Download 44. Mapping of neutralising sites on FMDV type Asia 1

Appendix 44
Mapping of neutralising sites on FMD virus type Asia 1 and relationships with sites described
in other serotypes
Santina Grazioli, Francesca Fallacara and Emiliana Brocchi*
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy
Abstract
Knowledge on the antigenic structure of foot-and-mouth disease virus (FMDV) has, besides a scientific
value, several practical applications: it may have relevance in the development of diagnostic assays, as
well as the evaluation of the antigenic variability and the selection of appropriate vaccine strains.
Antigenic sites have been studied only in FMDV type O, A and C, while it would be worthwhile to extend
studies also to other serotypes. This paper reports on the identification of neutralising sites in FMDV type
Asia 1 using a new panel of monoclonal antibodies (MAbs) and discuss their relation with sites described
in other types.
MAbs specific for FMDV type Asia 1 were produced and characterised using different immunoassays.
FMDV mutants resistant to MAbs neutralisation were selected and sequenced.
Out of 24 MAbs produced, 10 neutralise viral infectivity and 14 do not. The 10 neutralising MAbs, all typespecific, identify 4 independent antigenic sites on the basis of the reactivity profile with MAR-mutants. By
comparing the amino acids sequence of the parental virus and of virus mutants, the amino acids crucial
for the 4 sites were mapped at the following positions: VP1 142, VP2 67-79, VP3 58/59, VP3 218.
MAbs confirmed their powerful for making light insight the antigenic structure of FMDV. Three of the 4
neutralising sites identified and mapped on FMDV type Asia 1 correspond structurally and functionally to
analogous sites described in FMDV type O, A and C, enforcing the evidence that these are dominant
antigen sites in FMDV structure. One site, located at C-terminus of VP3, is a new independent site,
described for the first time in FMDV.
Further sites, not involved in neutralisation, were detected by 14 more MAbs.
Introduction
Foot-and-mouth disease (FMD) viruses constitute one genus in the family Picornaviridae, but are
classified in seven serotypes, each of which shows considerable intra-typic variation. This antigenic
variation leads to continuing difficulties in controlling the disease.
Studies on the antigenicity of FMD virus, complemented with crystallographic analysis of the threedimensional structure of the virus and of virus-antibody complexes, have improved understanding of the
interaction between virus and the host immune system, including the mechanism of virus escape from
neutralisation, which is also responsible for the high antigenic diversification of FMD viruses. Such studies
may contribute to a better control of the disease.
The identification of antigenic sites in FMDV has mainly relied on the use of monoclonal antibodies
(MAbs), and partly also on the evaluation of immunogenicity and antigenicity of viral peptides. However,
synthetic peptides only allow the study and characterisation of sequential epitopes, while the use of
MAbs, combined with detection of amino acids substitutions in virus mutants resistant to MAb-induced
neutralisation, allows the identification of any kind of antigenic sites, either linear or dependent on
conformation, provided their involvement in virus neutralisation processes.
Using this approach, antigenic sites have been identified and mapped on FMD viruses type O (Xie et al.
1987; Pfaff et al., 1988; Barnett et al., 1989; Kitson et al.,1990), type A (Thomas et al., 1988; Baxt et
al., 1989; Bolwell et al., 1989) and type C (Mateu et al., 1990; Lea et al., 1994). Results of these studies
agree in the evidence that, besides the continuous epitope termed site A or site 1, located within the
large and flexible G-H loop of VP1 and for a long time considered the main if not the single
immunodominant site, other epitopes, not found in continuous sequences but dependent on capsid
conformation, also exist in all the three serotypes studied (reviewed in Mateu, 1995). Amino acid
residues in the three structural proteins VP1-3 exposing loops on the virus surface have been indicated as
crucial elements for the site antigenicity.
These studies were conducted on isolates of FMDV types O, A, C which are extinct from the field.
Furthermore, it would be worthwhile to extend investigations also to other serotypes of FMDV, either to
improve basic knowledge on the virus structure and antigenicity, and for the benefits that this knowledge
may reflect on the selection of appropriate strategies for the disease control. Further knowledge on the
antigenic structure and the availability of well characterised MAbs have useful applications also in the
design of functional diagnostic assays and in studies on the antigenic evolution of FMD viruses. In this
report we describe the identification and characterisation of neutralising sites on FMDV type Asia 1 by a
new panel of MAbs. Four independent sites have been demonstrated, three of which, located on VP1, VP2
and VP3 respectively, correspond to sites previously determined in FMDV serotypes O, A and C, while the
fourth seems to be a new site described for the first time in FMDV.
277
Materials and Methods
Viruses
FMDV isolates used were received from the World Reference Laboratory, Pirbright, UK; they are listed in
table 1. Viruses were propagated in IBRS-2 cells monolayers and harvested when cytopathic effect was
maximum. The strain used for mice immunisation and SDS-polyacrilamide gel electrophoresis was
preliminarily inactivated with binary ethylenimine and purified by ultracentrifugation through a 25%
(w/w) sucrose cushion.
Monoclonal antibodies (MAbs)
The production of MAbs against FMDV Asia 1, strain Nepal 29/97 was described in Grazioli et al., 2002.
Trapping ELISA
The assay used for MAbs titration and for the evaluation of their intra- and inter-types reactivity was a
trapping ELISA (Samuel et al., 1991). Essentially, each MAb was reacted with pre-titrated concentrations
of viruses (supernatant of infected cells) which had been trapped using a polyclonal rabbit Asia 1
antiserum. Titres of MAbs were expressed as the reciprocal of the saturating dilution, while the reactivity
of mutants and field isolates with each MAb was expressed as a percentage of the corresponding reaction
with the parental strain, assumed to be 100%.
Virus Neutralization test (VNT)
VNT was carried out in microplates against 100 TCID50 of the homologous FMDV Asia 1 or MAR-mutants
and IBRS-2 as substrate. The final dilution required to neutralise 50% of the inoculated cultures was
calculated.
MAb neutralization-resistant (MAR) mutants
The selection of mutants resistant to neutralization by MAbs was carried out as described previously
(Borrego et al. 2002).
Immunoblotting analysis
Hybridoma supernatants were assayed against the purified homologous FMDV, resolved by SDSpolyacrilamide gel electrophoresis 10% or 12% and transferred to nitrocellulose filters, following standard
procedures (Harlow & Lane, 1988; Towbin et al., 1979).
Sequencing
RNA extracted from the supernatant of infected cultures, using a commercial kit (Qiagen RNeasy Mini kit,
Qiagen, Inc.), was used as the template for RT-PCR. Reverse transcription was carried out using AMV
reverse transcriptase with a hexanucleotide mixture of all possible sequences (“Random” primer)
(Roche). The cDNA produced was used as the template for PCR amplification of the region encoding the
structural proteins using the primers described in Table 1. The polyprotein P1 was amplified in five
fragments which included also some flanking nucleotides from both the 5’ non-translated region (NTR)
and protein 2A. Sequencing of PCR fragments and sequence analysis were carried out using an ABI 310
Automatic Sequencer and LaserGene software (DNASTAR Inc., Madison, WI, USA).
Results
Preliminary characterisation of MAbs
A preliminary characterisation of 24 MAbs raised against FMDV type Asia1, strain Nepal 29/97, was
achieved by analysing the immunoglobulin class, the capability to neutralise viral infectivity, the reactivity
with separated, denatured viral proteins and the level of cross-reactivity with the other six heterologous
FMDV types. Results of these preliminary analyses had been reported at the open session of the EUFMD
Research Group held in Turkey, 2002 (Grazioli et al., 2002); these results have now been updated with
missing information and summarised in table 2.
According results of VNT, MAbs were divided in two main categories: one including 10 MAbs that
neutralise virus infectivity, another composed by 14 MAbs that do not.
Although the major objective was focussed on mapping of neutralising MAbs, basic information were also
collected for non-neutralising antibodies. Among them, 4 MAbs (4G6, 3D8, 4B1, 4B2) recognised only the
homologous type Asia 1, while the other 10 MAbs showed different patterns of cross-reactivity with
heterologous serotypes: the most common profile, shown by four MAbs (3G3, 2A4, 3B6, 5H5), was crossreactivity with 4 serotypes, namely the homologous Asia 1 and the heterologous O, A, C types, but also 2
MAbs reacting with all the seven serotypes (4G2, 3H12) and another one recognising all serotypes but
SAT 1 (5F10) were detected. Non-neutralizing MAbs displayed a broad reactivity also with 11 isolates of
type Asia 1, representative of a thirty-year period, being the oldest virus of this panel originated in 1973
(Asia 1 Turkey 15/73) and the most recent one in the year 2000 (Asia 1 Greece 1/2000); the antigenic
stability of the relevant epitopes was expected, particularly for those conserved among different
serotypes.
Results of imunoblotting proved that one MAb (5F10) identifies a linear epitope in VP2 (figure 1),
subjected to cleavage by trypsin treatment (not shown). In contrast, all other 13 non-neutralising MAbs
were negative in immunoblotting, thus recognise conformation-dependent epitopes.
The 10 neutralising MAbs scored virus neutralising titres from 200 to more than 20000 (in ascitic fluids).
Three of them reacted with denatured VP1 in immunoblotting (table 2, figure 1); trypsin treatment of the
virus prevented this reactivity (not shown), suggesting that their target site should correspond to the
linear amino acid sequence containing a trypsin-sensitive site and designing the flexible G-H loop of VP1,
278
well described in the FMDV structure. Interestingly, these three MAbs showed a high degree of crossreactivity with the SAT 3 serotype, indicating a strong similarity of the target neutralising site between
the Asia 1 and SAT 3 serotypes. However, the other 7 neutralising MAbs were all type-specific and all
negative in imunoblotting with separated viral proteins: this implies they are likely directed against
conformational sites, but does not allow to differentiate them.
MAb-resistant mutants (MAR-mutants)
The selection, characterisation and sequencing of viral mutants resistant to neutralisation by each of the
neutralising MAbs was the system adopted to identify and then map the relevant target sites. From 1 to 9
MAR-mutants were independently selected for each MAb by serial passages of the parental virus in the
presence of high concentration of MAb. After stabilisation, mutants were analysed by both ELISA and VNT
against the 10 MAbs, in order to detect reciprocal relationships on the basis of similar patterns of
reactivity.
The antigenic profile of MAR-mutants, determined by ELISA (figure 2) lead to the definition of 4 distinct
neutralizing sites; sites were denominated I, II, IV, V to maintain a common denomination in relation
with analogous sites previously described in other FMDV types; substantially the 4 sites are independent
each other, as mutations in one site did not alter the reactivity of the other sites.
In particular, site I is defined by the 3 MAbs (5C12, 4E10, 4F10) reacting with a linear sequence of VP1:
mutations induced in any of the corresponding escape mutants annulled the reaction of the three MAbs,
without affecting the binding of the other seven MAbs. Another site, called site V, is defined by the unique
MAb 5G4: it is clearly distinct in that the five mutants selected with this antibody were still recognised by
all the other MAbs. Site IV includes three additional MAbs, 5E10, 3C6, 1F10: in fact, the 13 mutants
selected with any of the three MAbs lost reactivity with all of them. Similarly, the remaining three MAbs,
2C3, 2G1, 4D8, correspond to a distinct site called site II. A structural relation could occur between site II
and IV, since variations in one site may partially alter the reactivity of the other.
In order to evaluate the correlation between ELISA binding and neutralisation pattern, one or two
representative mutants for each MAb were also tested for the susceptibility to neutralisation by MAbs; the
neutralisation titres of the MAbs towards 100 TCID50 of the parent virus and the mutant viruses were
compared; it was arbitrarily assumed that differences in titres > 2 log10 units indicate resistance,
differences between 1 and 2 log10 units indicate partial resistance and < 1 log10 are indicative of
sensitivity to MAb-induced neutralisation.
Results of these cross-neutralisation assays, shown in figure 3 fully confirmed the findings obtained by
ELISA, providing further evidence of the four distinct sites, with a partial relation between sites II and IV.
Mapping of neutralising sites
The sequence of the capsid coding region of some mutants was also determined and compared to the
parental sequence, in order to identify amino acid substitutions responsible for the antigenic variation
(Figure 2 and Table 3).
The amino acids crucial for antigenic sites organization were determined, enabling us to map the 4
antigenic sites on the capsid proteins and identify the secondary structural element involved.
Sites I, IV and V could be related to a single amino acid change, while site II was associated to multiple
simultaneous changes. According to results of mutants sequencing,
Mutants selected with MAbs to site I repeatedly showed substitutions at residue 142 of VP1
(corresponding to residue 144 in type O), flanking the conserved RGD motif and included within the
flexible G-H loop of VP1;
site II is located in VP2 and involves multiple amino acid positions in the B-C loop: namely residues
67, 72, 74, 77, 79. Mutants in this site showed each three simultaneous changes; one of them
presented also two further changes at amino acids 49 and 207 of VP1;
site IV maps at the amino acid positions 58 or 59 of VP3, located within the secondary structural
element B-B knob. However, a further mutation at amino acid 67 of VP2 was detected in two
sequenced mutants: this second change is typical of site II and can justify the partially altered
reactivity of some MAbs of site II, confirming the structural relationship between site II and IV;
Site V maps on VP3, involving the amino acid 218, that corresponds to the VP3-carboxyl end.
Consistently with ELISA and neutralisation profiles, mutants selected with different MAbs defining the
same site showed amino acid changes at the same or contiguous residues; however, residues involved in
site IV may be substituted by different amino acids causing similar effects.
The profile of reactivity with MAbs of 11 FMDV isolates of type Asia 1, chronologically distant, proved
evidence that site I and IV are stable in the majority of isolates, whilst sites II and V are subject to
frequent antigenic variation: in fact MAbs to site I and IV broadly reacted with all but 1 isolate (Cambodia
3/93), while MAbs to site II and V reacted almost exclusively with the homologous strain (table 2).
279
Discussion
Mapping of antigenic sites by a new panel of MAbs, with focus on the sites involved in virus neutralisation
processes, has provided a better understanding of the antigenic structure of FMDV type Asia 1, allowing
to study the relationships with antigenic sites previously described in other FMDV types, more extensively
studied.
Recently, the use of two panels of MAbs lead to the identification of independent antigenic sites also in
the type Asia 1 of FMDV (Sanyal et al., 1997, Marquardt et al., 2000). The MAbs provided evidence of
antigen variability among field isolates (Sanyal et al., 1997; Sanyal et al., 2003; Marquardt et al., 2000),
but mapping of the relevant sites was only attempted on the basis of the correspondence observed
between MAbs-profiling and variations in the amino acid sequence of few isolates (Marquardt et al.,
2000). The characterisation of our new panel of MAbs, combined with sequencing of MAR-mutants,
enabled us to identify and map four independent neutralising sites. Interestingly, three of the sites
detected on type Asia 1 correspond structurally and functionally to analogous antigenic sites described in
types O, A and C (as shown in figure 3).
In particular, site I, defined by three MAbs of our panel, was previously called site 1 or A in the other
three serotypes; in all serotypes this site is located within the surface exposed G-H loop of VP1, that
contains one trypsin cleavage site and the highly conserved RGD (Arg-Gly-Asp) amino acid triplet,
corresponding to the presumed site of cell attachment (Fox et al., 1989; Mason et al., 1994). Several
amino acid residues in the sequential segment 138-154 of the capsid protein are involved in determining
site antigenicity. Substitutions detected either in MAR-mutants, or in field variants occur in residues
flanking both sides of the RGD motif (Stave et al., 1988; Bolwell et al., 1989; Mateu et al., 1990;
Marquard and Freiberg, 2000; Marquardt et al., 2000). Consistently, also in the FMDV type Asia 1 the
position 142, which was found repeatedly substituted in MAR-mutants, precedes the RGD motif. The G-H
loop is easily accessible on the virus surface and is characterised by high variability (reviewed in
Palmenberg, 1989 and Domingo et al., 1990). The antigenic diversity in this region relays on its
flexibility, as indicated by the disorder at G-H loop observed in crystal structures of both types O
(Acharya et al., 1989) and C (Lea et al., 1994). In fact a flexible loop can accept different amino acid
sequences while preserving a functional capsid structure.
In spite of this concept, and in contrast with the sequence variability observed in some field isolates of
Asia 1 type in the corresponding region (Marquard et al. 2000), our three MAbs to site I show a high level
of conservation among the field isolates tested. Furthermore, unexpectedly, all of them recognise also
the FMDV serotype SAT 3; the molecular bases that may explain cross-reactivity at this level should be
further investigated.
It has been reported that VP1 C-terminus contributes to the formation of a discontinuous site together
with site 1/A in the FMDV type O (Xie et al 1987, Parry et al., 1989), but represents a topologically
independent site in type C (site C, Mateu et al., 1990). In type A both situations have been observed in
different strains (Baxt et al., 1989, Thomas et al., 1988). The epitope included in VP1 C-terminus in the
sequence 200-213 of the serotypes O, A; C (figure 3). VP1 C-terminus was described as a trypsinsensitive, linear epitope of minor importance, given the weak neutralising capability of MAbs towards it
(Mateu et al. 1990, Thomas et al.,1988), their failure to compete with sera from convalescent animals
(Thomas et al., 1988) and the poor capacity of VP1 C-terminus to generate protecting antibodies (Meloen
and Barteling, 1986). Our MAb panel does not provide evidence of antigenicity of VP1 C-terminus in
FMDV type Asia 1; however a substitution at residue 207 was detected in one MAR-mutant of site II;
apparently it is not structurally related to site II itself, but rather indicates the occurrence of variability in
this region.
Site II involves multiple amino acid positions in the structural protein VP2, ranging from 67 to 79; the
same or contiguous residues were found crucial for the conformation of the analogous site in FMDV type
O (site 2, Kitson et al., 1990), A (Ag-site III, Thomas et al.,1988) and C (site D2, Lea et al., 1994). All
concerned residues lie in the exposed B-C loop of VP2. Frequent amino acids substitutions were
demonstrated in this region in field isolates of type Asia 1 (Marquardt et al., 2000), indicating its
susceptibility to variation. Consistently, our three MAbs to site II do not recognise most of the isolates
examined.
Site IV maps in the type Asia 1 at positions 58/59 of VP3, located within the B-B knob structural element;
in contrast to site II, this is a conserved region, as proved by the broad intra-typic reactivity of the three
target MAbs and in agreement with the absence of substitutions observed in the amino acid sequence of
field isolates (Marquardt et al., 2000). The same epitope was found also in types O (site 4), A (described
as part of Ag-site III) and C (site D3). While the same and unique residue 58 was found crucial for this
site in the serotypes O and C (Kitson et al., 1990; Lea et al., 1994), in the serotype A several contiguous
or close residues, namely 58 to 61, 69/70 and in addition two more distant positions 139 and 195, seem
to be part of this site (Thomas et al., 1988).
Evidence of structural relationship between site II and IV in the type Asia 1, based on the profile of
reactivity and also on amino acids replacements of the relevant MAR-mutants, confirmed previous
280
findings. In fact, in type A, like in type Asia 1, mutants induced with MAbs against the two sites showed a
certain level of reciprocal cross-neutralisation (Thomas et al., 1988), in type C they are considered parts
of a unique complex antigenic site (Lea et al., 1994). Furthermore, in the three dimensional structure of
the capsid, residues involved in formation of the two sites lie close each other on the surface of the
virion, around the three-fold axes of symmetry (Kitson et al., 1990; Lea et al., 1994).
The detection of equivalent sites in four different serotypes, detected through independent studies,
enforces the evidence that these are dominant antigenic sites in the FMDV structure. However, there are
further conformational sites, implicated in neutralisation processes, each described only in one FMDV
serotype.
In type A a site of minor importance was found at position 169 (Baxt et al., 1988) and 163 (Thomas et
al., 1998) of VP1; in type O the so called site 3 was mapped at the amino acid residues 43 to 45 and 48
of VP1 (Barnett et al., 1989; Kitson et al., 1990). It was supposed that these two sites could be related
each other, as residues involved are located within two different loops (H-I and B-C loops respectively) of
VP1 lying adjacent on the virus surface. We did not find these positions as part of any antigenic site in
type Asia 1, however one mutant obtained with site II MAbs presented, beside changes related to site II,
a substitution in position 43, overlapping to site 3 of type O. This could suggest that the corresponding
region is subjected to variability also in type Asia 1.
A further position in VP1, mapping at residues 193 was recognised as another neutralising site, named
D1, only in type C (Lea et al., 1994).
Finally the site called V, described here in type Asia 1, maps at position 218 of VP3 (VP3 C- terminus)
and is reported for the first time in FMDV antigenic structure. The profile of reactivity of several isolates
of type Asia 1 indicates variability in correspondence of this site, in accordance with the frequent
substitution rate detected in position VP3-218 by sequencing field isolates (Marquardt et al., 2000).
Further sites not involved in neutralisation were detected by another group of 14 MAbs, reacting with all
Asia 1 field isolates and presenting different degrees of even inter-types cross reactivity. One of these
MAbs (5F10) recognises a linear epitope in VP2, as proven by its profile of reactivity in imunoblotting; the
epitope is susceptible to trypsin cleavage and is common to at least six serotypes. MAbs with analogous
reactivity were independently selected by other authors from mice immunised with either FMDV type O,
SAT 1 or Asia 1. The epitope target of these antibodies resides at the intertypically conserved N-terminus
of VP2, as demonstrated by the reactivity profile with synthetic peptides. (Freiberg et al., 2001).
The remaining 13 not neutralising MAbs recognise conformational epitopes, indistinguishable each other,
except for different patterns of inter-types cross reactivity.
Also non neutralising MAbs may have a potential value for the development of diagnostic assays, but they
are more difficult to characterise than neutralising MAbs. Therefore, there is a requirement to investigate
alternative methods and strategies for their characterisation.
In conclusion, our results have improved understanding of the antigenic structure of FMDV, through the
description of a new neutralising site and providing more evidence of the immunodominant character of
three antigenic sites detected in 4 different serotypes. These data may have useful application in
diagnostic and epidemiological investigations, as MAbs directed against conserved epitopes provide
universal reagents for FMDV detection systems, while MAbs against known variable sites readily allow the
identification of antigenic variants. The development of diagnostic immunoassays using these MAbs is
providing satisfactory perspectives.
Conclusions:
1) Three of the 4 neutralising sites identified and mapped on FMDV type Asia 1 correspond
structurally and functionally to analogous sites described in FMDV type O, A and C, enforcing the
evidence that these are dominant antigen sites in FMDV structure.
2) One site, located at C-terminus of VP3, is a new independent site, described for the first time in
FMDV. Further antigenic sites, not involved in neutralisation, were detected by 14 more MAbs.
3) MAbs confirmed their powerful for a better understanding of the antigenic structure of FMDV.
Recommendations:
1) A detailed characterization of MAbs should be achieved in order to select appropriate panels for
different applications, such as diagnostic tests or antigen profiling, and to guide to a correct
interpretation of results based on MAbs use (it is important to predict which antigenic sites are
being analyzed and their relevance for purpose).
2) In the antigenic structure of FMDV, sites not involved in neutralization are usually under
evaluated, despite they could be useful target for diagnostic purposes. Few tools and systems
that allow their study are known; more research should be encouraged also in these aspects.
3) Criteria and funding to create a bank of available MAbs should be defined. Only well characterized
MAbs should be maintained within the MAbs bank.
281
Acknowledgments: we thank D. Gamba for hybridoma cultures, M. Bugnetti for virus cultures, F. De
Simone for the critical reading of the manuscript. Work was supported by Italian Ministry of Health (grant
IZSLER PRC 98/004) and by EU (grant CT98 4032).
References
Acharya, R. Fry, E. Stuart, D. Fox, G. Rowlands, D. & Brown, F. 1989. The three-dimensional
structure of foot-and-mouth disease virus at 2.9 A resolution. Nature 337:709-716.
Barnett, P.V. Ouldridge, E.J. Rowlands, D.J. Brown, F. & Parry, N.R. 1989. Neutralizing epitopes on
type O foot-and-mouth disease virus. I. Identification and characterization of three functionally
independent, conformational sites. Journal of General Virology 70:1483-1491.
Baxt, B. Vakharia, V. Moore, D. M. Franke, A. J. & Morgan, D. O. 1989. Analysis of neutralizing
antigenic sites on the surface of type A12 foot-and-mouth disease virus. Journal of Virology, 63(5): 21432151.
Bolwell, C. Clarke, B.E. Parry, N.R. Ouldridge, E.J. Brown, F. & Rowlands, D.J. 1989. Epitope
mapping of foot-and-mouth disease virus with neutralizing monoclonal antibodies. Journal of General
Virology, 70:59-68.
Borrego, B. Carra, E. Garcìa-Ranea, J. A. & Brocchi, E. 2002. Characterization of neutralization sites
on the circulating variant of swine vesicular disease virus (SVDV): a new site is shared by SVDV and the
related coxsackie B5 virus. Journal of General Virology, 83: 35-44.
Domingo, E. Mateu, M.G. Martìnez, M.A. Dopazo, J. Moja, A. & Sobrino, F. 1990. Genetic variability
and antigenic diversity in foot-and-mouth disease virus. In Applied Virology Research, Volume 2, pp 233266, Plemum Publishing, New York.
Fox , G. Parry, N. Barnett, P.V. McGinn, B. Rowlands, D.J. & Brown, F. 1989. The cell attachment
site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic
acid). Journal of General Virology 70:625-637.
Freiberg, B. Hohlich, B. Haas, B. Saalmuller, A. Pfaff. E. & Marquardt, O. 2001. Type-independent
detection of foot-and-mouth disease virus by monoclonal antibodies that bind to amino-terminal residues
of capsid protein VP2. Journal of Virological Methods, 92: 199-205.
Harlow, E. & Lane, D. 1988. Immunoblottibg protocols. In Antibodies: a Laboratoryy manual, p 479510. Cold Spring Harbor, NY: Cold Sring Harbor laboratory.
Kitson, J.D.A. McCahon, D. & Belsham, G. J. 1990. Sequence analysis of monoclonal antibody
resistant mutants of type O foot and mouth disease virus: evidence for the involvement of the three
surface exposed capsid proteins in four antigenic sites. Virology, 179: 26-34
Grazioli, S. Fallacara, F. & Brocchi, E. Monoclonal Antibodies against FMDV type Asia 1: preliminary
characterization and potential use in diagnostic assays. Report of the Sess. Res. Gr. St. Tech. Committee
of the Europ. Comm. Control FMD, Izmir, Turkey 17-20 September 2002, p.194-202
Lea, S. Hernàndez, J. Blakemore, W. Brocchi, E. Curry, S. Domingo, E. Fry, E. Abu-Ghazaleh, R.
King, A. Newman, J. Stuart, D. & Mateu, M. G. 1994. The structure and antigenicity of a type C footand-mouth disease virus. Structure 2: 123-139.
Marquardt, O. & Freiberg, B. 2000a. Antigenic variation among foot-and-mouth disease virus type A
field isolates of 1997-1999 from Iran. Veterinary Microbiology, 74: 377-386
Marquardt, O. Rahman, M.M. & Freiberg, B. 2000b. Genetic and antigenic variance of foot-and-mouth
disease virus type Asia 1. Archives of Virology, 145: 149-157.
Mason, P.W. Rieder, E. & Baxt, B. 1994. RGD sequence of foot-and-mouth disease virus is essential
for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement
pathway. Proc. Natl. Acad. Sci. USA 91:1932-1936.
Mateu, M. G. Martìnez, M. A. Capucci, L. Andreu, D. Giralt, E. Sobrino, F. Brocchi, E. & Domingo,
E. 1990. A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site
of foot-and-mouth disease virus of serotype C. Journal of General Virology, 71: 629-637.
Mateu, M. G. 1995. Antibody recognition of picornaviruses and escape from neutralization: a structural
view. Virus Research, 38: 1-24.
Meloen, R.H. & Barteling, S.J. 1986. An epitope located at the C-terminus of isolated VP1 of foot-andmouth disease virus type O induces neutralizing activity but poor protection. Journal of General Virology
67:289-294.
Palmenberg, A.C. 1989. Molecular aspects of picornavirus infection and detection. American Society for
Microbiology, Washington D.C.
Parry, N.R. Barnett, P.V. Ouldridge, E.J. Rowlands, D.J. & Brown, F. 1989. Neutralizing epitopes of
type O foot-and-mouth disease virus. II. Mapping three conformational sites with synthetic peptide
reagents. Journal of General Virology, 70:1493-1503.
Pfaff, E., Thiel, H.J., Beck, E., Strohmaier, K., & Schaller, H. 1988. Analysis of neutralizing epitopes
on foot-and-mouth disease virus. Journal of Virology 62:2033-2040.
Samuel, A.R. Knowles, N.J. Samuel, G.D. & Crowther, J.R. 1991. Evaluation of a trapping ELISA for
the differentiation of FMDV using MAbs. Biologicals 19: 299-310.
282
Sanyal, A. Venkataramanan, R. & Pattnaik, B. 1997. Antigenic features of foot-and-mouth disease
virus serotype Asia1 as releaved by monoclonal antibodies and neutralization-escape mutans. Virus
Research 50:107-117.
Sanyal, A. Gurumurthy, C. B. Venkataramanan. R, Hemadri, D. & Tosh, C. 2003. Antigenic
characterization of foot-and-mouth disease virus serotype Asia1 field isolates using polyclonal and
monoclonal antibodies. Veterinary Microbiology, 93: 1-11.
Stave, J.W. Card, J.L. Morgan, D.O. & Vakharia, V.N. 1988. Neutralization sites of type O1 foot-andmouth disease virus defined by monoclonal antibodies and neutralization-escape virus variants. Virology,
162: 21-29.
Stram, Y. Laor, O. Molad, T. Chai, D. Moore, D. Yadin, H. & Becker Y. 1994. Nucleotide sequence of
the P1 region of serotype Asia1 foot-and-mouth disease virus. Virus Genes 8:275-278.
Thomas, A.A.M. Woortmeijer, R. J. Puijk, W. & Barteling. S. J. 1988. Antigenic sites on foot-andmouth disease virus type A10. Journal of Virology, 62(8): 2782-2789.
Tjissen P. 1985. Preparation of enzyme-antibody or other enzyme-macromolecule conjugates. In
Laboratory techniques in biochemistry and molecular biology. Practice and theory of enzyme
immunoassays (R.H. Burdon & P.H. van Knippenberg, eds). Elsevier, Amsterdam, 221-277.
Table 1. Primers used for PCR amplification and sequencing of the capsid-coding region
(polyprotein P1)
Position
Primers
Nucleotide
Gene
1D
50-70
5’ NTR
564D
564-587
VP2
Sequenze 5’ → 3’
GATCAGAGACCACTCAACGGA
CACCGAACTTGGCATTTGGACACT
612D
665-685
VP2
GGGTGGGACATAGAGGTGACT
674R
674-696
VP2
TTTCCAACAGCAGTCACCTCTAT
1026D
1079-1098
VP3
CCAGTGTACGGGAAAGTGTT
GCCCCGCAGCGAACGAGACA
1206R
1237-1256
VP3
1632D
1682-1702
VP1
TCAGCGGACCCGGTGACAACC
1716R
1748-1766
VP1
CAAAGGCAACGTCAGTGTG
2313R
2313-2333
2A
ACTCAACGTCTCCTGCCAACT
(D): direct primers, sequence identical to the viral genome; (R): reverse sequence complementary to the viral
genome. Nucleotide numbering is according to Stram Y. et al (1994). All primer sequences correspond to FMDV Nepal
29/97 determined in this work, except for primers 564D and 2313R, which were designed according to the sequence of
serotype Asia 1, isolate L83 (Stram Y. et al, 1994, Accession N° U01207).
Table 3: Amino acid substitutions found in MAR-mutants
Ag site MAR-mutant
4E10/A7+A3
I
II
IV
V
Substituted amino acid
VP1 142 (R → Q)
4F10/A7
VP1 142 (R → Q)
5C12/A3
VP1 142 (R → Q); (VP1 121 A → G)
5E10/A11
VP3 58 (G → E); VP2 67 (F → L)
1F10/B2
VP3 59 (E → D); VP2 77 (H → D)
2C3/A7-A11
VP2 67 (F → L); VP2 77 (H → R); VP2 79 (Y → H)
2C3/A10
VP2 72 (D → N); VP2 74 (A → T); VP2 79 (Y → N)
2G1/A9
VP2 72 (D → G); VP2 77 (H → R); VP2 79 (Y → H) (VP1 48, VP1 207)
5G4/C9
VP3 218 (R → Q)
5G4/A2-A6
VP3 218 (R → Q)
283
Table 2: Reactivity of MAbs raised against FMDV type Asia 1, strain Nepal 29/97
Kuwait 2/81
Ag site
Tur 15/73
Gre 1/2000
SAU 39/94
Iran 58/99
Pak 3/98
Pak 2/98
India 10/82
0
Cam 9/80
100 100 100 100 100 100 100 100 100 70 100
(homologous)
+
Nepal 29/97
Cam 3/93
-
SAT 3 Zim 4/81
O1 Switz. 65
homologous
A 5 Italy 62
C1 Italy 64
SAT 1 Bot 1/68
SAT 2 Zim 5/81
-
percentage reactivity (ELISA)
-
-
+ 1
100 100 100 100 100 100 100 100 100 70 100
0
-
-
-
-
-
+
100 75 100 100 100 100 100 100 100 25
20
0
IgG1 5E10 24
3840
-
125
-
-
-
-
-
-
100 100 50
20
0
IgG1 3C6
3840
-
125
-
-
-
-
-
-
100 100 100 100 100 100 100 100 75 100 40
0
IgG1 1F10 >32 20480
-
625
-
-
-
-
-
-
100 100 100 100 100 100 100 100 100 100 100
0
IgG1 2C3 12
2560
-
375
-
-
-
-
-
-
100 50
10
10
10
10
10
10
10
10
10
5
IgG1 2G1 24
2560
-
375
-
-
-
-
-
-
100 50
0
0
0
0
0
0
0
0
0
0
100
0
0
0
0
0
0
0
0
0
0
0
100
0
50
50
35
20
0
0
0
0
0
0
3
25
4
2
based on MAR-Mutants
- +/- -
IgG1 5C12 >32 >20480 VP1 625
50
50
50
50
35
40
50
IgG2a 4D8
1
nd
-
5
-
-
-
-
-
-
IgM 5G4
6
240
-
125
-
-
-
-
-
-
IgG1 4G6
-
-
-
125
-
-
-
-
-
-
100 100 100 100 100 100 100 100 100 100 100
IgG1 3D8
-
-
-
3000 -
-
-
-
-
-
100 100 100 100 100 100 100 100 100 100 100 100
IgG1 4B1
-
nd
-
375 -/+ - -/+ -
-
-
IgG1 4B2
-
nd
-
625 -/+ - -/+ -
-
-
IgG1 2F7
-
nd
-
625 (+) (+) - -/+ -/+ -/+
IgG1 2G8
-
-
-
625 (+) (+) - -/+ -/+ -/+
IgG1 2B11
-
nd
-
625
+ +
IgG1 3G3
-
nd
-
625
+ + +
-
-
-
IgG1 2A4
-
-
-
125
+ + +
-
-
-
IgG1 3B6
-
nd
-
625
+ + +
-
-
-
IgG1 5H5
-
nd
-
5
+ + +
-
- -/+
IgG1 4G2
-
nd
-
2
+ + + + + +
IgG1 3H12
-
nd
-
125
+ + + + + +
IgG1 5F10
-
-
VP2
75
+ + + -/+ + + lin. site 100 100 100 100 100 100 100 100 100 100 100 100
- -/+ -/+
based on FMDV inter-types reactivity
-
5
multiple epitopes
NEUTRALIZING MAbS
Asia 1
5120 VP1 125 a) - +/- -
IgM 4F10 >32 20480 VP1
NON-NEUTRALISING MAbS
ELISA Trapping
Nepal 58/88
IgM 4E10 12
ascitic fluid
hybrid. culture
MAb
Ig class
VNT titre
Immunoblotting
MAbs reactivity against field isolates of type Asia 1
0
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100
a) : highest saturating dilution of hybridoma supernatant; + : same reactivity as with the homologous virus type; (+):
reduced signal with respect to the homologous virus type; -/+ : traces of reactivity; - : negative
284
Figure 2. ELISA reactivity profile of MAR-mutants with neutralizing anti-Asia 1 MAbs and
location of amino acid substitutions
Monoclonal Antibodies
site
5G4
V
4D8
2G1
2C3
site II
1F10
3C6
5E10
site IV
5C12
4F10
MAR-Mutants
4E10
site I
4E10 A9+A10
4E10 A7+A3
4F10 A7
4F10 B3
5C12 A3
5C12 B10
5C12 A10
5C12 A11
5C12 A7
Amino acid
Struct
substitutions in the
element
corresp. mutant
VP1 142
VP1 142
VP1 142 (VP1 121)
5E10 A11
3C6 pool B2-B6
3C6 pool A7-A11
3C6 A2+A6
1F10 B7-B11
1F10 A9-A10
1F10 D2-D4
1F10 D5
1F10 D3
1F10 B2
1F10 B9
1F10 A7
1F10 A3
VP1
G-H
loop
VP3 58 ; VP2 67
VP3
B-B
knob
VP3 59 ; VP2 77
2C3 C9-C11
2C3 pool A7-A11
2C3 pool B2-B6
2C3 pool B7-B11
2C3 pool A2-A6
2C3 D4
2C3 A10
2G1 A9
2G1 A8
2G1 A10
2G1 A11
VP2 67, 77, 79
VP2
B-C
loop
VP2 72, 74, 79
VP2 72, 74, 79 (VP1 48, 207)
5G4 pool B9-B11
5G4 C9
5G4 B7-B8-B10
5G4 A7-A11
5G4 A2-A6
VP3 218 (C-term)
VP3
C-term
VP3 218 (C-term)
Percentage of reactivity related to the parental virus
< 10%
10-30%
30-60%
60-80%
80-100%
285
Figure 1. Profile of reactivity of anti-FMDV type Asia 1 MAbs in immunoblotting
Figure 3. Neutralisation resistance pattern of MAR-mutants with MAbs
Monoclonal Antibodies
Site IV
Site I
MAR-mutants
Parental virus
4E10 A7-A3
4F10 A7
5C12 A3
5E10 A11
3C6 A2-A9
1F10 B2
1F10 A3
2C3 A7-A11
2C3 D4
2G1 A8
5G4 C9
Site II
Site V
4E10
4F10
5C12
5E10
3C6
1F10
2C3
2G1
5G4
4,186
4,01
4,311
3,885
3,885
4,612
2,38
2,681
2,204
>3
>3
>3
nd
0,78
0,3
0
0
0,3
>3
>3
>3
0,61
0,47
0,11
0
0
0,42
>3
>3
>3
0,31
0,3
0,42
0,48
1,2
0,72
0
0,1
0
2,03
2,6
2,8
0
0
0
0,78
0,3
0,1
2
>3
3
0,18
0,6
0,6
0,1
0
nd
0,1
2,3
2,6
>3
0,48
0,6
0,48
nd
0,1
2,6
>3
>3
>2
>2
0
0,78
nd
0,6
1,5
1,1
0,7
>2
>2
0,1
0,78
0,7
0,1
2
0,78
0,9
>2
>2
0,1
0,6
nd
0,42
2
0,9
1,2
>2
>2
0,1
1
0,89
0,2
0,78
0,3
0,42
0
0
>2
Numbers in cells express differences in VNT titres (log10) to the parental and mutants viruses;
white cells: differences > 2 log10 indicate resistance to neutralisation; grey cells: differences between 1 and 2 log10
indicate partial resistance; black cells: differences < 1 log10 indicate susceptibility to neutralisation.
286
Figure 4: Mapping of neutralising sites on FMDV structural proteins
Correspondence between antigenic sites described in different FMD virus types
1
85 /1
VP4
218 /1
58, 59
Site IV
67, 72, 74, 77, 79
Site II
1
218 /1
VP2
85/1
VP4
58
Site 4
218 /1
VP2
72, 74, 79
Site D2
213
FMDV-A10/12
138 Æ 154
200 Æ 213
169, 173
220/1
VP3
70, 71, 72, 75, 77, 131
Site 2
1
(207)
142
Site I
VP1
195
69 /1
VP4
FMDV-ASIA
(48)
58 Æ 61, 69, 70 139
Ag-Site III
80
Ag-Site III
1
218
Site V
VP3
VP2
211
VP 1
221 /1
218 /1
69 /1
VP4
219 /1
VP3
VP2
213
58
Site D3
200 Æ 213
43 Æ 45, 48
144 Æ 154
Site 3
Site 1
220/1
VP3
FMDV-O1
VP1
213
FMDV-C1
VP1
138 Æ 150
Site A
200 Æ 213
193
Site D1
287