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CHAPTER TWO DETERMINATION OF COMMON GENETIC VARIANTS WITHIN THE NON STRUCTURAL PROTEINS OF FOOT-AND-MOUTH DISEASE VIRUSES ISOLATED IN SUB SAHARAN AFRICA
Acknowledgements
This work has been submitted for publication in the Journal Veterinary Microbiology (VETMIC-D-14-9420). Funding was received from THRIP of the National Research Foundation of South Africa. I wish to express my gratitude to the following scientists; T.A.P. de Beer of European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, United Kingdom for the structural modelling; M. Chitray of Agriculture Research Council, Onderstepoort Veterinary Institute for availing the sequences for the Leader region of the African FMDV isolates for serotypes A and O; Agriculture Research Council, Onderstepoort Veterinary Institute for availing partial genome sequences for some of the SATs for the following genome regions, Leader-2A, 3C and 3D.
INTRODUCTION
Foot-and-mouth disease (FMD) is widely considered the most economically important disease of livestock, and is a controlled disease in many countries due to its highly contagious nature and associated productivity losses among even-toed ungulates (Artiodactyla). The disease is endemic to large parts of the African continent and an impediment to lucrative export markets for animal products (Vosloo et al., 2002). The different serotypes of FMD virus (FMDV) cause a clinically indistinguishable vesicular disease in cloven-hoofed animals and display different geographical distributions and epidemiology (Bastos et al., 2001; Samuel & Knowles, 2001; Bastos et al., 2003a; Bastos et al., 2003b; Knowles & Samuel, 2003; Bronsvoort et al., 2004). Of the seven serotypes, the South African Territories (SAT) types 1, 2 and 3 are confined to sub-Saharan Africa, although incursions into the Middle East by SAT1 (1961-65 and 1970) and SAT2 (1990, 2000 and currently in North Africa) viruses have been recorded (Ferris & Donaldson, 1992; Bastos et al., 2001; Valdazo-González et al., 2012; records of the Office International des Épizooties or OIE). In contrast, serotypes A and O occur globally (Samuel & Knowles, 2001) with the exception of southern Africa (Vosloo et al., 2002).
FMDV is a small non-enveloped virus, a member of the Aphthovirus genus within the family Picornaviridae. The icosahedral capsid consists of 60 copies of four structural proteins, VP1 to 4, arranged in a pseudo T=3 composition. The three surface-exposed proteins, VP1 (1D), VP2 (1B) and VP3 (1C), assemble into a protomeric subunit, with the smaller VP4 (1A) located internally (Acharya et al., 1989; Curry et al., 1995; Sobrino et al., 2001). Despite the high levels of genetic and antigenic variation (Vosloo et al., 1995; Reeve et al., 2010; Maree et al., 2011b), a consequence of the high mutation rate of the virus, the structural arrangement of the capsid is remarkably conserved, indicating plasticity within the three-dimensional structure of the capsid proteins (Acharya et al., 1989; Lea et al., 1994; Curry et al., 1995; Fry et al., 1999). The capsid encloses a ca. 8.5 kilobase, positive-sense, single-stranded RNA genome with a single open reading frame and two in-frame translation-initiation codons. Covalently linked to the 5’ end of the genome is the small viral protein 3B (or VPg), while the 3’ end is poly-adenylated (Carrillo et al., 2005). Upon virus infection, the interactions between VP2 and VP3 at the pentameric interfaces are disrupted by acidification within cellular endosomes, thereby releasing the viral RNA (Knipe et al., 1997; Ellard et al., 1999). The viral genome is rapidly translated into a polyprotein, which is co- and post-translationally cleaved by viral proteinases into several partially cleaved intermediates and ultimately into 12 mature proteins (Pereira, 1981; Rueckert, 1996).
In addition to the capsid proteins, the ORF of the viral RNA genome encodes eight non-structural proteins, each with its unique function within the replication cycle of FMDV (Belsham, 1993; Belsham, 2005). The non-structural proteins include three proteases, i.e. Lpro, 2A and 3Cpro, responsible for cleavage of the viral polyprotein and shut-down of the host cap-dependent translational system (Bablanian & Grubman, 1993; Martinez-Salas et al., 1996). Although several of the picornavirus proteins involved in RNA replication (2B, 2C and 3A) have membrane binding properties and disrupt protein trafficking in the cell (Moffat et al., 2005; Moffat et al., 2007), their particular functions during viral replication are still unknown. The 2B protein has been implicated in virus-induced cytopathic effect (CPE) (van Kuppeveld et al., 1997), while the 2C protein has recently been classified as an AAA+ ATPase enzyme that may act as an RNA helicase (Sweeney et al., 2010). The 3D gene encodes the viral RNA-dependent RNA polymerase (RDRP), and together with the 3A co-localizes with ER membrane-associated replication complexes (Lama et al., 1994).
Based on the genetic variability of the VP1-coding region, the FMDV strains that exist among the serotypes, group into topotypes that are geographically specific (Samuel & Knowles, 2001; Knowles & Samuel, 2003). Serotype A has three topotypes, of which the Africa topotype is endemic to sub-Saharan Africa. Of the eleven topotypes defined for serotype O, five are endemic in Africa, the East Africa (EA1 to EA4) and West Africa (WA) topotypes (Di Nardo et al., 2011). On the other hand, the SAT serotypes are more diverse genetically, and nine, fourteen and five topotypes have been defined for SAT1, SAT2 and SAT3 respectively (Bastos et al., 2001; Bastos et al., 2003a; Bastos et al., 2003b; Knowles et al., 2010b).
A few studies have looked at genome comparisons mainly focusing on serotype A, O, C and Asia-1 with a geographic distribution in Euro-Asia and South America (Pereda et al., 2002; Mason et al., 2003b; Carrillo et al., 2005). However, a limited number of complete non-structural protein analyses for viruses belonging to the SAT serotypes have been described (Carrillo et al., 2005). Here we have compared the non-structural proteins and its coding regions for the three SAT serotypes and viruses from serotype A and O found in sub-Saharan Africa from 1974 to 2006. Additionally, the natural variation found within the non-structural protein sequences of 79 viruses to identify structurally and possibly functionally conserved regions were mapped. Variation in the 3Cpro and 3Dpol was also mapped onto the protein structures to improve understanding of the plasticity of these enzymes. The deduced amino acid sequences of the non-structural proteins of two closely related SAT2 viruses causing an upsurge of outbreaks in North Africa and the Middle East in 2012 were also included (Valdazo-González et al., 2012).
MATERIALS AND METHODS
Virus isolates
The viruses included in this study were either supplied by the World Reference Laboratory (WRL) for FMD at the Pirbright Institute (United Kingdom) or form part of the virus bank at the Transboundary Animal Diseases Programme, Onderstepoort (South Africa). The SAT1 (n=30), SAT2 (n=26), SAT3 (n=7), serotype A (n=7) and serotype O (n=10) FMDV isolates from 17 countries in sub-Saharan Africa were selected for genomic characterisation. The isolates span a 32-year time period and represent various geographic locations and animal species. The viruses were propagated in IB-RS-2 cells prior to RNA extraction, cDNA synthesis and amplification of the relevant genome regions by PCR (Maree et al., 2011b). A description of the passage histories, host species and representative topotypes can be found in Table 2.1.
RT-PCR, sequencing and analysis
Viral RNA was extracted from infected cell culture supernatant using a modification (Bastos, 1998) of the guanidinium-silica based method described by Boom et al., (1990). To facilitate amplification of the Leader-P1-2A and P2-P3-coding regions, the viral genomic RNA was reverse transcribed with SuperScript IIITM (Life Technologies) using either the oligonucleotide 2B-208R (Bastos et al., 2001) for the Leader-P1-2A coding region or a modified oligo-dT, CCATGGCGGCC GCTTTTTT -TTTTTTTTT, for the P2-P3 coding region. The non-structural coding region and 3’UTR were subsequently amplified by three or four separate PCR reactions using the oligonucleotides detailed in Table 2.2 and Expand Long template Taq DNA polymeraseTM (Roche). The cycling conditions were 95°C for 20 s, 56°C for 20 s, 68°C for 2-3 min (30 cycles). Direct DNA sequencing of amplicons, using the ABI PRISMTM BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.0 (Perkin Elmer Applied Biosystems) and genome-specific primers (Table I; supplementary data, Appendix1), yielded a consensus sequence representing the most probable nucleotide for each position. For accuracy, at least two sequence reactions (forward and reverse) were generated for each amplicon ensuring that there was more than 50% overlap in the sequencing data. Sequences of the ca. 2.8 kb Leader-P1-2A and 4.2 kb P2-P3-coding regions were compiled and edited using BioEdit 5.0.9 software (Hall, 1999). The nucleotide data for eight non-structural protein coding regions (Leader, 2A, 2B, 2C, 3A, 3B, 3C, 3D), were determined in this study, while the nucleotide data for the P1 region have been described (Maree et al., 2011b).
The nucleotide and deduced amino acid sequences were aligned using ClustalX (Thompson et al., 1997). Phylogenetic relationships were inferred using Maximum-Likelihood (ML), Neighbour Joining (NJ), Minimum Evolution (ME), and Maximum Parsimony (MP) methods conducted in MEGA5 (Tamura et al., 2011), with the realiability of the nodes assessed using 1000 bootstrap replications in each method. For the phylogeny using ML method, the jModelTest 2.1.3 (Darriba et al., 2012), was used to predict the most appropriate model of evolution. It was found that the General Time Reversible model with gamma distribution and invariable rates (GIR+I+G) best described the pattern of nucleotide substitution. With the ME and NJ methods, the phylogenetic trees were constructed using the Kimura 2-parameter nucleotide substitution model. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. While the MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm, with search level 1, in which the initial trees were obtained by the random addition of sequences (10 replicates).
MEGA4 software (Tamura et al., 2007) was also used to identify hypervariable amino acid regions in a total alignment of the deduced amino acid sequences, defined as regions with more than 60% variable residues within a window of 10 residues. Entropy plots were drawn from the deduced amino acid alignments using BioEdit 5.0.9 software (Hall, 1999) and were defined as the uncertainty at each amino acid position with high values as an indication of high variation (Schneider & Stephens, 1990). The relative hydrophobicity of the peptides were predicted using the Kyte and Doolittle (1982) method operated in the BioEdit 5.0.9 software (Hall, 1999).
Structural modelling
Homology models of the 3C protease (3Cpro) and 3D RNA-polymerase (3Dpol) of SAT1 and SAT2 were built using Modeller 9v3 (Sali & Blundell, 1993) with FMDV A10 3Cpro (pdb id: 2j92) or type C RNA-polymerase (1UO9) as templates. Alignments were performed with ClustalX. Structures were visualised and the surface-exposed residues identified with PyMol v1.1 (Schrödinger, LLC, New York, NY).
Nucleotide sequence accession numbers
All nucleotide sequences determined in this study have been submitted to GenBank under the accession numbers indicated in Table 2.1.
Cell Types: BHK – baby hamster kidney cells; BOV – bovine; BTT – bovine tongue tissue; BTY- bovine thyroid; BVF – bovine vesicular fluid, CFK – calf foetal kidney; CK – calf kidney; GP – guinea pig vesicular fluid; LK – lamb kidney cells; PK – pig kidney; RS /IBRS – Instituto Biologico Renal Suino-2 Cells.
RESULTS
Phylogenetic trees of the various non-structural protein coding regions
For the 79 sub-Saharan African FMDV from 5 serotypes (SAT1, 2, 3, A and O) studied, 606, 1416 and 2721 nucleotide positions were aligned for the Lpro- P2- and P3-coding regions, respectively. The resultant deduced amino acid alignments, i.e. 202, 472 and 907 positions for the Lpro- P2- and P3 peptides respectively, are illustrated in Appendix 2. The Lpro-coding region displayed greater nucleotide variability i.e. 66.2% as compared to 46.7% and 47.9%, observed for the P2- and P3-coding regions, respectively.
Phylogenetic analyses of the Lpro-coding region and the P3-coding regions for the sub-Saharan African FMDV included sequences of two recent SAT2 viruses from the 2012 FMD outbreak in Egypt and the Middle East (Valdazo-González et al., 2012) obtained from GenBank (JX014255 and JX014256). The Maximum-Likelihood, Neighbour Joining, Minimum Evolution, and Maximum Parsimony methods yielded trees with almost similar topologies. Described below are midpoint rooted dendograms based on Maximum-likelihood methods, while the phylogeny depicted by the other methods is illustrated in Appendix 3.
Based on a cut-off value of less than 16% nucleotide difference, three non-serotype specific clusters were observed for the Lpro-coding region (Fig. 2.1) and four clusters for the P3-coding regions (Fig. 2.2), supported by strong bootstrap values (over 75%). Based on phylogeny of the Lpro- and P3-coding regions, the SAT1 and SAT2 viruses from southern Africa i.e. Angola, Botswana, Malawi, Mozambique, Namibia, Zambia, Zimbabwe and South Africa and the SAT3 viruses (from southern and East Africa), as well as two isolates from Kenya (SAT1/KEN/5/98 and SAT2/KEN/8/99) and one from Tanzania (SAT1/TAN/1/99), grouped together (i.e. cluster I; Fig. 2.1 and Fig. 2.2). The A and O type viruses that originated from Côte d’Ivoire, Senegal (western Africa), Eritrea, Ethiopia, Kenya, Somalia, Sudan, Tanzania and Uganda (eastern Africa), grouped together with SAT1 and SAT2 viruses from Eritrea, Nigeria, Rwanda, Sudan and Saudi Arabia (i.e. cluster II; Fig. 2.1 and 2.2). Three SAT2 viruses, two from Kenya (KEN/3/57 and KEN/11/60), and one from Uganda (UGA/MBF/4/02) were also found in cluster II, based on Lpro and P3 phylogeny (Fig. 2.1 and 2.2). It is interesting to note that three SAT2 viruses from Senegal and Ghana in West Africa (SAT2/SEN/5/75, SAT2/SEN/7/83, and SAT2/GHA/8/91) and a SAT1 virus from Uganda (SAT1/UGA/3/99) formed a strongly supported cluster based on Lpro-sequence phylogeny with 20.4% nucleotide differences within the group (cluster III; Fig. 2.1). Based on the P3-coding region, these West African SAT2 viruses shared a separate cluster III (Fig. 2.2), while three Ugandan isolates grouped together (SAT1/UGA/1/97, SAT1/UGA/3/99 and SAT2/UGA/2/02) (cluster IV; Fig. 2.2). Two virus strains from the recent SAT2 FMD outbreak in Egypt and the Palestinian Autonomous Territories (PAT), (EGY/9/12 and SAT2/PAT/1/12) grouped in cluster II, demonstrating a close genetic relationship with SAT2 viruses from Saudi Arabia and Eritrea (Fig. 2.1 and 2.2).
DECLARATION
ACKNOWLEDGEMENTS
SUMMARY
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
LIST OF ABBREVIATIONS AND SYMBOLS
CHAPTER ONE: LITERATURE REVIEW
1.1 GENERAL INTRODUCTION
1.2 EPIDEMIOLOGY OF FMD: A GLOBAL PERSPECTIVE
1.2.1 Global distribution of FMD
1.2.2 Serotypes of FMD virus, their distribution and epidemiological patterns
1.2.3 Topotype distribution of the various serotypes in Africa
1.2.3.1 Topotypes for Serotype O
1.2.3.2 Topotypes for Serotype A
1.2.3.3 Topotypes for SAT serotypes
1.2.4 Epidemiological patterns for FMDV in Africa
1.2.5 Implications of the genetic and topotype diversity in the FMDV on control using vaccination
1.2.6 Global Perspective on Control of FMD
1.3 CLASSIFICATION AND PHYSICAL PROPERTIES OF FMDV
1.3.1 Classification
1.3.2 Physical properties
1.4 VIRAL RNA GENOME ORGANISATION, FUNCTION AND STRUCTURE, CAPSID STRUCTURE AND ANTIGENIC PROPERTIES
1.4.1 Organisation, structure and function of the RNA genome
1.4.2 The viral structural and non-structural proteins
1.4.3 Structure of the FMDV capsid
1.4.4 Antigenic properties
1.5 INFECTIOUS CYCLE OF FMDV
1.5.1 Cell receptor recognition
1.5.2 Viral replication
1.6 FMD TRANSMISSION, PATHOGENESIS AND DISEASE MANIFESTATION
1.6.1. Transmission of virus
1.6.2 Incubation period and pathogenesis
1.6.3. Clinical FMD
1.6.4 Sub-clinical FMD
1.6.5 Persistent FMD infection
1.7 IMMUNE RESPONSES
1.7.1 Humoral immune response
1.7.2 Cell mediated immune response
1.7.3 Innate immune response
1.7.3.1 Cytokines
1.7.3.2 Phagocytosis
1.8 CONTROL OF FMD BY VACCINATION
1.9 NEW GENERATION FMD VACCINES
1.10 OBJECTIVES OF THE STUDY
CHAPTER TWO: DETERMINATION OF COMMON GENETIC VARIANTS WITHIN THE NON-STRUCTURAL PROTEINS OF FOOT-AND-MOUTH DISEASE VIRUSES ISOLATED IN SUB-SAHARAN AFRICA
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.3 RESULTS
2.4. DISCUSSION
CHAPTER THREE: ADAPTATION OF FOOT-AND-MOUTH DISEASE SAT TYPE VIRUSES TO CELLS IN CULTURE RESULTS IN THE FORMATION OF HEPARIN SULPHATE BINDING SITES AROUND THE FIVE-FOLDPORE OF THE CAPSID
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.3 RESULTS
3.4 DISCUSSION
CHAPTER FOUR: ANTIGENICITY OF AN INTRA-SEROTYPE CHIMERIC FOOT-AND-MOUTH DISEASE VACCINE
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.3 RESULTS
4.4 DISCUSSION
CHAPTER FIVE: CONCLUDING REMARKS AND FUTURE PROSPECTS
REFERENCES
APPENDICES
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