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Foot-and-mouth disease virus
Comparative Immunology, Microbiology & Infectious Diseases 25 (2002) 297–308 www.elsevier.com/locate/cimid
Foot-and-mouth disease virus Esteban Domingoa,*, Eric Baranowskib, Cristina Escarmı´sa, Francisco Sobrinoa,b a
Centro de Biologı´a Molecular “Severo Ochoa”, Universidad Auto´noma de Madrid, Cantoblanco, Madrid, Spain b Centro de Investigacio´n en Sanidad Animal (INIA), 28130 Valdeolmos, Madrid, Spain
Abstract Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Quasispecies; Picornavirus; Vaccine
Re´sume´ Le virus de la fie`vre aphteuse est un aphtovirus de la famille des Picornaviridae et l’agent de la maladie animale la plus importante sur le plan e´conomique. En tant que picornavirus typique, le virus de la fie`vre aphteuse est nu, sous forme d’icosae`dre et son ge´nome comprend un acide ribonucle´ique monobrin avec environ 8500 nucle´otides et une polarite´ positive. L’acide ribonucle´ique de ce virus est infectieux et il se re´plique par l’interme´diaire d’un brin d’ARN moins, comple´mentaire. La re´plication de l’acide nucle´ique de ce virus conduit a` des erreurs, de telle sorte que les populations virales comprennent un ensemble de mutants (quasi espe`ce) plutoˆt qu’une se´quence ge´nomique bien de´finie. Par suite, le virus de la fie`vre aphteuse est ge´ne´tiquement et antige´niquement varie´. Ceci entraıˆne des difficulte´s importantes pour le diagnostic, la pre´vention et la maıˆtrise de la fie`vre aphteuse. Une connaissance plus approfondie de la complexite´ et de l’e´volution de la population de ce virus a conduit a` des besoins pour une nouvelle ge´ne´ration de
* Corresponding author. Tel.: þ 34-91-3978485; fax: þ 34-91-3974799. E-mail address: [email protected] (E. Domingo). 0147-9571/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 7 - 9 5 7 1 ( 0 2 ) 0 0 0 2 7 - 9
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vaccines aphteux. Ceci est lie´ au de´bat actuel sur le choix d’une politique de vaccination ou de nonvaccination dans la lutte contre la fie`vre aphteuse. q 2002 Elsevier Science Ltd. All rights reserved. Mots-cle´: Fie`vre aphteuse; Virus; Picornavirus; E´volution virale; Quasi espe`ce; Vaccin; Maıˆtrise de maladie
1. Introduction Foot-and-mouth disease virus (FMDV) is the causative agent of the economically most important animal viral disease world-wide. Although mortality associated with FMD is usually low, the disease decreases livestock productivity, and affected countries cannot participate in international trade of animals and animal products. Unfortunately, in many underdeveloped regions of Asia, Africa and South America FMD is enzootic, preventing a possible source of economic development. For reviews of FMD and its world-wide impact, see Refs. [1 –3]. The 2001/2002 European outbreak of FMD which has affected mainly the UK will have an estimated cost of 6000 million Euros [4,5]. Because of the difficulties for its effective control and its economic impact, FMD ranks first in the A list of infectious diseases of animals, published by the Office International des Epizooties. A number of review articles [3 – 5] and web sites (http://www.oie.int;http://iah.bbsrc.ac.uk/ virus/Picornaviridae/Aphthovirus/fmd.htm) provide updated information on FMD. This article reviews the structure and genome organization of FMDV and the application of new technologies to the diagnosis and prevention of FMD.
2. FMDV particles FMDV is a non-enveloped virus with icosahedral symmetry that belongs to the aphthovirus genus of the Picornaviridae family [6]. Its genome is a single stranded RNA molecule of about 8500 nucleotides of positive polarity. Crystallographic studies have allowed the elucidation at atomic resolution of the three-dimensional structure of several FMDV isolates and antigenic variants [7 – 11]. Particles are about 30 nm in diameter and are composed of 60 copies of each of four capsid proteins termed VP1, VP2, VP3 and VP4. VP1, VP2, VP3 are external and VP4 is internal, in contact with the RNA and modified by a myristate group at its amino terminus. One copy of each capsid protein assembles to produce a protomer; in turn, five protomers form a pentamer, and 12 pentamers conform the complete capsid (Fig. 1). The individual surface capsid proteins share a structure consisting of an eight-stranded b-barrel. Surface loops connecting the b-strands include antigenic determinants (B-cell epitopes) involved in neutralization of infectivity by antibodies. Four major antigenic sites have been identified by combining immunologic, genetic and biochemical procedures [11]. Particularly interesting is a major, immunodominant site located within the G –H loop of VP1. This loop appears as highly disordered according to the X-ray diffraction patterns of crystal of native virions. A structure for the loop was obtained by crystallographic analysis of chemically modified FMDV [12], and it consists of a short b-strand which precedes an Arg-Gly-Asp (RGD) which adopts an openturn conformation, followed by a short helical region at the carboxy side of the RGD. This
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Fig. 1. The FMDV capsid and location of antigenic sites. In the upper part the relative position of the three surface proteins VP1, VP2 and VP3 in the particle of icosahedral symmetry is indicated [based on Ref. [7]]. In the lower part the location of the major antigenic sites in FMDV of serotypes O, A and C is given. The three boxes (not drawn to scale) correspond to capsid proteins VP2, VP3 and VP1. The letters on top of each box indicate the antigenic loops (Ct is the carboxy-terminal region of VP1). Antigenic sites are indicated in gray boxes. The letters and numbers inside these boxes refer to site designation for FMDV serotypes O, A and C; ( þ ) indicates sites not specifically named in the original references. The numbers at the bottom indicate the amino acid positions (counted individually for each protein) at which the different loops and domains are located (based on Ref. [11]).
loop structure is very similar to that found in crystals of complexes between the Fab fragment of MAbs which recognize this antigenic site and synthetic peptides representing several variant versions of the loop residues [11–15]. The loop appears to display a hinge movement about the capsid surface and it occupies different positions when bound to different antibodies [16–18]; its being exposed and mobile may contribute to its immunodominance. A remarkable feature of the G–H loop of VP1 is that the RGD has a dual function in recognition of integrins that serve as cellular receptors for FMDV and in antibody binding [11, 13,19,20]. The RGD is a critical part of several epitopes involved in FMDV neutralization that have been mapped within the loop [11,14]. An overlap between antigenic sites and receptor recognition sites is not unique to FMDV since it was first observed with human influenza virus and more recently with a variety of animal viruses [19]. Such an overlap permits coevolution of antigenicity and host cell tropism, and this is particularly relevant for highly variable RNA viruses since it favors virus adaptability and complicates viral disease prevention and control.
3. The FMDV genome FMDV RNA is polyadenylated at its 30 -end, and has a small protein, VPg, covalently
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Fig. 2. Scheme of the FMDV genome. Regulatory regions (50 UTR or 50 non-coding region, and 30 UTR or 30 noncoding region) are indicated by horizontal lines. Different subregions within the 50 UTR have been expanded. The coding region (single open-reading frame, with two functional AUG initiation codons) is depicted as a horizontal box with indication of the encoded proteins: L to 3D. VPg is the protein covalently linked to the 50 -end of the RNA and (A)n is the 30 terminal polyadenylate tract (based on Ref. [3] and references therein).
linked to its 50 -end. The FMDV genome can be divided into three main functional regions: (i) the 50 non-coding, regulatory region; (ii) the protein-coding region (subdivided in L/P1, P2 and P3); and (iii) the 30 non-coding, regulatory region (Fig. 2). For reviews of the picornavirus and aphthovirus genome organization and expression see Refs. [3,21,22]. The 50 non-coding region includes from the 50 -end a highly structured S fragment of about 370 residues followed by an internal polyribocytidylate (polyC) tract of variable length (usually 100– 400 residues). Downstream of the polyC tract there is a pseudoknot region which precedes the internal ribosome entry site (IRES), a stretch of about 440 residues which serves for the internal initiation of protein synthesis in a CAP-independent fashion [23]. The roles of the RNA domains preceding the IRES are poorly understood. Protein synthesis starts at two functional, in-frame AUG codons separated by about 80 nucleotides that delimit a long open-reading frame to encode a polyprotein of about 2330 amino acids. Differences in length of coding and non-coding regions are observed among natural isolates of FMDV and some times among viruses with a different passage history in cell culture. Because of the double initiation of protein synthesis, two forms of protease L are made. They both catalyze their own cleavage from the rest of the polyprotein, and also the cleavage of eIF-4G of the CAP complex contributing to the shut-off of host cell protein synthesis. P1 encodes the four capsid proteins, and P2 – P3 encode non-structural proteins involved in RNA genome replication and viral maturation. The function of several nonstructural proteins is still poorly understood although some of their homologous proteins in poliovirus have been studied in some detail. Protein 3C is a serin protease that catalyzes most of the cleavages necessary for polyprotein processing; 2C is involved in RNA synthesis and is the site of mutations that confer FMDV resistance to guanidine hydrochloride; 3B encodes three copies of VPg, the protein covalently linked to the 50 -end of the RNA (Fig. 2). Protein 3D is the viral RNA-dependent RNA polymerase (or viral replicase), which shows 34% amino acid sequence identity with poliovirus 3D, an enzyme whose three-dimensional structure has been resolved by X-ray crystallography [24]. Finally, the 30 non-coding region of about 90 residues is likely to be a site of interaction with viral and host proteins for RNA replication; the 30 -end contains a polyA tract which is genetically coded and heterogeneous in length [3,21,22]. Despite the classically established distinction between regulatory and coding regions, there is increasing evidence that coding regions of the picornaviral genomes may also be involved in
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regulatory functions [for example, Ref. [25]]. This dual role may be of relevance for the interpretation of evolutionary parameters of FMDV. Purified genomic FMDV RNA can act as messenger RNA in vitro and in vivo [5,21, 22]. The RNA extracted from virions or transcribed from full-length cDNA copies of the viral genome is infectious. This permits manipulation of DNA copies of specific FMDV genomic segments to study the phenotypic effects of mutations and other genomic alterations. The application of reverse genetics has allowed the constructions of chimeric FMDVs to define determinants of viral replication, cell recognition and virulence [5,21].
4. FMDV genome replication and generation of heterogeneity and diversity Replication of FMDV RNA follows the same general pattern as replication of other picornaviruses and although some features have been established with FMDV, others are assumed to be those established for poliovirus and other members of the same family [5, 21,22,26]. The site of RNA genome replication is a membrane-bound replication complex at the cell cytoplasm. Genome copying occurs via a complementary negative (or minus) strand RNA and the formation of double stranded replicative form, and perhaps partially double-stranded replicative intermediates. Minus strands are found in hundredfold lower concentrations than plus strands in infected cells, suggesting that each minus strand may serve as template for the synthesis of many plus strands [26]. The kinetics of RNA synthesis are not well understood and the number of rounds of template copying in infected cells is unknown. This is true of most RNA viruses and it is of relevance to interpret viral evolution [27]. In particular it is difficult to relate a rate of occurrence of mutations (either advantageous or detrimental) and their frequency in viral populations. Many such calculations require a number of assumptions and are based on inferences that are distant from the actual experimental results; they are of limited value. As for other RNA viruses (or viruses which include in their replication cycle an RNA intermediate), FMDV RNA genome replication is error-prone. This has been documented experimentally by determining nucleotide sequences of progeny from a single genome, and also from the frequencies of MAb-resistant mutants in clonal preparations and natural isolates of FMDV, among other lines of evidence (Refs. [27 – 29] and references therein). Low fidelity copying is expected from the absence of proofreading – repair activity in the viral replicase [24,27,30]. Because of high mutation rates (between 0.2 and 1 mutations are introduced every time a plus or minus strand is copied into an RNA product) FMDV populations consist of distributions of related but non-identical genomes. The consensus nucleotide sequence (the one determined by current sequencing methodology) is but an average of many different sequences, and a genome with a nucleotide sequence identical to the consensus may not exist in the population. Thus, the FMDV genome is statistically defined but individually undetermined. This population structure is shared with other RNA viruses and it is termed the quasispecies nature of RNA viruses, in recognition of the first theory that regarded ensembles of replicons as the target of selection and considered the wild type as an average of many different sequences [31]. Quasispecies can be applied to RNA virus populations [32 – 34] and it has provided an adequate theoretical framework to understand RNA viruses at the population level, including FMDV [3,22,27,30 –34]. Since
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a viral population consists of a swarm of genetic and phenotypic variants in perpetual renewal as genome replication proceeds in infected hosts, the quasispecies structure and dynamics of FMDV has implications not only for its adaptability and evolution but also for viral pathogenesis [5,19,26 – 30]. Current concepts on RNA virus evolution provide the following picture for generation of FMDV diversity: (i) Replication unavoidably results in the generation of FMDV quasispecies. Therefore infection of an animal, even in those cases in which infection is initiated by a single FMDV genome, will result in mutant swarms which will be subjected to positive selection, negative selection (ranking of variant frequencies according to relative fitness) and random drift within the animal (for example in the invasion of a new tissue or organ by one infectious mutant taken by chance from a pool of mutants). (ii) Transmission of FMDV from an infected animal to a susceptible host will initiate a new round of evolutionary events summarized in (i). This short-term (intra-host) FMDV evolution is essentially the result of differential growth of subpopulations of mutant FMDVs [3,28]. (iii) Successive rounds of intra-host evolution and transmission events lead to the progressive divergence of FMDV in nature. Because of the complexity of selective constraints encountered by the virus in different host species, the mechanisms of FMDV diversification in nature are largely unknown. (iv) Functional and structural constraints at the RNA and protein levels modulate variation despite high mutation rates [27,35]. Rates of evolution of FMDV in nature are not constant with time and they range between 1021 and 1024 substitutions per nucleotide per year [[5,27] and references therein]. (v) In FMDV there is an overlap between some antigenic sites and some cell receptor recognition sites, which may result in coevolution of antigenicity and host range [19].
5. FMDV types, subtypes and isolates The FMDVs isolated over the 20th century were grouped in seven serological types, termed A, O, C, Asia 1, SAT1, SAT2 and SAT3 [1 – 3]. FMDVs were assigned to a different serotype on the basis of lack of cross-protection following infection (convalescent animals) or vaccination. Viruses showing partial cross-protection were assigned to the same serotype but to a different subtype. About 65 subtypes of FMDV were defined, but about two decades ago it was realized that with the use of increasing numbers of MAbs virtually each isolate could be regarded as an antigenic variant [3,36,37]. Antigenic diversity is a direct consequence of genetic variation. A significant observation was the generation of an epitope previously assigned to a FMDV subtype as a consequence of an amino acid replacement in a FMDV of another subtype [38]. Classifications of FMDV isolates based on phylogenetic methods (Fig. 3) are gradually replacing the traditional groupings based on serological criteria. Phylogenies are based on RT-PCR amplification of genomic FMDV RNA isolates and nucleotide sequencing of specific genomic regions, usually those encoding capsid proteins, particularly VP1 [3,36]. Such procedures have established, for example, the relatedness among the O type FMDV isolates that originate from some initial strain in the center of Asia, expanded westward and eastward, causing the European outbreak of 2001/2002. This outbreak has represented
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Fig. 3. Phylogenetic relationships among FMDV isolates of seven serotypes, based on amino acid sequences of capsid protein VP1. The segment indicates a 10 amino acid distance. The spheres at the tip of each branch emphasize that each virus isolate is in reality a cloud of mutants, as discussed in the text (adapted from Ref. [3] and references therein).
the unprecedented penetration of FMDV of Asiatic origin into Europe, contributing to concerns on further FMDV spread in the face of an increasingly global economy [3 –5]. The potential for rapid evolution and the antigenic diversity of FMDV are complicating factors for the diagnosis, prevention and control of FMD.
6. Diagnosis and control of FMD: a growing challenge Intensive farm animal breeding and livestock production in the context of global trade connections necessitate rapid and reliable diagnosis of FMD to distinguish it from other vesicular diseases, notably swine vesicular disease or vesicular stomatitis [1 – 3]. The classical complement fixation and serum neutralization tests have been largely replaced by ELISA and genetic typing techniques [3]. ELISA employs serotype-specific sera or MAbs
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[39]. However, use of MAbs meets with the epitopic diversity among FMDV isolates [3, 36 –38]. Perhaps multinational collaborations to investigate cocktails of neutralizing and non-neutralizing MAbs [which often define conserved epitopes [37]] could produce reliable reagents for ELISA typing. For trade purposes, diagnostic procedures should distinguish animals that have been vaccinated from those that have been infected with FMDV. This distinction is now feasible by detecting antibodies against some of the nonstructural proteins by ELISA, in particular antibodies against 3AB and 3ABC. Such antibodies are found in animals which at some time have been infected with FMDV but not in vaccinated animals (Ref. [3] and references therein). RT-PCR amplification techniques are increasingly providing a versatile tool for an efficient and rapid diagnosis of FMD [3,40,41]. Coupled to automated nucleotide sequencing and phylogenetic analysis, these techniques can achieve a detailed virus identification to trace the origin of FMDVs associated with new outbreaks [3,4]. Reliability and rapidity in FMD diagnosis are a priority since a delay in the implementation of control or preventive measures may result in the uncontrollable spread of disease with devastating economic consequences [1 – 4]. Control of FMD is based on two major strategies: the slaughtering of affected and contact animals (the so called ‘stamping out’ procedure) or the regular vaccination of the major host species for FMDV (always cattle and when indicated also swine). Both strategies can be combined so that a ring vaccination can be established around a focus area where affected and contact animals have been slaughtered [1 – 5]. As a consequence of the massive killing of animals during the 2001/2002 European outbreak, and the increasing awareness of the community on animal welfare, there is presently a vivid debate towards whether a vaccination strategy may not be preferable to the stamping-out and non-vaccination procedure in operation in the EU since 1991. Such regulation was adopted for economic considerations, but left susceptible animals in a vast territory defenseless in the face of an accidental introduction of the virus in the region, a situation that historically has been known to favor the occurrence of large epizootics [1 –5]. In the event of a return to a vaccination policy, basic investigations over the last decade should provide the basis for manufacturing more effective and safer vaccines than those presently available. Such new vaccines could replace current vaccines in those territories where a vaccination policy is presently in operation. The basic rules that should be considered for the development of new anti-FMD vaccines are [3,42]: (i) Vaccines should include multiple B-cell and T-cell epitopes to produce an ample humoral (antibody) and cellular immune response, in particular, T helper responses for antibody production [3,43]. Such an ample response is required to delay, and ideally abolish, a possible selection in the field of FMDV variants showing partial resistance to the immune response evoked by the vaccine [3,5,19,42]. The B cell epitope map has been investigated with considerable detail [11], and a number of T-cell epitopes have been identified in structural and non-structural proteins of FMDV [[3,43 – 45] and references therein]. Therefore, it should be possible to present an ample epitopic repertoire either with synthetic constructs or with complete, non-infectious particles. Research along these lines is currently in progress. (ii) Although FMDV infection is usually acute, and viremia and lesions occur within days after infection, ruminants can establish an asymptomatic carrier state in which the virus replicates in the oesopharyngeal region of the animals [46,47]. The presence of carriers
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jeopardizes animal trade since it cannot be excluded that carrier animals could on occasions originate acute disease when in contact with susceptible animals. Classical vaccines cannot prevent the establishment of persistent FMDV infection in cattle, and a new vaccine should target systemic and mucosal immunity, hoping that the latter may minimize chances of establishing the carrier state. (iii) Vaccine manufacturing should not require the handling of virus because of the danger of virus escaping from vaccine factories. Also, classical, inactivated whole-virus vaccines may be at the origin of outbreaks if inactivation prior to vaccine formulation was not complete. There is good evidence that some FMD outbreaks probably had a vaccine origin [3]. These constitute powerful arguments to design vaccines that do not require infectious virus at any stage of their preparation [48,49]. (iv) Finally, vaccines must be marked to distinguish vaccination from infection, as discussed above for diagnostic purposes. A vaccine could be marked positively by the presence of some genetic tag or negatively by the absence of a gene product systematically found during a natural infection [3,40]. The recent European epizootic of FMD at the onset of the 21 century has made us aware of the great economic losses to be endured because no effective preventive and control measures are available for FMD. We should not forget, however, that underdeveloped countries in need for active livestock trade have suffered FMD-related losses for decades. Interest on new vaccines and basic epidemiology to define the strategies for a more effective control of FMD has been boosted as a consequence of the recent European epizootic. Yet, active research on FMD and FMDV should have never been interrupted [5]. It is hoped that application of new molecular techniques and increasing computation capacities for epidemiological modeling, will contribute to a more effective control of FMD in Europe and also in underdeveloped countries. Experience tells us that what we learn with FMDV is likely to be of value also to understand and control some other viruses.
Acknowledgments We thank many colleagues for important contributions to FMDV research. Unavoidably, space limitations did not allow quoting all relevant work. Research supported by grants DGES PM97-0060-C02-01, BIO 99-0833-01, BMC 2001-1823-C0201, and Fundacio´n Ramo´n Areces from Spain, and FAIR 5 PL97-3665, CT97-3665 and 341 from the EU.
References [1] Bachrach HL. Foot-and-mouth disease virus. Annu Rev Microbiol 1968;22:201– 44. [2] Pereira HG. Foot-and-mouth disease virus. In: Gibbs RPG, editor. Virus diseases of food animals, vol. 2. New York: Academic Press; 1981. p. 333– 63. [3] Sobrino F, Sa´iz M, Jime´nez-Clavero MA, Nu´n˜ez JI, Rosas MF, Baranowski E, Ley V. Footand-mouth disease virus: a long known virus, but a current threat. Vet Res 2001;32:1– 30. [4] Samuel AR, Knowles NJ. Foot-and-mouth disease virus: cause of the recent crisis for the UK livestock industry. Trends Genet 2001;17:421– 4.
306
E. Domingo et al. / Comp. Immun. Microbiol. Infect. Dis. 25 (2002) 297–308
[5] Sobrino F, Domingo E. Foot-and-mouth disease in Europe. EMBO Rep 2001;2:459– 61. [6] van Regenmortel MHV, Fauquet CM, Bishop DH, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, Mc Geoch DJ, Pringle CR, Wickner RB. Virus taxonomy. San Diego: Academic Press; 2000. [7] Acharya R, Fry E, Stuart D, Fox G, Rowlands D, Brown F. The three-dimensional structure of ˚ resolution. Nature 1989;337:709 –16. foot-and-mouth disease virus at 2.9 A ´ [8] Lea S, Hernandez J, Blakemore W, Brocchi E, Curry S, Domingo E, Fry E, Abu-Ghazaleh R, King A, Newman J, Stuart D, Mateu MG. The structure and antigenicity of a type C foot-andmouth disease virus. Structure 1994;2:123– 39. [9] Lea S, Abu-Ghazaleh R, Blakemore W, Curry S, Fry E, Jackson T, King A, Logan D, Newman J, Stuart D. Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant, G67. Structure 1995;3:571– 80. [10] Curry S, Fry E, Blakemore W, Abu-Ghazaleh R, Jackson T, King A, Lea S, Newman J, Rowlands D, Stuart D. Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity. Structure 1996;4:135– 45. [11] Mateu MG. Antibody recognition of picornaviruses and escape from neutralization: a structural view. Virus Res 1995;38:1– 24. [12] Logan D, Abu-Ghazaleh R, Blakemore W, Curry S, Jackson T, King A, Lea S, Lewis R, Newman J, Parry N, Rowlands D, Stuart D, Fry E. Structure of a major immunogenic site on foot-and-mouth disease virus. Nature 1993;362:566– 8. [13] Verdaguer N, Mateu MG, Andreu D, Giralt E, Domingo E, Fita I. Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction. EMBO J 1995;14:1690– 6. [14] Verdaguer N, Sevilla N, Valero ML, Stuart D, Brocchi E, Andreu D, Giralt E, Domingo E, Mateu MG, Fita I. A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation. J Virol 1998;72:739– 48. [15] Ochoa WF, Kalko SG, Mateu MG, Gomes P, Andreu D, Domingo E, Fita I, Verdaguer N. A multiply substituted G– H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules. J Gen Virol 2000;81:1495 – 505. [16] Parry N, Fox G, Rowlands D, Brown F, Fry E, Acharya R, Logan D, Stuart D. Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth disease virus. Nature 1990;347:569– 72. [17] Hewat EA, Verdaguer N, Fita I, Blakemore W, Brookes S, King A, Newman J, Domingo E, Mateu MG, Stuart DI. Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop. EMBO J 1997;16:1492– 500. [18] Verdaguer N, Schoehn G, Ochoa WF, Fita I, Brookes S, King A, Domingo E, Mateu MG, Stuart D, Hewat EA. Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation. Virology 1999; 255:260– 8. [19] Baranowski E, Ruiz-Jarabo CM, Domingo E. Evolution of cell recognition by viruses. Science 2001;292:1102– 5. [20] Nemerow GR, Stewart PL. Antibody neutralization epitopes and integrin binding sites on nonenveloped viruses. Virology 2001;288:189– 91. [21] Belsham GJ. Distinctive features of foot-and-mouth disease virus, a member of the picornavirus family; aspects of virus protein synthesis, protein processing and structure. Prog Biophys Mol Biol 1993;60:241– 60.
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[22] Flint SJ, Enquist LW, Krug RM, Racaniello VR, Skalka AM. Molecular biology, pathogenesis and control. Virology, Washington, DC: ASM Press; 2000. [23] Martı´nez-Salas E. Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotechnol 1999;10:458– 64. [24] Hansen J, Long AM, Schultz S. Structure of the RNA-dependent RNA polymerase of poliovirus. Structure 1997;15:1109– 22. [25] McKnight KL, Lemon SM. Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA. J Virol 1996;70:1941 –52. [26] Gromeier M, Wimmer E, Gorbalenya AE. In: Domingo E, Webster RG, Holland JJS, editors. Origin and evolution of viruses. San Diego: Academic Press; 1999. p. 287– 343. [27] Domingo E, Webster RG, Holland JJ, editors. Origin and evolution of viruses. San Diego: Academic Press; 1999. [28] Nu´n˜ez JI, Baranowski E, Molina N, Ruiz-Jarabo CM, Sa´nchez C, Domingo E, Sobrino F. A single amino acid substitution in nonstructural protein 3A can mediate adaptation of foot-andmouth disease virus to the guinea pig. J Virol 2001;75:3977– 83. [29] Escarmı´s C, Go´mez-Mariano G, Da´vila M, La´zaro E, Domingo E. Resistance to extinction of low fitness virus subjected to plaque-to-plaque transfers: diversification by mutation clustering. J Mol Biol 2002;315:647– 61. [30] Drake JW, Holland JJ. Mutation rates among RNA viruses. Proc Natl Acad Sci USA 1999;96: 13910– 3. [31] Eigen E, Schuster P. The hypercycle. A principle of natural self-organization. Berlin: Springer; 1979. [32] Eigen M, Biebricher CK. Sequence space and quasispecies distribution. In: Domingo E, Ahlquist P, Holland JJS, editors. RNA genetics, vol. 3. Boca Raton, FL: CRC Press; 1988. p. 211– 45. [33] Eigen M. Viral quasispecies. Sci Am 1993;269:42 –9. [34] Eigen M. Natural selection: a phase transition? Biophys Chem 2000;85:101– 23. [35] Martı´nez MA, Dopazo J, Herna´ndez J, Mateu MG, Sobrino F, Domingo E, Knowles NJ. Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades. J Virol 1992;66:3557 – 65. [36] Martı´nez MA, Carrillo C, Plana J, Mascarella R, Bergada J, Palma EL, Domingo E, Sobrino F. Genetic and immunogenic variations among closely related isolates of foot-and-mouth disease virus. Gene 1988;62:75– 84. [37] Mateu MG, Da Silva JL, Rocha E, De Brum DL, Alonso A, Enjuanes L, Domingo E, Barahona H. Extensive antigenic heterogeneity of foot-and-mouth disease virus of serotype C. Virology 1988;167:113– 24. [38] Herna´ndez J, Martı´nez MA, Rocha E, Domingo E, Mateu MG. Generation of a subtype-specific neutralization epitope in foot-and-mouth disease virus of a different subtype. J Gen Virol 1992; 73:213– 6. [39] Roeder PL, Le Blanc Smith PM. Detection and typing of foot-and-mouth disease virus by enzyme linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis. Res Vet Sci 1987;45:225– 30. [40] Office international des epizooties WOfAHF-a-mdOSC, Manual of standards for diagnostic tests and vaccines. Paris, France: OIE; 1996. [41] Reid SM, Hutchings GH, Ferris NP, De Clercq K. Diagnosis of foot-and-mouth disease by RTPCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples. J Virol Meth 1999;83:113– 23. [42] Domingo E, Holland JJ. Complications of RNA heterogeneity for the engineering of virus vaccines and antiviral agents. Genet Engng (NY) 1992;14:13– 31.
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[43] Collen T. In: Goddeev BML, Morrison I, editors. Foot-and-mouth disease virus (aphthovirus): viral T cell epitopes. Cell mediated immunity in ruminants, Boca Raton: CRC Press; 1994. p. 173– 97. [44] Blanco E, McCullough K, Summerfield A, Fiorini J, Andreu D, Chiva C, Borras E, Barnett P, Sobrino F. Interspecies major histocompatibility complex-restricted Th cell epitope on footand-mouth disease virus capsid protein VP4. J Virol 2000;74:4902– 7. [45] Blanco E, Garcia-Briones M, Sanz-Parra A, Gomes P, De Oliveira E, Valero ML, Andreu D, Ley V, Sobrino F. Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus. J Virol 2001;75:3164 – 74. [46] van Bekkum JG, Frenke HS, Frederiks HHJ, Frenkel S. Observations on the carrier state of cattle exposed to foot-and-mouth disease virus. Tijdschr Diergeneeskd 1959;84:1159 –64. [47] Salt JS. The carrier state in foot and mouth disease—an immunological review. Br Vet J 1993; 149:207– 23. [48] Brown F. New approaches to vaccination against foot-and-mouth disease. Vaccine 1992;10: 1022– 6. [49] Brown F. Foot-and-mouth disease. In: Nicholson BH, editor. Synthetic vaccines. Oxford: Blackwell; 1994. p. 416– 32.
Foot-and-mouth disease virus Esteban Domingoa,*, Eric Baranowskib, Cristina Escarmı´sa, Francisco Sobrinoa,b a
Centro de Biologı´a Molecular “Severo Ochoa”, Universidad Auto´noma de Madrid, Cantoblanco, Madrid, Spain b Centro de Investigacio´n en Sanidad Animal (INIA), 28130 Valdeolmos, Madrid, Spain
Abstract Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Quasispecies; Picornavirus; Vaccine
Re´sume´ Le virus de la fie`vre aphteuse est un aphtovirus de la famille des Picornaviridae et l’agent de la maladie animale la plus importante sur le plan e´conomique. En tant que picornavirus typique, le virus de la fie`vre aphteuse est nu, sous forme d’icosae`dre et son ge´nome comprend un acide ribonucle´ique monobrin avec environ 8500 nucle´otides et une polarite´ positive. L’acide ribonucle´ique de ce virus est infectieux et il se re´plique par l’interme´diaire d’un brin d’ARN moins, comple´mentaire. La re´plication de l’acide nucle´ique de ce virus conduit a` des erreurs, de telle sorte que les populations virales comprennent un ensemble de mutants (quasi espe`ce) plutoˆt qu’une se´quence ge´nomique bien de´finie. Par suite, le virus de la fie`vre aphteuse est ge´ne´tiquement et antige´niquement varie´. Ceci entraıˆne des difficulte´s importantes pour le diagnostic, la pre´vention et la maıˆtrise de la fie`vre aphteuse. Une connaissance plus approfondie de la complexite´ et de l’e´volution de la population de ce virus a conduit a` des besoins pour une nouvelle ge´ne´ration de
* Corresponding author. Tel.: þ 34-91-3978485; fax: þ 34-91-3974799. E-mail address: [email protected] (E. Domingo). 0147-9571/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 7 - 9 5 7 1 ( 0 2 ) 0 0 0 2 7 - 9
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vaccines aphteux. Ceci est lie´ au de´bat actuel sur le choix d’une politique de vaccination ou de nonvaccination dans la lutte contre la fie`vre aphteuse. q 2002 Elsevier Science Ltd. All rights reserved. Mots-cle´: Fie`vre aphteuse; Virus; Picornavirus; E´volution virale; Quasi espe`ce; Vaccin; Maıˆtrise de maladie
1. Introduction Foot-and-mouth disease virus (FMDV) is the causative agent of the economically most important animal viral disease world-wide. Although mortality associated with FMD is usually low, the disease decreases livestock productivity, and affected countries cannot participate in international trade of animals and animal products. Unfortunately, in many underdeveloped regions of Asia, Africa and South America FMD is enzootic, preventing a possible source of economic development. For reviews of FMD and its world-wide impact, see Refs. [1 –3]. The 2001/2002 European outbreak of FMD which has affected mainly the UK will have an estimated cost of 6000 million Euros [4,5]. Because of the difficulties for its effective control and its economic impact, FMD ranks first in the A list of infectious diseases of animals, published by the Office International des Epizooties. A number of review articles [3 – 5] and web sites (http://www.oie.int;http://iah.bbsrc.ac.uk/ virus/Picornaviridae/Aphthovirus/fmd.htm) provide updated information on FMD. This article reviews the structure and genome organization of FMDV and the application of new technologies to the diagnosis and prevention of FMD.
2. FMDV particles FMDV is a non-enveloped virus with icosahedral symmetry that belongs to the aphthovirus genus of the Picornaviridae family [6]. Its genome is a single stranded RNA molecule of about 8500 nucleotides of positive polarity. Crystallographic studies have allowed the elucidation at atomic resolution of the three-dimensional structure of several FMDV isolates and antigenic variants [7 – 11]. Particles are about 30 nm in diameter and are composed of 60 copies of each of four capsid proteins termed VP1, VP2, VP3 and VP4. VP1, VP2, VP3 are external and VP4 is internal, in contact with the RNA and modified by a myristate group at its amino terminus. One copy of each capsid protein assembles to produce a protomer; in turn, five protomers form a pentamer, and 12 pentamers conform the complete capsid (Fig. 1). The individual surface capsid proteins share a structure consisting of an eight-stranded b-barrel. Surface loops connecting the b-strands include antigenic determinants (B-cell epitopes) involved in neutralization of infectivity by antibodies. Four major antigenic sites have been identified by combining immunologic, genetic and biochemical procedures [11]. Particularly interesting is a major, immunodominant site located within the G –H loop of VP1. This loop appears as highly disordered according to the X-ray diffraction patterns of crystal of native virions. A structure for the loop was obtained by crystallographic analysis of chemically modified FMDV [12], and it consists of a short b-strand which precedes an Arg-Gly-Asp (RGD) which adopts an openturn conformation, followed by a short helical region at the carboxy side of the RGD. This
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Fig. 1. The FMDV capsid and location of antigenic sites. In the upper part the relative position of the three surface proteins VP1, VP2 and VP3 in the particle of icosahedral symmetry is indicated [based on Ref. [7]]. In the lower part the location of the major antigenic sites in FMDV of serotypes O, A and C is given. The three boxes (not drawn to scale) correspond to capsid proteins VP2, VP3 and VP1. The letters on top of each box indicate the antigenic loops (Ct is the carboxy-terminal region of VP1). Antigenic sites are indicated in gray boxes. The letters and numbers inside these boxes refer to site designation for FMDV serotypes O, A and C; ( þ ) indicates sites not specifically named in the original references. The numbers at the bottom indicate the amino acid positions (counted individually for each protein) at which the different loops and domains are located (based on Ref. [11]).
loop structure is very similar to that found in crystals of complexes between the Fab fragment of MAbs which recognize this antigenic site and synthetic peptides representing several variant versions of the loop residues [11–15]. The loop appears to display a hinge movement about the capsid surface and it occupies different positions when bound to different antibodies [16–18]; its being exposed and mobile may contribute to its immunodominance. A remarkable feature of the G–H loop of VP1 is that the RGD has a dual function in recognition of integrins that serve as cellular receptors for FMDV and in antibody binding [11, 13,19,20]. The RGD is a critical part of several epitopes involved in FMDV neutralization that have been mapped within the loop [11,14]. An overlap between antigenic sites and receptor recognition sites is not unique to FMDV since it was first observed with human influenza virus and more recently with a variety of animal viruses [19]. Such an overlap permits coevolution of antigenicity and host cell tropism, and this is particularly relevant for highly variable RNA viruses since it favors virus adaptability and complicates viral disease prevention and control.
3. The FMDV genome FMDV RNA is polyadenylated at its 30 -end, and has a small protein, VPg, covalently
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Fig. 2. Scheme of the FMDV genome. Regulatory regions (50 UTR or 50 non-coding region, and 30 UTR or 30 noncoding region) are indicated by horizontal lines. Different subregions within the 50 UTR have been expanded. The coding region (single open-reading frame, with two functional AUG initiation codons) is depicted as a horizontal box with indication of the encoded proteins: L to 3D. VPg is the protein covalently linked to the 50 -end of the RNA and (A)n is the 30 terminal polyadenylate tract (based on Ref. [3] and references therein).
linked to its 50 -end. The FMDV genome can be divided into three main functional regions: (i) the 50 non-coding, regulatory region; (ii) the protein-coding region (subdivided in L/P1, P2 and P3); and (iii) the 30 non-coding, regulatory region (Fig. 2). For reviews of the picornavirus and aphthovirus genome organization and expression see Refs. [3,21,22]. The 50 non-coding region includes from the 50 -end a highly structured S fragment of about 370 residues followed by an internal polyribocytidylate (polyC) tract of variable length (usually 100– 400 residues). Downstream of the polyC tract there is a pseudoknot region which precedes the internal ribosome entry site (IRES), a stretch of about 440 residues which serves for the internal initiation of protein synthesis in a CAP-independent fashion [23]. The roles of the RNA domains preceding the IRES are poorly understood. Protein synthesis starts at two functional, in-frame AUG codons separated by about 80 nucleotides that delimit a long open-reading frame to encode a polyprotein of about 2330 amino acids. Differences in length of coding and non-coding regions are observed among natural isolates of FMDV and some times among viruses with a different passage history in cell culture. Because of the double initiation of protein synthesis, two forms of protease L are made. They both catalyze their own cleavage from the rest of the polyprotein, and also the cleavage of eIF-4G of the CAP complex contributing to the shut-off of host cell protein synthesis. P1 encodes the four capsid proteins, and P2 – P3 encode non-structural proteins involved in RNA genome replication and viral maturation. The function of several nonstructural proteins is still poorly understood although some of their homologous proteins in poliovirus have been studied in some detail. Protein 3C is a serin protease that catalyzes most of the cleavages necessary for polyprotein processing; 2C is involved in RNA synthesis and is the site of mutations that confer FMDV resistance to guanidine hydrochloride; 3B encodes three copies of VPg, the protein covalently linked to the 50 -end of the RNA (Fig. 2). Protein 3D is the viral RNA-dependent RNA polymerase (or viral replicase), which shows 34% amino acid sequence identity with poliovirus 3D, an enzyme whose three-dimensional structure has been resolved by X-ray crystallography [24]. Finally, the 30 non-coding region of about 90 residues is likely to be a site of interaction with viral and host proteins for RNA replication; the 30 -end contains a polyA tract which is genetically coded and heterogeneous in length [3,21,22]. Despite the classically established distinction between regulatory and coding regions, there is increasing evidence that coding regions of the picornaviral genomes may also be involved in
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regulatory functions [for example, Ref. [25]]. This dual role may be of relevance for the interpretation of evolutionary parameters of FMDV. Purified genomic FMDV RNA can act as messenger RNA in vitro and in vivo [5,21, 22]. The RNA extracted from virions or transcribed from full-length cDNA copies of the viral genome is infectious. This permits manipulation of DNA copies of specific FMDV genomic segments to study the phenotypic effects of mutations and other genomic alterations. The application of reverse genetics has allowed the constructions of chimeric FMDVs to define determinants of viral replication, cell recognition and virulence [5,21].
4. FMDV genome replication and generation of heterogeneity and diversity Replication of FMDV RNA follows the same general pattern as replication of other picornaviruses and although some features have been established with FMDV, others are assumed to be those established for poliovirus and other members of the same family [5, 21,22,26]. The site of RNA genome replication is a membrane-bound replication complex at the cell cytoplasm. Genome copying occurs via a complementary negative (or minus) strand RNA and the formation of double stranded replicative form, and perhaps partially double-stranded replicative intermediates. Minus strands are found in hundredfold lower concentrations than plus strands in infected cells, suggesting that each minus strand may serve as template for the synthesis of many plus strands [26]. The kinetics of RNA synthesis are not well understood and the number of rounds of template copying in infected cells is unknown. This is true of most RNA viruses and it is of relevance to interpret viral evolution [27]. In particular it is difficult to relate a rate of occurrence of mutations (either advantageous or detrimental) and their frequency in viral populations. Many such calculations require a number of assumptions and are based on inferences that are distant from the actual experimental results; they are of limited value. As for other RNA viruses (or viruses which include in their replication cycle an RNA intermediate), FMDV RNA genome replication is error-prone. This has been documented experimentally by determining nucleotide sequences of progeny from a single genome, and also from the frequencies of MAb-resistant mutants in clonal preparations and natural isolates of FMDV, among other lines of evidence (Refs. [27 – 29] and references therein). Low fidelity copying is expected from the absence of proofreading – repair activity in the viral replicase [24,27,30]. Because of high mutation rates (between 0.2 and 1 mutations are introduced every time a plus or minus strand is copied into an RNA product) FMDV populations consist of distributions of related but non-identical genomes. The consensus nucleotide sequence (the one determined by current sequencing methodology) is but an average of many different sequences, and a genome with a nucleotide sequence identical to the consensus may not exist in the population. Thus, the FMDV genome is statistically defined but individually undetermined. This population structure is shared with other RNA viruses and it is termed the quasispecies nature of RNA viruses, in recognition of the first theory that regarded ensembles of replicons as the target of selection and considered the wild type as an average of many different sequences [31]. Quasispecies can be applied to RNA virus populations [32 – 34] and it has provided an adequate theoretical framework to understand RNA viruses at the population level, including FMDV [3,22,27,30 –34]. Since
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a viral population consists of a swarm of genetic and phenotypic variants in perpetual renewal as genome replication proceeds in infected hosts, the quasispecies structure and dynamics of FMDV has implications not only for its adaptability and evolution but also for viral pathogenesis [5,19,26 – 30]. Current concepts on RNA virus evolution provide the following picture for generation of FMDV diversity: (i) Replication unavoidably results in the generation of FMDV quasispecies. Therefore infection of an animal, even in those cases in which infection is initiated by a single FMDV genome, will result in mutant swarms which will be subjected to positive selection, negative selection (ranking of variant frequencies according to relative fitness) and random drift within the animal (for example in the invasion of a new tissue or organ by one infectious mutant taken by chance from a pool of mutants). (ii) Transmission of FMDV from an infected animal to a susceptible host will initiate a new round of evolutionary events summarized in (i). This short-term (intra-host) FMDV evolution is essentially the result of differential growth of subpopulations of mutant FMDVs [3,28]. (iii) Successive rounds of intra-host evolution and transmission events lead to the progressive divergence of FMDV in nature. Because of the complexity of selective constraints encountered by the virus in different host species, the mechanisms of FMDV diversification in nature are largely unknown. (iv) Functional and structural constraints at the RNA and protein levels modulate variation despite high mutation rates [27,35]. Rates of evolution of FMDV in nature are not constant with time and they range between 1021 and 1024 substitutions per nucleotide per year [[5,27] and references therein]. (v) In FMDV there is an overlap between some antigenic sites and some cell receptor recognition sites, which may result in coevolution of antigenicity and host range [19].
5. FMDV types, subtypes and isolates The FMDVs isolated over the 20th century were grouped in seven serological types, termed A, O, C, Asia 1, SAT1, SAT2 and SAT3 [1 – 3]. FMDVs were assigned to a different serotype on the basis of lack of cross-protection following infection (convalescent animals) or vaccination. Viruses showing partial cross-protection were assigned to the same serotype but to a different subtype. About 65 subtypes of FMDV were defined, but about two decades ago it was realized that with the use of increasing numbers of MAbs virtually each isolate could be regarded as an antigenic variant [3,36,37]. Antigenic diversity is a direct consequence of genetic variation. A significant observation was the generation of an epitope previously assigned to a FMDV subtype as a consequence of an amino acid replacement in a FMDV of another subtype [38]. Classifications of FMDV isolates based on phylogenetic methods (Fig. 3) are gradually replacing the traditional groupings based on serological criteria. Phylogenies are based on RT-PCR amplification of genomic FMDV RNA isolates and nucleotide sequencing of specific genomic regions, usually those encoding capsid proteins, particularly VP1 [3,36]. Such procedures have established, for example, the relatedness among the O type FMDV isolates that originate from some initial strain in the center of Asia, expanded westward and eastward, causing the European outbreak of 2001/2002. This outbreak has represented
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Fig. 3. Phylogenetic relationships among FMDV isolates of seven serotypes, based on amino acid sequences of capsid protein VP1. The segment indicates a 10 amino acid distance. The spheres at the tip of each branch emphasize that each virus isolate is in reality a cloud of mutants, as discussed in the text (adapted from Ref. [3] and references therein).
the unprecedented penetration of FMDV of Asiatic origin into Europe, contributing to concerns on further FMDV spread in the face of an increasingly global economy [3 –5]. The potential for rapid evolution and the antigenic diversity of FMDV are complicating factors for the diagnosis, prevention and control of FMD.
6. Diagnosis and control of FMD: a growing challenge Intensive farm animal breeding and livestock production in the context of global trade connections necessitate rapid and reliable diagnosis of FMD to distinguish it from other vesicular diseases, notably swine vesicular disease or vesicular stomatitis [1 – 3]. The classical complement fixation and serum neutralization tests have been largely replaced by ELISA and genetic typing techniques [3]. ELISA employs serotype-specific sera or MAbs
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[39]. However, use of MAbs meets with the epitopic diversity among FMDV isolates [3, 36 –38]. Perhaps multinational collaborations to investigate cocktails of neutralizing and non-neutralizing MAbs [which often define conserved epitopes [37]] could produce reliable reagents for ELISA typing. For trade purposes, diagnostic procedures should distinguish animals that have been vaccinated from those that have been infected with FMDV. This distinction is now feasible by detecting antibodies against some of the nonstructural proteins by ELISA, in particular antibodies against 3AB and 3ABC. Such antibodies are found in animals which at some time have been infected with FMDV but not in vaccinated animals (Ref. [3] and references therein). RT-PCR amplification techniques are increasingly providing a versatile tool for an efficient and rapid diagnosis of FMD [3,40,41]. Coupled to automated nucleotide sequencing and phylogenetic analysis, these techniques can achieve a detailed virus identification to trace the origin of FMDVs associated with new outbreaks [3,4]. Reliability and rapidity in FMD diagnosis are a priority since a delay in the implementation of control or preventive measures may result in the uncontrollable spread of disease with devastating economic consequences [1 – 4]. Control of FMD is based on two major strategies: the slaughtering of affected and contact animals (the so called ‘stamping out’ procedure) or the regular vaccination of the major host species for FMDV (always cattle and when indicated also swine). Both strategies can be combined so that a ring vaccination can be established around a focus area where affected and contact animals have been slaughtered [1 – 5]. As a consequence of the massive killing of animals during the 2001/2002 European outbreak, and the increasing awareness of the community on animal welfare, there is presently a vivid debate towards whether a vaccination strategy may not be preferable to the stamping-out and non-vaccination procedure in operation in the EU since 1991. Such regulation was adopted for economic considerations, but left susceptible animals in a vast territory defenseless in the face of an accidental introduction of the virus in the region, a situation that historically has been known to favor the occurrence of large epizootics [1 –5]. In the event of a return to a vaccination policy, basic investigations over the last decade should provide the basis for manufacturing more effective and safer vaccines than those presently available. Such new vaccines could replace current vaccines in those territories where a vaccination policy is presently in operation. The basic rules that should be considered for the development of new anti-FMD vaccines are [3,42]: (i) Vaccines should include multiple B-cell and T-cell epitopes to produce an ample humoral (antibody) and cellular immune response, in particular, T helper responses for antibody production [3,43]. Such an ample response is required to delay, and ideally abolish, a possible selection in the field of FMDV variants showing partial resistance to the immune response evoked by the vaccine [3,5,19,42]. The B cell epitope map has been investigated with considerable detail [11], and a number of T-cell epitopes have been identified in structural and non-structural proteins of FMDV [[3,43 – 45] and references therein]. Therefore, it should be possible to present an ample epitopic repertoire either with synthetic constructs or with complete, non-infectious particles. Research along these lines is currently in progress. (ii) Although FMDV infection is usually acute, and viremia and lesions occur within days after infection, ruminants can establish an asymptomatic carrier state in which the virus replicates in the oesopharyngeal region of the animals [46,47]. The presence of carriers
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jeopardizes animal trade since it cannot be excluded that carrier animals could on occasions originate acute disease when in contact with susceptible animals. Classical vaccines cannot prevent the establishment of persistent FMDV infection in cattle, and a new vaccine should target systemic and mucosal immunity, hoping that the latter may minimize chances of establishing the carrier state. (iii) Vaccine manufacturing should not require the handling of virus because of the danger of virus escaping from vaccine factories. Also, classical, inactivated whole-virus vaccines may be at the origin of outbreaks if inactivation prior to vaccine formulation was not complete. There is good evidence that some FMD outbreaks probably had a vaccine origin [3]. These constitute powerful arguments to design vaccines that do not require infectious virus at any stage of their preparation [48,49]. (iv) Finally, vaccines must be marked to distinguish vaccination from infection, as discussed above for diagnostic purposes. A vaccine could be marked positively by the presence of some genetic tag or negatively by the absence of a gene product systematically found during a natural infection [3,40]. The recent European epizootic of FMD at the onset of the 21 century has made us aware of the great economic losses to be endured because no effective preventive and control measures are available for FMD. We should not forget, however, that underdeveloped countries in need for active livestock trade have suffered FMD-related losses for decades. Interest on new vaccines and basic epidemiology to define the strategies for a more effective control of FMD has been boosted as a consequence of the recent European epizootic. Yet, active research on FMD and FMDV should have never been interrupted [5]. It is hoped that application of new molecular techniques and increasing computation capacities for epidemiological modeling, will contribute to a more effective control of FMD in Europe and also in underdeveloped countries. Experience tells us that what we learn with FMDV is likely to be of value also to understand and control some other viruses.
Acknowledgments We thank many colleagues for important contributions to FMDV research. Unavoidably, space limitations did not allow quoting all relevant work. Research supported by grants DGES PM97-0060-C02-01, BIO 99-0833-01, BMC 2001-1823-C0201, and Fundacio´n Ramo´n Areces from Spain, and FAIR 5 PL97-3665, CT97-3665 and 341 from the EU.
References [1] Bachrach HL. Foot-and-mouth disease virus. Annu Rev Microbiol 1968;22:201– 44. [2] Pereira HG. Foot-and-mouth disease virus. In: Gibbs RPG, editor. Virus diseases of food animals, vol. 2. New York: Academic Press; 1981. p. 333– 63. [3] Sobrino F, Sa´iz M, Jime´nez-Clavero MA, Nu´n˜ez JI, Rosas MF, Baranowski E, Ley V. Footand-mouth disease virus: a long known virus, but a current threat. Vet Res 2001;32:1– 30. [4] Samuel AR, Knowles NJ. Foot-and-mouth disease virus: cause of the recent crisis for the UK livestock industry. Trends Genet 2001;17:421– 4.
306
E. Domingo et al. / Comp. Immun. Microbiol. Infect. Dis. 25 (2002) 297–308
[5] Sobrino F, Domingo E. Foot-and-mouth disease in Europe. EMBO Rep 2001;2:459– 61. [6] van Regenmortel MHV, Fauquet CM, Bishop DH, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, Mc Geoch DJ, Pringle CR, Wickner RB. Virus taxonomy. San Diego: Academic Press; 2000. [7] Acharya R, Fry E, Stuart D, Fox G, Rowlands D, Brown F. The three-dimensional structure of ˚ resolution. Nature 1989;337:709 –16. foot-and-mouth disease virus at 2.9 A ´ [8] Lea S, Hernandez J, Blakemore W, Brocchi E, Curry S, Domingo E, Fry E, Abu-Ghazaleh R, King A, Newman J, Stuart D, Mateu MG. The structure and antigenicity of a type C foot-andmouth disease virus. Structure 1994;2:123– 39. [9] Lea S, Abu-Ghazaleh R, Blakemore W, Curry S, Fry E, Jackson T, King A, Logan D, Newman J, Stuart D. Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant, G67. Structure 1995;3:571– 80. [10] Curry S, Fry E, Blakemore W, Abu-Ghazaleh R, Jackson T, King A, Lea S, Newman J, Rowlands D, Stuart D. Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity. Structure 1996;4:135– 45. [11] Mateu MG. Antibody recognition of picornaviruses and escape from neutralization: a structural view. Virus Res 1995;38:1– 24. [12] Logan D, Abu-Ghazaleh R, Blakemore W, Curry S, Jackson T, King A, Lea S, Lewis R, Newman J, Parry N, Rowlands D, Stuart D, Fry E. Structure of a major immunogenic site on foot-and-mouth disease virus. Nature 1993;362:566– 8. [13] Verdaguer N, Mateu MG, Andreu D, Giralt E, Domingo E, Fita I. Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction. EMBO J 1995;14:1690– 6. [14] Verdaguer N, Sevilla N, Valero ML, Stuart D, Brocchi E, Andreu D, Giralt E, Domingo E, Mateu MG, Fita I. A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation. J Virol 1998;72:739– 48. [15] Ochoa WF, Kalko SG, Mateu MG, Gomes P, Andreu D, Domingo E, Fita I, Verdaguer N. A multiply substituted G– H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules. J Gen Virol 2000;81:1495 – 505. [16] Parry N, Fox G, Rowlands D, Brown F, Fry E, Acharya R, Logan D, Stuart D. Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth disease virus. Nature 1990;347:569– 72. [17] Hewat EA, Verdaguer N, Fita I, Blakemore W, Brookes S, King A, Newman J, Domingo E, Mateu MG, Stuart DI. Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop. EMBO J 1997;16:1492– 500. [18] Verdaguer N, Schoehn G, Ochoa WF, Fita I, Brookes S, King A, Domingo E, Mateu MG, Stuart D, Hewat EA. Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation. Virology 1999; 255:260– 8. [19] Baranowski E, Ruiz-Jarabo CM, Domingo E. Evolution of cell recognition by viruses. Science 2001;292:1102– 5. [20] Nemerow GR, Stewart PL. Antibody neutralization epitopes and integrin binding sites on nonenveloped viruses. Virology 2001;288:189– 91. [21] Belsham GJ. Distinctive features of foot-and-mouth disease virus, a member of the picornavirus family; aspects of virus protein synthesis, protein processing and structure. Prog Biophys Mol Biol 1993;60:241– 60.
E. Domingo et al. / Comp. Immun. Microbiol. Infect. Dis. 25 (2002) 297–308
307
[22] Flint SJ, Enquist LW, Krug RM, Racaniello VR, Skalka AM. Molecular biology, pathogenesis and control. Virology, Washington, DC: ASM Press; 2000. [23] Martı´nez-Salas E. Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotechnol 1999;10:458– 64. [24] Hansen J, Long AM, Schultz S. Structure of the RNA-dependent RNA polymerase of poliovirus. Structure 1997;15:1109– 22. [25] McKnight KL, Lemon SM. Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA. J Virol 1996;70:1941 –52. [26] Gromeier M, Wimmer E, Gorbalenya AE. In: Domingo E, Webster RG, Holland JJS, editors. Origin and evolution of viruses. San Diego: Academic Press; 1999. p. 287– 343. [27] Domingo E, Webster RG, Holland JJ, editors. Origin and evolution of viruses. San Diego: Academic Press; 1999. [28] Nu´n˜ez JI, Baranowski E, Molina N, Ruiz-Jarabo CM, Sa´nchez C, Domingo E, Sobrino F. A single amino acid substitution in nonstructural protein 3A can mediate adaptation of foot-andmouth disease virus to the guinea pig. J Virol 2001;75:3977– 83. [29] Escarmı´s C, Go´mez-Mariano G, Da´vila M, La´zaro E, Domingo E. Resistance to extinction of low fitness virus subjected to plaque-to-plaque transfers: diversification by mutation clustering. J Mol Biol 2002;315:647– 61. [30] Drake JW, Holland JJ. Mutation rates among RNA viruses. Proc Natl Acad Sci USA 1999;96: 13910– 3. [31] Eigen E, Schuster P. The hypercycle. A principle of natural self-organization. Berlin: Springer; 1979. [32] Eigen M, Biebricher CK. Sequence space and quasispecies distribution. In: Domingo E, Ahlquist P, Holland JJS, editors. RNA genetics, vol. 3. Boca Raton, FL: CRC Press; 1988. p. 211– 45. [33] Eigen M. Viral quasispecies. Sci Am 1993;269:42 –9. [34] Eigen M. Natural selection: a phase transition? Biophys Chem 2000;85:101– 23. [35] Martı´nez MA, Dopazo J, Herna´ndez J, Mateu MG, Sobrino F, Domingo E, Knowles NJ. Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades. J Virol 1992;66:3557 – 65. [36] Martı´nez MA, Carrillo C, Plana J, Mascarella R, Bergada J, Palma EL, Domingo E, Sobrino F. Genetic and immunogenic variations among closely related isolates of foot-and-mouth disease virus. Gene 1988;62:75– 84. [37] Mateu MG, Da Silva JL, Rocha E, De Brum DL, Alonso A, Enjuanes L, Domingo E, Barahona H. Extensive antigenic heterogeneity of foot-and-mouth disease virus of serotype C. Virology 1988;167:113– 24. [38] Herna´ndez J, Martı´nez MA, Rocha E, Domingo E, Mateu MG. Generation of a subtype-specific neutralization epitope in foot-and-mouth disease virus of a different subtype. J Gen Virol 1992; 73:213– 6. [39] Roeder PL, Le Blanc Smith PM. Detection and typing of foot-and-mouth disease virus by enzyme linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis. Res Vet Sci 1987;45:225– 30. [40] Office international des epizooties WOfAHF-a-mdOSC, Manual of standards for diagnostic tests and vaccines. Paris, France: OIE; 1996. [41] Reid SM, Hutchings GH, Ferris NP, De Clercq K. Diagnosis of foot-and-mouth disease by RTPCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples. J Virol Meth 1999;83:113– 23. [42] Domingo E, Holland JJ. Complications of RNA heterogeneity for the engineering of virus vaccines and antiviral agents. Genet Engng (NY) 1992;14:13– 31.
308
E. Domingo et al. / Comp. Immun. Microbiol. Infect. Dis. 25 (2002) 297–308
[43] Collen T. In: Goddeev BML, Morrison I, editors. Foot-and-mouth disease virus (aphthovirus): viral T cell epitopes. Cell mediated immunity in ruminants, Boca Raton: CRC Press; 1994. p. 173– 97. [44] Blanco E, McCullough K, Summerfield A, Fiorini J, Andreu D, Chiva C, Borras E, Barnett P, Sobrino F. Interspecies major histocompatibility complex-restricted Th cell epitope on footand-mouth disease virus capsid protein VP4. J Virol 2000;74:4902– 7. [45] Blanco E, Garcia-Briones M, Sanz-Parra A, Gomes P, De Oliveira E, Valero ML, Andreu D, Ley V, Sobrino F. Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus. J Virol 2001;75:3164 – 74. [46] van Bekkum JG, Frenke HS, Frederiks HHJ, Frenkel S. Observations on the carrier state of cattle exposed to foot-and-mouth disease virus. Tijdschr Diergeneeskd 1959;84:1159 –64. [47] Salt JS. The carrier state in foot and mouth disease—an immunological review. Br Vet J 1993; 149:207– 23. [48] Brown F. New approaches to vaccination against foot-and-mouth disease. Vaccine 1992;10: 1022– 6. [49] Brown F. Foot-and-mouth disease. In: Nicholson BH, editor. Synthetic vaccines. Oxford: Blackwell; 1994. p. 416– 32.