Rift Valley Fever virus (RVFV) causes an infection with severe impact on animal and human health. The disease is endemic throughout almost the entire African continent and large regions of the Arabian Peninsula. During epidemics, high mortality is observed in animals, especially among cattle, goats, and sheep. In humans, the symptoms vary from a benign influenza-like disease to a life-threatening hemorrhagic fever. Due to the devastating effect on communities in endemic regions and the possibility of further spread of this virus, there is an imperative need to improve and develop control measurements against this emerging disease. Therefore, this thesis focuses on diagnostics and vaccines against RVFV. RVFV infection kinetics was studied in a mouse model system by detection and quantification of viral genomes, using a developed quantitative real-time PCR (QRT-PCR) method. This novel QRT-PCR method proved to be reliable and serves as a supplement to standard diagnostics, direct virus isolation and serological methods. High levels of viral RNA were found in blood and liver samples from experimentally infected mice during the first days post infection. Thereafter the levels declined rapidly and dropped below detection limit approximately seven days post infection. The QRT-PCR technique was also used in a study aimed to improve diagnosis of RVFV from field samples collected on filter strips. Today, the available RVFV vaccines are only approved for animal use and these vaccines have several shortcomings. Since RVFV is a highly pathogenic organism requiring bio-safety level 3 laboratories, two different none-replicating vaccine approaches have been applied and evaluated using a mouse model. A DNA based vaccine, administered via gene-gun, and the use of virus-like particles (VLP), by the intra-peritoneal route. RVFV specific and neutralising antibodies were raised with both vaccine approaches. However, VLP vaccination against Rift valley Fever proved to be more promising as a future vaccine, since higher titres of neutralising antibodies and improved survival rate were found upon a lethal RVFV challenge in mice. In conclusion, a sensitive and specific method for quantifying RVFV infection and a promising vaccine candidate against RVFV were developed.

At present, the human adenoviruses (Ads) comprise 51 members, which have been classified into six species (A to F). In general, adenovirus (Ad) tissue tropism or disease patterns vary according to species, although adenoviruses from different species can sometimes cause the same symptoms. The current interest in adenoviruses is partly due to the aim of using them as vectors for gene therapy. Hematopoietic cells are attractive targets for gene therapy and the transductions can be performed ex vivo. However, the most commonly used adenovirus vectors, based on Ad2 or Ad5, are inefficient in their transduction of hematopoietic cells since they attach poorly to these cells. Most Ads, including Ad2 and Ad5, appear to use the coxsackie-adenovirus receptor (CAR) (a component of tight junctions), for attachment to host cells. However, species B Ads do not bind to CAR and several studies have indicated that species B-based vectors would be more suitable for hematopoietic cells. Species B Ads can be further divided into species B1 and B2, which display different tissue tropisms. Species B1 Ads mostly cause acute respiratory infections whereas species B2 Ads have been associated with persistent infections of the kidney and urinary tract. One of the key determinants of tropism is believed to be the initial high-affinity attachment of the virion to host cell fiber receptors. By reciprocal blocking experiments and different ways of characterizing the species B attachment receptors, we have shown that the species B2 serotypes Ad11p and Ad35 and the species B1 serotypes Ad3p and Ad7p also differ in receptor usage. There are at least two different Ad species B receptors. Since one of these receptors appeared to be used by all four serotypes, we designated this receptor sBAR (species B adenovirus receptor). The other receptor appeared to be used exclusively by the two species B2 serotypes and was therefore designated sB2AR (species B2 adenovirus receptor). Binding to sBAR can be abolished by EDTA and restored with Mn2+ or Ca2+, whereas binding (of Ad11p and Ad35) to sB2AR is independent of divalent cations. Furthermore, sBAR appears to be trypsin sensitive whereas sB2AR is not. We also identified CD46 as a receptor for Ad11p. Even so, CD46 does not appear to be a functional receptor for Ad7p. Both Ad7p and Ad11p attached to CD46-transfected Chinese hamster ovary (CHO) cells more efficiently than to control CHO cells. However, only Ad11p (selectively) infected CD46-transfected CHO cells. Anti-CD46 antibodies inhibited Ad7p and Ad11p from binding to, and Ad11p from infecting, CD46-transfected CHO cells. However, in human cells, anti-CD46 antibodies had an inhibitory effect only on Ad11p binding (~30%) but did not affect Ad7p binding. In binding experiments with EDTA, divalent cations and pretrypsinized cells, Ad11p and Ad7p showed the same pattern in their binding to CHO-CD46 cells as in the previous study. Since Ad7p interacted almost as efficiently with control CHO cells as with CHO-CD46 cells after addition of Mn2+, it seems that Ad7p mainly addressed an endogenously expressed hamster receptor on CHO-CD46, the properties of which resemble sBAR. In addition, Ad3p and Ad7p attach poorly to PBMCs and CD46 is expressed on all nucleated cells. Thus, CD46 appears to correspond to sB2AR rather than to sBAR. With these differences in receptor usage in mind, we studied the binding and infectious capacity of these species B Ads in various hematopoietic cells. We found that all species B serotypes bound efficiently to CD34+ hematopoietic stem cells (HSCs) and also productively infected HSCs. However, only the sB2AR binding Ad serotypes Ad11p and Ad35 could attach primary PBMCs efficiently. Our results regarding the subsequent steps in infection of PBMCs suggest that both Ad11p and Ad35 enter PBMCs and deliver viral DNA to the nuclei of most PBMC cell types. However, productive infections were only clearly detected in stimulated T-cells (most frequently) and monocytes, whereas Ad infection seemed eclipsed in unstimulated lymphocytes. Replication of Ad DNA seemed seriously impaired in at least T-cells, indicating limited production of infectious particles in PBMCs. The capacity of species C Ads to establish persistent infections in lymphatic tissues has been described previously. These Ads also persistently infect various transformed hematopoietic cell lines in vitro. Our studies indicate that replication of the species B2 Ads is also restricted in cells of hematopoietic origin (both in primary and transformed cells). Taken together, the results indicate that species B2 Ads (as compared to other Ads) seem to enter and infect most hematopoietic cells efficiently, which is in line with the persistent nature of these Ads. They would presumably act as suitable vectors for efficient transduction of most cells of hematopoietic origin, as has already been shown for e.g. HSCs and dendritic cells. The finding that replication of Ads in T-cells appears to depend on the level of T-cell activation, strengthens the hypothesis that T-cells may serve as a reservoir for human Ads and raises possible safety issues for usage of species B-based vectors in hematopoietic cells.

Trace elements are essential for the host defence against infections, and during common infections, the balance of trace elements is changed in serum and tissues. Supplementation with selenium (Se), an essential trace element, is known to decrease the severity of coxsackievirus B3 (CVB3) infection in mice. Even the non-essential trace element arsenic (As) seems to influence the replication of some viruses. During the course of an acute CVB3 infection in mice, Se concentrations decreased in most tissues and were negatively correlated to viral load in our study. However, As concomitantly decreased in most tissues. As has previously been shown to interfere with the balance of essential trace elements. However, in the present study As supplementation in healthy mice resulted in minor effects on seven studied trace elements in serum and tissues. The effects of As supplementation were more pronounced in CVB3-infected mice, with an increase in As, but a decrease in Se in most tissues when compared with non-infected mice. As supplementation during CVB3 infection in mice decreased viral RNA concentrations in the brain (97%) and pancreas (75%), two of the target organs of this infection. In vitro experiments indicate that As caused an impaired virion assembly or release. In vivo, infection-induced expression of the host defence-associated genes nuclear factor κB (NFκB) and interferon γ (IFN-γ) were unaffected by As supplementation, except for an earlier increase in IFN-γ in the brain. In conclusion, a clinically relevant dose of As decreased the replication of CVB3 in vitro and in vivo. This antiviral effect in vivo was not related to changes in specific trace elements or in the host’s immune-mediated defence. Although the mechanism underlying the observed effect on viral replication remains to be further elucidated, As seems to be an intriguing trace element to study in the pursuit of new antiviral drugs.

A vast number of studies have been completed on the virulence determinants of Yersinia spp.; however, the focus of many of these studies has been on the virulence plasmid and the plasmid-encoded Type three secretion system. Nevertheless, many chromosomal genes whose products are directly involved in virulence have also been identified. Some of these critical virulence determinants are outer membrane proteins. Outer membrane proteins of Gram-negative bacteria often have important physiological roles; however, some have also been found to be important for pathogenesis. In this thesis, we investigated two Yersinia. pestis outer membrane proteins, Ail and OmpA, and their roles in virulence. We provide evidence that Y. pestis Ail is a highly expressed outer membrane protein that is absolutely essential for Y. pestis to resist the killing action of the complement system present in human blood and tissues, as well as the blood and tissues of other mammalian hosts. Furthermore, Ail was important for virulence in a Y. pestis-Canorhabditis elegans model of infection.The work in this thesis also provided the first evidence that another surface-exposed outer membrane protein, termed OmpA, is required for both Yersinia pseudotuberculosis and Y. pestis to survive and proliferate intracellularly in macrophages. Finally, we provide evidence that Y. pestis has a functional small RNA MicA that controls the expression of OmpA. This is the first demonstration of sRNA-mediated regulation of a Yersinia virulence factor. This work has paved the way for future studies on the role of outer membrane proteins in virulence, particularly the role of Ail and OmpA.

En 1998, le traitement de la co-infection VHC rarement discuté avant l'ère des HAART, compte tenu d'une réponse médiocre à la monothérapie par IFNα et d'un pronostic de vie lié au VIH estimé en moyenne à 10 ans, fût reconsidéré. C'est ainsi que débuta en 2000, l'essai RIBAVIC HC02, essai randomisé et multicentrique comparant l'association de la ribavirine 800 mg/j à l'Interféron 3 MUI x3/semaine ou au PEG-α-2b Interféron 1,5 μg/kg/semaine pendant 48 semaines. Une cohorte des patients inclus dans l'essai RIBAVIC (cohorte RIBAVIC EP10) débuta en 2001 pour évaluer le devenir à long terme de ces patients. L'essai RIBAVIC et la cohorte RIBAVIC ont apporté les enseignements suivants : - la cinétique de la charge virale VHC peut différer selon la nature du traitement antirétroviral. - la prévalence et les facteurs de risque de la stéatose sont similaires à ceux observés dans la population mono-infectée VHC - le taux de réponse virologique soutenue est inférieur chez les patients co-infectés (27%) comparé aux patients mono-infectés VHC (50%) - le taux de non réponse virologique (diminution de la charge virale VHC inférieure à 2 log à S12) sous traitement par pegIFN plus ribavirine est plus élevé (33%) comparé aux patients monoinfectés VHC (14%). L'interaction entre la ribavirine et l'abacavir pourrait être un facteur de risque. - l'indétectabilité de l'ARN VHC dès S4 est prédictive de la réponse à long terme (valeur prédictive positive 97%) et la décroissance de la charge virale VHC est significativement plus lente chez les patients rechuteurs comparée aux patients répondeurs long terme à S2 et à S4 - Au cours du traitement anti-VHC :1- le risque d'anémie est élevé et majoré par la coprescription de zidovudine et de ribavirine ; 2- l'amaigrissement est fréquent et sévère et peut être révélateur d'une toxicité mitochondriale ; 3- le risque bactérien n'est pas lié au taux des polynucléaires neutrophiles mais à la fibrose hépatique ; 4- le risque de toxicité mitochondriale, d'aggravation de la fibrose et de décompensation hépatique est majoré par l'interaction entre la didanosine et la ribavirine - une réponse virologique soutenue est associée à un bénéfice histologique et clinique.

The extent to which economic evaluations of public health programs in Ontario are conducted and used by decision makers is currently very limited. This thesis supports public health decision-making through applied and methodological work. The applied work demonstrates different methods to evaluate the cost-effectiveness of public health interventions using the examples of seasonal and pandemic influenza immunization programs. The methodological component explores whether time horizon choice, one methodological consideration in economic evaluations, introduces bias. The economic evaluation of Ontario’s universal influenza immunization program (UIIP) uses primarily provincial health administrative databases to assess the impact of UIIP on health outcomes (quality-adjusted life years (QALYs), mortality), health care resource use (physician office visits, emergency department visits, and hospitalizations), and costs due to seasonal influenza. Ontario’s UIIP was found to be cost-effective compared to a targeted program. The economic evaluation of Ontario’s H1N1 (2009) mass immunization program uses a mathematical modeling approach to describe the pandemic as observed in Ontario. By removing immunization from the simulation, the impact of the program was evaluated. Outcome measures include health outcomes (attack rate, deaths, QALYs), resource use, and cost (physician office visits, emergency department visits, hospitalizations). The analysis found Ontario’s mass immunization program to be highly cost-effective despite high program cost. The methodological component investigates whether time horizon choice, a major methodological choice, introduces bias to economic evaluations. The existence, magnitude and direction of time horizon bias are demonstrated using a formal model. This work supports current guidelines in recommending a lifetime time horizon and provides a framework to discuss bias in economic evaluations. This thesis demonstrates different approaches to evaluate the cost-effectiveness of public health interventions, informs decision-making, and establishes the groundwork to guide future economic evaluations of public health interventions.

The extent to which economic evaluations of public health programs in Ontario are conducted and used by decision makers is currently very limited. This thesis supports public health decision-making through applied and methodological work. The applied work demonstrates different methods to evaluate the cost-effectiveness of public health interventions using the examples of seasonal and pandemic influenza immunization programs. The methodological component explores whether time horizon choice, one methodological consideration in economic evaluations, introduces bias. The economic evaluation of Ontario’s universal influenza immunization program (UIIP) uses primarily provincial health administrative databases to assess the impact of UIIP on health outcomes (quality-adjusted life years (QALYs), mortality), health care resource use (physician office visits, emergency department visits, and hospitalizations), and costs due to seasonal influenza. Ontario’s UIIP was found to be cost-effective compared to a targeted program. The economic evaluation of Ontario’s H1N1 (2009) mass immunization program uses a mathematical modeling approach to describe the pandemic as observed in Ontario. By removing immunization from the simulation, the impact of the program was evaluated. Outcome measures include health outcomes (attack rate, deaths, QALYs), resource use, and cost (physician office visits, emergency department visits, hospitalizations). The analysis found Ontario’s mass immunization program to be highly cost-effective despite high program cost. The methodological component investigates whether time horizon choice, a major methodological choice, introduces bias to economic evaluations. The existence, magnitude and direction of time horizon bias are demonstrated using a formal model. This work supports current guidelines in recommending a lifetime time horizon and provides a framework to discuss bias in economic evaluations. This thesis demonstrates different approaches to evaluate the cost-effectiveness of public health interventions, informs decision-making, and establishes the groundwork to guide future economic evaluations of public health interventions.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2009. / Härtill 3 uppsatser.

published_or_final_version / Public Health / Master / Master of Public Health

This work uses mathematical models of evolutionary dynamics to address clinical questions about HIV treatment, public health questions about vaccination, and theoretical questions about evolution of high mutation rates.