Host tissue specificity of selected South African isolates of Rift Valley fever virus

Abstract

Rift Valley fever (RVF), is a mosquito-borne viral disease affecting humans and some species of ruminants including sheep, cattle, goats, buffalos and to a lesser extent wild animals. It is a re-emerging disease responsible for major losses in livestock production, with negative impacts on livelihoods of both commercial and resource- poor farmers in sub-Saharan African and some countries in the Middle East. It remains a threat to both endemic and non-endemic countries where competent mosquito vectors exist. The RVF virus (RVFV) causes the disease and though only a single serotype exists, differences in virulence and pathogenicity of the virus have been observed in a wide range of affected mammalian host species. This necessitates the need for a detailed genetic characterization of various isolates of the virus and whether the causal factors for host tissue tropism can be explained. Therefore, the aims of this study were to obtain comprehensive information on the genetic composition of the RVFVs circulating in South Africa between 2008 and 2010 and to differentiate these isolates based on cell infectivity and genomic parameters. In the first chapter the status of some published literature on the disease as well as the virus are reviewed. Viral characteristics, replication, assembly and release of the viral particle from the cell as well as virus-host receptors documented are also mentioned in this chapter. Chapter two focused on the genetic composition of RVFVs that caused outbreaks during 2008- 2010 in South Africa. Complete genome sequence analysis of isolates from different hosts and tissues collected at discrete foci of outbreaks were analysed and compared with virus sequences from earlier outbreaks in South Africa and from other countries. Phylogenetic analysis indicated that viruses that caused outbreaks during 2008-2010 were most probably reassortants, resulting from exchange of portions of the genome of different isolates, particularly of Segment M. In addition, the analysis indicated that the viruses were not introduced from outside the country but mutated in time and caused the outbreaks when the environmental conditions became favourable. Although no clear association between the virus genotype and phenotype has been established, various amino acid substitutions have been implicated for changes in the phenotype. The third chapter describes the characterization of isolates derived from different hosts (bovine and ovine), but from the same tissue (liver). The isolates from bovine liver presented a different growth phenotype in a cell culture-based system as well as some amino acid substitutions when compared with isolates from ovine livers. Although the codon usage patterns of the six isolates were the same, they differed with those of their hosts. Further investigation of the coding regions of the genome, molecular modelling of glycoproteins and codon usage bias failed to explain the phenotypic changes. The fourth chapter focused on an attempt to identify RVFV glycoprotein receptors using the yeast two-hybrid (Y2H) system. Baby hamster kidney cells were chosen as host cells in the laboratory because hamsters are known to be highly susceptible to RVFV. The complexity of the cDNA library constructed from BHK cells were assessed by random sequencing of 100 clones and revealed that 51 clones were genes from mRNA from the Syrian/Golden hamster using BLAST. The constructed library can also be used to study other animal pathogens such as bluetongue virus and African horse sickness virus. The constructed bait plasmids did not show any autoactivation or toxicity in yeast, thus making them suitable to be used in the Y2H system. Twelve unique clones (4 clones using transformants of the glycoprotein Gn and 8 clones using transformants of glycoprotein Gc) were screened from the cDNA library. Identification and further characterization of the clones is necessary. Sampling of the isolates that caused the 2008-2010 outbreaks in South Africa and full genome sequencing indicated that the isolates were genetically distinct, grouping in different clades, namely C and H. Reassortment have been identified in some of these isolates, particularly in their M segments. The majority of isolates that emerged in the outbreaks accumulated mutations over time while circulating in South Africa. The impact of these mutations on the pathogenicity of RVFV should be further investigated. Sequencing should be done on clinical samples directly to have a better idea of the phenotype and the effect of amino acid substitutions. Different phenotypes observed between cattle and sheep in tissue culture systems should be further investigated including investigation of different phenotypes in vivo using small experimental animals. The study has laid a foundation in understanding the pathogenicity of RVFV and necessitates the importance of understanding molecular mechanisms of the virus.