity towards leukemia cell lines. In this work we explored the mechanism of the antileukemic activity of NCP in T-cell lymphoblast cells (CCRF-CEM). Specifically, we searched for a potential link between its ability to induce cell death on the one hand and to modulate intracellular glutathione (GSH) that is necessary to its metabolic transformation via glutathione-S-transferase on the other hand. We have observed that GSH levels decreased rapidly in NCP-treated cells. Despite a complete regeneration following 24h of incubation with NCP, this profound drop in cellular GSH content triggered ER stress, ROS production and lipid peroxidation leading to the loss of mitochondrial membrane potential (MMP). These events induced concentration-dependent cell cycle arrest in G2/M phase and apoptosis. Both MMP loss and apoptosis were reversed by sulfhydryl-containing compounds (GSH, N-acetyl-l-cysteine). Furthermore, we have also shown that NCP-induced GSH decrease activated the Nrf2 pathway and its downstream targets NAD(P)H:quinone oxidoreductase (NQO-1) and glutamate cysteine ligase modifier subunit (GCLm), thus explaining the fast restoration of GSH pool and ROS decrease. Importantly, we confirmed that the cell death-inducing properties of the compounds were co-dependent on their ability to diminish cellular GSH level by analyzing the relationships between the GSH-depleting potency and cytotoxicity in a series of other norbornylpurine analogs. Altogether, the results demonstrated that in CCRF-CEM cells NCP triggered apoptosis through GSH depletion-associated oxidative and ER stress and mitochondrial depolarization
Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion
No full textCleavage of the mouse hepatitis coronavirus strain A59 spike protein was blocked in a concentration-dependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements
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