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Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.

In this thesis we have focussed on the RVFV Gn and Gc glycoproteins, the keys for virus entry and virus control. RVFV is an emerging virus that has already spread across the African continent and to the Arabian Peninsula. Outbreaks of RVFV are characterized by abortion storms and high mortality, particularly among young animals. Humans can be infected by exposure to blood or other body fluids of viremic animals or via mosquito bite. RVFV has the potency to spread across large distances by as yet unknown mechanisms, explaining the need for contingency plans in currently unaffected areas. We aimed to develop control measures to protect livestock against RVFV infection. Antibodies directed against the Gn and Gc glycoproteins of RVFV are able to neutralize the virus, which is the only established correlate of protection against RVFV infection. We have developed and characterized different Gn/Gc-based vaccines that are considered safe for animals of all ages and evaluated their efficacy in mouse and sheep animal models. Registration trials with one of our vaccines are currently being performed. We have also investigated the RVFV cell entry mechanism and paid specific attention to the membrane fusion process. We demonstrate that the glycosaminoglycan heparan sulfate is required for efficient RVFV infection of different mammalian cells. Our studies have also confirmed the hypothesis that RVFV membrane fusion occurs in endosomes and is triggered by low pH. The activating pH and the kinetics of virus entry suggest that RVFV penetrates from late endosomal compartments. We further demonstrate that exposure of virus particles to low pH results in the formation of highly stable multimers of the RVFV Gc glycoprotein. Mutagenesis of Gc in combination with biochemical and functional analyses suggest that protonation of specific histidines in Gc plays a crucial role in the conformational rearrangements of the Gc fusion protein that lead to virus entry.

Most human cases of highly pathogenic H5N1 avian influenza virus (HPAIV) infection are the result of direct contact with infected poultry. Therefore, infection of poultry should be prevented to avoid human exposure. One method to combat HPAIV outbreaks relies on depopulation. An alternative or supplementary method is the use of DIVA (discriminating infected from vaccinated animals) vaccines to prevent infection of animals on holdings surrounding an outbreak. Discrimination between infected and vaccinated animals is often based on the ‘heterologous neuraminidase’ strategy. This implies that a suitable vaccine can only be selected when the N-subtype of the outbreak strain is known. Thus, at least two vaccines with different N-subtypes must be available, allowing a switch of vaccine in the event that one of them matches the outbreak strain. However, such vaccines cannot be used preventively in situations in which the N-subtype of the outbreak strain is unknown. In order to circumvent these drawbacks we generated a recombinant influenza virus containing the HA gene of a contemporary H5N1 HPAIV strain in combination with the NA gene of a human type B influenza virus. An inactivated vaccine based on this virus protected chickens against clinical disease, and completely prevented virus shedding after H5N1 HPAIV challenge infection. Serological analyses confirmed that the vaccine complied with the DIVA principle. Since NA of type B does not occur in avian influenza strains, this vaccine is suitable as a DIVA vaccine against any H5 HPAIV, and may be used preventively without compromising the DIVA principle.

A multiplex bead-based suspension array was developed that can be used for the simultaneous detection of antibodies against the surface glycoprotein Gn and the nucleocapsid protein N of Rift Valley fever virus (RVFV) in various animal species. The N protein and the purified ectodomain of the Gn protein were covalently linked to paramagnetic Luminex beads. The performance of the resulting multiplex immunoassay was evaluated by testing a comprehensive and well-characterized panel of sera from sheep, cattle and humans. The suitability of this multiplex immunoassay to differentiate infected from vaccinated animals (DIVA) was investigated by testing sera from lambs vaccinated with a paramyxovirus vaccine vector expressing the RVFV surface glycoproteins Gn and Gc. The results suggest that the bead-based suspension array can be used as a DIVA assay to accompany several recently developed experimental vaccines that are based on RVFV glycoproteins, and are devoid of the N protein. © 2012 Elsevier B.V.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

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