The Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tripartite negative-strand RNA virus (NSRV) capable of causing severe hemorrhagic fever within humans. The segmented RNA genome of CCHFV does not exist as naked RNA, but is instead completely encapsidated by a nucleocapsid protein (NP). Very little is known about the molecular mechanism underlying viral NP–RNA encapsidation among the Bunyaviridae. This thesis demonstrates that the CCHFV NP exists as a monomer in solution, an unusual characteristic among NSRVs, even in the absence of RNA. Molecular interactions between recombinant CCHFV NP and ssRNA/dsRNA versions of the short 5’–3’ panhandle formed by the small (S) segment of the CCHFV genome were analyzed by gel electrophoresis mobility shift assays (EMSA) and demonstrated that CCHFV NP failed to bind to the ssRNA version of the short 5’–3’ panhandle structure. This thesis research provides the basis for a broader understanding of the CCHFV NP structure, mechanisms behind RNA encapsidation and the molecular basis of interaction between the short 5’–3’ RNA S-segment panhandle and CCHFV NP.

The Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tripartite negative-strand RNA virus (NSRV) capable of causing severe hemorrhagic fever within humans. The segmented RNA genome of CCHFV does not exist as naked RNA, but is instead completely encapsidated by a nucleocapsid protein (NP). Very little is known about the molecular mechanism underlying viral NP–RNA encapsidation among the Bunyaviridae. This thesis demonstrates that the CCHFV NP exists as a monomer in solution, an unusual characteristic among NSRVs, even in the absence of RNA. Molecular interactions between recombinant CCHFV NP and ssRNA/dsRNA versions of the short 5’–3’ panhandle formed by the small (S) segment of the CCHFV genome were analyzed by gel electrophoresis mobility shift assays (EMSA) and demonstrated that CCHFV NP failed to bind to the ssRNA version of the short 5’–3’ panhandle structure. This thesis research provides the basis for a broader understanding of the CCHFV NP structure, mechanisms behind RNA encapsidation and the molecular basis of interaction between the short 5’–3’ RNA S-segment panhandle and CCHFV NP.

The domestic dog is the principal reservoir of rabies in Nigeria and the source of infection for over 99% of human cases that have been documented. The first recorded cases of human and dog rabies were in 1912 and 1928 respectively. The disease has been continually diagnosed in the domestic dog until to date. One of the control measures practiced in this West African country includes the vaccination of domestic dogs with readily available rabies vaccines. However, trend analyses show that dog rabies is increasing probably indicating that the vaccination programmes are inadequate. Rabies is a member of the Lyssavirus genus and currently comprises of seven genotypes (GT 1-7) namely the classical rabies virus (RABV) GT1, Lagos bat virus (LBV) GT2, Mokola virus (MOKV) GT3, Duvenhage (DUVV) GT4, European bat lyssavirus type-1 (EBLV-1) GT5, European bat lyssavirus type-2 (EBLV-2) GT6 and Australian bat lyssavirus (ABL) GT7. Three of these have been identified in Nigeria (classical rabies (RABV) (GT 1), Lagos bat virus (LBV) (GT 2) and Mokola virus (MOKV) (GT 3). The domestic dog is the major maintenance and vector species of rabies in this country and the West Africa sub-region. This study was therefore undertaken to further elucidate the epidemiology of dog rabies in Nigeria. Secondly, it was the aim of this study to determine the phylogenetic relationships of dog rabies viruses and the distribution of the respective rabies variants. Finally, to assess the phylogenetic relationships of the viruses in the study sample with those of the neighbouring countries (Chad, Cameroon, Benin and Niger). A panel of 100 viruses recovered primarily from the domestic dog was included in the study. Partial regions of the nucleoprotein gene (n=100) and the cytoplasmic domain of the glycoprotein and the G-L intergenic region (n=80) were successfully amplified, sequenced and phylogenetically analysed. Nucleotide sequences of representative rabies viruses of the partial N gene of the neighbouring countries and elsewhere in Africa available in the GenBank were also included in the phylogenetic analysis. The phylogenetic analysis demonstrated that the rabies viruses from the study sample were closely related with a 99% sequence homology for both the N and G regions but despite the close homogeneity the viruses segregated into two major clusters. Within the major cluster 1, three sub-clades were identified comprising of rabies isolates from the northern part of Nigeria whereas cluster 2 was made of viruses from the southern part of the country together with an isolate from a stray dog. Further analysis of representative viruses from the study sample with viruses from the GenBank revealed an evolutionary link with the viruses from Chad, Benin, Cameroon and Niger with a ≥96% sequence homology. The demonstration of the evolutionary link of rabies viruses in the study sample and those from neighbouring countries indicates the transboundary nature of rabies and the existence of an active rabies cycle in the region. The study data revealed that a single major virus variant is circulating in domestic dogs in Nigeria belonging to the Africa 2 dog lineage. These data suggest that control strategies including mass vaccination with effective coverage of ≥70% and the control of stray dogs will contribute to the breaking of the rabies cycle. This will dramatically reduce the demand for post-exposure prophylaxis which is costly and not readily available in most states of the country. There is also a need to enforce strict movement of animals across international borders so as to diminish the spread of the infection from one area to another, as rabies still inflicts a considerable public health burden in the region and many parts of Africa. / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by viral nucleoprotein (NP) and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins (vRNPs) responsible for RNA synthesis. NP is a structural component of the vRNP but also interacts with both viral and host factors to regulate viral RNA expression. NP is conserved among influenza A isolates, making NP interactions compelling antiviral targets. Here I characterize mutations within 5 amino acids of NP that comprise an accessible region of the NP body domain, as determined by NP crystal structure. This region was selected for mutagenesis to target interaction between NP and RdRP. NPbd3 encodes glycine at 5 amino acids within the accessible NP body domain. Cellular fractionation and Western Blot, in addition to NP-GFP fusions and fluorescence, confirm NPbd3 was expressed and localized as WT-NP. Gel shift with purified NP protein confirm NPbd3 bound nucleic acids as WT-NP. Although NPbd3 was expressed, localized, and bound nucleic acid as WT-NP, I found NPbd3 was defective for RNA expression in reconstituted vRNPs, as evaluated by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). To investigate this NP body domain further, single and double amino acid mutations were cloned. Analysis of NP single mutants revealed that all were nearly as functional as WT-NP for RNA expression in reconstituted vRNPs, suggesting these accessible amino acids in the NP body domain play a redundant role. However, four different combinations of two amino acid mutations resulted in NP double mutants that displayed a significant defect in RNA expression in reconstituted vRNPs, confirming these accessible amino acids in the NP body domain play a significant role for viral RNA synthesis. A disruption in an essential NP interaction with the RdRP is likely the explanation for the RNA defect observed. In support of this, avian influenza virus passaged in human cells resulted in virus with one NP amino acid change in this domain consistently paired with specific changes in the PB2 subunit of the RdRP. I reason this accessible body domain of NP is a viable antiviral target. Indeed, two amino acids in this NP body domain comprise a novel groove implicated in binding the small molecule inhibitor nucleozin. My thesis highlights this conserved NP body domain as an important interaction surface essential for viral RNA synthesis and support further investigation of antiviral drugs that target this region of NP.

The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay.

Genetic immunization, also known as DNA or polynucleotide immunisation, is well documented to induce broad-based immunity in various animal models of infectious and non-infectious diseases. However, the low potency of DNA vaccines has to date precluded the development of commercial vaccines. The aim of this study was to systematically investigate a number of parameters to improve the potency of DNA vaccines for use in chickens, using a low pathogenic avian influenza (LPAI) virus as a proof-of-concept for their ability to produce a humoral immune response. The index virus used in the study was avian influenza virus A/coot/WA/2727/79 (H6N2), isolated from an apparently healthy Eurasian coot in 1979. Prior to any DNA experiments the virus was rigorously characterized. The virus strain was shown to be an H6 subtype by haemaglutination inhibition (HI) testing and as an N2 subtype by gene sequence analysis. The isolate was shown to be able to grow on MDCK cells in the absence of exogenous trypsin. It was further biologically characterized as LPAI with an intravenous pathogenicity index (IVPI) of 0.15 and a motif of 321PQAETRG328 at the cleavage site of the haemagglutinin (HA) protein. It was capable of infecting domestic chickens under experimental conditions with a low level of virus excretion via the cloaca and oropharynx following intravenous or oral and oculonasal inoculation. The full-length HA and nucleoprotein (NP) genes of this H6N2 virus were subsequently cloned into the eukaryotic expression vector VR1012 to generate VR-HA and VR-NP constructs. Six-week-old Hy-Line chickens were intramuscularly injected with either the VR-HA or VR-NP vaccine at different dose rates, with or without lipofectin as adjuvant. Minimal or no detectable antibody was produced, as measured by HI, ELISA and Western blotting-based assay, but high titres of H6-specific HI antibodies appeared 10 days after homologous virus challenge. In contrast to the empty vector controls, there was a significant difference in HI antibody titre between pre- and post-challenge in vaccinated birds, indicating some evidence for the priming effect of the DNA vaccines. Using the frequency of virus shedding as an indicator of protection, lower doses (50 or 100 ¦Ìg per chicken) of either adjuvanted VR-HA or VR-NP vaccine significantly reduced virus shedding in oropharyngeal and cloacal swabs compared to higher doses (300 or 500 ¦Ìg per chicken ) or empty vector control chickens. Although two vaccinations with naked VR-HA alone were not sufficient to induce an effective immune response against a homologous virus challenge, further repeat vaccinations and incorporation of adjuvant did lead to the generation of low to moderate HI antibody titres in some chickens and resulted in no or reduced virus shedding after challenge. Next, to examine the effect of expression vector, three different DNA vectors, pCI, pCI-neo and pVAX1 were used to clone the same HA gene and generate three DNA vaccine constructs. Once again, direct intramuscular injection of the three DNA constructs did not elicit measurable H6-specific HA antibody response in Hy-Line chickens but the 100 µg pCI-HA lipofectin adjuvanted vaccine group showed a significant increase in post-challenge HI titres from the naive control group, indicating that an anamnestic antibody response had been induced by the pCI-HA DNA vaccination. Compared with the controls, the three DNA constructs showed significantly reduced virus shedding in cloacal swabs post virus challenge, suggesting that the three DNA vaccines induced some level of immune response in vaccinated chickens. As with the VR-HA construct, the lower dose groups for each vaccine (50 or 100 g) were more effective at reducing virus shedding from the cloaca than the higher dose group (300 g). To further investigate why the DNA vaccines did not elicit a measurable antibody response, the HA gene incorporating a Kozak enhancer sequence was cloned into an alternative expression vector, pCAGGS, to produce the pCAG-HAk construct. Three-week-old SPF chickens were immunized with this construct either by the intramuscular route (IM) or electroporation (EP). H6 HI antibodies were present in some chickens by 3 weeks after the first IM vaccination and 75% of the chickens vaccinated with 10, 100 or 300 µg pCAG-HAk were antibody positive by 2 weeks after the second IM vaccination. For EP immunization, 87.5% of vaccinated birds seroconverted after the first vaccination and 100% seroconverted after the second vaccination and the H6 HI antibody titres were significantly higher than for chickens vaccinated by IM inoculation. Another group was given a single dose IM vaccination with 100 µg of the pCAG-HAk construct and showed a maximum sero-conversion rate of 53.3% with a peak H6 HI titre of 27 at 5 weeks post-vaccination. This demonstrated that optimization of the expression vector and insertion of a Kozak sequence could synergistically enhance expression of the H6 HA gene and result in a measurable H6 antibody response in SPF chickens. EP was also compared with IM inoculation with the 100 g pCI-HA construct in SPF chickens, resulting in a 50% sero-conversion rate and mean HI titre of 21.3 at 2 weeks after the second vaccination by EP. By comparison, only 25% chickens had trace HI titres by IM inoculation. This indicated that EP was more efficient than IM delivery for both constructs. A codon-optimized complete HA gene from A/coot/WA/2727/79 (H6N2) was then chemically synthesized and cloned into a pCAGGS vector to generate the pCAG-optiHAk construct. SPF chickens immunized twice with either 10 µg or 100 µg of pCAG-optHA showed 37.5% and 87.5% sero-conversion rates respectively, with a mean H6 HI tire of 21.4 and 22.6 at 3 weeks after the second immunization, but the differences were not statistically significant. There were also no significant differences in either the sero-conversion rate or the H6 HI titre between the pCAG-HAk and pCAG-optiHAk groups, suggesting that a codon-optimized HA DNA vaccine did not achieve significantly better immunogenicity than the pCAG-HAk vaccine. In vitro expression of the developed DNA constructs in chicken-, hamster-, monkey- and human-origin cells, as measured by Western blotting and immunofluorescence testing (IFT), showed the strength of H6 HA expression in the following descending order - pCAG-optiHAk/pCAG-HAk, pCI-HAk, VR-HA, pCI-HA, pCIneo-HA and pVAX-HA. The in vivo chicken vaccinations also showed that the pCI-HA construct was more effective than the pCI-neo-HA, and that the pCAG-optiHA or pCAG-HAk constructs were better than pCI-HAk in term of reduction in virus shedding after H6N2 virus challenge. Thus, in vitro HA gene expression directly correlated with the generation of immune responses in vivo, indicating that in vitro studies can be used for pre-selection of expression plasmids prior to development of avian influenza DNA vaccines. Lipofectin as a chemical adjuvant was shown to enhance the DNA-induced immune response but is prohibitively expensive for routine use in poultry vaccines. Thus, an experimental adjuvant for poultry DNA vaccines (Essai) and a new nanoparticle (Phema) adjuvant used for the first time in poultry were compared with conventional aluminum salts (alum) adjuvant in the present study. No HI antibody was detected in any adjuvant-vaccinated Hy-Line chickens following two immunizations. However, in comparison with the naive control group, the alum- and Phema adjuvanted pCAG-HAk groups significantly reduced the frequency of virus shedding in oropharyngeal swabs, but Essai adjuvant was not effective in augmenting the pCAG-HAk vaccine efficacy. This pilot study also emphasised that the traditional aluminum hydroxide adjuvant, either DNA binding or non-binding, may be useful as an adjuvant for enhancing DNA-induced immune responses in chickens owing to its low price and safety record. Overall, DNA immunization with various HA-expressing constructs was shown to be variably effective in inducing immune responses in chickens. The efficacy of DNA vaccines could be synergistically improved by taking appropriate approaches. With continuing research DNA vaccines have the potential to become an important tool for disease prevention and control.

Homologous recombination (HR) is an essential mechanism for accurate repair of DNA double-strand breaks (DSBs). However, HR must be tightly controlled because excessive or unwanted HR events can lead to genome instability, which is a prerequisite for premature aging and cancer development. A critical step of HR is the loading of RAD51 molecules onto single-stranded DNA regions generated in the vicinity of the DSB, leading to the formation of a nucleoprotein filament. Several DNA helicases have been involved in the regulation of the HR process. One of these is human FBH1 (F-box DNA helicase 1) that is a member of SF1 superfamily of helicases. As a unique DNA helicase, FBH1 additionally possesses a conserved F-box motif that allows it to assemble into an SCF complex, an E3 ubiquitin ligase that targets proteins for degradation. FBH1 has been implicated in the restriction of nucleoprotein filament stability. However, the exact mechanism of how FBH1 controls the RAD51 action is still not certain. In this work, we revealed that FBH1 actively disassembles RAD51 nucleoprotein filament. We also show that FBH1 interacts with RAD51 and RPA physically in vitro. Based on these data, we propose a potential mechanism of FBH1 antirecombinase function.

Os hantavírus, membros da família Bunyaviridae, são os agentes infecciosos responsáveis pela Febre Hemorrágica com Síndrome Renal e pela Síndrome Cardiopulmonar por Hantavírus. São vírus com genoma constituído por três segmentos de RNA fita simples, de polaridade negativa, designados como S, M e L, que codificam, respectivamente, a nucleoproteína, as glicoproteínas G1 e G2 e a RNA polimerase dependente de RNA. Com o objetivo de estudar a montagem de pseudopartículas quiméricas de hantavírus, a proteína N do vírus Araraquara e as glicoproteínas G1 e G2 do vírus Andes foram expressas em sistema baculovírus. A microscopia confocal mostrou a colocalização das proteínas G1 e G2 com a proteína N. Pelos ensaios de imunoprecipitação e de centrifugação em gradiente de sacarose, foi observada a interação entre as proteínas N, G1 e G2. Nas análises por microscopia eletrônica de transmissão foi observada a montagem do pseudo-hantavírus quimera, com morfologia semelhante ao do vírion. O pseudo-hantavírus quimera obtido neste estudo poderá, no futuro, ser utilizado em estudos imunológicos, estruturais e morfológicos. / Hantaviruses, members of the Bunyaviridae family, are the infectious agents responsible for Hemorrhagic Fever with Renal Syndrome and the Hantavirus Cardiopulmonary Syndrome. The viral genome is composed by three segments of single-stranded negative-sense RNA, designated as S, M and L, which encode, respectively, the nucleoprotein, the G1 and G2 glycoproteins, and the RNA-dependent RNA polymerase. In order to study the assembly of a chimeric hantavirus-like particle, the Araraquara nucleoprotein and the Andes glycoproteins were expressed in a baculovirus system. Confocal microscopy showed the colocalization of G1 and G2 proteins with the N protein. Immunoprecipitation assay and sucrose density gradient showed the interaction among N, G1 and G2 proteins. The transmission electron microscopy showed the hantavirus-like particle with the same morphology of the virion. The chimeric hantavirus-like particle produced in this study could be used, in the future, in immunological, structural and morphological studies.

No Brasil a raiva dissemina-se de maneira insidiosa nos herbívoros domésticos, produzindo perdas à indústria pecuária. No estado de São Paulo, a última epizootia registrada ocorreu entre os anos 1997-2002, acometendo bovinos e equinos. Este estudo examinou a possível relação de alguns casos detectados no sul de Minas Gerais no período 2000 a 2009 com o foco paulista citado, mediante análise filogenética de segmentos do gene da glicoproteína (540 nucleotídeos) e nucleoproteína (416 nt) viral, usando o algoritmo de Neighbor-joining, modelo evolutivo Kimura 2- parâmetros com 1000 replicações, considerando sequências de isolados procedentes de diferentes regiões do interior paulista e do Brasil, tomadas do GenBank. Foi proposta uma análise geográfica mediante o programa ArcGis, localizando as coordenadas geográficas dos municípios de origem dos casos sobre mapas de relevo, bacias hidrográficas e distribuição de biomas. A análise filogenética dos dois genes estudados sugeriu que os focos mineiros podem ter a mesma origem genética da última epizootia paulista de raiva em herbívoros ocorrida entre 1997 e 2002. A análise filogenética baseada na nucleoproteína mostrou um maior nível de detalhamento sugerindo a ocorrência de diferentes eventos e/ou centros de dispersão de raiva em herbívoros na área de estudo. A análise geográfica insinuou que os casos aconteceram nas porções menos elevadas da Serra da Mantiqueira, na área de Mata Atlântica e em proximidades das bacias dos rios Piracicaba/Jaguarí, Paranaíba do Sul, Grande, Pardo e Mogi-Guaçu. / Bovine rabies is still spreading insidiously in Brazil, producing economic losses to livestock industry. A remarkable epizootics took place in São Paulo state between 1997 and 2002, affecting bovine and equines. This research was intended to examine genetic relations among some rabies outbreaks in Minas Gerais (MG), during 2000 and 2009 with São Paulo epizootics by phylogenetic analysis based on glycoprotein (540 nucleotides) and nucleoprotein (416 nt) genes partial sequences using Neighbor- joining algorithm, Kimura 2-parameters model, with 1000 bootstrap replications, considering sequences from different regions of São Paulo State (SP) and Brazil from GenBank. A geographic analysis was proposed, plotting geographic coordinates of municipalities with rabies cases on topographic, hydrographic and biome maps using ArcGis software. Phylogenetic analysis for both genes suggested that cases from MG can have the same genetic origin of SP epizootics. Phylogenetic analysis based on nucleoprotein gene showed a richer level of detailing than glycoprotein gene, suggesting different events and/or dispersion centers of livestock rabies in the studied area. Geographic analysis proposed that MG cases occurred at less elevated portions of Serra da Mantiqueira mountains in the area of Atlantic forest, near Piracicaba/Jaguarí, Paranaíba do Sul, Grande, Pardo and Mogi-Guaçu rivers.

The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4 % in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.