INVESTIGATING RECOMBINATION IN AHSV GENOMES

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Total dsRNA extraction

Total dsRNA extraction was carried-out as described by Potgieter et al., 2009. Briefly; infected cells of 4+ CPE were scraped from the flask and centrifuged at 3000xg for 5 minutes. The centrifugation was followed by RNA extraction using a commercial guanidinium isothiocyanate reagent (TRI-Reagent) (Ambion). The aqueous phase was mixed with CHCl3 prior to centrifugation at 12 000 x g for 40 minutes at 4oC. The supernatants were placed into a clean tube followed by the addition of an equal volume of isopropanol and centrifuged at 12 000 x g for 45 minutes at 4oC to precipitate the RNA. The RNA was dissolved in 90 μl elution buffer. Single-stranded RNA (ssRNA) was precipitated by adding LiCl to a final concentration of 2.6M and incubation at 4oC overnight. The ssRNA was pelleted by centrifugation for 40 minutes at 4oC. The centrifugation step was followed by dsRNA purification from collecting supernatants using a MiniElute gel extraction kit (Qiagen), according the manufacturer’s specifications). After extraction, the dsRNA genome was visualised on 1% agarose gel.

Oligo ligation

The.‘anchor.primer synthesized by Tib Molbiol was ligated to the dsRNA as previously outlined by Potgieter et al (2009). Briefly; The anchor primer, PC3-T7 loop, was added to the reaction containing 0.5 – 200 ng RNA, 3 μl DMSO (Sigma), PC3-T7 loop primer (300 ng/μl), 1 Weiss unit T4 RNA ligase (Thermo Scientific) in a 10X ligation buffer consisting of 10 μl of 60% PEG 6000; 0.5M HEPES, pH8.0; 0.18M MgCl2; 10mM ATP; 30.6mM DTT with BSA 0.1 % (Takara) for a final volume of 30 μl and incubated at 37°C overnight.

Oligo-Ligation products purification

The dsRNA was purified using the MinElute Gel extraction kit as per manufacturer specifications (Qiagen). Briefly; the volume of the oligo-ligation reaction mixture was increased to 100 μl with Ultra-pure water. Three times buffer QC (Qiagen) and an equal volume of isopropanol was added. The solution was centrifuged through a column for 1 minute at 12 000 x g. The column was w Sequence–independent cDNA synthesis The dsRNA was denatured using 300 mM methyl mercury hydroxide (MMOH) and reverse transcribed into complementary DNA (cDNA) as described previously by Potgieter et al (2009). The dsRNA was denatured using 300 mM mercury hydroxide (MMOH) and incubated at room temperature for 30 minutes. After denaturation of dsRNA, a cDNA mixture was prepared by adding 2.5mM dNTP mix (Takara), cDNA buffer consisting of 300 mM MMOH, 1M Tris-Cl pH 8.3, 3M KCL, 1M β-M ethanol, 15U/μl Cloned Avian Myeloblastosis Virus (AMV) Reverse Transcriptase (Invitrogen) and denatured RNA into 0.5 ml tube. The mixture was incubated at 42oC for 55 minutes, at 55°C for about 15 minutes and allowed to cool to 20°C before adding 1N NaOH. After adding 1N NaOH, the content was incubation at 65°C for 30 minutes and cooling at 20 °C to remove RNA strands. After the removal of RNA, Tris buffer (pH 8.3) was added to the 0.1M final concentration and followed by adding HCl to the 0.1M final concentration and incubated at 65°C for 60 minutes followed by cooling at 20°C.

PCR amplification and cDNA purification

The Phusion® High-Fidelity PCR kit (NEB) was used to amplify the cDNA as described by Potgieter et al (2009). A PCR mixture was prepared by adding a 5x Phusion buffer, 10 mM dNTPs (Phusion HF PCR kit- NEB), 50 pmol/μl PC2 primer (5’ PO4-CCGAATTCCCGGGATCC-OH 3’), H2O, cDNA and Phusion Polymerase (2U/μl) making a final volume of 50 μl. The reaction had an initial denaturation at 98°C for 1.5 min, followed by 20 cycles of 94°C for 15 sec, 65oC for 30 seconds, 72°C for 4 minutes and a final elongation step at 72°C for 5 min. The amplified cDNA was visualized and evaluated on a 1.2 % agarose gel that was stained with ethidium bromide. The cDNA products were subsequently purified using the GeneJET Gel Extraction kit (Thermo Scientific) as per manufacturer specifications.

AHSV ds-cDNA sequencing

Purified PCR products were commercially sequenced using an Illumina MiSeq instrument (Illumina, Inc, CA, USA) at Inqaba Biotec (Pretoria, South Africa). Libraries were put together using the Nextera DNA Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA) using sequencing adapters and barcodes with the Illumina MiSeq sequencing reagent v3. The purified AHSV dsRNA PCR products were sequenced on a single run. Approximately 75Mb of data was obtained per virus consisting of 300-bp paired-end reads.

AHSV sequence assembly

The forward and reverse reads of each sample were imported into CLC Genomics Workbench v7.0 (CLC bio, Aarhus, Denmark). Pair-end sequence reads were trimmed at the 5’ and 3’ end in order to remove the sequencing adapters. All the reads below 50 and above 1000 bp were discarded. The trimmed sequences were assembled using de novo assembly and best coverages contigs were selected and the consensus sequences extracted. The minimum contig length of 500 bp was set in order to accommodate the smaller genome segment of about 756 to 763 bp. The reads were subsequently mapped to reference virus sequences obtained from NCBI. Consensus sequences obtained via de novo assembly and mapping to reference genomes were assembled and new contigs obtained for each of the 10 genome segments. The contigs were trimmed to include the orbivirus start position (GTT) and the end position (TAC). The conflicts were resolved to obtain the final consensus of each genome segment for all the AHSV isolates. The final consensus sequence of each African horse sickness virus genome segment was submitted to GenBank and accession numbers assigned (KP939368- KP940236).

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TABLE OF CONTENTS :

DECLARATION
DEDICATION
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
THESIS SUMMARY
LIST OF FIGURES
LIST OF TABLES
LIST OF ABBREVIATIONS AND SYMBOLS
CHAPTER 1 LITERATURE REVIEW
1.1 INTRODUCTION
1 1.2 AFRICAN HORSE SICKNESS (AHS)
1.2.1 African horse sickness historical perspective of outbreaks
1.2.2 AHSV transmission
1.2.3 Epidemiology
1.2.4 Pathogenesis and disease forms
1.2.5 Prevention and control of disease
1.3 AFRICAN HORSE SICKNESS VIRUS (AHSV)
1.3.1 Taxonomic classification of AHSV
1.3.2 The AHSV Viron
1.3.3 Viral genome
1.3.4 The AHSV Proteins
1.3.5 Replication overview
1.3.6 Diagnosis and Laboratory confirmation
1.4 VIRUS EVOLUTION
1.4.1 Mechanism of RNA genetic variation
1.4.2 Arbovirus evolution and their host factors
1.4.3 Orbiviruses evolution
1.4.4 Implication of AHSV genetic variation
1.5 AIMS AND OBJECTIVES
1.6 STUDY PROBLEM
CHAPTER 2 DETERMINING THE SUBSTITUTION RATES AND SELECTIONPRESSURE USINGCOMPLETE GENOME SEQUENCES FROMREPRESENTATIVES OF ALL SEROTYPES OF AFRICAN HORSE SICKNESSVIRUS COLLECTED BETWEEN 1960 AND
2.1 BACKGROUND
2.2 AIMS OF THE STUDY
2.3 MATERIAL AND METHODS
2.3.1 Viruses
2.3.2 Tissue culture and virus infection
2.3.3 Total dsRNA extraction
2.3.4 Oligo ligation
2.3.5 Oligo-Ligation products purification
2.3.6 Sequence–independent cDNA synthesis
2.3.7 PCR amplification and cDNA purification
2.3.8 AHSV ds-cDNA sequencing
2.3.9 AHSV sequence assembly
2.3.10 Phylogenetic analysis
2.3.11 Estimation of evolutionary dynamics
2.4 RESULTS
2.4.1 The dsRNA extractions
2.4.2 Synthesis and amplification of cDNA
2.4.3 Whole genome sequence analysis
2.4.4 Evolutionary rate and selection pressure on AHSV genome
2.5 DISCUSSION
CHAPTER 3 INVESTIGATING RECOMBINATION IN AHSV GENOMES
3.1 BACKGROUND
3.2 AIM OF STUDY
3.3 MATERIAL AND METHODS
3.3.1 Datasets
3.3.2 Genomic recombination analysis
3.4 RESULTS
3.4.1 Segment -1 (VP1).
3.4.2 Segment-6 (VP5).
3.4.3 Segment-7 (VP7).
3.4.4 Segment-10 (NS3)
3.5 DISCUSSION
CHAPTER 4 INVESTIGATING GENOMIC REASSORTMENT IN AHSV
4.1 BACKGROUND
4.2 AIMS OF STUDY
4.3 MATERIALS AND METHODS
4.3.1 Determining the effect of genomic reassortment on AHSV evolution by analysing 101 viral genomes isolated over 60 years
4.3.2 Experimentally investigating reassortment by co-infecting BSR cells with two different AHSV serotypes
4.4 RESULTS
4.4.1 Detecting naturally occurring genome reassortment
4.4.2 Induced reassortment
4.5 DISCUSSION
CHAPTER 5 CONCLUDING REMARKS
CHAPTER 6 REFERENCES
APPENDIX
APPENDIX 2. Phylogenies and ethics