Project topics and materials
Get Complete Project Material File(s) Now! »
CHAPTER THREE PYROSEQUENCING ANALYSES REVEALS COMPARABLE POPULATION DIVERSITY OF CHIMERA AND HOST-ADAPTED FOOT-AND-MOUTH DISEASE VIRUSES
INTRODUCTION
Foot-and-mouth disease (FMD), of which FMD virus (FMDV) is the aetiological agent, is a contagious, acute infection of cloven-hoofed animals, including cattle, pigs, goats, sheep and several wildlife species. Despite the restricted global distribution of FMD, it remains a compulsory notifiable disease of the OIE. FMD not only poses a constant threat world-wide as was evidenced by its extensive spread in South Korea and Japan in 2010, but it also has a major economic impact on the livestock industry as has been illustrated by recent FMD outbreaks in South Africa from 2000 to 2012 (Records of the OIE). Rapid and accurate diagnosis of FMD is a prerequisite for effective control of the disease and is based on a combination of clinical, epidemiological and laboratory observations.
FMDV belongs to the Picornaviridae family, which consists of numerous widely-studied RNA viruses, including important human and animal pathogens. FMDV, together with equine rhinitis A virus (Li et al., 1996; King, 2000), is classified under the genus Aphthovirus and is characterised by high genetic and antigenic variation (Rueckert, 1996). The positive-sense single-stranded RNA genome of FMDV is ca. 8 500 nucleotides in length. During viral RNA replication high mutation rates occur (0.2-1 mutation per plus or minus strand copied) (Drake and Holland, 1999). A consequence of the high mutation rate is a continuous generation of genetic variants that are observed in infected hosts or cell culture. Thus, diversity within the FMDV population is generated as new genetic and phenotypic variants arise during the process of genome replication (Domingo et al., 2003). Many of the progeny positive-sense RNA strands are initially recruited as viral mRNA, of which almost 50% of these molecules are packaged into virions (Rueckert, 1996). Virion assembly involves the formation of capsid protomers and association of five such protomers results in the formation of a pentamer, which is followed by assembly into either empty capsids or provirions (Rueckert, 1996). Autolytic cleavage of the 1AB polypeptide present in the provirion is required for the generation of virus particles (about 104-105 viral particles per cell), of which 0.1-1% are infectious (Palmenberg, 1990; Belsham, 1993; Rueckert, 1996).
High variability in the RNA genome population gives rise to distinct but related viruses, referred to as the quasispecies of FMDV (Domingo et al., 2003). In vivo and in vitro the quasispecies exist due to mutation, recombination and selection. Sanger sequencing using conventional methods allows for sequence determination of the virus at a most-represented basis of the entire population present. The consensus nucleotide sequence is an average determination of many different sequences and it is possible that such a genome does not exist in a viral population. Since the quasispecies of RNA viruses consists of viruses where the genome is statistically defined but not determined on an individual level (Domingo et al., 2002), it therefore does not discriminate between variants in the population that may be present at a lower frequency. Consequently, the true sequence diversity in the viral population is not always accounted for. This makes next-generation sequencing (NGS) (Ronaghi, 2001) unique in its ability to decipher the genetic identity of any genomic region of several numbers and types of viruses. Several studies have applied next-generation sequencing to detect sequence variation in human viruses such as HIV (Knapp et al., 2011), hepatitis virus (Lindström et al., 2004; Solmone et al., 2009; Wang et al., 2010), cytomegalovirus (Görzer et al., 2010) and rotavirus (Jere et al., 2011). This sequencing technology has not yet been applied extensively in the veterinary field (Leifer et al., 2010; Wright et al., 2011).
The application of next-generation sequencing to study viral population diversity in picornaviruses is currently limited (Silva et al., 2008; Wright et al., 2011). Host infection with FMDV results in fixation of variants different to viruses that have been cultured in vitro in cells. In addition, a chimeric virus that has its origin as a plasmid DNA clone is potentially less variable due to the narrow representation of the population, which resembles passages of small viral populations or repeated bottleneck effects (such as plaque-to-plaque transfers) (Escarmís et al., 2008). The 454 Life Sciences platform allows for pyrosequencing of individual DNA molecules using the Genome Sequencer (GS) FLX. Next-generation sequencing technology provides rapid and simple methods to characterise and investigate complex virus populations through in-depth sequence data analyses where genetic identity is represented in large numbers.
In this study the population diversity of two dissimilar FMD viruses, i.e. a chimera derived from a cloned population characterised by cell adaptation to BHK-21 cells and an animal-adapted virus isolated from pigs, was characterised. Next-generation sequencing was applied to investigate the variation present in the capsid sequences and to assess key viral properties. Direct PCR sequencing was used as reference in the analysis and polymorphisms in the P1 region were determined. Pyrosequencing detected considerable heterogeneity in the diversity of the two FMD populations. An improved understanding of the FMDV population diversity may impact positively on aspects relating to disease control such as the genetic and antigenic characteristics of circulating field strains, as well as the choice of appropriate vaccine antigens and diagnostic reagents that could be applied.
MATERIALS AND METHODS
Cell lines
Baby hamster kidney cells-21 clone 13 (BHK-21, ATCC CCL-10) were maintained in Eagle’s basal medium (BME; Invitrogen) supplemented with 10% (v/v) tryptose phosphate broth (TPB; Sigma-Aldrich), 1 mM L-glutamine (Invitrogen), antibiotics and 10% (v/v) fetal calf serum (FCS; Delta Bioproducts). Both sources of pig cells, primary pig kidney cells 337 (PK) and Instituto Biologico Renal Suino-2 cells (IB-RS-2), were maintained in RPMI medium (Sigma-Aldrich) supplemented with 10% FCS and antibiotics. Chinese hamster ovary cells (CHO strain K1, ATCC CCL-61) were grown in Ham’s F-12 medium supplemented with 10% FCS and antibiotics.
Viruses and plasmids
The SAT1/KNP/196/91 strain (designated KNP/196/91) was isolated at the Transboundary Animal Diseases Programme (ARC-OVI) from a buffalo sample received from the Kruger National Park, South Africa. The full spectrum of the virus population was recovered by passage once in PK (KNPPK1) and four times in IB-RS-2 (KNPPK1RS4) cells. For vaccine production purposes, KNPPK1RS4 was adapted to the FMDV host-species of choice, cattle (B = bovine), and subsequently propagated in BHK-21 cells. This virus, KNPPK1RS4B1BHK4, was used to infect pigs (P = pig) and the recovered host-adapted viruses KNPPK1RS4B1BHK4P2 and KNPPK1RS4B1BHK4P3 are henceforth referred to as KNPP2 and KNPP3, respectively.
The external capsid-coding region (1B-1D/2A) of plasmid pSAT2, a genome-length infectious cDNA clone of SAT2/ZIM/7/83 (van Rensburg et al., 2004), was replaced with that of KNP/196/91 to yield plasmid pKNP/SAT2 (constructed by H.G. van Rensburg). In constructing the recombinant clone, pSAT was digested with endonucleases SspI and XmaI to excise the ca. 2-kb external capsid-coding region from the pSAT2 clone to facilitate cloning of the corresponding KNP/196/91 amplicon containing the same restriction endonuclease sites.
Transcription of viral RNA, transfection and amplification of chimera viruses
Plasmid DNA for use as template in in vitro transcription reactions was obtained by plasmid extraction using the QIAprep Spin Miniprep kit (Qiagen). For RNA synthesis, plasmid pKNP/SAT2 was linearised with SwaI and in vitro transcribed using the MEGAscript T7 kit (Ambion). In vitro-transcribed RNA was transfected into cells using Lipofectamine 2000 reagent (Invitrogen). BHK-21, CHO-K1 and IB-RS-2 cell monolayers in 35-mm wells were maintained at 37°C for 48 h in virus growth medium (VGM: the appropriate medium supplemented with 1% HEPES and 1% FCS) and passaged as described previously (van Rensburg et al., 2004). The transfected cell cultures were harvested and aliquots of the supernatant were stored at -80°C. Ten percent of the thawed supernatant was used to infect freshly prepared cells and observed for cytopathic effect (CPE) up to 48 h post-infection. The same procedure was followed for serial passage of the viruses in the respective cells, and the viruses were designated vKNP/SAT2RS3, vKNP/SAT2BHK5 and vKNP/SAT2CHO4.
Viral RNA extraction, cDNA synthesis and DNA sequencing
The FMDV were characterised by RT-PCR of the capsid-coding region (P1), followed by nucleotide sequencing of the amplicon. The viral RNA was extracted with TRIzol® reagent (Life Technologies) according to the specifications of the manufacturer and used as template for cDNA synthesis. The viral RNA was reverse-transcribed using AMV-Reverse Transcriptase (Promega) and the antisense oligonucleotide 2B208R, which is situated in the 2B region. The Leader-P1-2A-coding region was amplified using Expand Long Template polymerase (Roche), as described previously (Chapter 2, Section 2.2.2). The PCR-amplified products were purified from the agarose gel with the Nucleospin Extract kit (Macherey-Nagel) and the nucleotide sequence was determined with an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit v3.0 (Applied Biosystems). SAT-specific oligonucleotides (Chapter 2, Table 2.2) were used to determine the sequence of the full-length P1 and to obtain good overlaps. After cycle sequencing, the extension products were purified and resolved on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The sequence data was analysed using BioEdit 5.0.9 software (Hall, 1999) and Sequencher 5.0 DNA sequencing software (Gene Codes Corporation). All nucleotide possibilities were taken in consideration, which resulted in polymorphisms in the deduced amino acid sequences. The nucleotide sequences were submitted to the NCBI GenBank under the accession numbers JQ692595 for KNPP3 and JQ692596 for vKNP/SAT2BHK5.
Plaque assays
Plaque titration assays were performed on BHK-21, IB-RS-2 and CHO-K1 cells. VGM was used to prepare log10 dilutions of each virus. The medium was aspirated from the cell monolayers and 250 µl of each virus dilution added to the cells. Following incubation for 1 h at 37ºC, 2 ml of tragacanth overlay was added to each well and incubated for 48 h (Rieder et al., 1993). The cell monolayers were then stained with 1% (w/v) methylene blue in 10% ethanol and 10% formaldehyde, prepared with PBS (pH 7.4). All plaque assays were performed in duplicate.
Pyrosequencing
Amplicons for pyrosequencing were generated by using cDNA from vKNP/SAT2BHK5 and KNPP3 as template. Amplicon sequencing fusion oligonucleotides were designed to anneal to the P1 region of vKNP/SAT2BHK5 and KNPP3 with overlaps, and rendering products of 200 to 300 bp (Appendix B to this thesis). In addition, the fusion oligonucleotides for the respective viruses included a 19-mer adapter sequence at the 5’-end (“A” and “B”) to match the components for forward and reverse reactions, and a 4-bp tag sequence allowing for the recognition of the two viruses upon sequence analysis. The tag sequences were “ACGT” and “CATG” for KNPP3 and vKNP/SAT2BHK5, respectively. The 3’ 20-29 nucleotides of the fusion oligonucleotides were complimentary to the FMDV genomes. PCR was performed using the Expand Long Template system (Roche Diagnostics) with added Pfu DNA polymerase (Promega). PCR reactions consisted of 30 cycles of denaturation at 95°C for 20 s, annealing at 56°C for 20 s and extension at 68°C for 3 min, with a final extension step of 68°C for 7 min. The PCR products were purified and then quantified using the Picogreen DNA Quantification kit (Invitrogen) on a fluorometer (BMG Labtech), as well as a DNA Pico chip on a 2100 Bioanalyzer (Agilent Technologies). Large volume emulsion-based clonal amplification (EmPCR) was performed at predetermined DNA copies per bead and the sequencing run was performed using the GS FLX instrument (Roche, 454 Life Sciences, Branford, CT, USA) at Inqaba Biotechnical Industries, Pretoria, South Africa.
Pyrosequencing data analysis
Low-quality reads were removed and the remaining sequences were built into a multi-sequence assembly using the Sanger-obtained sequences as references. The assemblies were performed using CLC Genomic Workbench (CLC bio). Single-nucleotide polymorphisms (SNPs) detected between pyrosequencing data reads and the reference sequences were exported to Excel® (Microsoft Windows) where the frequency of each SNP was calculated. Antoinette van Schalkwyk is thankfully acknowledged for assisting with data analysis.
RESULTS
Generation and characterisation of inter-serotypic chimera progeny
Several FMDV variants representing populations of the KNP/196/91 virus were recovered by propagation in PK, IB-RS-2 and BHK-21 cell cultures, as well as FMDV host-species such as cattle and pigs (Fig. 3.1). In order to obtain infectious vKNP/SAT2 chimeric virus, BHK-21, IB-RS-2 and CHO-K1 cells were transfected with in vitro-synthesised RNA transcripts and the results obtained following repeated passaging in cells are shown in Table 3.1. Initially, 80% CPE was observed for BHK-21 cells transfected with RNA derived from the chimeric pKNP/SAT2 clone, compared to 20% CPE at the second passage (after 48 h) in IB-RS-2 cells. Recovery of viable virus was less effective in the CHO-K1 cells where 5% CPE was only observed after four passages and 48 h. In agreement with the aforementioned recovery results, virus titres were the highest on BHK-21 and IB-RS-2 cells, followed by 10-fold lower titres on CHO-K1 cells, which have a restricted receptor phenotype (Jackson et al., 2003).
Phenotypic properties of the virus populations recovered from an infectious clone and from pigs were compared to other KNP/196/91 viruses, and are described in Table 3.2. The parental KNP/196/91 virus (KNPPK1) was unable to replicate in CHO-K1 cells. However, during four subsequent passages in IB-RS-2 cells, KNPPK1RS4 acquired the necessary adaptations to produce plaques on CHO-K1 cells, a characteristic that has been linked to the ability to interact with heparan sulfate proteoglycan (HSPG) receptors during cell entry (Jackson et al., 1996; Fry et al., 1999; Zhoa et al., 2003). Interestingly, the original viruses (KNPPK1 and KNPPK1RS4) and pig-adapted viruses (KNPP2 and KNPP3) produced larger sized, turbid plaques on BHK-21 and IB-RS-2 cells. In contrast, the vaccine seed stock (KNPPK1RS4B1BHK5) and chimera viruses produced clear plaques on the above-mentioned cells in addition to displaying similar infectious titres and plaque morphology (Tables 3.1 and 3.2).
DECLARATION
ACKNOWLEDGEMENTS
SUMMARY
LIST OF ABBREVIATIONS
LIST OF FIGURES
LIST OF TABLES
CHAPTER ONE: LITERATURE REVIEW
1.1 GENERAL INTRODUCTION
1.2 FMD: A GLOBAL PERSPECTIVE
1.3 CLASSIFICATION AND PHYSICAL PROPERTIES OF FMDV
1.3.1 Classification
1.3.2 Physical properties
1.4 VIRAL RNA GENOME, CAPSID AND ANTIGENIC PROPERTIES
1.4.1 Structure of the RNA genome
1.4.2 Structure of the FMDV capsid
1.4.3 Antigenic properties
1.5 INFECTIOUS CYCLE OF FMDV
1.5.1 Cell recognition
1.5.1.1 RGD-dependant mechanism of cell binding
1.5.1.2 RGD-independant mechanisms of cell binding
1.5.2 Protein synthesis and processing of the FMDV polyprotein
1.6 PATHOGENESIS
1.7 IMMUNE RESPONSES
1.7.1 Humoral immune response
1.7.2 Cellular immune response
1.8 CONTROL OF FMD BY VACCINATION
1.8.1 Conventional vaccines
1.8.1.1 Production of FMDV in cell lines
1.8.1.2 Inactivation of FMDV
1.8.1.3 Purification and concentration of the FMDV antigen
1.8.1.4 Formulation of the antigen
1.8.1.5 Assessment of vaccine potency
1.8.2 Emergency and high potency vaccines
1.9 NEW GENERATION FMD VACCINES
1.9.1 Protein and peptide vaccines
1.9.2 Genetically engineered attenuated strains
1.9.3 DNA vaccines
1.9.4 Vector-associated vaccines
1.9.5 Marker vaccines
1.10 VACCINE MATCHING
1.10.1 Classical techniques
1.10.2 Antigenic cartography using multi-dimensional scaling
1.10.3 Linear mixed-effects models
1.11 VACCINATION IN SOUTH AFRICA: AIMS OF THIS INVESTIGATION
1.12 REFERENCES
CHAPTER TWO: PREDICTING ANTIGENIC SITES ON THE FOOT-ANDMOUTH DISEASE VIRUS CAPSID OF THE SOUTH AFRICAN TERRITORIES TYPES USING VIRUS NEUTRALISATION DATA
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.3 RESULTS
2.4 DISCUSSION
2.5 REFERENCES
CHAPTER THREE: PYROSEQUENCING ANALYSES REVEALS COMPARABLE POPULATION DIVERSITY OF CHIMERA AND HOST-ADAPTED FOOT-ANDMOUTH DISEASE VIRUSES
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.3 RESULTS
3.4 DISCUSSION
3.5 REFERENCES
CHAPTER FOUR: CUSTOM-ENGINEERED CHIMERIC FOOT-AND-MOUTH DISEASE VACCINE ELICITS PROTECTIVE IMMUNE RESPONSES IN PIGS
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.3 RESULTS
4.4 DISCUSSION
4.5 REFERENCES
CHAPTER FIVE: CONCLUDING REMARKS
PUBLICATIONS AND CONGRESS CONTRIBUTIONS 195
APPENDICES
GET THE COMPLETE PROJECT
