FOOT-AND-MOUTH DISEASE VIRUS

Get Complete Project Material File(s) Now! »

CHAPTER 2 SYMMETRICAL ARRANGEMENT OF POSITIVELY CHARGED RESIDUES AROUND THE FIVEFOLD PORE OF FOOTANDMOUTH DISEASE SAT TYPE VIRUS CAPSIDS RESULTS IN THE ENHANCED AFFINITY TO HEPARAN SULPHATE

INTRODUCTION

Foot-and-mouth disease virus (FMDV) is a small, non-enveloped, icosahedral virus with a polyadenylated, single-stranded, positive-sense RNA genome belonging to the Aphthovirus genus in the Picornaviridae family (Knowles and Samuel, 2003). The virus capsid comprises 60 copies each of four virus-encoded structural proteins, VP1 to VP4. The outer shell of the capsid contains VP1, VP2 and VP3, whilst VP4 lines the interior surface (Acharya et al., 1989). FMDV is an important pathogen that causes a highly contagious, vesicular disease affecting cloven-hoofed animals, including cattle, pigs, goats, sheep and buffalo, with severe economic consequences worldwide (Alexandersen and Mowat, 2005).
FMDV naturally infects epithelial cells and to date studies have shown FMDV can adhere to any of four members of the αV subgroup of the integrin family of cellular receptors, i.e. αVβ1, αVβ3, αVβ6 and αVβ8 (Berinstein et al., 1995; Jackson et al., 1997, 2000a, 2002, 2004; Neff et al., 1998; 2000; Duque and Baxt, 2003). Attachment to these receptors is mediated via a highly conserved Arg-Gly-Asp (RGD) motif (Fox et al., 1989; Baxt and Becker, 1990; Mason et al., 1994; Leippert et al., 1997), located within the structurally disordered G- H loop of VP1 (Acharya et al., 1989; Curry et al., 1995; Lea et al., 1995). Following FMDV-receptor interactions, the virus is internalized, and the viral genome released into the cytosol after acid-induced capsid dissociation (Cavanagh et al., 1978; Grubman and Baxt, 2004).
Although FMDV infection is mediated by the RGD motif, RGD-independent infection can also occur (Baranowski et al., 1998; Zhao et al., 2003; Rieder et al., 2005; Maree et al., 2011; Chamberlain et al., 2015; Lawrence et al., 2016a). Molecules such as cell-surface glycosaminoglycans (GAGs) have been implicated in FMDV infections in cultured cells and may be involved in RGD-independent infection (Jackson et al., 1996; Sa-Carvalho et al., 1997; Zhao et al., 2003). However, whilst the interactions between FMDV variants and heparin is known at atomic resolution for two strains of the virus, the molecular basis of this interaction and mechanism of cell entry by other strains are largely not well understood (Fry et al., 1999; 2005). O’Donnell et al., 2008 showed that heparan sulphate (HS) binding to FMDV enters via caveola-mediated endocytosis whilst recently, Lawrence et al. (2016a) demonstrated that FMDV can utilize the Jumonji C-domain containing protein (JMJD6) as part of an RGD-independent infection.
It has been documented that variants of FMDV, virulent to cultured cells, emerge following serial cytolytic infections (Charpentier et al., 1996; Sevilla and Domingo, 1996; Martínez et al., 1997). By comparison to parental field virus, these culture-adapted, virulent FMDV strains displayed a shorter replication cycle in BHK-21 cells and an enhanced ability to kill cells (Sevilla and Domingo, 1996). It is thought that FMDV adaptation to cell culture is made possible by the selective pressure of the viral quasi-species, exerted by cell surface molecules that may act as virus receptors (Baranowski et al., 1998). However, it has been noted that field Southern African Territories (SAT) serotype viruses are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen (Pay et al., 1978; Preston et al., 1982).
Adaptation of FMDV field isolates to enable efficient replication in cultured cells is accompanied by changes in viral properties, including the acquisition of the ability to bind to alternative cellular receptors such as cell-surface GAGs (Jackson et al., 1996; Sa-Carvalho et al., 1997; Zhao et al., 2003; Maree et al., 2011). The interactions of a diverse group of ligands, such as growth factors, chemokines, herpes simplex virus (HSV), human immunodeficiency virus (HIV), respiratory syncytial virus, alphaviruses, dengue virus, adeno-associated virus and FMDV, to highly sulphated GAGs are typically via a positively charged domain (Fromm et al., 1995; Jackson et al., 1996; Sa-Carvalho et al., 1997; Chen et al., 1997; Krusat and Streckert, 1997; Byrnes and Griffin, 1998; Klimstra et al., 1998; Summerford and Samulski, 1998; Fry et al., 1999; 2005; Zhao et al., 2003). Zhao et al. (2003) reported that cell culture adaptation of FMDV serotype O selects for variant viruses with positively charged residues situated at antigenically relevant positions in the VP1 capsid protein, showing that an increase in positively charged residues at the 5-fold axis plays a role during virus cell culture adaptation. Although the genetic alterations associated with increased virulence during cytolytic passages of the SAT serotype FMD viruses in BHK-21 cells are largely unknown, it has been noted that amino acid substitutions accumulate in their capsids during serial passaging (Maree et al., 2010; Berryman et al., 2013; Sarangi et al., 2015). Surprisingly there is a common site of attachment between A10 and O1BFS (Fry et al., 1999; 2005).
In a study by Nsamba 2015a, amino acid substitutions within the capsid proteins of SAT1 and SAT2 viruses that are consistent with a binding site, at a position close to the icosahedral five-fold axis, for a moiety of roughly the size and charge of a sulphated GAG was identified. Heparan sulphate proteoglycan (HSPG) is an example of such a GAG. We extended these investigations in this study and by utilizing infectious chimeric, genome-length clones, containing the outer capsid-coding sequence of either SAT2/SAU/6/00 or SAT1/NAM/307/98, we investigated some of these mutations in detail with respect to their effect on infectivity, cell binding and HS dependence in cultured cells. The results of this study combined with Nsamba, 2015a, proposes that these mutant viruses have a specific affinity for HS, thus implying that binding to cell surface HSPG is most likely required for entry into cultured cells.

MATERIALS AND METHODS

Cells, viruses and plasmids

Baby hamster kidney (BHK) cells, strain 21, clone 13 (ATCC CCL-10), used during virus passage and plaque assays, was maintained in Glasgow minimum essential medium (GMEM, Sigma), supplemented with 10% (v/v) foetal bovine serum (FBS, Hyclone), 1× antibiotic-antimycotic solution (Invitrogen), 1 mM L-glutamine (Invitrogen) and 10% (v/v) tryptose phosphate broth (TPB, Sigma-Aldrich). Wild-type Chinese hamster ovary (CHO) cells, strain K1 (ATCC CCL-61), were grown in Ham’s F-12 nutrient medium (GIBCO ), supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. The same maintenance medium was used for the CHO-K1 derivative cell lines, CHO-677 (pgsD-677) (ATCC CRL-2244), which is HSPG deficient (HS), and CHO-745 (pgsA-745) (ATCC CRL-2242), which is HS and chondroitin sulphate (CS) deficient. The CHO-Lec2 (Pro-5WgaRII6A) (ATCC CRL-1736) cell line, which is sialic acid (SA) deficient, was grown in Alpha minimum essential medium (MEMAlpha) (GIBCO) supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics.
A part of this study is a continuation of the PhD work done by Nsamba, 2015a where 15 FMDV isolates, belonging to the SAT1 and SAT2, serotypes were isolated on either primary pig kidney or bovine thyroid cells, followed by amplification on Instituto Biologico Renal Suino-2 (IB-RS-2) cell monolayers. These viruses were either supplied by the World Reference Laboratory (WRL) for FMD at the Institute for Animal Health, Pirbright (United Kingdom), or are part of the virus bank at the Agricultural Research Council (ARC), Onderstepoort Veterinary Research (OVR) (South Africa) and were further analysed for the purposes of this study. The viruses were collected from either cattle or wildlife (Impala, Aepyceros melampus and African Buffalo, Syncerus caffer), and of the 15 virus isolates, 14 viruses were serially passaged eight times on BHK-21 cells and the SAT2/SAU/6/00 virus (from this study) was passaged 58 times on BHK-21 cells.
The nucleotide sequences generated for the fifteen cell culture-adapted FMDV isolates, were submitted to GenBank by Nsamba, 2015a. The respective accession numbers are as follows: GU194495 (SAT1/ KNP/148/91), GU194498 (SAT1/KNP/41/95), GU194497 (SAT1/ZIM/13/90), DQ009721 (SAT1/KEN/05/98), AF378302 (SAT1/TAN/1/99), AY442012 (SAT1/UGA/01/97), DQ009725 (SAT1/SUD/03/76), DQ009723 (SAT1/NIG/5/81), DQ009724 (SAT1/NIG/15/75), GU194502 (SAT1/NIG/06/76), GU194488 (SAT2/KNP/02/89), GU194489 (SAT2/KNP/51/93), AF367113 (SAT2/ZIM/10/91), DQ009731 (SAT2/UGA/02/02) and AY297948 (SAT2/SAU/6/00).
The construction of infectious genome-length plasmids for this study i.e. pSAT2, pNAMSAT2 and pSAUSAT2 (superscript indicates the donor capsid-coding sequence) has been described previously (Sa-Carvalho et al., 1997; Van Rensburg et al., 2004; Maree et al., 2010; Berryman et al., 2013). In order to construct the chimeric cDNA clones, the outer capsid-coding region of pSAT2 was replaced with the corresponding regions of SAT1/NAM/307/98 or SAT2/SAU/6/00 by making use of the flanking unique restriction enzyme sites, SspI and XmaI, in the VP2 and 2A-coding regions, respectively (Storey et al., 2007; Maree et al., 2010). The viruses recovered from pSAT2, pNAMSAT2 and pSAUSAT2 recombinant plasmid DNA were designated vSAT2, vNAMSAT2 (inter-serotype chimera) and vSAUSAT2 (intra-serotype chimera).

READ  Cell preparation for immunofluorescence microscopy

Plaque titration

Titrations were performed by making use of a standard plaque assay method. Monolayers of BHK-21, CHO-K1, CHO-677, CHO-745 or CHO-Lec2 cells in 35-mm cell culture plates (Nunc) were infected with serially diluted viruses for 1 h, followed by the addition of a 2 ml tragacanth overlay (Grubman and Baxt 1979; Rieder et al., 1993) and incubation for 48 h or 72 h at 37ºC. The overlayed, infected monolayers were stained with 1% (w/v) methylene blue in 10% ethanol and 10% formaldehyde in phosphate buffered saline (PBS) (pH 7.4). Virus titres were calculated and expressed as plaque forming units per millilitre (PFU/ml).

RNA extraction, cDNA synthesis, PCR amplification and nucleotide sequencing

RNA was extracted from 200 µl infected cell lysates using a guanidium-based nucleic acid extraction method (Bastos, 1998) and utilized as templates for cDNA synthesis. Viral cDNA was synthesized with SuperScript III (Life Technologies) and oligonucleotide 2B208R (Reeve et al., 2010). The ca. 3.0 kb leader and capsid-coding regions of the viral isolates were obtained by PCR amplification using Expand Long template Taq DNA polymerase (Roche) and SAT genome-specific oligonucleotides (Sa-Carvalho et al., 1997). The consensus nucleotide sequences of the amplicons were determined using a primer-walking approach and the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.0 (Perkin Elmer Applied Biosystems). The extension products were resolved on an ABI 3100 Genetic Analyzer (Applied Biosystems). Sequences were compiled and edited using Sequencher v5.4.6 (Gene Codes Corporation, Ann Arbor, MI, USA) sequence analysis software for Windows. The nucleotide and deduced amino acid sequences were aligned with ClustalX (Thompson et al., 1997)).

Site-directed mutagenesis and sub-cloning

The P1-2A region of SAT1/NAM/307/98 and SAT2/SAU/6/00 was cloned into pBlueScript (Stratagene) and the respective recombinant plasmids were designated pNAM-P1 and pSAU-P1. Mutagenesis primers, complementary to the P1-2A regions, were designed, containing the nucleotide substitutions (lower case, bold) to be introduced i.e.
The QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) enabled the introduction of the desired mutations by an inverse PCR method, according to the manufacturer’s instructions. The resulting plasmid DNA amplicons were transformed into XL10-Gold Ultra competent cells (Stratagene). The extracted plasmids were characterized by restriction enzyme digestion, followed by automated sequencing and selection of plasmid DNA containing the respective mutations.
The mutated P1-2A region of pNAM-P1 was cloned into the corresponding region of pSAT2 using the unique restriction sites, SspI and XmaI. Cloning and transformation procedures were performed as described previously (Sa-Carvalho et al., 1997; Maree et al., 2010). For the mutated P1-2A region of pSAU-P1, cloning into the corresponding region of pSAT2 was achieved with the In-Fusion HD Cloning Kit (Clontech). Briefly, pSAT2 was linearized by digestion with SspI and XmaI.

DECLARATION 
ACKNOWLEDGEMENTS 
SUMMARY 
LIST OF FIGURES 
LIST OF TABLES 
LIST OF ABBREVIATIONS 
CHAPTER ONE: LITERATURE REVIEW 
1.1 INTRODUCTION
1.2 FOOT-AND-MOUTH DISEASE VIRUS
1.3 EPIDEMIOLOGY OF FMDV
1.3.1 DISTRIBUTION
1.3.2 EPIDEMIOLOGICAL PATTERNS FOR FMDV IN AFRICA
1.3.3 THE ROLE OF CARRIERS IN THE EPIDEMIOLOGY OF FMDV
1.3.4 TRANSMISSION OF FMD
1.4 CONTROL AND ERADICATION OF FMDV
1.5 PATHOGENESIS OF THE DISEASE
1.6 THE VIRION
1.7 FMD VIRAL STRUCTURAL PROTEINS
1.8 FMDV GENOME
1.9 VIRUS REPLICATION AND TRANSLATION
1.10 THE CELLULAR RECEPTORS OF FMDV
1.11 DESIGN OF IMPROVED INACTIVATED VACCINES
1.12 FMDV DIAGNOSTIC ASSAYS
1.13 THE IMMUNE RESPONSE TO FMDV
1.13.1 THE INNATE IMMUNE SYSTEM
1.13.2 THE ADAPTIVE IMMUNE SYSTEM
1.13.2.1 HUMORAL IMMUNE RESPONSES
1.13.2.2 CELLULAR IMMUNE RESPONSES
1.14 ANTIBODIES
1.14.1 ANTIBODY STRUCTURE AND DIVERSITY
1.16 AIMS OF THE STUDY
CHAPTER TWO: SYMMETRICAL ARRANGEMENT OF POSITIVELY CHARGED RESIDUES AROUND THE FIVE-FOLD PORE OF FOOT-AND-MOUTH DISEASESAT TYPE VIRUS CAPSIDS RESULTS IN THE ENHANCED AFFINITY TO HEPARAN SULPHATE 
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.3 RESULTS
2.4 DISCUSSION
CHAPTER THREE: DEVELOPMENT AND VALIDATION OF A FOOT-ANDMOUTH DISEASE VIRUS SAT SEROTYPE-SPECIFIC 3ABC ASSAY TO DIFFERENTIATE INFECTED FROM VACCINATED ANIMALS 
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.3 RESULTS
3.4 DISCUSSION
CHAPTER FOUR: NOVEL SINGLE-CHAIN ANTIBODY FRAGMENTS AGAINST FOOT-AND-MOUTH DISEASE SEROTYPE A, SAT1 AND SAT3 VIRUSES USING A NKUKU® PHAGE DISPLAY LIBRARY 
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.3 RESULTS
4.4 DISCUSSION
CHAPTER FIVE: CONCLUDING REMARKS 
REFERENCES 
APPENDICES
GET THE COMPLETE PROJECT

Related Posts