Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

The 5’ untranslated region (UTR) of coronaviral genomes contains cis-acting sequences necessary for replication, transcription and translation. A consensus secondary structural model of the 5' 140 nucleotides of the 5' UTRs of nine coronaviruses (CoVs) derived from all three major CoV groups is presented and characterized by three major stem loops, SL1, SL2 and SL4. SL2 is conserved in all CoVs, typically containing a pentaloop (C47-U48-U49-G50-U51 in MHV) stacked on a 5-bp stem, with some sequences containing an additional U 3' to U51. NMR structural studies of SL2 hairpin reveal that SL2 adopts a U-turn-like conformation. Parallel molecular genetic experiments reveal that SL2 plays an essential role in sgRNA synthesis as does SL1. We observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a collection of non-canonical base pairs near the deleted A35. Viruses containing destabilizing SL1-∆A35 mutations also contain one of two specific single nucleotide mutations in the 3' UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-∆A35 substitutions recover one or more features of the wild-type SL1. We propose a "dynamic SL1" model that supports viral replication; these characteristics of SL1 appear to be conserved in other coronaviral genomes. The coronaviral nucleocapsid (N) protein contains two or more RNA binding domains. We investigated the RNA-binding properties of the N-terminal (NTD) and Cterminal (CTD) domain of MHV N. Our results reveal that the NTD specifically interacts with the TRS-L3 sequence. The role of conserved residues (Y127, Y129 and R110) for this specific interaction were systematically investigated. In contrast to the NTD, the MHV CTD is homodimeric in solution and binds single-strand RNA nonspecifically in a binding mode of the noncooperative large ligand lattice model. The CTD dimer binds with a site size, n=4 nucleotide and the appending of the NTD enhances the single-strand nucleic acid binding affinity.

Masters of Science / The Coronaviridae family consists of RNA viruses within the order Nidovirales. The family is classified into two genera, namely the corona- and toroviruses. Coronaviruses are enveloped, single stranded, positive sense RNA viruses with genomes ranging between 27-32kb in size. The 5’ two-thirds of the genome encodes for the 1a/b polyprotein, while the 3’ one-third of the genome encodes for the structural proteins that mediate viral entry into the host cell. These structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The nucleocapsid protein is expressed at high levels within an infected cell. Studies have shown that this protein plays a key regulatory role in different cellular pathways, including the inhibition of interferon production and the up-regulation of the AP1 signal transduction pathway, amongst others. Also, the N protein is vital in the formation of the ribonucleocapsid core by binding to the viral RNA during virion assembly. The focus of this study is the immune response in whole blood cultures to the presence of human coronavirus (HCoV) NL63 N protein. To characterise the stimulation of the immune activity against HCoV-NL63 N in blood cultures, the HCoV-NL63 N gene was expressed in a bacterial system. In this pilot study, GSTtagged N constructs were then purified and used to treat whole blood cultures from three volunteers. ELISAs were used to measure the cytokine response in these treated whole blood cultures. Results showed that the nucleocapsid protein has an inflammatory response on whole blood cultures. These results have generated vital information in the potential function of the HCoV-NL63 N protein on the immune system. It is suffice to say that the HCoV-NL63 N protein is able to elicit an effective inflammatory response within the host cell. Future studies into the cellular pathways affected by the HCoV-NL63 N protein will clarify its exact role in stimulating the host immune system.

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Gold compounds have been used in medicine dating back as early as 2500 BC. Over the years gold(I) and gold(III) compounds have been used and designed to target rheumatoid arthritis, cancer, and viral diseases. New drug targets have been found for gold compounds that give insight into their mechanisms of action. Here we focus on the synthesis of Au(III) compounds designed to selectively target zinc finger (ZF) proteins. ZF proteins exhibit a variety of functions, including transcription, DNA repair, and apoptosis. Displacement of the central zinc ion, along with mutation of coordinated amino acids can result in a loss of biological function. Synthesis of complexes that selectively target zinc finger proteins, in turn inhibiting DNA/ZF interactions and therefore resulting in loss of protein function, is of great interest. Of particular interest here is the Cys3His (Cys = cysteine, His = histidine) HIV nucleocapsid zinc finger protein, NCp7. NCp7 is involved in multiple steps of the HIV life cycle, thus making it a desirable drug target. Previous studies from our group show platinated nucleobases such as [Pt(dien)(9-EtG)]2+ (dien = diethylenetriamine; 9-EtG = 9-ethylguanine) to stack effectively in a non-covalent manner with tryptophan of the C-terminal finger of HIV Nucleocapsid, NCp7(F2), a key residue involved in nucleic acid recognition. Due to the isoelectronic and isostructural relationship of Au(III) to Pt(II), we have expanded this system to Au(III)-(nucleobase/N-heterocycle) compounds. Novel Au(III)(dien)(N-heterocycle) compounds, including the first Au(III)N3(N-purine) examples, were synthesized. As previously reported for [AuCl(dien)]Cl2, these compounds exhibit pH dependency of the 1H NMR chemical shifts of the dien ligand. The acidity of the dien ligand is affected by the nature of the fourth ligand as a leaving group. The presence of an inert nitrogen donor, compared to that of the more labile Cl-, as the leaving group stabilizes the Au(III) metal center towards reduction, resulting in significant enhancement of π−π stacking interactions with tryptophan relative to platinum(II) and palladium(II) compounds. The presence of a more inert N-donor as the leaving group slows down the reaction with the sulfur-containing amino acid N-Acetylmethionine (N-AcMet); essentially no reaction was observed for the Au(III)-N-heterocycle compounds. All compounds react readily with N-Acetylcysteine (N-AcCys), however lack of N-heterocycle ligand dissociation indicates, to our knowledge, the first long-lived N-heterocycle-Au-S species in solution. Electrospray ionization mass spectrometry (ESI-MS) studies with NCp7(F2) indicate [Au(dien)(DMAP)]3+ (DMAP = 4-dimethylaminopyridine) to be the least reactive of the Au(III) compounds studied, showing the presence of intact NCp7(F2) zinc finger at initial reaction times. Reactivity of the Au-compounds was compared with that of Sp1(F3), a Cys2His2 ZF; in contrast, no intact ZF was observed for any of the compounds studied, suggesting the mode of action of these compounds is dependent on the nature of the zinc binding core. ESI-MS studies were expanded to that of the full HIV NCp7 zinc finger. [Au(dien)(9-EtG)]3+ reacts quickly with NCp7, resulting in immediate zinc ejection and replacement with up to three gold ions. Unlike with [Au(dien)(DMAP)]3+, no intact NCp7 was observed. Addition of [Au(dien)(9-EtG)]3+ to preformed NC-SL2 complex results in release of free RNA; based on EMSA (electrophoretic mobility shift assay) studies, [Au(dien)(9-EtG)]3+ disrupts the NCp7-RNA complex with an IC50 of ~450 µM. It is possible that this HIV nucleocapsid-nucleic acid antagonism may result in a loss of viral activity.

O vírus Oropouche (OROV) é um arbovírus que ocorre na região amazônica causando surtos de doenças febris agudas e que, ocasionalmente, podem ser associados a meningoencefalite. Aproximadamente 500.000 casos de Oropouche teriam ocorrido no Brasil. Entretanto, não existe vacina contra o OROV. O objetivo deste trabalho foi desenvolver um modelo animal de infecção por OROV para estudar a patogênese da doença e um modelo para testar candidatas vacinais. Protótipo vacinal utilizando a proteína recombinante do nucleocapsídeo (N) de OROV (NrOROV), que é o principal antígeno viral, foi usado como potencial candidato para vacina. Neste estudo utilizou-se um modelo animal em camundongos Balb/c de 12 semanas de idade, inoculados intracerebralmente com 8x105 PFU de OROV, capaz de induzir 100% de letalidade após o terceiro dia da infecção. Altos títulos virais foram encontrados no cérebro e na medula espinhal dos animais. Surpreendentemente, 12 e 24 horas pós-infecção foi possível detectar vírus no fígado e baço (3 Log10 PFU/g) dos camundongos. Com este modelo foram testados os candidatos vacinais. Grupos de camundongos foram imunizados 3 vezes com OROV, OROV e FCA, NrOROV, NrOROV e FCA, NrOROV, Poli I:C e Montanide ISA 720. Após 3 imunizações, os animais foram desafiados com 10 LD50 de OROV e observados por 20 dias. Os animais imunizados com NrOROV e adjuvantes, não foram capazes de produzir anticorpos neutralizantes e adquirir imunidade protetora contra OROV enquanto que os imunizados com OROV apresentaram altos níveis de anticorpos neutralizantes e completa proteção in vivo. Ainda, os anticorpos produzidos pelos animais imunizados permitiram estudar o ciclo de replicação celular do OROV utilizando imunofluorescência. / Oropouche (OROV) is an arbovirus that occurs in the South American, Amazon region, producing outbreaks of acute febrile illness occasionally associated to meningoencephalitis. Approximately 500,000 cases of Oropouche have been reported in Brazil in the last 60 years. However, there is no available vaccine for OROV. We show here the development of an animal model of OROV suitable for studies on pathogenesis and vaccine testing. A vaccine prototype based on recombinant OROV nucleocapsid protein (NrOROV), an important viral antigen, was evaluated in the animal model. Initialy, we observed that all 12-week-old Balb/c mice inoculated intracerebrally with 8x105 PFU died after the third day of infection. Surprisingly, OROV genome was detectable in the liver as early as 12 hours post infection (pi) and in the spleen at 24 hours pi at 3 log10 PFU/g. Besides, high viral titers were found in brain and spinal cord. To test the NrOROV as a vaccine candidate, animals divided in 5 groups were immunized subcutaneously 3 times, two weeks apart with either OROV, OROV and Freud complete Adjuvant (FCA), NrOROV, NrOROV and FCA, NrOROV and Poly I:C and Montanide ISA 720. The experiment also included a group of naïve animals. After the third immunization, the animals were challenged with 10LD50 by intracerebral route and followed for 20 days. The animals immunized with NrOROV and adjuvants developed specific antibodies that were not able to neutralize the virus or confer protective immunity against OROV. Nevertheless, mice immunized with OROV showed high levels of neutralizing and protective antibodies. Despite the discouraging results with NrOROV as a vaccine, the mouse model is suitable to study pathogenesis, and to test other vaccines for OROV.

O vírus Oropouche (OROV) é um arbovírus que ocorre na região amazônica causando surtos de doenças febris agudas e que, ocasionalmente, podem ser associados a meningoencefalite. Aproximadamente 500.000 casos de Oropouche teriam ocorrido no Brasil. Entretanto, não existe vacina contra o OROV. O objetivo deste trabalho foi desenvolver um modelo animal de infecção por OROV para estudar a patogênese da doença e um modelo para testar candidatas vacinais. Protótipo vacinal utilizando a proteína recombinante do nucleocapsídeo (N) de OROV (NrOROV), que é o principal antígeno viral, foi usado como potencial candidato para vacina. Neste estudo utilizou-se um modelo animal em camundongos Balb/c de 12 semanas de idade, inoculados intracerebralmente com 8x105 PFU de OROV, capaz de induzir 100% de letalidade após o terceiro dia da infecção. Altos títulos virais foram encontrados no cérebro e na medula espinhal dos animais. Surpreendentemente, 12 e 24 horas pós-infecção foi possível detectar vírus no fígado e baço (3 Log10 PFU/g) dos camundongos. Com este modelo foram testados os candidatos vacinais. Grupos de camundongos foram imunizados 3 vezes com OROV, OROV e FCA, NrOROV, NrOROV e FCA, NrOROV, Poli I:C e Montanide ISA 720. Após 3 imunizações, os animais foram desafiados com 10 LD50 de OROV e observados por 20 dias. Os animais imunizados com NrOROV e adjuvantes, não foram capazes de produzir anticorpos neutralizantes e adquirir imunidade protetora contra OROV enquanto que os imunizados com OROV apresentaram altos níveis de anticorpos neutralizantes e completa proteção in vivo. Ainda, os anticorpos produzidos pelos animais imunizados permitiram estudar o ciclo de replicação celular do OROV utilizando imunofluorescência. / Oropouche (OROV) is an arbovirus that occurs in the South American, Amazon region, producing outbreaks of acute febrile illness occasionally associated to meningoencephalitis. Approximately 500,000 cases of Oropouche have been reported in Brazil in the last 60 years. However, there is no available vaccine for OROV. We show here the development of an animal model of OROV suitable for studies on pathogenesis and vaccine testing. A vaccine prototype based on recombinant OROV nucleocapsid protein (NrOROV), an important viral antigen, was evaluated in the animal model. Initialy, we observed that all 12-week-old Balb/c mice inoculated intracerebrally with 8x105 PFU died after the third day of infection. Surprisingly, OROV genome was detectable in the liver as early as 12 hours post infection (pi) and in the spleen at 24 hours pi at 3 log10 PFU/g. Besides, high viral titers were found in brain and spinal cord. To test the NrOROV as a vaccine candidate, animals divided in 5 groups were immunized subcutaneously 3 times, two weeks apart with either OROV, OROV and Freud complete Adjuvant (FCA), NrOROV, NrOROV and FCA, NrOROV and Poly I:C and Montanide ISA 720. The experiment also included a group of naïve animals. After the third immunization, the animals were challenged with 10LD50 by intracerebral route and followed for 20 days. The animals immunized with NrOROV and adjuvants developed specific antibodies that were not able to neutralize the virus or confer protective immunity against OROV. Nevertheless, mice immunized with OROV showed high levels of neutralizing and protective antibodies. Despite the discouraging results with NrOROV as a vaccine, the mouse model is suitable to study pathogenesis, and to test other vaccines for OROV.

The dissertation describes development and characterization of monoclonal antibodies against recombinant yeast-expressed antigens: nucleocapsid (N) proteins of human parainfluenza virus type 3, Menangle virus, hantavirus and rabies virus. The newly developed antibodies were investigated by different immunochemical assays for their specificity, affinity and ability to recognize native viruses in infected cells. It was determined that the antibodies raised against recombinant yeast-expressed viral proteins are suitable to identify virusinfected cells. These data confirmed that recombinant yeast-expressed viral N proteins possess antigenic properties similar to that of native viral nucleocapsids. The monoclonal antibodies were also used to study the antigenic structure of viral N proteins and localize their immunodominant regions. The obtained results may have impact on the development of new immunodiagnostic test systems for the detection of viral infections. The dissertation consists of the introduction, three sections, references, and the list of author’s publications. In the Introductory Chapter, the research topic, the actuality, the aim and tasks, scientific novelty and practical value of the dissertation are discussed. Author’s publications and conference reports are also presented. The first Chapter of the dissertation provides literature overview on the genome organization, structural proteins, pathogenesis and epidemiology of parainfluenza viruses, Menangle virus... [to full text] / Disertacijoje aprašomi monokloniniai antikūnai, sukurti prieš rekombinantinius mielėse susintetintus antigenus: žmogaus paragripo treciojo tipo viruso, Menangle viruso, hantavirusų bei pasiutligės viruso nukleokapsidės (N) baltymus. Sukurtieji antikūnai buvo visapusiškai charakterizuoti įvairiais imunocheminės analizės metodais, įvertintas jų specifiškumas, afiniškumas, sugebėjimas atpažinti natyvius virusus infekuotų ląstelių kultūrose. Buvo nustatyta, kad antikūnai, sukurti prieš rekombinantinius mielėse susintetintus virusų baltymus, tinka virusų nustatymui infekuotose ląstelėse. Šie tyrimai patvirtino, kad rekombinantiniai mielėse susintetinti virusu N baltymai turi panašias antigenines savybes, kaip natyvūs virusų N baltymai, formuojantys nukleokapsides. Sukurtieji monokloniniai antikūnai taip pat buvo panaudoti išsamiems minėtų virusų N baltymų antigeninės struktūros tyrimams bei imunodominuojančių sekų nustatymui. Disertaciniame darbe gauti duomenys svarbūs, kuriant naujas imunodiagnostikos sistemas, skirtas virusų infekcijoms nustatyti. Disertacija sudaro įvadas, trys skyriai, naudotos literatūros sąrašas ir autorės publikacijų sąrašas. Įvadiniame skyriuje aptariama tiriamoji problema, darbo aktualumas, formuluojamas darbo tikslas bei uždaviniai, darbo mokslinis naujumas ir praktinė reikšmė, pristatomos paskelbtos publikacijos ir pranešimai konferencijose. Pirmasis disertacijos skyrius skirtas literatūros apžvalgai: jame apibūdinamos paragripo virusų, Menangle viruso... [toliau žr. visą tekstą]