BACKGROUND
foot-and-mouth disease virus  is an important animal pathogen that causes a severe vesicular disease in cattle, swine, sheep and other cloven-hoofed animals  <cit> .
the virus belongs to the aphthovirus genus within the picornaviridae family  <cit> .
the genome is a positive-sense single-stranded rna molecule that is encapsidated by  <dig> copies of each of the four structural polypeptides of which vp <dig> is internal and the others  are exposed  <cit> .
it has been shown that vp <dig> is the most variable among the capsid polypeptides, and it is widely used to characterize field strains of fmdv to provide data to support epidemiological investigations of disease outbreaks among livestock.
a major, highly variable antigenic site of fmdv is located at the exposed g-h loop comprising amino acids 134- <dig> of the capsid protein vp <dig>  <cit> , which plays an important role in cell infection and is also a major target for protective host responses mediated via humoral immunity  <cit> .
this mobile loop contains a conserved arg-gly-asp  motif that has been shown to be a major determinant in the interaction of the virus with cell surface receptors of the integrin superfamily  <cit> .
indeed, previous studies, using different approaches, have indicated that naturally occurring field isolates of fmdv bind to cells via these highly conserved surface-exposed rgd residues  <cit> .
in particular, it has been reported that fmd viruses utilize multiple rgd-dependent integrins of the αv subgroup to initiate infection, including αvβ <dig>  αvβ <dig>  αvβ <dig> and αvβ <dig>  <cit> .
however, the rgd integrin recognition domain can become dispensable upon in-vitro passage of fmdv: multiple phenotypic changes that are associated with a limited number of amino acid substitutions at the capsid surface which may even include modifications within the rgd triplet  <cit> .
tissue culture-adapted viruses can use heparan sulfate  as a receptor to enter cells  <cit> , and can also dispense with their rgd integrin-binding motif  <cit> .
these findings indicate the existence of alternative rgd-independent pathways for fmdv entry into cell.
in the present study we report that two viruses harboring alternative receptor binding sites  were generated after short-term passage of an fmdv field isolate  in different environments.
the non-rgd receptor recognition motifs were stably maintained during subsequent passage in cell culture.
to study the ability of an rdd-containing fmd viral genome to accommodate substitution in receptor binding site and non-rgd viruses to cause disease in susceptible animals, we assembled an rdd-containing fmdv  full-length cdna clone and derived mutant clones harboring rgd or rsd motif with a single amino acid substitution  in the receptor binding site.
following transfection of bsr/t <dig> cell with three full-length plasmids, the resulting viruses were examined for their infectious potential in-vitro and in-vivo.
RESULTS
sequence analysis of asia1/js/cha/ <dig> and its derivatives
deduced amino acid sequence analysis of the 1d-encoding region showed that asia1/js/cha/ <dig> had a consensus rgd triplet at position 143- <dig> of vp <dig>  while asia1/jsp1c <dig> obtained an alternative rdd triplet at this position.
however, careful inspection of the electropherograms from the asia1/jsm <dig> vp <dig> gene sequencing reactions revealed the presence of two genetic subpopulations, one with rgd and the other with rsd at receptor binding site.
to further investigate the genetic heterogeneity within these samples,  <dig> biological clones containing vp <dig> genes of each asia1/js/cha/ <dig>  asia1/jsp1c <dig> and asia1/jsm <dig> were sequenced.
the  <dig> clones obtained from each of the asia1/js/cha/ <dig> and asia1/jsp1c <dig> viruses respectively encode rgd and rdd tripeptide at position 143- <dig> of vp <dig>  for asia1/jsm <dig>  four clones encoded rsd and six clones maintain the rgd motif at the same position.
these results were identical to the amino acid sequence analysis performed by direct sequencing of pcr amplicons.
additionally, amino acid sequence analysis of the capsid-coding regions of asia1/js/cha/ <dig>  asia1/jsp1c <dig>  and asia1/jsm <dig> showed that asia1/jsp1c <dig> had seven amino acid substitutions in the capsid region  compared with asia1/js/cha/ <dig> and asia1/jsm <dig>
a
a amino acid residues are numbered from the amino terminus to the carboxyl terminus.
single letter amino acid code is used.
taken together, these results suggested that the rdd mutants became dominant or obtained equilibrium with the rgd and rsd subpopulations at an early phase of asia1/js/cha/ <dig> quasispecies evolution.
since rdd and rsd motifs are unusual in lacking the rgd integrin-recognition sequence, additional multiple passages were performed to determine its stability.
amino acid sequence of the vp <dig> gene of the viruses obtained from different passages of asia1/jsp1c <dig> and asia1/jsm <dig> revealed that the rdd and rsd sequence were genetically stable for at least  <dig> passages .
the amino acid sequences of the g-h loop of viruses derived from different passages are summarized in table  <dig>  evidence that fmdvs can contain an rdd or rsd receptor-binding site increases the quasispecies complexity around the rgd-coding region.
c
a the rescued viruses were passaged  <dig> times in cell culture.
b virus recovered from vesicular lesions, away from the inoculation site.
c sequence data were obtained by rt-pcr of the vp <dig> capsid region.
the dashes represent receptor binding triplet of the viruses derived from different origins and full-length plasmids.
rescue of viable viruses from the full-length cdna clones
to examine the influence of single amino acid substitutions in the receptor binding site of the rdd-containing fmd viral genome on virus viability and the ability of non-rgd viruses to cause disease in susceptible animals, we assembled a full-length cdna clone of an rdd-containing fmdv and derived mutant clones containing rsd or rgd motifs with a single amino acid substitution in the receptor binding site .
bsr-t7/ <dig> cells were independently transfected with linearized-plasmids, prdd, prgd and prsd.
the typical fmdv cytopathic effect was clearly visible at  <dig> h post-transfection with prdd and prgd, and at  <dig> h post-transfection with prsd.
the three rescued viruses were named fmdv-rdd, fmdv-rgd, and fmdv-rsd, respectively.
to increase the virus titers, all rescued viruses were subjected to serial passage in bhk- <dig> cells, after which the vp <dig> sequence was analyzed to confirm that the recovered viruses had maintained the cdna-encoded receptor binding motifs .
when the growth characteristics of the rescued viruses were compared with the parental virus asia1/jsp1c <dig> by one-step growth kinetics assays, rescued viruses showed similar growth properties to the parental virus .
in addition, the plaque sizes of the parental virus and the rescued viruses were also similar .
these results suggest that single amino acid substitutions in the receptor binding site of asia1/jsp1c <dig> virus do not affect virus viability.
the pathogenicity of the rescued viruses in cattle and swine
to investigate the pathogenicity of the non-rgd viruses in the natural host, we performed direct inoculation of parental virus asia1/jsp1c <dig> and recombinant viruses  in cattle and pigs.
after inoculation, a number of disease parameters were analyzed, including fever, clinical score, and viremia.
the animals, except for the fmdv-rsd-inoculated animals, showed fever and extensive tissue damage at the inoculation sites by day  <dig> and achieved the maximal score of lesions on day 2- <dig>  some fmdv-rsd-inoculated animals developed fever and tissue damage by day  <dig> and achieved the maximal score of lesions on day 3- <dig>  two animals  had no evidence of tissue damage, except for occasional depression and anorexia when their body temperatures rose.
the asia1/jsp1c <dig> and fmdv-rdd viruses produced more extensive tissue damage at the injected sites and induced fever and vesicles a day earlier than in the fmdv-rsd-inoculated animals.
there were significant differences in lesion scores between rdd viruses  and rsd virus , however, no significant differences in lesion scores between cattle and pigs .
the lesion scores for the inoculated animals are summarized in table  <dig> and figure  <dig> shows the rectal temperature of all of the inoculated animals.
the disease was characterized by viremia in all inoculated animals, including the animals that did not generate vesicular lesions.
the level of viremia increased following inoculation, typically reaching a peak level after two or three days then decreasing to zero by day  <dig>  in contrast to the lesion scores, there were no significant differences caused by viruses or species in terms of the maximum viremia , time of maximum viremia  or time of maximum rate of increase in viremia .
virus rna copies in the blood of the inoculated animals are summarized in table  <dig>
a
a lesion scores were calculated as described by rieder et al.
.
b
b the amount of virus in the blood was measured by real-time quantitative rt-pcr assay as described in materials and methods.
blood samples were collected at 1- <dig> dpi in inoculated animals.
vesicular fluid was collected from inoculated animals, and each sample was separately processed for rt-pcr and nucleotide sequencing.
the results revealed that the original receptor-binding motif did not change during growth in vivo.
discussion
the rgd integrin-binding motif within vp <dig> is highly conserved among fmdv field isolates, and is generally considered essential for virus viability via its interaction with cellular integrin heterodimers  <cit> .
biochemical evidence that small peptides containing the rgd sequence inhibited the adsorption of the virus to tissue culture cells  <cit> , and genetically engineered virions containing either mutations or deletions of the rgd sequence were unable to bind to cells or cause disease in susceptible animals  <cit> .
however, the rgd triplet may be dispensable upon short-term evolution of the virus harboring it in a constant environment  <cit> .
in the present report, we have documented that non-rgd derivatives arose serendipitously during short-term passage of a fmdv field isolate in vitro and in vivo.
one derivative containing an rdd triplet in the receptor-binding site was obtained from the serotype asia  <dig> field isolate after a single cattle-to-pig transmission and subsequent bhk- <dig> in vitro passage.
sequence analysis of  <dig> biological clones of the vp <dig> encoding region of this population demonstrated that rdd viruses instead of the original rgd virus became predominant at an early phase of asia1/js/cha/ <dig> quasispecies evolution.
unexpectedly, however, both rgd and rsd viruses were obtained from the asia1/jsm <dig> population that were generated after four serial passages of the asia1/js/cha/ <dig> field isolate in suckling mice, via intraperitoneal inoculation.
the population equilibrium of rsd mutant and ancestor viruses was maintained after  <dig> passages of the asia1/jsm <dig> population in bhk- <dig> cells.
although rdd- or rsd-containing fmdv are unusual, they were genetically stable upon extended replication in cell culture.
our results suggest that, in the context of the capsid proteins of asia1/js/cha/ <dig>  a highly conserved rgd motif is not essential for replication in vitro and in vivo, suggesting functional flexibility of fmdv to enter cells in response to environmental modifications.
like other rna viruses, fmdv exists as closely related but non-identical genomes, termed viral quasispecies  <cit> .
genetic diversity is an intrinsic property of the quasispecies, which arise due to the lack of proofreading activity during viral genome replication, a short replication cycle, and other environmental selective pressures  <cit> .
our observations showed that evolution of fmdv population exhibited receptor binding motif diversity  subjected to short-term passage of field isolate in different environments.
from the standpoint of rna virus population evolution, one possible scenario could explain this observation.
the early interactions between viruses and host cells exert major selective force on virus populations, thus, the variants  may already be present at low frequency in the natural population that are possibly more fit in new environments and become dominant strains.
while this presumption is contrary to the view that the rgd triplet is highly conserved among natural isolates of fmdv, there is direct evidence that an rdd containing field virus was isolated from pigs during a type asia  <dig> fmd outbreak in china.
rdd-containing fmdv vp <dig> genes were amplified from sheep oesophageal-pharyngeal fluids  collected during  <dig> from a sheep herd in the region of china that had endemic asia  <dig> serotype fmdv  <cit> .
the emergence of these non-rgd mutants in nature is likely to be influenced by specific epidemiological and immunological aspects of host-virus interaction as well as the quasispecies composition of the viral population  <cit> .
the molecular basis for this apparent relaxation of the requirement for the precise rgd sequence is not well understood, but functional replacements within the rgd triplet allow the use of an alternative receptor for entry of fmdv in bhk- <dig> cells.
since other fmdv lack the rgd motif, host cell recognition may be mediated through another integrin receptor or a non-integrin pathway, or use a third receptor  for entry into the host cell  <cit> .
further studies are required to analyze the interaction of these mutants with the major fmdv integrin receptors αvβ <dig>  αvβ <dig>  αvβ <dig> and αvβ <dig> identified to date, and to understand whether these viruses obtain alteration of cell tropism, antigenicity, and virulence.
to examine the influence of single amino acid substitutions in the receptor binding site of rdd-containing fmd viral genome on virus viability and the ability of non-rgd viruses to cause disease in susceptible animals, we constructed an fmdv asia1/js/p1c <dig> full-length clone and derived mutant molecules with rgd or rsd receptor recognition motifs.
following transfection of bsr cells with these clones, three recombinant viruses were rescued, in particular, six other amino acid differences in the p <dig> capsid region of asia1/js/cha/ <dig> and asia1/jsm <dig>  did not affect rescue of viable rgd- and rsd-harboring viruses.
furthermore, in vitro growth properties of these viruses did not differ significantly.
our results showed that asia1/js/p1c <dig> viral genome can tolerate substitutions in the receptor binding site with no other changes in the capsid.
the ability of the asia1/js/p1c <dig> viral genome to tolerate substitution of receptor binding sites may depend on the capsid sequence, because the asp- <dig> gly change of receptor recognition site was lethal in the context of the capsid proteins of fmdv c-s8c <dig>  however, the same replacement yielded viable viruses in the context of the capsid protein of fmdv c-s8c1p <dig> and c-s8c1p <dig>  <cit> .
to assess the ability of non-rgd fmd viruses to cause disease in naturally susceptible animals, we performed experiment infections of cattle and pigs using the asia1/js/p1c <dig> and two non-rgd recombinant viruses.
subsequent experiments showed that all viruses were able to cause disease in cattle and pigs and produce rapid onset of clinical signs, characteristic of infection with rgd field strains.
the disease was characterized by viremia in all inoculated animals, including the individuals that did not generate vesicular lesions.
amongst these viruses, the rsd virus produced less tissue damage at the inoculation sites and induced fever and vesicles a day later than in the animals inoculated with rdd-containing viruses, which indicated a different degree of disease severity.
the different virulence of these viruses was also supported by the maintenance of original receptor recognition sequence in vesicle samples obtained from infected animals.
the reason for low virulence of rsd virus, which may bear on receptor usage, needs to be further explored.
these observations are particularly interesting, since the presence of an rgd motif is believed to be the main determinant to direct fmdv to integrin-containing target tissues during infection in the natural host  <cit> .
in addition, information currently available indicates that fmdv utilizes integrins for entry in the natural host, and there is no evidence of the use of alternative receptors in vivo  <cit> .
therefore, our results further support the possibility that a non-rgd-integrin interaction could be responsible for the generation of fmd in the natural host.
our study was the first to demonstrate the ability of an rdd containing natural isolate to cause disease in naturally susceptible animals, and will provide knowledge about the in vivo pathogenesis of non-rgd viruses.
CONCLUSIONS
fmdv quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition sites.
thus, the early interaction between the viruses and the host cells may exert major selective pressure on fmdv populations that contributes to the evolution and functional flexibility of fmdv to enter cells.
our studies using two non-rgd fmdvs not only show that there was an increase in the number of viable mutants with substitutions in the receptor-binding region, but also provide useful tools for studies of cell recognition by fmdv.
based on an rdd-containing full-length infectious cdna clone, the rsd- and rgd-containing recombinant viruses were rescued, and single amino acid substitutions in the receptor-binding site did not affect virus viability.
the viruses expressing non-rgd receptor binding sites can replicate stably in vitro and induce the disease in susceptible animals.

 foot-and-mouth disease virus is an important animal pathogen that causes a severe vesicular disease in cattle, swine, sheep and other cloven-hoofed animals. the virus belongs to the aphthovirus genus within the picornaviridae family. the genome is a positive-sense single-stranded rna molecule that is encapsidated by copies of each of the four structural polypeptides of which vp is internal and the others are exposed. it has been shown that vp is the most variable among the capsid polypeptides, and it is widely used to characterize field strains of fmdv to provide data to support epidemiological investigations of disease outbreaks among livestock. a major, highly variable antigenic site of fmdv is located at the exposed g-h loop comprising amino acids 134- of the capsid protein vp, which plays an important role in cell infection and is also a major target for protective host responses mediated via humoral immunity. this mobile loop contains a conserved arg-gly-asp motif that has been shown to be a major determinant in the interaction of the virus with cell surface receptors of the integrin superfamily. indeed, previous studies, using different approaches, have indicated that naturally occurring field isolates of fmdv bind to cells via these highly conserved surface-exposed rgd residues. in particular, it has been reported that fmd viruses utilize multiple rgd-dependent integrins of the αv subgroup to initiate infection, including αvβ αvβ αvβ and αvβ. however, the rgd integrin recognition domain can become dispensable upon in-vitro passage of fmdv: multiple phenotypic changes that are associated with a limited number of amino acid substitutions at the capsid surface which may even include modifications within the rgd triplet. tissue culture-adapted viruses can use heparan sulfate as a receptor to enter cells, and can also dispense with their rgd integrin-binding motif. these findings indicate the existence of alternative rgd-independent pathways for fmdv entry into cell. in the present study we report that two viruses harboring alternative receptor binding sites were generated after short-term passage of an fmdv field isolate in different environments. the non-rgd receptor recognition motifs were stably maintained during subsequent passage in cell culture. to study the ability of an rdd-containing fmd viral genome to accommodate substitution in receptor binding site and non-rgd viruses to cause disease in susceptible animals, we assembled an rdd-containing fmdv full-length cdna clone and derived mutant clones harboring rgd or rsd motif with a single amino acid substitution in the receptor binding site. following transfection of bsr/t cell with three full-length plasmids, the resulting viruses were examined for their infectious potential in-vitro and in-vivo.  sequence analysis of asia1/js/cha/ and its derivatives deduced amino acid sequence analysis of the 1d-encoding region showed that asia1/js/cha/ had a consensus rgd triplet at position 143- of vp while asia1/jsp1c obtained an alternative rdd triplet at this position. however, careful inspection of the electropherograms from the asia1/jsm vp gene sequencing reactions revealed the presence of two genetic subpopulations, one with rgd and the other with rsd at receptor binding site. to further investigate the genetic heterogeneity within these samples, biological clones containing vp genes of each asia1/js/cha/ asia1/jsp1c and asia1/jsm were sequenced. the clones obtained from each of the asia1/js/cha/ and asia1/jsp1c viruses respectively encode rgd and rdd tripeptide at position 143- of vp for asia1/jsm four clones encoded rsd and six clones maintain the rgd motif at the same position. these results were identical to the amino acid sequence analysis performed by direct sequencing of pcr amplicons. additionally, amino acid sequence analysis of the capsid-coding regions of asia1/js/cha/ asia1/jsp1c and asia1/jsm showed that asia1/jsp1c had seven amino acid substitutions in the capsid region compared with asia1/js/cha/ and asia1/jsm a a amino acid residues are numbered from the amino terminus to the carboxyl terminus. single letter amino acid code is used. taken together, these results suggested that the rdd mutants became dominant or obtained equilibrium with the rgd and rsd subpopulations at an early phase of asia1/js/cha/ quasispecies evolution. since rdd and rsd motifs are unusual in lacking the rgd integrin-recognition sequence, additional multiple passages were performed to determine its stability. amino acid sequence of the vp gene of the viruses obtained from different passages of asia1/jsp1c and asia1/jsm revealed that the rdd and rsd sequence were genetically stable for at least passages. the amino acid sequences of the g-h loop of viruses derived from different passages are summarized in table evidence that fmdvs can contain an rdd or rsd receptor-binding site increases the quasispecies complexity around the rgd-coding region. c a the rescued viruses were passaged times in cell culture. b virus recovered from vesicular lesions, away from the inoculation site. c sequence data were obtained by rt-pcr of the vp capsid region. the dashes represent receptor binding triplet of the viruses derived from different origins and full-length plasmids. rescue of viable viruses from the full-length cdna clones to examine the influence of single amino acid substitutions in the receptor binding site of the rdd-containing fmd viral genome on virus viability and the ability of non-rgd viruses to cause disease in susceptible animals, we assembled a full-length cdna clone of an rdd-containing fmdv and derived mutant clones containing rsd or rgd motifs with a single amino acid substitution in the receptor binding site. bsr-t7/ cells were independently transfected with linearized-plasmids, prdd, prgd and prsd. the typical fmdv cytopathic effect was clearly visible at h post-transfection with prdd and prgd, and at h post-transfection with prsd. the three rescued viruses were named fmdv-rdd, fmdv-rgd, and fmdv-rsd, respectively. to increase the virus titers, all rescued viruses were subjected to serial passage in bhk- cells, after which the vp sequence was analyzed to confirm that the recovered viruses had maintained the cdna-encoded receptor binding motifs. when the growth characteristics of the rescued viruses were compared with the parental virus asia1/jsp1c by one-step growth kinetics assays, rescued viruses showed similar growth properties to the parental virus. in addition, the plaque sizes of the parental virus and the rescued viruses were also similar. these results suggest that single amino acid substitutions in the receptor binding site of asia1/jsp1c virus do not affect virus viability. the pathogenicity of the rescued viruses in cattle and swine to investigate the pathogenicity of the non-rgd viruses in the natural host, we performed direct inoculation of parental virus asia1/jsp1c and recombinant viruses in cattle and pigs. after inoculation, a number of disease parameters were analyzed, including fever, clinical score, and viremia. the animals, except for the fmdv-rsd-inoculated animals, showed fever and extensive tissue damage at the inoculation sites by day and achieved the maximal score of lesions on day 2- some fmdv-rsd-inoculated animals developed fever and tissue damage by day and achieved the maximal score of lesions on day 3- two animals had no evidence of tissue damage, except for occasional depression and anorexia when their body temperatures rose. the asia1/jsp1c and fmdv-rdd viruses produced more extensive tissue damage at the injected sites and induced fever and vesicles a day earlier than in the fmdv-rsd-inoculated animals. there were significant differences in lesion scores between rdd viruses and rsd virus, however, no significant differences in lesion scores between cattle and pigs. the lesion scores for the inoculated animals are summarized in table and figure shows the rectal temperature of all of the inoculated animals. the disease was characterized by viremia in all inoculated animals, including the animals that did not generate vesicular lesions. the level of viremia increased following inoculation, typically reaching a peak level after two or three days then decreasing to zero by day in contrast to the lesion scores, there were no significant differences caused by viruses or species in terms of the maximum viremia, time of maximum viremia or time of maximum rate of increase in viremia. virus rna copies in the blood of the inoculated animals are summarized in table a a lesion scores were calculated as described by rieder et al.. b b the amount of virus in the blood was measured by real-time quantitative rt-pcr assay as described in materials and methods. blood samples were collected at 1- dpi in inoculated animals. vesicular fluid was collected from inoculated animals, and each sample was separately processed for rt-pcr and nucleotide sequencing. the results revealed that the original receptor-binding motif did not change during growth in vivo. discussion the rgd integrin-binding motif within vp is highly conserved among fmdv field isolates, and is generally considered essential for virus viability via its interaction with cellular integrin heterodimers. biochemical evidence that small peptides containing the rgd sequence inhibited the adsorption of the virus to tissue culture cells, and genetically engineered virions containing either mutations or deletions of the rgd sequence were unable to bind to cells or cause disease in susceptible animals. however, the rgd triplet may be dispensable upon short-term evolution of the virus harboring it in a constant environment. in the present report, we have documented that non-rgd derivatives arose serendipitously during short-term passage of a fmdv field isolate in vitro and in vivo. one derivative containing an rdd triplet in the receptor-binding site was obtained from the serotype asia field isolate after a single cattle-to-pig transmission and subsequent bhk- in vitro passage. sequence analysis of biological clones of the vp encoding region of this population demonstrated that rdd viruses instead of the original rgd virus became predominant at an early phase of asia1/js/cha/ quasispecies evolution. unexpectedly, however, both rgd and rsd viruses were obtained from the asia1/jsm population that were generated after four serial passages of the asia1/js/cha/ field isolate in suckling mice, via intraperitoneal inoculation. the population equilibrium of rsd mutant and ancestor viruses was maintained after passages of the asia1/jsm population in bhk- cells. although rdd- or rsd-containing fmdv are unusual, they were genetically stable upon extended replication in cell culture. our results suggest that, in the context of the capsid proteins of asia1/js/cha/ a highly conserved rgd motif is not essential for replication in vitro and in vivo, suggesting functional flexibility of fmdv to enter cells in response to environmental modifications. like other rna viruses, fmdv exists as closely related but non-identical genomes, termed viral quasispecies. genetic diversity is an intrinsic property of the quasispecies, which arise due to the lack of proofreading activity during viral genome replication, a short replication cycle, and other environmental selective pressures. our observations showed that evolution of fmdv population exhibited receptor binding motif diversity subjected to short-term passage of field isolate in different environments. from the standpoint of rna virus population evolution, one possible scenario could explain this observation. the early interactions between viruses and host cells exert major selective force on virus populations, thus, the variants may already be present at low frequency in the natural population that are possibly more fit in new environments and become dominant strains. while this presumption is contrary to the view that the rgd triplet is highly conserved among natural isolates of fmdv, there is direct evidence that an rdd containing field virus was isolated from pigs during a type asia fmd outbreak in china. rdd-containing fmdv vp genes were amplified from sheep oesophageal-pharyngeal fluids collected during from a sheep herd in the region of china that had endemic asia serotype fmdv. the emergence of these non-rgd mutants in nature is likely to be influenced by specific epidemiological and immunological aspects of host-virus interaction as well as the quasispecies composition of the viral population. the molecular basis for this apparent relaxation of the requirement for the precise rgd sequence is not well understood, but functional replacements within the rgd triplet allow the use of an alternative receptor for entry of fmdv in bhk- cells. since other fmdv lack the rgd motif, host cell recognition may be mediated through another integrin receptor or a non-integrin pathway, or use a third receptor for entry into the host cell. further studies are required to analyze the interaction of these mutants with the major fmdv integrin receptors αvβ αvβ αvβ and αvβ identified to date, and to understand whether these viruses obtain alteration of cell tropism, antigenicity, and virulence. to examine the influence of single amino acid substitutions in the receptor binding site of rdd-containing fmd viral genome on virus viability and the ability of non-rgd viruses to cause disease in susceptible animals, we constructed an fmdv asia1/js/p1c full-length clone and derived mutant molecules with rgd or rsd receptor recognition motifs. following transfection of bsr cells with these clones, three recombinant viruses were rescued, in particular, six other amino acid differences in the p capsid region of asia1/js/cha/ and asia1/jsm did not affect rescue of viable rgd- and rsd-harboring viruses. furthermore, in vitro growth properties of these viruses did not differ significantly. our results showed that asia1/js/p1c viral genome can tolerate substitutions in the receptor binding site with no other changes in the capsid. the ability of the asia1/js/p1c viral genome to tolerate substitution of receptor binding sites may depend on the capsid sequence, because the asp- gly change of receptor recognition site was lethal in the context of the capsid proteins of fmdv c-s8c however, the same replacement yielded viable viruses in the context of the capsid protein of fmdv c-s8c1p and c-s8c1p. to assess the ability of non-rgd fmd viruses to cause disease in naturally susceptible animals, we performed experiment infections of cattle and pigs using the asia1/js/p1c and two non-rgd recombinant viruses. subsequent experiments showed that all viruses were able to cause disease in cattle and pigs and produce rapid onset of clinical signs, characteristic of infection with rgd field strains. the disease was characterized by viremia in all inoculated animals, including the individuals that did not generate vesicular lesions. amongst these viruses, the rsd virus produced less tissue damage at the inoculation sites and induced fever and vesicles a day later than in the animals inoculated with rdd-containing viruses, which indicated a different degree of disease severity. the different virulence of these viruses was also supported by the maintenance of original receptor recognition sequence in vesicle samples obtained from infected animals. the reason for low virulence of rsd virus, which may bear on receptor usage, needs to be further explored. these observations are particularly interesting, since the presence of an rgd motif is believed to be the main determinant to direct fmdv to integrin-containing target tissues during infection in the natural host. in addition, information currently available indicates that fmdv utilizes integrins for entry in the natural host, and there is no evidence of the use of alternative receptors in vivo. therefore, our results further support the possibility that a non-rgd-integrin interaction could be responsible for the generation of fmd in the natural host. our study was the first to demonstrate the ability of an rdd containing natural isolate to cause disease in naturally susceptible animals, and will provide knowledge about the in vivo pathogenesis of non-rgd viruses.  fmdv quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition sites. thus, the early interaction between the viruses and the host cells may exert major selective pressure on fmdv populations that contributes to the evolution and functional flexibility of fmdv to enter cells. our studies using two non-rgd fmdvs not only show that there was an increase in the number of viable mutants with substitutions in the receptor-binding region, but also provide useful tools for studies of cell recognition by fmdv. based on an rdd-containing full-length infectious cdna clone, the rsd- and rgd-containing recombinant viruses were rescued, and single amino acid substitutions in the receptor-binding site did not affect virus viability. the viruses expressing non-rgd receptor binding sites can replicate stably in vitro and induce the disease in susceptible animals. 
amino acid sequence analysis of the 1d-coding region of different derivatives derived from the asia1/js/cha/ field isolate revealed that the rdd mutants became dominant or achieved population equilibrium with coexistence of the rgd and rsd subpopulations at an early phase of type asia fmdv quasispecies evolution. following transfection of bsr cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals. foot-and-mouth disease virus uses a highly conserved arg-gly-asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. to examine the influence of single amino acid substitutions in the receptor binding site of the rdd-containing fmd viral genome on virus viability and the ability of non-rgd fmdvs to cause disease in susceptible animals, we constructed an rdd-containing fmdv full-length cdna clone and derived mutant molecules with rgd or rsd receptor recognition motifs. using reverse genetics, the rdd-, rsd-, and rgd-containing fmd viruses were rescued from full-length cdna clones, and single amino acid substitution in rdd-containing fmd viral genome did not affect virus viability. the genetically engineered viruses replicated stably in bhk- cells and had similar growth properties to the parental virus. the viruses expressing non-rgd receptor binding sites can replicate stably in vitro and produce typical fmd clinical disease in susceptible animals. previous sequence analyses of the 1d-encoding region of an fmdv field isolate and its two derivatives indicated that two viruses, which contained an arg-asp-asp or an arg-ser-asp triplet instead of the rgd integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. the rdd-containing fmd viral genome can accommodate substitutions in the receptor binding site without additional changes in the capsid. the rdd parental virus and two non-rgd recombinant viruses were virulent to pigs and bovines that developed typical clinical disease and viremia. fmdv quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition site.
 foot-and-mouth disease virus uses a highly conserved arg-gly-asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. previous sequence analyses of the 1d-encoding region of an fmdv field isolate and its two derivatives indicated that two viruses, which contained an arg-asp-asp or an arg-ser-asp triplet instead of the rgd integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. to examine the influence of single amino acid substitutions in the receptor binding site of the rdd-containing fmd viral genome on virus viability and the ability of non-rgd fmdvs to cause disease in susceptible animals, we constructed an rdd-containing fmdv full-length cdna clone and derived mutant molecules with rgd or rsd receptor recognition motifs. following transfection of bsr cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals.  amino acid sequence analysis of the 1d-coding region of different derivatives derived from the asia1/js/cha/ field isolate revealed that the rdd mutants became dominant or achieved population equilibrium with coexistence of the rgd and rsd subpopulations at an early phase of type asia fmdv quasispecies evolution. furthermore, the rdd and rsd sequences remained genetically stable for at least passages. using reverse genetics, the rdd-, rsd-, and rgd-containing fmd viruses were rescued from full-length cdna clones, and single amino acid substitution in rdd-containing fmd viral genome did not affect virus viability. the genetically engineered viruses replicated stably in bhk- cells and had similar growth properties to the parental virus. the rdd parental virus and two non-rgd recombinant viruses were virulent to pigs and bovines that developed typical clinical disease and viremia.  fmdv quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition site. the rdd-containing fmd viral genome can accommodate substitutions in the receptor binding site without additional changes in the capsid. the viruses expressing non-rgd receptor binding sites can replicate stably in vitro and produce typical fmd clinical disease in susceptible animals. 