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Two highly pathogenic arthropod-borne (arbo)viruses, West Nile virus (WNV) and chikungunya virus (CHIKV), recently (re-)emerged in both Europe and the Americas. This resulted in large-scale epidemics of severe encephalitic and arthritogenic human disease, respectively. Both viruses replicate in their vertebrate hosts and mosquito vectors to complete their respective transmission cycles. In mosquitoes, arbovirus infections lead to relatively high viral titres without causing notable disease symptoms or fitness costs, whereas virus replication in the vertebrate host initiates strong antiviral responses and can be highly pathogenic and sometimes deadly.
WNV is a flavivirus (family Flaviviridae; genus Flavivirus), that finds its origin in Africa. The introduction of lineage 1 WNV into North America in 1999 caused the largest outbreak of human neuroinvasive disease to date. In southern Europe, a highly pathogenic lineage 2 strain has recently established itself in 2010, causing annual outbreaks. Additionally, the related flavivirus Usutu virus (USUV), has also emerged in Europe. Both WNV and USUV are transmitted by mainly Culex mosquitoes between avian amplifying hosts, but also frequently infect humans and horses. USUV and WNV co-circulate in parts of southern Europe, but the distribution of USUV extends further into central and north-western Europe.
In this thesis the potential spread of both WNV lineages through Europe is investigated by determining how effectively north-western European common house mosquitoes (Culex pipiens) transmit WNV. The results were compared to the transmission rates of USUV. North-western European mosquitoes were found to be highly competent vectors for both pathogenic lineages of WNV, which underscores the epidemic potential of WNV in Europe. Interestingly, American Culex pipiens only efficiently transmitted WNV lineage 1 but not the European lineage 2, which indicates a high degree of genotype-genotype specificity in the interaction between virus and vector. Furthermore, by comparing blood meal infection with intrathoracic injection of mosquitoes with WNV, the differential transmission rates of WNV lineage 2 could be attributed to infection barriers at the midgut level. In the vector competence studies, European mosquitoes were also found to be highly competent for USUV transmission. Interestingly, at higher temperatures USUV infected significantly more mosquitoes as compared to WNV. This indicates that mosquitoes from WNV-free areas are intrinsically capable of transmitting both pathogenic WNV lineages and explains the current localized WNV activity in southern Europe.
In addition, the infection rates of WNV and USUV were both enhanced at higher temperatures. This implies further epidemic spread of WNV and/or USUV during periods with favourable climatic conditions. Finally, as both viruses utilize the same vector and reservoir species, the higher infection rate of USUV suggests that this virus may precede WNV transmission in Europe. This presses the need for intensified surveillance of virus activity in current WNV-free regions and warrants increased awareness in the clinic throughout Europe
In contrast to WNV, CHIKV (Family Togaviridae; genus Alphavirus) is transmitted in an urban transmission cycle involving humans and two major mosquito species: Aedes aegypti and Aedes albopictus. These invasive, originally African and Asian mosquito species are the drivers of the recent CHIKV outbreaks in Europe and the Americas. The first autochthonous CHIKV transmission on the American continent was detected in late 2013 and by the end of 2014 over a million people were diagnosed with a CHIKV infection. In humans, CHIKV can cause high fever and incapacitating arthralgia. There are no vaccines or antiviral compounds available for human use against either CHIKV (or WNV) and broad-spectrum antiviral treatments have proven ineffective. To develop novel strategies that interrupt the CHIKV transmission cycle, it is key to understand how CHIKV replicates in both vertebrates host and the mosquito vector. The molecular mechanisms that determine effective viral replication in mosquitoes are largely unknown. By studying the intracellular localization of CHIKV non-structural protein 3 (nsP3) in insect cells, an interaction between nsP3 and the endogenous mosquito protein Rasputin was uncovered and elucidated. Both proteins were found to interact via two short amino acid repeats within the C-terminus of nsP3 and the NTF2-like domain of Rasputin, forming cytoplasmic nsP3-Rin granules. Silencing of endogenous Rasputin in live Ae. albopictus mosquitoes revealed that this protein is essential for CHIKV to effectively establish transmissible infections. This is the first reported function of mosquito Rasputin in arbovirus infection.
Vertebrate cells express two proteins that are homologue to mosquito Rasputin, namely Ras-GAP SH3 domain-binding protein (G3BP) 1 and 2. G3BP proteins are crucial components of mammalian stress granules (SG), which are RNA triage centers that form during environmental stress, leading to impaired translation of most mRNAs. In co-localization studies in mammalian cells it is shown how CHIKV nsP3 sequesters G3BP into viral nsP3-G3BP granules. By making G3BP unavailable, nsP3 inhibits a bona fide SG response. The evidence obtained in these studies contributes to the growing evidence that cellular SGs possess antiviral activity, yet at the same time indicate a novel, proviral role for Rasputin during infection of the mosquito vector.
In mammalian cells, cytoplasmic pattern recognition receptors (PRR) localize to SGs. These PRRs recognize specific viral molecular patterns and upregulate the expression of interferon (IFN), activating the most potent vertebrate antiviral response, the IFN-response. The IFN-response is sufficient to clear most arbovirus infections, but administering IFN in response to CHIKV infections is ineffective. Experiments in this thesis show that CHIKV replication is resistant to IFN once RNA replication has been established, because CHIKV actively prevents IFN-induced gene expression via the inhibition of the down-stream JAK-STAT signaling pathway. In response to extracellular IFNs, this pathway activates STAT proteins, which then dimerise and translocate to the nucleus to activate antiviral gene transcription. WNV and other flaviviruses have evolved specific mechanisms to evade and inhibit the IFN-response, while alphaviruses such as CHIKV cause general host shut-off to prevent antiviral gene expression. Clear evidence is now obtained that in addition to general host shut-off, CHIKV nsP2 inhibits the JAK-STAT signaling pathway in a specific manner. Genetic evidence is presented which reveals that nsP2 independently affects RNA replication, CHIKV induced host shut-off and cytopathicity, and JAK-STAT signaling. Additional data shows that the activation and nuclear translocation of STAT is unaffected by nsP2, but that the C-terminal domain of nsP2 within the nucleus is sufficient to quickly redirect STAT dimers out of the nucleus. This host shut-off-independent inhibition of IFN signaling by CHIKV nsP2 is likely to have an important role in viral pathogenesis.
In the final phase of viral replication, viral envelope proteins mature in the endoplasmic reticulum (ER) before they translocate to the plasma membrane. When the ER-protein folding load becomes too high, unfolded and misfolded proteins in the ER will activate the unfolded protein response (UPR). Transient expression of CHIKV envelope glycoproteins are now shown to have the potential to induce the UPR. The UPR aims to reduce general protein synthesis and increase the protein-folding capacity of the ER. CHIKV infection resulted in the phosphorylation of eukaryotic translation initiation factor 2, but did not increase the expression of well-known UPR target genes. In addition, functional X-box-binding protein 1 did not translocate into the nucleus during CHIKV infection. Individual expression of CHIKV nsPs revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
Finally, this thesis describes how effectively potential mosquito vectors transmit the flaviviruses WNV and USUV and provides novel insights on the underlying molecular mechanisms that enable CHIKV to accomplish successful infections in both its human host and mosquito vector. The effective inhibition of the JAK-STAT signaling pathway, combined with host shutoff, induced by nsP2 provides a rationale for the ineffectiveness of broad-spectrum antivirals against acute arbovirus infections and suggests directing future antiviral drug development to more specific compounds that directly interfere with viral replication and transmission. The uncovered interaction between CHIKV nsP3 and Rasputin/G3BP may provide such a target as disturbing this interaction could potentially re-instate cellular stress responses and interfere with viral replication and transmission.
|Doctor of Philosophy
|5 Jun 2015
|Place of Publication
|Published - 5 Jun 2015
- west nile virus
- chikungunya virus
- disease transmission
- mosquito-borne diseases