134 research outputs found

    Genomic Organization of Infectious Salmon Anemia Virus

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    Infectious salmon anemia virus (ISAV) is an emerging pathogen of farmed Atlantic salmon (Salmo salar). The development of an effective ISA virus vaccine is a high priority for salmon producers in the U.S. and elsewhere. The process of developing a recombinant vaccine requires complete genetic characterization of the virus. Toward this end we have cloned, sequenced and determined the organization of the eight segments of single-stranded RNA from ISA virus isolate CCBB. The virus was grown in cell culture and purified by density gradient ultracentrifugation. Viral RNA was isolated from purified ISAV and used in the construction of two different cDNA libraries. After screening the libraries, individual ISA virus-specific cDNA clones were placed into eight groups and the DNA from a representative clone from each group was sequenced. Using Northern blot hybridization results, the eight representative clones were assigned to specific RNA segments of the ISA virus genome and a genetic map of ISA virus strain CCBB was constructed. In addition, N-terminal amino acid sequence analyses of purified ISA virus proteins correlated protein product(s) to specific RNA segments and provided evidence for protein synthesis initiation sites. Finally, Western blot analysis identified viral proteins that were irnmunoreactive with ISA virus-specific serum from mice and Atlantic salmon

    Bioengineering viral subunits for influenza vaccine development

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    Tilapia lake virus (TiLV) : utvikling av PCRbaserte diagnostiske metoder og studier av infeksjonsmekanismer

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    Tilapia lake virus (TiLV) is an emerging virus of wild and farmed tilapiines responsible for causing mortalities and significant economic losses to the aquaculture industry. Since its first report in Israel, the virus has been reported in four continents (Asia, Africa, South and North America) to cause mortalities ranging from the lower extreme of 5-20% and higher extremes of up to 90%. The infection status in many countries is not yet known and implementation of surveillance programs has been recommended. Lake Victoria was selected for TiLV surveillance because it contains a large number of tilapiine species of economic importance to the surrounding East African countries. Well-optimized tools for rapid detection and quantification of the virus are also needed. Moreover, the mode of virus uptake in cells and importance for replication is not known. Therefore, this thesis aimed at understanding the possible presence of TiLV in Lake Victoria in East Africa, to develop tools for detection and quantification of the virus and to shed light of virus uptake mechanisms in permissive cells in vitro. In this thesis, a standard RT-PCR and a quantitative real-time RT-PCR (qRT-PCR) were developed based on all the ten segments of TiLV. The standard PCR was used to screen Nile tilapia (Oreochromis niloticus) from Lake Victoria in Eastern Africa. The quantitative real-time RT-PCR was developed based on virus supernatant of known titre and against samples of unknown virus titre originating from infected TFC #10 cells and fish organs. The virus’ ability to hemagglutinate avian and piscine erythrocytes was assessed, and the modulation of ammonium chloride on uptake and replication of TiLV in E-11 cells was studied. The findings reported in study I showed that primers designed from segment two of TiLV for a standard RT-PCR were the best at detecting TiLV in the infected cells and Nile tilapia organs. TiLV genome was detected in 28 Nile tilapias (14. 66%, N = 191) in which 17.78% (N=45) were from wild fish and 13.70% (N=146) from farmed fish (cage farming). The genomes of circulating TiLV in Nile tilapia from Lake Victoria were identical to those detected from Israel (98%), Ecuador (98%), Thailand (96%), Peru (96%) and USA (97%). TiLV was not grown from infected fish and thus its ability to cause disease in Nile tilapia was not studied. Therefore, I am recommending further studies to fulfil Koch’s postulates. The data reported in study II showed that the developed and optimized quantitative real-time RT-PCR detected TiLV in virus supernatants of known titre and in organs of unknown titre from infected Nile tilapia. The developed assay is sensitive and specific to TiLV with all the primers efficiency being within the range of 95-105%, except primers targeting segment ten that gave an efficiency of 93%. The intra- and inter-assay coefficient of variation ranged between 0.00% ~ 2.63% and 0.00% ~ 5.92%, respectively, which is within the recommended range (below 5%) for an assay to be repeatable and reproducible. The detection limit of 2 TCID50/ml was found for primers targeting segments 1, 2, 3, 4 and 9 while lower detection limit of 20 TCID50/ml was found for primers targeting segment 5, 6, 7, 8 and 10. Overall primers targeting segment 3 had the highest detection limit and primers targeting segment 7 had the lowest detection limit. Interestingly, despite the two primer sets (for segment 3 and 7) having different TiLV detection limits they had an equal amplification efficiency of 98%. Therefore, primer optimization for qRT-PCR is important to optimize assay sensitivity. The study reported in paper III was directed at understanding the hemagglutination property of TiLV using avian and piscine erythrocytes, and the infection mechanisms in E-11 cells. TiLV did not hemagglutinate erythrocytes from any of the species tested indicating that the virus lack hemagglutinin. Further, the study has shown that ammonium chloride does not affect the replication of TiLV in E-11 cells indicating that the virus is not using the endocytic pathway during internalization. Taken together, the two observations suggest that, TiLV is not taken up by receptor-mediated endocytosis during internalization into E-11 cells. Thus, further studies are needed to unravel the uptake mechanism(s), which is the important information for controlling the virus by antiviral agents or immunoprophylaxis.Tilapia lake virus (TiLV) er et fremvoksende virus som infiserer ville arter og oppdrettsarter av tilapia og gir dødelighet og betydelige økonomiske tap i oppdrett. Siden den første beskrivelsen av sykdommen fra Israel, har viruset blitt påvist på fire kontinenter (Asia, Afrika, Sør- og Nord-Amerika) og gir dødelighet fra 5-20% opp mot 90%. Forekomst av viruset er ikke kjent i mange av de landene som driver tilapiaoppdrett, og det er nødvendig å etablere bedre overvåknings- og kontrollprogrammer i disse landene. I denne studien ble Lake Victoria valgt for TiLV-screening fordi den inneholder et stort antall tilapia-arter som er av økonomisk betydning for de omkringliggende østafrikanske landene. Det er også behov for optimaliserte metoder for rask deteksjon og kvantifisering av viruset. Hvordan viruset tas opp i cellene og hvordan det replikerer er ikke kjent. I denne avhandlingen ble det gjennomført studier for å forstå forekomst av TiLV i Lake Victoria i Øst-Afrika, det ble etablert verktøy for påvisning og kvantifisering av viruset med molekylærbiologiske metoder, og det ble gjennomført studier for å bedre forstå opptaksmekanismer i celler under infeksjonen. Det ble utviklet en standard RT-PCR og en kvantitativ RT-PCR (qRT-PCR) basert på alle de ti segmentene til viruset. Standard PCR ble brukt til å undersøke Nile tilapia (Oreochromis niloticus) fra Victoriasjøen. Den kvantitative RT-PCR metoden ble testet mot kjent og ukjent virustiter fra henholdsvis infiserte TFC# 10 celler og organer fra infiserte fisk. Hemagglutinering av røde blodlegemer fra hønsefugl og fisk ble også undersøkt, samt effekten av ammoniumklorid på replikasjonen av TiLV i E-11-celler. Resultatene i studie I viste at primere designet fra segment 2 benyttet for påvisning med standard RT-PCR var best egnet til å påvise TiLV i infiserte cellene og organer fra infisert fisk. TiLV fra Victoriasjøen ble påvist i 28 fisk (14. 66%, N = 191) hvor 17,78% (N = 45) var fra villfisk og 13,70% (N = 146) fra oppdrettsfisk. De sekvensene som ble påvist i Nil-tilapia fra Victoriasjøen var tilnærmet identiske med de som ble påvist i Israel (98%), Ecuador (98%), Thailand (96%), Peru (96%) og USA (97%). TiLV ble ikke dyrket eller testet med tanke på virulens/evne til å forårsake sykdom i Nil-tilapia, og derfor anbefaler jeg videre studier for å oppfylle Kochs postulater. I studie II ble det etablert en ny og optimalisert kvantitativ RT-PCR metode for påvisning av TiLV genom i prøver fra infiserte celler med kjent titer (mengde virus) og fra organer fra infisert Nil-tilapia uten kjent titer. Metoden som ble utviklet er sensitiv og spesifikk for TiLV, og primer-effektiviteten var innenfor et akseptabelt område, 95-105%, bortsett fra primere rettet mot segment 10 (93%). Variasjonskoeffisienten for intra- og inter-analyse varierte mellom henholdsvis 0,00% ~ 2,63% og 0,00% ~ 5,92%, som er innenfor det anbefalte området (under 5%) for at en analyse skal anses som repeterbar og reproduserbar. Sensitiviteten til metoden var 2 TCID50/ml, og primere spesifikke for segmentene 1, 2, 3, 4 og 9 gav samme resultat. En nedre deteksjonsgrense på 20 TCID50/ml ble påvist for primere rettet mot segment 5, 6, 7, 8 og 10. Primere spesifikke for mot segment 3 gav høyest sensitivitet og primere segment 7 den laveste. Begge primersettene hadde en effektivitet på 98%. I artikkel III var målsettingen å forstå hemagglutinasjonsegenskapen til TiLV ved bruk av erythrocytter fra hønsefugl, tilapia og laks, samt betydningen av endocytose i tidlig fase av infeksjonen i E-11-celler. TiLV gir ikke hemagglutinering av erytrocytter fra noen av de testede artene, noe som indikerer at viruset mangler hemagglutinin. Studien har også vist at ammoniumklorid ikke påvirker, dvs. hemmer eller forsinker replikasjonen av TiLV i E-11-celler, noe som indikerer at viruset ikke tas opp ved endocytose. Samlet antyder de to observasjonene at TiLV ikke blir tatt opp av reseptormediert endocytose i E-11-celler. De gjennomførte studiene viser at det er behov for å forstå opptaksmekanismen(e) til virus, som er en viktig informasjonen for å kontrollere virusinfeksjonen med anti-virale midler eller vaksiner

    Integrating glycomics, proteomics and glycoproteomics to understand the structural basis for influenza a virus evolution and glycan mediated immune interactions

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    Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins

    Novel Cationic Pentablock Copolymers as a Nanovaccine Delivery Platform

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    The main objective of this project is the development of a safe and effective vaccine against respiratory pathogens including the highly pathogenic avian influenza (HPAI) H5N1. Amphiphilic pentablock copolymers based on Pluronic F127 and poly(2-diethylaminoethyl methacrylate (PDEAEM) have several characteristics that make them promising candidates as injectable vaccine carriers and adjuvants. Individual block copolymer molecules self-assemble into micelles in aqueous solutions. These micelles can be used to encapsulate protein for vaccine delivery. As the concentration of the block copolymer increases, the micelles form a temperature dependent gel and the length of the PDEAEM blocks control the dissolution rate of the gels. An injectable formulation can be designed to gel at physiological temperatures and form an antigenic depot. In addition, these biocompatible pentablock copolymers are based on pluronic, which is FDA approved as an injectable material. In order to design an efficacious next-generation vaccine against HPAI H5N1 several specific research goals were laid out including: (1) Design, synthesis, and optimization of the pentablock copolymer vaccine platform; (2) Evaluation of vaccine efficacy in vitro and in vivo; and (3) Immunization of mice with the pentablock copolymer vaccine to asses an immune response. Our block copolymer injectable delivery platform demonstrates the ability to sustain the release of antigen with minimal effects on protein stability or antigenicity and persist at the injection site. We have also successfully modified the polymers through an azide-alkyne click reaction to include mannose moieties that act as ligands for pattern recognition receptors on antigen presenting cells. These data coupled with the strong immune response demonstrate the potential for block copolymers for use simultaneously for injectable delivery and as a vaccine adjuvant platform

    A Difference in Heterosubtypic Immunity Induced by a Modified Live Attenuated Avian Influenza Backbone in Mice and Ferrets

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    The unprecedented emergence of multiple avian influenza virus (AIV) subtypes with a broad host range poses a major challenge in the design of vaccination strategies that are effective against multiple subtypes of influenza. The present study focused on the protective effects of a modified AIV as a backbone for epidemic and pandemic influenza. In addition, the ability of this backbone to induce heterosubtypic immunity (Het-I) was also analyzed. Het-I is the ability of one influenza subtype to protect against a different influenza subtype. Previously, a live attenuated AIV with the internal backbone of A/guinea fowl/Hong Kong/WF10/99 (H9N2) (WF10), called WF10att, protected chickens against a lethal influenza challenge. To characterize the WF10att backbone as a master donor strain and determine its ability to induce Het-I, we evaluated its protective efficacy in mice and ferrets. Vaccinated mice were protected against homologous challenge with A/WSN/1933 (H1N1) (WSN), mouse-adapted A/California/04/2009 (pH1N1) and A/Vietnam/1203/2004 (H5N1) (HPAI H5N1) viruses, and ferrets survived homologous challenge with HPAI H5N1. H7N2att vaccinated mice were protected against both H1N1 and HPAI H5N1 challenge; however, Het-I was observed in H9N2att vaccinated ferrets challenged with HPAI H5N1. We found that both B and T cells are involved in the Het-I induced by our WF10att backbone. Cross-reactive non-neutralizing antibodies to viral proteins were detected. JhD-/- mice, which lack mature B-lymphocytes, were vaccinated with the recombinant vaccines and challenged with HPAI H5N1. None of the vaccinated mice survived challenge further suggesting a role for Het-I. In addition, cells isolated from the lungs of H7N2att vaccinated mice had cross-reactive antibody-secreting cells targeted to HPAI H5N1. Together, these results suggest a role for B cells in Het-I. Although B cells are important, T cells may also play a role in Het-I. Both IFN-γ and Granzyme B secreting cells were detected in lung and spleen cells isolated from H7N2att vaccinated mice and stimulated with HPAI H5N1 suggesting a role for T cells in Het-I. The ability of our WF10att backbone to induce Het-I depends on the surface glycoproteins expressed and the challenge virus subtype. In addition, WF10att uses both B and T cells to induce Het-I

    Multiplex point-of-care detection of human and avian influenza viruses using an antibody microarray technology

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    Over the last two decades concern about influenza has increased worldwide due to the rising number of human infections caused by an avian virus, H5N1, and the 2009-pandemic of swine-origin H1N1 virus. Counter-measures, for example the selective administration of antivirals to infected individuals, their hospitalization, and the culling of large numbers of infected animals require a prompt and reliable diagnosis, that ideally identifies the type and subtype of the virus and can be carried out at the Point-of-Care (POC). Nowadays instead diagnosis is still largely laboratory-bound, as the available POC-suitable tests are hampered by poor and very variable clinical sensitivity. This PhD was developed in the frame of a European joint effort (Fluarray) that aimed at constructing an automated diagnostic system for the rapid influenza diagnosis. Key technology of the system is an antibody microarray technology that permits the simultaneous analysis of hundreds of antigen-antibody interactions. This research investigated ways to detect the influenza virus using a panel of monoclonal antibodies that were purified, characterized and integrated into the microarray platform. The optimised immunoassay detects and differentiates the influenza nucleoproteins of type A and B at concentrations in the order of few nanograms/ml. A panel of influenza viruses were detected and their type identified within 1 hour hands-on time. The developed microarray platform will soon be integrated into an Automated Diagnostic Analyser for Microarrays (ADAM) developed by the collaborating partner Microtest Matrices. It is envisaged that the system will be a consistent aid in the influenza diagnosis and sensibly facilitate and speed up diagnostic procedures

    In vitro and in silico epitope-paratope mapping

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    Disulfides as redox switches : from molecular mechanisms to functional significance

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    The molecular mechanisms underlying thiol-based redox control are poorly defined. Disulfide bonds between Cys residues are commonly thought to confer extra rigidity and stability to their resident protein, forming a type of proteinaceous spot weld. Redox biologists have been redefining the role of disulfides over the last 30&ndash;40 years. Disulfides are now known to form in the cytosol under conditions of oxidative stress. Isomerization of extracellular disulfides is also emerging as an important regulator of protein function. The current paradigm is that the disulfide proteome consists of two subproteomes: a structural group and a redox-sensitive group. The redoxsensitive group is less stable and often associated with regions of stress in protein structures. Some characterized redox-active disulfides are the helical CXXC motif, often associated with thioredoxin-fold proteins; and forbidden disulfides, a group of metastable disulfides that disobey elucidated rules of protein stereochemistry. Here we discuss the role of redox-active disulfides as switches in proteins.<br /
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