Fish nodavirus causes the death of several high economic fish. For studying the package and stability of dragon grouper necrosis virus (DGNNV), wild type virus-like particles (wt-VLPs) and £GN25-VLPs were employed. After experiments of disassembly and assembly, the result showed that DNA of 608 bp was able to be packaged by wt-VLPs. The sedimentation of VLPs was affected by different pH buffers during the process of purification: the VLPs at alkaline conditions behaved faster than those at the acidic. The stability of pure VLPs was pH-independent. The micrographs of the wt-VLPs in alkaline buffers showed that particles were rough and irregular in shapes, some of which were stain-permeable. However, the wt-VLPs in acidic buffers were morphologically indistinguishable from the untreated VLPs. The Western blotting for gradiently purified miture of VLPs and lysozyme revealed that lysozyme was co-precipitated with wt-VLPs.

Piscine nodaviruses are members of genus Betanodavirus, which infect more than 30 species of fish and cause massive mortality in larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the dissociation and reassembly of betanodavirus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. The experiments with calcium-chelating or reducing/oxidizing reagents elicited that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. In the mutational analysis of DxxDxD that is putatively involved in the coordination of calcium ions, the results showed that the D133N mutation significantly disrupted the assembly of VLPs while D130N and D135N mutants produced heterogeneous particles with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of D135N mutant were stable at a temperature of 85¢XC, which is slightly higher than that for wild-type, whereas VLPs of D130N mutant could not tolerate the thermal effects at a temperature higher than 60¢XC. It is deduced that three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and, among them, the DxxD provides a more stable coordinate of calcium-ligand than DxD.

Dragon grouper nervous necrosis virus (DGNNV), a betanodavirus, is the causative agent of viral nervous necrosis (VNN) in dragon grouper (Epinephelus lanceolatus). In our study, capsid protein of DGNNV was expressed in Escherichia coli. We mutated arginines at N-termini capsid protein to investigate the role of arginines at 29-31th position. When capsid protein lost 25 amino acids at N-termini VLPs form, mutation in any two arginines at 29-31 position to alanine could the prohibit VLPs formation. Another extending three arginines at 23-25 position wouldn¡¦t increase the RNA encapsulation into VLPs. Furthermore, N-termini mutated VLPs were all RNase resistance like wt-VLPs, but the yield was distinctly less than wt-VLPs. In the single point alanine mutations, the VLPs yield of R29A was apparently higher than others (R30A and R31A). Using circular dichroism to observe the thermal denature process and thermal stability of DGNNV VLPs, we found the Tm about 60¢J of VLPs wouldn¡¦t alter even if arginines at 23-31 position were mutated. The findings suggested the VLPs of mutated arginines at 23-31 position wouldn¡¦t affect RNase resistance and thermal stability, but the yield were lower. Another, the arginines at the 30 and 31 position is more important than at 29 position for formation of VLPs.

Molecular imaging involves the development of probes which can specifically label a certain object in the body at cellular or subcellular level. This thesis consists of three parts, each involving the development of novel labelling methods for viruses or virus-like particles with specific applications. Virus-like particles (VLP) derived from the E. coli bacteriophage Qβ are widely employed as a nano-carrier for drugs and vaccines, but a powerful method for tracing its circulation without affecting its structure is yet to be developed. In the first part of the thesis, the electrophilic fluorine source ¹⁹F-Selectfluor^TM was employed for introducing single fluorine atoms on Qβ VLPs. For the 'tag-and-modify' approach, site-selective electrophilic C-F bond formation was achieved on the dehydroalanine (Dha) amino acid tag of VLPs under aqueous conditions. Chemoselective electrophilic aromatic fluorination on tyrosine residues were also achieved using the same reagent by manipulating the amino acid sequence. Similar results were observed in conditions required for ¹⁸F-Selectfluor™ reaction, indicating the potential of this technique for positron emission tomography (PET) imaging. In addition, there is a lack of in situ technique for tracking the functional status of Qβ VLPs and hence the release of cargos. In the second part of the thesis, a simple way to monitor the disassembly of ¹⁹F-labelled Qβ VLPs by ¹⁹F NMR spectrosocpy is reported. Analysis of resonances, using experiments under a range of conditions, allowed determination not only of the intact particle but also the disassembled multimeric species and even smaller peptides upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems. In the third part of the thesis, a new type of rhodamine B fluorescent dye functionalised with a 2-imino-2-methoxyethyl (IME) group is reported. The amidine linkage formed between the IME group and lysine residue retains the pKaH of the original side chain, which cannot be achieved using commercially available conjugating dyes. This in turn minimises the change in net charge hence virus infectivity following virus labelling. By employing adenovirus (AV) as an example, the IME dye was shown to be a better choice in retaining virus infectivity compared to dyes linked with other coupling groups. In addition, preliminary experiments on dengue virus with the synthesised dyes were also performed.

The groupers are vital fish in Taiwan, the market of grouper fry over 300 million dollars. While grouper nervous necrosis virus (NNV) has caused mass mortality, especially 100% in larvae and juveniles, which economically impacts on culture of marine fish. The vaccination is one of the best methods to against viral diseases. The dragon grouper (Epinephelus lanceolatus), malabar grouper (E. malabaricus) and brown-marbled grouper (E. fuscoguttatus) were injected with different dosages and injection frequencies of virus-like particles (VLPs) of DGNNV, which is by the first claimed. The anti-sera of vaccinated fish were analyzed with eight kinds of immunology methods, among which antigen-capture ELISA was the best choice for qualitative and quantitative assays. The signal of antibodies in the vaccinated fish was detected in all groupers in one week after primary immunization, and the antibody titers increased markedly in one month. In dragon grouper, fish was injected with 10 £gg of VLPs, the antibody titer reached 1.05. To given booster injection once, antibody titers were raised to 35.7%. In malabar grouper, after injected twice with 50 £gg of VLPs, the antibody titer raised 33.3% than inoculation once in six weeks. After brown-marbled grouper was injected with 450 £gg of VLPs, the high antibody titer reached to 1.57 at five weeks post primary immunization. Specific antibodies still can be detected after seven months. In the in vitro assay with MGNNV of 103.5 TCID50/mL, neutralizing antibody titer of control fish were all lower than 1:50. The neutralizing antibody titer of anti-serum of dragon grouper was detected at 1:200 at one week, and raised to 1:1600 at four weeks and 1:6400 at eleven weeks after primary vaccination. In malabar grouper and brown-marbled grouper, the neutralizing antibody titers were 1:3200 and 1:400, respectively, in one month. The antibody titer can not increased by Freund¡¦s complete adjuvant. The fish produced high antibody titer and high protection by immunization with VLPs.

Recentemente, vários arbovírus têm acometido a população de países emergentes ocasionando sérios problemas de saúde pública, como as doenças causadas pelos vírus da dengue, Chikungunya, Zika e febre amarela. Um vírus emergente e já circulante no Brasil, chamado Mayaro (MAYV), do mesmo gênero do Chikungunya (Alphavirus), possui potencial prejudicial semelhante a esses já estabelecidos. Seu vetor de transmissão é o mosquito do gênero Haemagogus, característico de regiões isoladas, principalmente florestas. Entretanto, estudos demonstraram que o Aedes aegypti é um competente vetor desse agente, o que possibilita sua disseminação em regiões urbanas. O presente trabalho avaliou a expressão das proteínas estruturais do vírus Mayaro (E1, E2, E3, C e 6K), utilizando dois sistemas de expressão distintos, um baseado na levedura Pichia pastoris, e outro derivado de Baculovírus (BEVS). Essa estratégia foi estabelecida para que a expressão dessas proteínas promova a formação de partículas semelhantes ao vírus (virus like particles), estruturas multiprotéicas que mimetizam a conformação de uma partícula viral podendo ser utilizada como um candidato vacinal. O trabalho evidenciou a correta obtenção de organismos recombinantes em ambos os sistemas, com a avaliação da expressão sendo feita com técnicas de dot blot, western blot e imunofluorescência indireta (IFI). Com o sistema baculovírus, foram avaliadas as linhagens Sf-9 e Hi-5, sendo evidenciada a expressão de proteínas do MAYV em ambas, utilizando MOI 10 e tempos pós-infecção de 96 e 72 h, respectivamente. A correta expressão das proteínas de MAYV também foi evidenciada com a levedura Pichia pastoris, com cultivo a 30 °C e tempo de análise 48 h após indução. A geração de VLPs foi avaliada em amostras de sobrenadantes de ambos os sistemasapós a concentração por ultracentrifugação em gradiente de iodixanol, e análise por microscopia eletrônica de transmissão, sendo observadas nos dois sistemas com tamanhos variando entre 30-60 nm. Os resultados desse projeto podem gerar ferramentas importantes no desenvolvimento de kits diagnósticos e métodos vacinais contra o MAYV. / Recently, several arboviruses have affected emerging countries causing serious public health problems, such as diseases caused by dengue viruses, Chikungunya, Zika and yellow fever. An emerging and already circulating virus in Brazil, called Mayaro (MAYV), of the same genus of Chikungunya (Alphavirus), has harmful potential similar to those already established. Its transmission vector is the mosquito of the genus Haemagogus, characteristic of isolated regions, mainly forests. However, studies have demonstrated that Aedes aegypti is a competent vector of this agent, which would allow its dissemination in urban areas. The present work evaluated the expression of the structural proteins of the Mayaro virus (E1, E2, E3, C and 6K) using two distinct expression systems, one based on the yeast Pichia pastoris and another derived from Baculovirus (BEVS). The strategy of expressing structural proteins has been established so that the expression of these proteins promotes the formation of viruslike particles or VLPs, multiprotein structures that mimic the conformation of a viral particle and can be used as a vaccine candidate. The work evidenced the correct obtaining of recombinant organisms in both systems, with the evaluation of the expression being done with dot blot, western blot and indirect immunofluorescence (IFI) techniques. With the baculovirus system, the Sf-9 and Hi-5 strains were evaluated, and the expression of the MAYV proteins was evidenced in both, using MOI 10 and the time of post-infection analysis of 96 and 72 h, respectively. Correct expression of MAYV proteins was also evidenced with yeast Pichia pastoris, with culture at 30 ° C and analysis time 48 h after induction. The generation of VLPs was evaluated in both supernatants after concentration by iodixanol gradient ultracentrifugation and transmission electron microscopy analysis, being observed in both systems with sizes ranging from 30-60 nm. The results of this project can generate important tools in the development of diagnostic kits and vaccine methods against the MAYV.

The L1 and L2 capsid proteins of papillomaviruses are characterized by the ability to self- assemble into viral capsids, which can be divided into pseudovirions (PsVs) and virus-like particles (VLPs) by inner content. In addition to the fact that such particles can serve as "nano-containers" for diagnostic and therapeutic agents, it has also been shown that papillomaviruses, whether wild, PsVs or VLPs have a higher affinity for tumor tissue than non-tumor tissue. This thesis deals with relatively newly discovered (2011) mouse papillomavirus (MusPV) and nanoparticles derived from this virus. This papillomavirus has been chosen for its positives, including easy preparation of VLPs and PsVs, as well as an available model organism for possible testing. Furthermore, MusPV has the potential for use in gene therapy and cancer diagnosis, because there is no immune response in the human population. The aim of this diploma thesis is to prepare an expression system for the production of PsVs and VLPs. In additional it will also look at the quality and quantity of PsVs and VLPs, characterization of these particles and verification of existing postulates regarding higher affinity of papillomaviruses for tumor cells. Finally, it will also to verify whether the same effect is observed in MusPV. In the results of...

Virus-like particles (VLPs) are elegant functional architectures formed by the self-assembly of viral structural proteins. VLPs have been developed as vaccines against hepatitis B and cervical cancer, and have recently been shown in animal studies to provide protection against both seasonal and avian influenza following intranasal administration. This new class of vaccines offers unprecedented immunoprotection, inherent safety, and a simple route of administration. To realize the full potential of VLP technology as an efficient and responsive vaccine platform, this project exploits the parallel advancements in recombinant technology, analytical techniques and colloidal science to facilitate the swift and economical delivery of candidate VLP vaccines from laboratory to clinical trials, and ultimately into commercial production. Three areas of VLP production are specifically targeted in this work, i.e., VLP subunit production, particle characterisation and assembly. The major research outcomes in this work are: (i) establishment of a simple and economical VLP subunit production method which eliminates inefficient and complicated purification procedures necessitated by the current in vivo production methods; (ii) development of a high-resolution and high-throughput analytical method for rapid and reliable quality control check of VLP products; and (iii) establishment of the foundation to predict optimal VLP self-assembly conditions through molecular thermodynamics. These research outcomes collectively enhance the quantitative knowledge base in VLP assembly and may ultimately enable the development of a mechanistic and descriptive modelling approach to optimize VLP production. From a fundamental perspective, this work introduces the first experimental technique to measure protein interactions of viral subunits undergoing rapid, irreversible assembly reaction. Such information, when correlated with molecular details and assembly conditions may provide unique insights into the molecular switches responsible for viral assembly, unveiling the fundamental mechanism underpinning viral self-assembly.

Grouper in Taiwan is of high value, but nervous necrosis virus infection causes 100% mortality. Our laboratory had developed a good expression system to produce virus-like particles that induced immune functions. In this study, cells producing monoclonal antibody against the virus-like particles were used to induce BALB/c mice production of high titer ascites. The ascite from the H1 cells generated 6000-fold of antibodies higher than cell culture, using enzyme-linked immune-sorbent assay, although relatively less than the previous monoclonal cells of mX, 8000-fold. In the electron microscopy, the ascite antibody bound to various mutants of virus-like particles. Using the polymerase chain reaction to amplify fragments of IgG cDNAs, we will clone and express such cDNAs to efficiently produce the desired monoclonal antibodies.

abstract: The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus-based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2016