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INTRODUCTION
In 1898 researchers identified infectious agents that were smaller than the smallest known bacteria. Viruses are obligate intracellular parasites that use the machinery of the host cell to replicate. They consist of a DNA or RNA genome surrounded by a protein capsid, which may be surrounded by an envelope in some viruses (Fields, 1998, Gale et al., 2000). Virus classification rests largely on the characteristics of the viral genome and virus structure.
Numerous viruses perform virion associated enzymatic activities, which vary according to the strategy for replication of their nucleic acid. Genome transcription is an important stage in the life cycle of a virus as the product of this process must be recognized by the machinery of the host cell for the production of viral proteins necessary for genome replication and the assembly of progeny virions. In the case of viruses with dsRNA genomes, host cells do not have the endogenous enzymes for transcribing from a dsRNA template. Also, dsRNA is never released into the cell because it would evoke a cellular defence response (Bamford, 2000). As a result, dsRNA viruses produce their own enzymes to synthesize mRNA transcripts within the core particle (Lawton et al., 2000).
It is thought that the mechanisms involved in the production of mRNA are similar in most viruses having dsRNA genomes. Functional similarities include a RNA-dependent RNA polymerase that produces single-stranded mRNA transcripts from the dsRNA genome as well as replicating the viral genome from single-stranded RNA templates.Viruses infecting eukaryotes have a capping enzyme for synthesizing 5’ capped mRNA for translation by the eukaryotic machinery (Lawton et al., 2000). Double-stranded RNA viruses require unwinding of the genome segments by a helicase prior to transcription (Kadaré and Haenni, 1997).
The International Committee for the Taxonomy of Viruses (ICTV) recognizes eight families of dsRNA viruses (Mertens, 2004). This includes the family Reoviridae, members of which infect mammals, other vertebrates, insects and plants. Within the Reoviridae family is the genus Orbivirus (Levy et al., 1994). Virus species within the Orbivirus genus that are known to infect equids include African horsesickness virus (AHSV), equine encephalosis virus (EEV) (Mertens et al., 2000) and Peruvian horse sickness virus (PHSV) which was recently assigned to the genus.
African horsesickness virus causes an infectious but non-contagious disease of equines (Coetzer and Erasmus, 1994). Mortality rates in naive populations of horses can exceed 98% (Mertens et al., 2000). It is accordingly considered to be one of the most lethal diseases of horses and has been given Office International des Epizooties (OIE) list A University of Pretoria etd – De Waal, P J (2006) status. Although mortality of horses as a result of AHS occurs annually in South Africa, major epidemics of the disease also occur (Coetzer and Erasmus, 1994).
CHAPTER 1: LITERATURE REVIEW
1.1 INTRODUCTION
1.2 FAMILY REOVIRIDAE
1.3 ORBIVIRUSES
1.4 AHSV AND BTV EPIDEMIOLOY, TRANSMISSION AND GEOGRAPHICAL
DISTRIBUTION
1.5 ORBIVIRUS INFECTION
1.6 AFRICAN HORSESICKNESS PATHOGENESIS
1.7 DETECTION OF AHSV IN INFECTED AND VACCINATED HORSES
1.8 ORBIVIRUS MORPHOLOGY
1.9 STRUCTURE AND FUNCTION RELATIONSHIPS OF ORBIVIRUS GENES AND GENE PRODUCTS
1.9.1 The outer capsid: VP2 and VP5
1.9.2 The core
1.9.3 The inner core
1.9.4 The nonstructural proteins: NS1, NS2, NS3 and NS3A
1.10 VIRAL ENZYMATIC FUNCTIONS
1.10.1 Viral Helicases
1.10.1.1 Structural features of helicases
1.10.1.2 Helicase families
1.10.1.3 Function of the conserved motifs
1.10.1.5 Models of helicase activity
1.10.1.6 Prevalence and role of helicases in viruses
1.10.1.7 Past and future aspects of viral helicases
1.10.2 Viral Transcriptase Activities
1.10.3 Replication and transcription in BTV
1.11 AIMS
CHAPTER 2: CLONING AND CHARACTERIZATION OF THE GENOME SEGMENT
ENCODING VP6 OF AHSV
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.2.1 Preparation of dsRNA for cDNA synthesis
2.2.2 Sephadex column chromatography
2.2.3 cDNA synthesis
2.2.4 Alkaline agarose gel electrophoresis
2.2.5 Glassmilk purification
2.2.6 Preparation and Transformation of competent E. coli cells
2.2.7 Plasmid isolation
2.2.8 Preparation of Recombinant Plasmid DNA by Cesium Chloride gradient purification
2.2.9 Amplification by PCR
2.2.10 Cloning of the PCR product
2.2.11 Subcloning of the genome segment encoding VP6
2.2.11.1 Subcloning into M13
2.2.11.2 Subcloning into pBS
2.2.12 Manual sequencing
2.2.13 Sequence analysis
2.2.14 Phylogenetic analysis
2.2.15 Primary Structure Analysis
2.2.16 Hydrophilicity and Secondary Structure
2.3 RESULTS
2.3.1 Cloning of AHSV serotype 9
2.3.2 Amplification of the genome segment encoding VP6 of AHSV by polymerase chain reaction from pools of cDNA
2.3.3 Subcloning and sequencing
2.3.4 Sequence analysis
2.3.5 Phylogenetic analysis
2.3.6 Amino Acid Sequence Analysis
2.3.7 Hydrophilicity and Secondary Structure
2.4 DISCUSSION
CHAPTER 3: CHARACTERIZATION OF THE VP6 PROTEIN OF AHSV
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 In vitro expression
3.2.2 In vitro translation
3.2.3 Polyacrylamide gel electrophoresis
3.2.4 In vivo expression using the BAC-to-BAC system
3.2.5 Preparation of competent cells by the DMSO method
3.2.6 Generation of recombinant bacmids in DH10BAC cells by transposition
3.2.7 Isolation of composite bacmid DNA
3.2.8 Transfection into Spodoptera frugiperda cells
3.2.9 Infection of Sf9 cells
3.2.10 Virus titration by plaque assay
3.2.11 Infection of monolayers for virus stocks and protein
3.2.12 Western immunoblo
3.2.13 5’ modification by PCR
3.2.14 Automated sequencing
3.2.15 Cloning and expression in A baculovirus System
3.2.16 Cloning and expression using A bacterial system
3.2.17 In vivo protein labelling
3.2.18 Sucrose gradient analysis
3.2.19 Ni-NTA column purification
3.2.20 Glycosylation assay by PAS staining
3.3 RESULTS
3.3.1 In vitro translation of VP6 mRNA
3.3.2 In vivo baculovirus expression
3.3.3 Bacterial Expression
3.3.4 Western immunoblot analysis
3.3.5 Protein solubility studies
3.3.6 Protein purification
3.3.7 Glycosylation assay by PAS staining
3.4 DISCUSSION
CHAPTER 4: ANALYSIS OF NUCLEIC ACID BINDING ACTIVITY OF AHSV-6 VP6
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.2.1 Nucleic acid overlay protein blot assays
4.2.2 Competition assays
4.2.3 Preparation of single and double-stranded nucleic acid probes
4.2.4 Specific activity calculations
4.2.5 N-Glycosidase F deglycosylation
4.2.6 Tunicamycin deglycosylation
4.2.7 Deletion mutation analysis
4.2.8 Screening of composite bacmid DNA by PCR
4.2.9 Electrophoretic mobility shift assays (EMSA)
4.3 RESULTS
4.4 DISCUSSION
CHAPTER 5: CONCLUDING REMARKS
CHAPTER 6: RESEARCH OUTPUT
CHAPTER 7: REFERENCES
