Virus replication occurs mostly in tongue and mouth cells [5]. After the virus attaches to its host cell, it enters by endocytosis and its coating from around the genome is removed [6].
The exact receptors for FMDV are not quite known, but beta integrins that are used for adhering to other cells and the cell matrix as well as interacting with extracellular proteins to perform physiological processes have been identified as FMD receptors; a receptor known as a heparan sulfate, a glycosaminoglycan, also shows affinity for FMDV [6]. After the viral RNA is in the cytoplasm of the cell, ribosomes are recruited to make negative-strand RNA to serve as viral templates.
Much like other picornaviruses, the virus 'hijacks' the cell and redirects versicles to form reproduction sites within the cell. Vesicles redirected from the endoplasmic reticulum are housing the process where new viral RNA is being made, while ribosomes are synthesizing P1-encoded proteins.
Once the capsids are ready, their proteins are quickly rearranged so that the positive-strand RNA can be inserted [7]. From there, the virions burst out of the cell and travel all around the body, or if they were being produced in a blister, the fluid released by the blister exposes the virions to the air, feed, and other animals. Infected animals can carry the virus prior to showing symptoms and for up to four years afterwards [1].
The graphic renderings were done by using RasMole. The reason for this instability is thought to be a cluster of His residues at the interface between VP2 and VP3 which become protonated at low pH, weakening the capsid through electrostatic repulsion , FMDV, like other members of the Picornaviridae , has a relatively short infectious cycle in cultured cells. Depending on the multiplicity of infection, newly formed infectious virions begin to appear at between 4 and 6 h after infection.
The virus is cytocidal, and infected cells exhibit morphological alterations, commonly called cytopathic effects, which include cell rounding and alteration and redistribution of internal cellular membranes.
The virus also causes biochemical alterations, including inhibition of host translation and transcription The interactions of FMDV with cells have been extensively studied for many years. It is generally accepted that FMDV receptors, as well as other picornavirus receptors, play a role in tissue and organ tropism which leads to disease pathogenesis , , , These studies also suggested that while six of the seven serotypes bound to a single class of receptor site, some of the serotypes bound to a second class of receptors which were present at a high copy number 39 , Early studies showed that limited trypsin digestion of virus resulted in viral particles which were noninfectious due to the inability to bind to cells in culture 22 , 30 , 38 , 90 , These results suggested that this region of the VP1 protein interacted with the cell surface receptor.
The fibronectin receptor was subsequently shown to be part of a large family of transmembrane glycoproteins called integrins They are involved in cell adhesion, cell migration, thrombosis, and lymphocyte interactions , , The first indication that the RGD sequences might be involved in the virus-receptor interaction came from studies showing that small peptides containing RGD could inhibit the binding of virus to cells 40 , Direct genetic evidence for this interaction was obtained by mutating or deleting the RGD sequence in infectious cDNA clones, resulting in viral particles which were noninfectious, could not adsorb to susceptible cells, and could not cause disease in susceptible animals , , The first identification of the integrin receptor for FMDV was made based on comparison of the receptor specificity of the virus with that of a human enterovirus, coxsackievirus A9 CAV9.
This virus contains an extension in its VP1 protein that includes an RGD sequence 92 , 93 , which was shown to be involved in virus binding to cells in culture A number of alternative receptors have been shown to mediate FMDV infection in vitro. Antibody-complexed virus can infect cells via Fc receptor-mediated adsorption 42 , , In addition, an artificial receptor which consists of a single-chain anti-FMDV monoclonal antibody fused to intercellular adhesion molecule 1 ICAM-1 has been engineered, and this receptor was also able to mediate infection with RGD-deleted virus In , Jackson and coworkers reported that a type O 1 virus was able to utilize the glycosaminoglycan heparan sulfate HS as a coreceptor.
We had previously shown that there appeared to be a second, unidentified receptor present at a high copy number 39 , , and the report by Jackson et al. We reconciled these seemingly opposite findings by showing that tissue culture adaptation of a type O 1 virus selects a variant which has a positively charged Arg at residue 56 of VP3 and can grow in CHO cells Interestingly, this variant was relatively avirulent in cattle In contrast, a second variant of this virus containing a His at residue 56 of VP3 could not grow in CHO cells and was relatively virulent in cattle We expanded these studies to show that the virus with the Arg residue required only HS to replicate in CHO cells but that the variant with the His residue required the integrin to replicate in cell culture A similar result was also noted for type C viruses, where multiple passages in tissue culture selected viruses with positive surface charges which can replicate in the absence of the known integrin receptors 26 , 27 , While the penetration and uncoating of FMDV have not been studied in great detail, there have been some observations which suggest possible mechanisms of how they might occur.
We and others have shown that after adsorption to the cell surface, the S virion breaks down into 12S pentameric subunits, releasing the RNA 37 - 39 , This breakdown does not occur at the cell surface, since particles which are eluted from the cell after adsorption are fully infectious and still sediment at S By using a series of lysosomotropic agents, which raise the pH of intracellular endosomes, it has been demonstrated that the virus probably breaks down upon entering an acidic endosome 37 , 87 , Thus, the breakdown of S virus to pentameric subunits by itself does not lead to productive infection, but there must be other events after the breakdown.
These results indicate that the viral receptor is responsible only for docking the virus to the membrane of the susceptible cell and plays no role in viral uncoating, which is consistent with the ability of FMDV to utilize multiple receptors for infection in cell culture.
Following uncoating, the RNA is released into the cytoplasm by an as-yet-unknown mechanism and begins a round of viral translation. The genome-linked protein VPg is cleaved by a cellular enzyme prior to translation of the incoming RNA 10 , 11 ; however, protein synthesis initiation complexes can be formed with mRNA containing VPg Cap-dependent mRNA translation is inhibited in infected cells as the result of the cleavage of the protein synthesis initiation factor eIF4G by L pro , A host factor of 57 kDa, subsequently identified as the nuclear polypyrimidine tract binding protein PTBP , , , , was shown to interact with at least two regions of the IRES , Deletion of these two sites inhibited both the binding of the protein and in vitro translation It has also been postulated that PCBP facilitates a circularization of the poliovirus genome to modulate the balance between translation and RNA replication 33 , Following initiation, translation results in the production of a single polypeptide which undergoes a series of cleavages leading to the production of both structural and NS proteins Fig.
The primary cleavage reactions are performed by three different proteases. As discussed above, L pro autocatalytically cleaves itself from polyprotein. There has been a suggestion that this cleavage is not a proteolytic event but rather is a modification of the translational machinery by the 2A peptide which allows the release of PA from the ribosome while permitting the synthesis of the downstream proteins to proceed , This hypothesis, however, has not been confirmed by other laboratories.
Nevertheless, the 2A peptide has been used in nonviral systems to cleave foreign genes from polyproteins , All of the other cleavages of the polyprotein, as outlined in Fig. This protein is related to the trypsin family of serine proteases 18 , 45 , , and Grubman and coworkers have mapped the active site of FMDV 3C pro to Cys, His46, and Asp84 Picornavirus RNA replication presents a number of unique challenges.
In addition, since the mRNA and the genome RNA are the same molecule, with the exception of the genome-linked VPg, there must be a mechanism to distinguish RNAs which are bound for the ribosome and those which will be packaged into virion particles.
While there have been very few studies on transcription and replication in the FMDV system, extensive studies on these activities have been performed with enteroviruses. This system has not been studied in FMDV; however, the models of RNA replication developed for poliovirus are probably quite similar It is thought that translation of the plus-strand RNA must cease before minus-strand synthesis begins There is still controversy about the initiation of picornavirus minus-strand RNA synthesis.
Since initiation of minus-strand synthesis occurs in the cytoplasm in the presence of cellular mRNAs, which also contain poly A tails, picornaviruses must have developed mechanisms enabling the polymerase to recognize viral RNA. The discovery of the cre provided a rational mechanism for both the uridylylation of VPg and the ability of the polymerase to discriminate viral RNA from cellular mRNAs.
This conserved motif is required for poliovirus and rhinovirus minus-strand synthesis , , and the first two A's serve as the template for the synthesis of VPgpU and VPgpUpU and for viral replication initiation in enteroviruses , Mutations in this motif within the FMDV cre severely reduced viral replication in cell culture; however, the cre is positionally independent within picornavirus genomes , , , The second model, discussed below, postulates that minus-strand synthesis is primed on the poly A tail.
Following the initiation reaction, elongation of the minus strand begins, catalyzed by 3D pol. The mechanism by which this occurs is unknown, but one hypothesis suggests that binding of PABP to the poly A tract positions this region of the plus strand near the cre The elongation of the nascent strands results in the formation of a double-stranded molecule, the replicative form RF 3 , Free minus strands are not detectable in vivo. After formation of the RF, new plus-strand synthesis can begin.
In poliovirus-infected cells, the ratio of plus to minus strands is about , indicating that a single minus strand can be a template for the synthesis of numerous plus strands, resulting in the formation of a partially double-stranded RNA molecule, the replicative intermediate 3. The initiation of plus-strand synthesis from the RF has not been elucidated; however, two possible mechanisms to generate VPgpUp have been suggested.
Since the data which led to the latter hypothesis was generated totally with cell-free systems, it is still uncertain whether these mechanisms are utilized in infected cells. In addition, it has recently been suggested that FMDV cre function can be complemented in trans While more studies are necessary to confirm this result, it should be noted that cre function could not be complemented in trans in either the human rhinovirus or the poliovirus , system.
For plus-strand synthesis to proceed, the RF must be unwound. The mechanism for this is also unclear. The picornavirus 2C protein both has ATPase activity , , and contains helicase motifs , , , , , but helicase activity has not been demonstrated The possibility of involvement of either a cellular helicase or a nuclear protein has also been suggested, since the RF is infectious when transfected into whole cells but not when transfected into enucleated cells The elongation of the plus strand by 3D pol also occurs by an unknown mechanism.
The complete replication of a picornaviral RNA in a cell-free system, including de novo protein synthesis, genome replication, and encapsidation to produce infectious virus, has been accomplished for poliovirus 32 , , , and EMCV RNA synthesis occurs within a membranous replication complex, which is derived from membranes of the endoplasmic reticulum and Golgi and contains viral NS proteins encoded by both the P2 2B, 2BC, and 2C and P3 3A and its precursors, 3C pro , and 3D pol regions 58 - 61 , 66 , , , , , , , , , The 2B protein has been shown to enhance membrane permeability and block protein secretion , , , , but its role in RNA synthesis is not clear.
FMDV mutants that are resistant to guanidine inhibition had an altered 2C isoelectric point , and changes in the viral 2C protein in resistant mutants were directly shown by using recombination and RNase T 1 oligonucleotide mapping Infection with picornaviruses results in a rapid inhibition of host cell transcription, which does not appear to be related to the inhibition of host cell translation In the case of enterovirus infections, transcription catalyzed by RNA polymerases I, II, and III is inhibited, and this inhibition requires the synthesis of 3C pro , which appears to cleave cellular transcription factors required for the activity of these three enzymes see reference and references therein.
Thus, FMDV may inhibit host cell transcription by a mechanism different from that of the entero- and cardioviruses. The mechanisms of encapsidation and maturation are still unresolved and are probably the least studied of all of the steps in the replication cycle. Again, most of the studies have been done with the enteroviruses, and therefore analogies must be drawn with FMDV.
In broad terms, the 3C pro cleavage products of the P1 region are assembled into a protomer structure containing one copy of each of the proteins VP0, VP1, and VP3 Fig. Five protomers can assemble into a pentamer Fig. A number of intermediate particles have been identified in picornavirus-infected cells, including protomers, pentamers, a particle containing RNA with an uncleaved VP0 provirion , , and a particle with an uncleaved VP0 lacking RNA empty capsid , Two unresolved issues in picornaviral maturation are what signals are necessary for encapsidation of the RNA and what are the roles of the empty capsid and provirion.
In addition, only newly synthesized plus-strand RNAs are encapsidated, indicating that there is a link between active RNA replication and encapsidation , Thus, there may be cis -acting packaging signals present within the plus-strand RNA to facilitate encapsidation. The putative packaging signal does not appear to reside within the P1 region of the genome, since defective-interfering particles have been demonstrated in poliovirus-infected cells , , , all of which have deletions within the P1 region.
Interestingly, no defective-interfering particles have been detected in FMDV-infected cells , In addition, the complete P1 regions of a number of picornaviruses, including FMDV , can be deleted and replaced with a reporter gene generating a replicon, which, in the case of poliovirus, can be encapsidated when capsid proteins are provided in trans by a coinfecting virus 15 , 28 , , Further studies, using the poliovirus replicon system, have shown that if the structural proteins of a heterologous picornavirus are provided in trans , packaging of the replicon RNA does not occur 28 , These data provide additional evidence for a packaging element, specific for each individual picornavirus, located within the genome outside the P1 region.
There has been a single unconfirmed report of heterologous trans encapsidation of the FMDV genome with bovine enterovirus structural proteins This is the first report of such an element within the Picornaviridae. While naturally occurring provirions have not been demonstrated in FMDV-infected cells, they have been shown to occur during the replication of other picornaviruses 63 , , There are currently two models of picornavirus assembly.
One proposes that pentamers assemble into empty capsids, followed by insertion of the RNA, and the second proposes that pentamers directly interact with the RNA to form the provirion. In either case, it is known that myristylation of the N terminus of VP0 is necessary for capsid formation 16 , , Studies with FMDV have demonstrated that radioactive label can be chased from structural proteins into protomers, pentamers, empty capsids, and finally virions In addition, poliovirus pentamers can self-assemble into empty capsids in vitro in the absence of viral RNA , , More recently, however, it has been shown in a cell-free replication system that only poliovirus pentamer structures can interact with newly synthesized RNA to form virions , indicating that empty capsids may be either a storage particle for pentamers or a by-product of the assembly reaction.
Cleavage is thought to be autocatalytic and results from a conserved His residue in VP2 which activates local water molecules, leading to a nucleophilic attack on the scissile bond and cleavage 34 , , Maturation cleavage is required for the generation of infectious virus , , In FMDV, site-directed mutations within VP0 led to the formation of noninfectious provirions that exhibited receptor binding and acid sensitivity, similar to the case for infectious virus Upon acid dissociation, however, the generated pentamers were more hydrophobic than those from mature virions, suggesting that VP0 cleavage may be necessary for release of the RNA into the cytoplasm The presence of seven serotypes and multiple subtypes and variants has added to the difficulty of laboratory diagnosis and control of FMD.
The rise of new variants is inevitably caused by continued circulation of the virus in the field and the quasispecies nature of the RNA genome , This high error rate leads to differences of FMDV replicated genomes from the original parental genome of 0. In its simplest terms, the concept envisions that within any population of virus, all genome sequences are not identical, and that selection occurs at the population level rather than at the individual level The environment can be in either tissue culture or a particular host species, and in either situation, immunologic pressure or physical conditions such as temperature or pH are influential.
The quasispecies nature of the FMDV genome was described over 20 years ago , , and while the concept is studied at the nucleotide level, the variability of FMDV populations is manifested when mutations lead to codon changes resulting in a change in the viral phenotype.
Most of this variation occurs within the capsid-coding region of the genome the P1 region [Fig. While mutations also occur within the NS protein-coding regions of the genome, they are probably less tolerated, since proteins encoded by these regions are necessary for viral replication and changes are more likely to be lethal. Interestingly, these studies indicated that recombination was more likely to occur within the regions of the genome coding for the NS proteins; however, a more recent study has suggested that RNA recombination within the capsid-coding P1 region of the genome may contribute to genetic diversity in FMDVs isolated from the field Antigenic variation in the field increases with time and most probably results from immunologic pressure placed on the virus by either the infected or vaccinated host species , In addition, antigenic variation in FMDV has also been observed in tissue culture in the absence of immunologic pressure 67 , , , , , , indicating that antigenic sites on the virion may also be involved in other virus functions.
Regardless of the mechanism, analysis of both genome sequence and antigenic variation has been invaluable in epidemiological studies of outbreaks and analysis of virus within countries where the disease is enzootic, and, in the case of a possible deliberate introduction of virus, it will also have forensic value in tracking the source 17 , , , , , Antigenic sites on the surface of the FMD virion have been identified for five of the seven serotypes of the virus South African Territories 1 and 3 being the only exceptions 41 , 43 , 68 , 80 , , , , , , , , , At least four antigenic sites have been identified, involving one or more of the capsid proteins, VP1, VP2, and VP3; however each serotype may not contain all four sites.
While all of the sites appear to be necessary for a complete immunologic response to either infection or vaccination, the major antigenic site, to which most of the immune response is directed and which is common to all of the serotypes, is located within the G-H loop of VP1 Fig. While this site is clearly the major antigenic site, FMDV antigenic variation is associated with mutations leading to amino acid replacements within all of the known antigenic sites , Nevertheless, even though there is extensive antigenic variation within FMDV, the changes are limited to very specific regions of the viral surface.
This may be because changes within other regions of the capsid would compromise either viral structural integrity or virus identity The antigenic variation within FMDV makes control extremely difficult, since even the best vaccine may induce immunologic pressure within the population that results in the emergence of a new variant.
Furthermore, the observation that antigenic variation can also occur in tissue culture has implications for vaccine production, since a number of tissue culture passages are required to produce vaccine for a new variant, leading to the possibility that the virus eventually utilized as antigen may not provide the antigenic coverage needed.
While FMD affects a wide variety of cloven-hoofed animals, pathogenesis has been studied mainly in cattle and pigs. Infection of cattle generally occurs via the respiratory route by aerosolized virus Infection can also occur through abrasions on the skin or mucous membranes, but is very inefficient, requiring almost 10, times more virus Virus is excreted into the milk of dairy cattle 78 , , as well as in semen, urine, and feces , , and calves can become infected by inhaling milk droplets.
Infected cattle also aerosolize large amounts of virus, which can infect other cattle in addition to other species In cattle experimentally infected via aerosol, it was found, by in situ hybridization ISH , that within the first 24 h, virus was present in respiratory bronchiolar epithelium, subepithelium, and interstitial areas of the lung By 72 h, signal was detected in epithelial cells of the tongue, soft palate, feet, tonsils, and tracheobronchial lymph nodes Other studies, however, have suggested that the pharynx, and not the lungs, may be the initial site of viral replication in infected cattle 8 , 79 , The conflicting observations about the region of the respiratory tract that is initially infected in cattle exposed to aerosols may be the result of a number of variables, including aerosol particle size, strain of virus, or how the aerosol was generated 8.
Vesicles develop at multiple sites, generally on the feet and tongue, and are usually preceded by fever. Severe lesions often occur in areas subjected to trauma or physical stress, and most animals develop viremia. The incubation period can be between 2 and 14 days, depending on the infecting dose and route of infection Pigs usually become infected either by eating FMDV-contaminated food, by direct contact with infected animals, or by being placed into areas that had once housed FMDV-infected animals.
They are, however, much less susceptible to aerosol infection than cattle 5 , 6 , yet they excrete far more aerosolized virus than cattle or sheep 6 , 7. As in cattle, the incubation period is dependent on the amount of infecting virus and the route of infection, but it is generally 2 days or more. Animals develop fever, viremia, and lesions on the feet and tongue. Foot lesions are the most common finding in pigs, while lesions at other sites occur less frequently Tongue lesions are usually small and less noticeable than those in cattle In young piglets, the infection may be fatal due to myocarditis.
Initial replication of the virus occurs at the site through which the virus gains entry, followed by rapid dissemination to most of the epithelial sites within the animal 73 , Interestingly, virus can be found at sites where clinical lesions either were not present or do not form 73 , While pigs excrete large amounts of aerosolized virus, recent evidence suggests that much more viral replication takes place in the nasal mucosa than in the lungs Of all of the important livestock species, sheep played the major role in the United Kingdom outbreak of see below.
Because it is very difficult to make a clinical diagnosis of FMD in sheep R. De la Rua, G. Watkins, and P. Watson, Letter, Vet. Sheep are highly susceptible to virus infection via aerosol and can excrete airborne virus; however, during outbreaks they are most likely infected by contact with infected animals Clinical disease in sheep is characterized by lesions on the feet and mouth, fever, and viremia.
FMDV can also infect a wide variety of wildlife. The risk of spread of the infection by wildlife is controversial and is discussed in two recent review articles , In theory, any of the viral structural and NS proteins, elements of the viral RNA, and host proteins and membranes that participate in the viral replication cycle can be considered a virulence factor, since defects in the factor or its absence in the cell may lead to the virus' inability to replicate and cause disease in the host species.
It is beyond the scope of this review to examine the roles of all of these factors, so we will discuss only those that have been shown to be directly involved in virulence in susceptible animals. It has been recognized for many years that viral receptors play a role in tissue tropism and disease pathogenesis , , , It is known that virus which has had the RGD sequence of the VP1 G-H loop either mutated or deleted cannot replicate in tissue culture or cause disease in animals , , This virus was shown to be relatively avirulent in cattle, while the wild-type virus, which required only integrin receptors to initiate infection in vitro , was virulent in bovines More interestingly, two bovines inoculated with large amounts of the HS binding virus eventually showed signs of FMD.
Virus isolated from these animals could only utilize the integrin as a receptor and had lost the ability to interact with HS in vitro , It is not clear why FMDV should need three integrin receptors to cause disease or whether it utilizes all or some the integrins in the susceptible hosts.
We have recently shown in tissue culture that different serotypes of the virus exhibit altered efficiencies of integrin utilization It is possible that the virus uses different receptors during various stages of the disease. While it appears that the disease process in susceptible animals is mediated by the virus-integrin interaction, a type C virus containing an RGGD sequence has been isolated from a bovine which was not protected from virus challenge following immunization with an experimental peptide vaccine , In addition, a tissue culture-adapted type C virus with a genetically engineered RGG sequence, which was unable to bind to heparin, was able to infect cells expressing both HS and integrin receptors and cells which do not express FMDV integrin receptors and HS 26 , The ability of these two viruses to cause disease in susceptible animals has not been demonstrated.
More interestingly, a tissue culture-adapted derivative of a type O 1 virus, isolated in China, was able to replicate in tissue culture in both an integrin- and HS-independent manner and to cause mild disease in pigs Thus, there is a possibility that nonintegrin receptors may be involved in disease pathogenesis. However, with the exception of the virus isolated after challenge from the peptide-vaccinated bovine , , no natural isolate of FMDV which does not contain the RGD sequence within the VP1 G-H loop has been identified.
L pro was shown to be a virulence determinant based on experiments with animals with genetically engineered type A 12 virus with L pro deleted leaderless 98 , , It was thought that this virus would be less virulent than the wild-type virus, since the lack of L pro would lead to the inability of the virus to cleave eIF4G and shut off cellular protein synthesis.
While this virus replicated at only a slightly lower rate than wild-type virus in BHK cells , it was markedly avirulent when injected into cattle and pigs and was unable to spread to cohoused animals 98 , The mechanism of attenuation of leaderless type A 12 virus was examined by aerosol exposure of cattle.
Wild-type-infected cattle had histologically altered respiratory bronchioles and virus-specific ISH signals in bronchioles by 24 h, and by 72 h they developed clinical disease, including fever and vesicles on the feet and positive ISH signals in epidermal sites corresponding to visible lesion development.
In contrast, cattle infected with leaderless virus showed no clinical disease at 72 h and no pulmonary changes at either 24 or 72 h. These animals had only limited positive virus-specific ISH signals in respiratory bronchioles by 24 h and had no evidence of lesions or ISH signals in epithelial tissue by 72 h Thus, the leaderless virus did not appear to replicate well at the site of primary infection and was not able to spread to other sites within the host.
The latter observation can be attributed to the inability of the leaderless virus to inhibit host translation, including IFN synthesis, and the production of IFN within the infected animal probably inhibited initial amplification and spread of the virus. In contrast, wild-type virus infection blocks capped IFN mRNA translation, allowing the virus to rapidly spread to neighboring cells and systemically prior to the induction of the adaptive immune response.
This outbreak was unusual in that only pigs, and not cattle, were affected, and the disease had an unusually high mortality rate in pigs , Molecular characterization of the virus revealed that it contained a codon deletion in the C-terminal half of the 3A protein The location of this deletion was similar to that of a to codon deletion found in FMDV passaged in chicken embryos.
This virus also exhibited reduced virulence in bovines , The role of this deletion in the bovine-attenuated phenotype was confirmed by using reverse genetic analysis 47 , and an analysis of viruses circulating in the region for the last 30 years suggested that in addition to the deletion, mutations in the 3A protein in the region surrounding the deletion may also be responsible for the observed phenotype The molecular basis for the porcinophilic phenotype appears to be related to a reduction in viral RNA synthesis, which is manifested to a greater degree in bovine cells than in swine cells The 3A proteins from either the porcinophilic or a bovine-virulent isolate colocalized to RNA replication complexes in either bovine or porcine cells and also caused a disruption of the Golgi apparatus However, as yet, there is no clear picture as to why the 3A deletion should affect FMDV replication in bovine cells more than in swine cells.
Nunez and coworkers have also shown that a single amino acid change in the 3A protein was responsible for adaptation of FMDV to guinea pigs; however, the mutation was located in a different region than the deletion associated with the porcinophilic phenotype It has been suggested that IRES elements, and possibly the host factors that bind to them, affect the pathogenicity and virulence of other picornaviruses , , , , , In FMDV, a virus rescued from persistently infected BHK cells had two mutations within the IRES, which the authors suggest might have resulted in increased virulence of the virus in tissue culture O'Donnell, E.
Pilipenko, E. Roos, and P. Mason, Abstr. Study Group Mol. Picornaviruses, abstr. K11, The virus elicits a rapid humoral response in either infected or vaccinated animals.
Virus-specific antibodies protect animals in a serotype-specific manner against reinfection, or against infection in the case of vaccination, and protection is generally correlated with high levels of neutralizing antibodies reviewed in references and In cattle, the immunoglobulin G1 IgG1 response predominates over IgG2 86 , , , and antibody, including IgA, can be detected in upper respiratory secretions early in infection , The neutralization of virus within the host may occur by mechanisms similar to those occurring in in vitro neutralization; however, there is a suggestion that macrophages may play a role in clearing the virus from the infected animal by phagocytosis of opsonized virus , , The role of cellular immunity in the protection of animals from FMD is still a matter of some controversy.
The induction of anti-FMDV antibody correlates with a lymphoproliferative response in cattle and swine , and is T-cell dependent in mice In addition, T-cell function, as measured by response to mitogens, is either reduced or eliminated Both the number of lymphocytes and the altered T-cell function return to normal levels by 4 days after infection.
These results suggest that T cells play a role in virus protection and that the reduction of both T-cell numbers and function enhances viral pathogenesis by allowing the virus to spread within the host, leading to increased viral shedding into the environment. In addition, immunization of both cattle and swine with a replication-competent human adenovirus 5 Ad5 vector expressing the P1 capsid precursor did not result in the generation of virus-specific neutralizing antibody in the serum but partially protected animals from FMDV challenge , The swine developed FMDV-specific T-cell responses , but these assays were not performed on the immunized cattle.
These results may further indicate a role for cellular immunity in protection from FMDV infection; however, it also possible that innate immune responses may be responsible for the protection seen in these studies.
There has been increasing interest recently concerning the role of the innate immune response of the host to both FMDV infection and vaccination. In addition to the IFNs, other cytokines may also play a role in the host response. In studies of swine which were immunized with a conventional FMD vaccine, it was shown that vaccinated pigs did not appear to exhibit a systemic inflammatory response, but chemotactic activity of plasma on peripheral blood leukocytes increased within the first week after immunization Although the levels of IL-6 and IL-8 did not appear to be related to protection of pigs upon challenge, IL levels were higher in vaccinated pigs, which were protected from contact challenge, suggesting a role for cytokine-induced monocytic cell activity in protection from acute-phase disease Following the acute phase of FMDV infection in ruminants, some animals may experience a long asymptomatic persistent infection.
In addition, animals which have been successfully vaccinated may also become persistently infected if exposed to infectious virus.
These animals are referred to as carrier animals, and the carrier state is a complication which can occur during outbreak situations. In this section we briefly discuss what is known about the carrier state.
For more in-depth information, a number of excellent reviews on this topic have been written 7 , , Van Bekkum and colleagues first demonstrated that live FMDV could be recovered from esophageal-pharyngeal fluids of cattle during the convalescent phase of FMD Currently, carrier animals are defined as those from which live virus can be isolated at 28 days, or later, after infection In domestic cattle, the carrier state can last as long as 3.
African buffalo have been reported to carry live virus for up to 5 years , and other cloven-hoofed wildlife may become carriers see reference 7 and references therein. The number of carrier animals in a population depends on the species, the incidence of infection, and the immune state of the herd i.
In general, the titer of virus in the esophageal-pharyngeal fluids of carrier animals is low, and virus is not consistently recovered from individual animals. Currently, virus isolation from esophageal-pharyngeal fluids is the most sensitive method to detect carrier animals, but reverse transcription-PCR RT-PCR assays are being developed to attempt to increase sensitivity. The recovered virus probably originates in the pharynx, which appears to be the target region for persistent infection in cattle 7 , , The role of carrier animals in the spread of virus in the field is still controversial.
The only direct evidence is that of transmission from African buffalo to cattle during outbreaks in Zimbabwe in the late s and early s In addition, transmission from buffalo to cattle has been obtained experimentally , and it has recently been proposed that such transmission might occur through sexual contact There has been no experimental evidence to date indicating that carrier cattle or sheep can transmit virus to uninfected animals.
The presence of live virus in esophageal-pharyngeal fluids, however, does make this a real possibility. In addition, the long persistence and replication of the virus in the host animals can lead to genetic variation in the field, possibly being responsible for the generation of new viral variants , , The mechanisms for the establishment and maintenance of the carrier state are not well understood, since persistence can occur in animals exposed to virus after either acute disease or vaccination.
It does appear that the immune status of the animal probably controls the level of virus replication 7. Alexandersen and colleagues 7 have proposed two mechanisms for the development of persistence in the pharynx. One suggests that FMDV can infect immune system cells, such as macrophages, or other immunologically privileged sites, leading to evasion of the immune response.
Baxt and Mason 42 examined viral replication in porcine peripheral blood macrophages and found that virus can infect such cells only when presented as an immune complex, presumably by Fc receptor-mediated adsorption.
Furthermore, the infection was abortive and did not lead to the production of new infectious virus. In a more recent study, Rigden and colleagues found that porcine alveolar macrophages also were not able to support viral replication; however, the virus bound to macrophages in the absence of specific antibody.
In addition, virus appeared to be internalized by phagocytosis but remained infectious for at least 12 h. The second mechanism proposes that the virus exploits the host response to provide favorable intracellular conditions for long-term persistence, possibly by utilizing cytokine signaling. Since the beginning of the 20th century, FMD has been of considerable concern to many countries, and outbreaks or the fear of disease incursions have led to the establishment of institutes to investigate methods to control the disease.
In the late 19th and early 20th centuries, FMD outbreaks occurred sporadically in Europe, but their occurrence had devastating consequences By the early s however, some countries in Western Europe were experiencing 10 4 to 10 5 outbreaks per year At that time, disease control consisted of inhibition of animal movement, slaughter of infected animals, and disinfection. As a result of a concerted effort in the s, especially by Waldmann and colleagues in Germany , an inactivated FMD vaccine was developed Galan A.
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