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RVFV pathology and epidemiology 383

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus 384 (RVFV), a member of the Phlebovirus genus, family Phenuiviridae of the recently 385 established order Bunyavirales (Maes et al., 2018, Briese et al., 2016). RVFV was first 386 isolated in 1930, during an outbreak with sudden deaths and abortions among sheep 387 along the lake Naivaska in the Rift Valley of Kenya (Daubney et al., 1931). The first 388 human disease caused by RVFV was reported in 1951 from South Africa (Woods et 389 al., 2002; Mundel and Gear, 1951). Outbreaks then occurred in Egypt 1977/1978 390 (between 18,000 and 200,000 human infections and 598 deaths), Mauritania 1987 391 (approximately 200 human deaths), East Africa and Madagascar 1991 (89,000 human 392 infections and more than 500 deaths) and East Africa 1998 (98,000 human infections 393 and 250 deaths) (Swanepoel and Paweska, 2011; Swanepoel and Coetzer, 2004). 394 The disease emerged for the first time outside Africa on the Arabian Peninsula in 2000- 395 2001 and caused an outbreak among livestock and humans (Balkhy and Memish, 396 2003). RVF outbreaks in Saudi Arabia and Yemen in 2000 affected 882 humans and 397 resulted in 124 deaths (Balkhy and Memish, 2003; Jupp et al, 2002).
More recently, 398 RVF outbreaks in humans were recorded in East Africa between 2006 and 2011 399 (Archer et al., 2011; Mohamed et al., 2010; Nguku et al., 2010; Shieh et al., 2010), 400 Madagascar in 2008 (Andriamandimby et al., 2010; Swanepoel and Coetzer., 2004) 401 South Africa in 2008 and between 2009 and 2011 (Metras et al., 2012; Archer et al., 402 2011; Swanepoel and Paweska., 2011) and for the first time on the Archipelago of 403 Comoros on the French Island of Mayotte in 2012 (Lernout et al., 2013, Cetre-Sossah 404 et al., 2012; Sissoko et al., 2009). Outbreaks of RVF are usually associated with 405 prolonged and heavy rainfall occurring at irregular intervals, leading to an upsurge in 406 mosquito populations (Swanepoel and Paweska, 2011; Swanepoel and Coetzer, 407 2004). Mosquitoes of the Aedes and Culex genera are usually involved in transmission 408 of RVFV, but other mosquito genera (Anopheles, Eretmapoites and Mansonia) have 409 also been shown to be potential vectors of the virus (Turell et al., 2008).

Virus structure and genome organization 453

RVFV has a negative-sense, single-stranded RNA genome, divided into three 454 segments. The large segment (L) encodes the RNA-dependent RNA polymerase (L- 455 protein) in the negative sense orientation. The medium (M) segment encodes the 456 precursor of the envelope glycoproteins Gn and Gc, a 78-kilodalton (kDa) minor 457 structural glycoprotein and a non-structural 14-kDa protein, named NSm, in the 458 negative sense orientation. The small segment (S) employs an ambisense coding 459 strategy, with the open reading frame (ORF) of the nucleocapsid (N) protein in the 460 negative sense orientation and a non-structural protein (NSs) in the positive sense 461 orientation (Giorgi et al., 1991). The nucleocapsid protein and viral polymerase protein 462 associate with the viral genome segments to form ribonucleoproteins (RNPs), which 463 make up the replication machinery of the virus. The three genome segments contain 464 untranslated regions (UTRs) that contain promoters for transcription and replication by 465 the viral polymerase. The UTRs have complementary 5’-3’ ends with conserved 466 phlebovirus-specific sequences that base-pair to form panhandle structures that play 467 a role in replication, transcription and packaging (Kolakofsky and Hacker, 1991, 468 Simons and Petterson, 1991).

Viral entry, packaging and release 517

Although the complete life cycle of RVFV has not been fully elucidated, reverse 518 genetics, non-spreading virus particles, and minigenome systems have helped define 519 critical processes of attachment, RNA synthesis, packaging and release ( Wichgers 520 Schreur and Kortekaas 2016; Kortekaas, 2011; Lopez et al., 1995). Virions have an 521 icosahedral symmetry with a spherical shell in a T=12 lattice (Sherman et al., 2009; 522 Freiberg et al., 2008). There are 122 capsomeres consisting of hexamers and 523 pentamers of Gn-Gc heterodimers (Huiskonen et al., 2009), which comprise a Gn 524 head and Gc base (Rusu et al., 2012). Gn is most likely the binding partner of the 525 surface receptor while Gc facilitates membrane fusion through acid-triggered type-II 526 fusion (Halldorsson et al., 2018). Due to their location at the site of initial infection, 527 dermal macrophages and dendritic cells (DCs) are believed to be responsible for 528 phlebovirus spread through the host, including RVFV (Léger et al., 2015). The 529 Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC- 530 SIGN) is a C-type lectin highly expressed on dermal dendritic cells and macrophages, 531 and was found to act as an authentic entry receptor for phleboviruses including RVFV 532 (Léger et al., 2016).

Reverse-genetics of bunyaviruses 571

The study of molecular biology of negative strand RNA viruses has been 572 revolutionized by the development of reverse genetics techniques. These techniques 573 comprise two approaches. The first approach involves a “rescue system” where 574 changes are introduced in the viral genome and the effects of these changes on viral 575 phenotype and biology are directly studied. In this approach, a cDNA copy of the 576 altered viral RNA genome is generated and delivered into permissive cells, where it 577 serves as a template for the production of new viral proteins and new virus particles. 578 The second approach involves “minigenome systems”, in which genome analogues 579 containing a reduced number of viral genes and an easily measurable reporter gene 580 are transcribed and replicated by coexpressed viral proteins (Reviewed in Bouloy and 581 Weber, 2010).

Natural variation in the RVFV genome 620

Viral genome characterization is essential to improve understanding of the role 621 sequence variation has on virulence. The RVFV genome is overall highly conserved 622 and stable (Grobbelaar et al., 2011; Aitken., 2008; Bird et al., 2007b; Sall et al., 1997; 623 Battles and Dalrymple, 1988). Despite their geographical dispersion, all strains of 624 RVFV remain closely related at the nucleotide and amino acid level. Sequencing of 625 geographically diverse RVFV isolates revealed only a 4% and 1% variation in the S- 626 segment, a 5% and 2% variation in the M-segment and a 4% and 1% variation in the 627 L-segment at nucleotide and deduced amino acid sequences, respectively (Bird et al., 628 2007b). Similar results were obtained from partial M-segment sequences (Grobbelaar 629 et al., 2011). This is likely because the time to most recent common ancestor (TMRCA) 630 for RVFV is recent, with an estimated time of 120-130 years (Grobbelaar et al., 2011; 631 Bird et al., 2007b). Another explanation for this low divergence is the so called “double- 632 filter” hypothesis, which suggests that arbovirus genomes are subject to selective 633 pressures in both the mammalian and insect host, thus leading to tighter genomic 634 constraint (Bird et al., 2007b). This hypothesis has been supported for RVFV in vitro 635 in which the genomic stability of NSs was dependent upon alternative passage 636 between insect and mammalian cells (Moutailler et al., 2011). Initial phylogenetic 637 studies indicated that RVFV groups into two major lineages (Egyptian and sub- 638 Saharan), based on sequences of the NSs gene (Sall et al., 1997).

TABLE OF CONTENTS :

• Declaration
• Acknowledgements
• Thesis summary
• Table of contents
• List of tables
• List of figures
• List of abbreviations
• 1 General introduction
  • 1.1 RVFV pathology and epidemiology
  • 1.2 Virus structure and genome organization
  • 1.3 Viral entry, packaging and release
  • 1.4 Reverse-genetics of bunyaviruses
  • 1.5 Natural variation in the RVFV genome
  • 1.6 Virus host interactions
  • 1.6.1 Interferon pathways
  • 1.6.2 Interferon-stimulated genes and restriction factors
  • 1.7 RVFV virulence factors
  • 1.7.1 RVFV NSs protein
  • 1.8 Animal models for Rift Valley fever virus
  • 1.8.1 Mice
  • 1.8.2 Rats
  • 1.8.3 Ruminants
  • 1.8.4 Non-human primates
  • 1.9 Thesis objectives
  • 1.9.1 General objective:
  • 1.9.2 Specific objectives:
  • 1.10 Hypothesis
• 2 Materials and methods
  • 2.1 Ethics statement
  • 2.2 Experimental animals
  • 2.3 Viruses
  • 2.4 Cell lines
  • 2.5 Mutagenesis of the five cysteine residues of the NSs protein
  • 2.5.1 Extraction of the DNA Plasmid pUC
  • 2.6 Synthesis of the mutant strand
  • 2.6.1 Digestion of the parental supercoiled plasmid DNA
  • 2.6.2 Transformation of the mutated plasmid in ultracompetent cells
  • 2.6.3 Extraction of mutated plasmids
  • 2.6.4 Viruses rescue
  • 2.6.5 Sequencing analysis of the rescued viruses
  • 2.7 Characterization of the rescued virus
  • 2.7.1 Viral growth kinetics analysis
  • 2.7.2 Characterization of viral foci morphology by plaque assay
  • 2.7.3 Western blot analysis
  • 2.7.4 Immunofluorescence analysis
  • 2.7.5 Two-step quantitative TaqMan RT-PCR assays
  • 2.8 Study of the virulence of the mutated viruses in BALB/c mice
  • 2.8.1 Virus inoculation in BALB/c mice and collection of samples
  • 2.8.2 Extraction of RNA
  • 2.8.3 Viral loads
  • 2.9 Sequencing analysis of the virus extracted from infected organs
  • 2.10 Histopathology examination
  • 2.11 Immunohistochemistry examination
  • 2.11.1 Inflammatory cytokines and receptors and Type-I inflammatory response
  • 2.12 Relative quantification data analysis
  • 2.13 Statistical analysis
• 3 Rescue and characterization of wtRVFV and NSs mutants
  • 3.1 Introduction
  • 3.2 Results
  • 3.2.1 Rescue of recombinant RVFV from transcription plasmids
  • 3.2.2 Characterization of the rescued virus
  • 3.3 Discussion
• 4 Virulence study in BALB/c mice
  • 4.1 Introduction
  • 4.2 Results
  • 4.2.1 Evaluation of RVFV virulence in BALB/c mice
  • 4.2.2 Survival of mice
  • 4.2.3 Detection of viral RNA by Q-RT-PCR in infected mice
  • 4.2.4 Histopathology
  • 4.3 Immunohistochemistry
  • 4.4 Discussion
• 5 Cytokine gene expression studies
  • 5.1 Introduction
  • 5.2 Results
  • 5.2.1 Cytokine gene expression in RVFV infected mice
  • 5.3 Discussion
• 6 Conclusions and future directions
• 7 References
• 8 APPENDICES

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Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in a murine model infection