Mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease SAT type viruses

Abstract

Foot-and-mouth disease virus (FMDV) infects host cells by adhering to the α_V subgroup of the integrin family of cellular receptors in a Arg–Gly–Asp (RGD) dependent manner. FMD viruses, propagated in non-host cell cultures are reported to acquire the ability to enter cells via alternative cell surface molecules. Sequencing analysis of SAT1 and SAT2 cell culture-adapted variants showed acquisition of positively charged amino acid residues within surface-exposed loops of the outer capsid structural proteins. The fixation of positively charged residues at position 110–112 in the βF–βG loop of VP1 of SAT1 isolates is thought to correlate with the acquisition of the ability to utilise alternative glycosaminoglycan (GAG) molecules for cell entry. Similarly, two SAT2 viruses that adapted readily to BHK-21 cells accumulated positively charged residues at positions 83 and 85 of the βD–βE loop of VP1. Both regions surround the fivefold axis of the virion. Recombinant viruses containing positively charged residues at position 110 and 112 of VP1 were able to infect CHO-K1 cells (that expresses GAG) and demonstrated increased infectivity in BHK-21 cells. Therefore, recombinant SAT viruses engineered to express substitutions that induce GAG-binding could be exploited in the rational design of vaccine seed stocks with improved growth properties in cell cultures.

Introduction

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals causing significant distress and suffering in animals. Although mortality is usually low (<5%) (Thomson, 1995), morbidity can reach 100% and the impact can be catastrophical when an outbreak occurs in a FMD-free region with immunologically naïve population of livestock. Consequently FMD is classified by the OIE as one of the most important infectious diseases of livestock (Office International des Épizooties Terrestrial Manual, 2009). The economically critical effects to livestock farming due to the high cost of disease control and international trade restrictions (Sellers and Daggupaty, 1990) was evidenced during the 2000–2001 outbreaks in Europe and the virus escape that occurred more recently, in 2007 in the United Kingdom (Samuel and Knowles, 2001, Cottam et al., 2008). In endemic regions, FMD is controlled by restricting animal movement, the implementation of vaccination programmes and biosecurity measures. In disease-free countries where vaccination is normally not applied, ring-vaccination is only used in an emerging outbreak with subsequent slaughtering of vaccinated animals (Müller et al., 2001, Tomassen et al., 2002).

In South Africa, other regions of the African continent, as well as in some Asian and South American countries, regular immunisation is essential for disease control, and in maintaining FMD-free status. Current FMD vaccines are chemically inactivated preparations of concentrated, virus-infected cell culture supernatants (Office International des Épizooties Terrestrial Manual, 2009). Therefore, large-scale vaccine production utilize a suitable cell line, like BHK-21 cells, and requires that the vaccine strain is adapted and propagated in cell culture (Amadori et al., 1994, Amadori et al., 1997). However, these cell lines have limited (monolayers) or no (in suspension) expression of the required primary receptor for infection by FMDV (Amadori et al., 1994). In the early 1980s it was noted that viruses of the three SAT serotypes, endemic in Africa, are notorious for their difficulty to adapt to BHK-21 cells (Pay et al., 1978, Preston et al., 1982). It is thought that the cell surface molecules, which may act as virus receptors, exert an important selective pressure on viral RNA quasi-species, thereby enabling adaptation. Studies have shown that repeated passaging of FMDV in cultured cells rapidly gave rise to mutant viruses within the population (Rieder et al., 1994, Herrera et al., 2007). Adaptation of wild-type SAT viruses in cell culture to produce high yields of stable antigen is an intricate and time-consuming process that is often associated with a low success rate.

FMD virus (FMDV), the type species of the Aphtovirus genus in the family Picornaviridae (Racaniello, 2006), infects epithelial cells by adhering to any of four members of the α_V subgroup of the integrin family of cellular receptors, i.e. α_Vβ1, α_Vβ3, α_Vβ6 and α_Vβ8 (Berinstein et al., 1995, Neff et al., 1998, Neff et al., 2000, Jackson et al., 1997, Jackson et al., 2000, Jackson et al., 2002, Jackson et al., 2004, Duque and Baxt, 2003). Attachment to the 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 βG–βH loop of VP1 (Acharya et al., 1989, Lea et al., 1995, Curry et al., 1997). Following FMDV–receptor interactions, the virus is internalised and the viral genome is released in the cytosol. Adaptation of FMD 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 glycosaminoglycans (GAGs) (Jackson et al., 1996, Jackson et al., 2001, Sa-Carvalho et al., 1997, Zhao et al., 2003). The interactions of a diverse group of ligands, such as growth factors, chemokines, herpes simplex virus (HSV), human immunodeficiency virus, respiratory syncytial virus, alphaviruses, dengue virus, adeno-associated virus and FMDV, to the highly sulfated GAGs (also known as heparan sulfate proteoglycans, HSPG) is typically via a positively charged domain on these proteins (Patel et al., 1993, Gromm et al., 1995, Jackson et al., 1996, Chen et al., 1997, Krusat and Streckert, 1997, Sa-Carvalho et al., 1997, Byrnes and Griffin, 1998, Klimstra et al., 1998, Summerford and Samulski, 1998, Fry et al., 1999, Fry et al., 2005, Zhao et al., 2003). The ability of a type O virus to enter cells following adhesion to HSPG is thought to be dependent on the presence of the positively charged Arg residue at position 56 of VP3 which results in a net gain of positive charge on the virion surface (Sa-Carvalho et al., 1997, Fry et al., 1999).

Replacement of the external capsid-coding sequence of an infectious cDNA clone with the corresponding region of an outbreak virus results in the transfer of surface-exposed epitopes from the aetiological agent to the recombinant virus (Zibert et al., 1990, Rieder et al., 1993, Almeida et al., 1998, Van Rensburg and Mason, 2002, Van Rensburg et al., 2004). The chimeric viruses, produced in this manner, induce a protective immune response in animals similar to that of the outbreak virus. However, co-transferral of undesirable traits, such as capsid instability and poor cell culture adaptation of the field virus may also occur. Therefore, application of reverse genetics technology in FMD vaccinology includes the identification of amino acid sequences associated with the acquisition of HSPG-binding during cell culture adaptation of SAT viruses and the introduction of these changes into chimeric constructs.

In this report we identify novel amino acid residues within the capsid proteins of SAT1 and SAT2 viruses that affect the virus’ ability to grow in different cell lines. We demonstrated that cell culture adaptation to BHK-21 cells is acquired following repeated passaging of the field viruses in these cells. Furthermore, we illustrated that this phenotype can be transferred to an infectious cDNA clone of a FMD field virus from which viable cell culture-adapted viruses were recovered.