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The aim of this study was to identify respiratory viruses circulating amongst elite racehorses in a training yard by serological testing of serial samples and to determine their impact on health status and ability to race. A six-month longitudinal study was conducted in 30 Thoroughbred racehorses (21 two-year-olds, five three-year-olds and four four-year-olds) during the Flat racing season. Sera were tested for the presence of antibodies against equine herpesvirus 1 and 4 (EHV-1 and EHV-4) and equine rhinitis viruses A and B (ERAV and ERBV) by complement fixation (CF) and equine arteritis virus (EAV) by ELISA. Antibodies against equine influenza (EI) were measured by haemagglutination inhibition (HI). Only ERAV was circulating in the yard throughout the six-month study period. Seroconversion to ERAV frequently correlated with clinical respiratory disease and was significantly associated with subsequent failure to race (p = 0.0009). Over 55% of the two-year-olds in the study seroconverted to ERAV in May and June. In contrast, only one seroconversion to ERAV was observed in the older horses. They remained free of any signs of respiratory disease and raced successfully throughout the study period. The importance of ERAV as a contributory factor in the interruption of training programmes for young horses may be underestimated., Competing Interests: The authors declare no conflict of interest.

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: To develop a method for experimental induction of equine rhinitis A virus (ERAV) infection in equids and to determine the clinical characteristics of such infection., Animals: 8 ponies (age, 8 to 12 months) seronegative for antibodies against ERAV. PROCEDURES-Nebulization was used to administer ERAV (strain ERAV/ON/05; n = 4 ponies) or cell culture medium (control ponies; 4) into airways of ponies; 4 previously ERAV-inoculated ponies were reinoculated 1 year later. Physical examinations and pulmonary function testing were performed at various times for 21 days after ERAV or mock inoculation. Various types of samples were obtained for virus isolation, blood samples were obtained for serologic testing, and clinical scores were determined for various variables., Results: ERAV-inoculated ponies developed respiratory tract disease characterized by pyrexia, nasal discharge, adventitious lung sounds, and enlarged mandibular lymph nodes. Additionally, these animals had purulent mucus in lower airways up to the last evaluation time 21 days after inoculation (detected endoscopically). The virus was isolated from various samples obtained from lower and upper airways of ERAV-inoculated ponies up to 7 days after exposure; this time corresponded with an increase in serum titers of neutralizing antibodies against ERAV. None of the ponies developed clinical signs of disease after reinoculation 1 year later., Conclusions and Clinical Relevance: Results of this study indicated ERAV induced respiratory tract disease in seronegative ponies. However, ponies with neutralizing antibodies against ERAV did not develop clinical signs of disease when reinoculated with the virus. Therefore, immunization of ponies against ERAV could prevent respiratory tract disease attributable to that virus in such animals.

Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.

Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses. Candidate vaccines to date have been hindered by low expression levels and the induction of non-neutralising antibodies. The immunodominant epitope of ERAV is conformational and is located within the quaternary structure of the capsid. This site should be retained in ERAV virus-like particles (VLPs) to stimulate the induction of neutralising antibodies. The immunogenicity of a plasmid-based DNA vaccine designed to express ERAV VLPs was assessed. The plasmid construct, pcD.P12A.3C, contained the capsid precursor (P1-2A) and the viral protease 3C, under the transcriptional control of a cytomegalovirus (CMV) promoter. Mature viral capsid proteins and VLPs were detected in vitro in transfected COS7 cells. Immunisation of BALB/c mice with pcD.P12A.3C induced virus neutralising antibodies and enhanced the virus neutralising antibody response to purified, UV-inactivated ERAV. This study further supports the use of DNA vaccines to elicit neutralising antibodies to complex antigenic proteins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Equine rhinitis A virus (ERAV) is a picornavirus which causes an acute respiratory infection in horses worldwide, and virus neutralization (VN) has been the standard method for the detection of ERAV antibody in horse serum. Previous studies have identified recombinant virion protein VP1 (rVP1) purified under native conditions to be of high potential for the development of a diagnostic ERAV enzyme-linked immunosorbent assay (ELISA). This study presents an optimized protocol for the expression and purification of native full-length rVP1. Furthermore, we demonstrate that, upon denaturation, rVP1 no longer reacts to ERAV antibody in a prototype ELISA. Size exclusion chromatography (SEC) performed on native and denatured rVP1 indicates that denatured rVP1 forms multimeric aggregates that may causally connect to the loss of antigenicity observed in the ELISA.

The objective of this study was to determine the incidence of serum neutralising (SN) antibody to ERAV, ERBV1 and ERBV2 in a population of horses from birth to 22 years of age. The prevalences of ERAV, ERBV1 and ERBV2 SN antibodies in 381 sera obtained from 291 horses were 37%, 83% and 66%, respectively. ERAV, ERBV1 and ERBV2 maternal antibody was present in foals 12 h postsuckling but by 10-12 months, ERAV SN antibody was not detected in any of the horses, while ERBV1 and ERBV2 SN antibodies were common (83% and 100%, respectively). Sera were obtained from 44 Thoroughbred horses when they were newly introduced into a training centre when their average age was 23 months and a second sample was obtained approximately 7 months later. ERAV SN antibody was present in 8 (18%) when first bled and in 27 (61%) when tested 7 months later. Accordingly 19 of the 44 horses (43%) seroconverted to ERAV within 7 months of entering the training stable. Among all the horses the average ERAV SN antibody titre was relatively high (3796) and in contrast, ERBV1 and ERBV2 titres were relatively low (average 84 and 45, respectively) and often fell to below detectable levels over time and at a rate comparable to new seroconversions in the same group of horses.

Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

In the present report, we characterized the immune response and the protection conferred by recombinant baculoviruses or infected insect cells expressing the fusions gp64-P1 and gp64-site A FMDV antigens. Mice, vaccinated intraperitoneally with gp64-P1 immunogens, showed a low-antibody response and a variable degree of protection. However, when mice received recombinant baculoviruses or infected insect cells expressing the fusion protein gp64-site A, high-ELISA and seroneutralizing titers (SNT) against FMDV were elicited. All mice immunized with Sf9 cells expressing FMDV site A developed a protective immune response against challenge with virulent FMDV, indicating that the baculovirus display of foreign epitopes is a promising approach to biosynthetic vaccines.

The use of foot-and-mouth disease (FMD) vaccines that do not induce antibodies against non-structural proteins (NSP) is extremely relevant for the demonstration of regions "free of FMDV infection" and control strategies. In this study cattle were primed and boosted with five doses of oil vaccines containing high antigenic payloads on days 0, 90, 130, 160 and 200. The serological response against NSP was measured using four commercially available assays: two 3ABC-ELISAs; one 3B-ELISA (and complementary 3A-ELISA) and an enzyme-immunotransfer blot assay (EITB). Additionally, locally produced NSP antibodies detection reagents and VIAA antibodies were evaluated. A high level of specific immune response against vaccine strains was shown. After four doses of vaccine, non-reactive animals were detected by any of the NSP assays. After the fifth immunization, 2 of 17 animals were reactive in one ELISA kit, but these samples proved negative by confirmatory tests. Antibodies against NSP were not detected in single dose immunized cattle. The principle of the NSP-ELISA used as a screening test for large sero-surveys in South America is established and this paper emphasizes the importance of using vaccines that have demonstrated no interference with NSP antibodies detection assays.

Mouse monoclonal antibodies (mAbs) were employed to select neutralization escape mutants of equine rhinitis A virus (ERAV). Amino acid changes in the ERAV mutants resulting in resistance to neutralization were identified in capsid protein VP1 at Lys-114, Pro-240 and Thr-241. Although the changes were located in different parts of the polypeptide chain, these mutants exhibited cross-resistance against all four mAbs employed, indicating that these residues contribute to a single immunogenic site. To explain this result, we constructed a model of the three-dimensional structure of the ERAV capsid based on comparison with the closely related foot-and-mouth disease virus (FMDV O(1)). According to this model, VP1 is folded so that Lys-114 is in the beta E-beta F loop of the polypeptide chain at a considerable distance from Pro-240 and Trp-241 in the C-terminal region. However, around the fivefold axis of symmetry, the C terminus of VP1 in each protomer extends to the beta E-beta F loop of the adjacent VP1 in the next protomer. We therefore propose that the immunogenic site in ERAV is formed as a result of the close proximity of the Lys-114 residue in the beta E-beta F loop of one VP1 molecule and of the Pro-240/Thr-241 residues in the adjacent VP1 polypeptide chain. In terms of the overall architecture of the viral capsid structure, this site in ERAV most closely resembles the immunogenic site 1 of FMDV O(1).

Equine rhinitis A virus (ERAV) is an important respiratory pathogen of horses and is of additional interest because of its close relationship and common classification with foot-and-mouth disease virus (FMDV). As is the case with FMDV, the VP1 capsid protein of ERAV has been shown to be a target of neutralizing antibodies. In FMDV VP1, such antibodies commonly recognize linear epitopes present in the betaG-betaH loop region. To map linear B cell epitopes in ERAV VP1, overlapping fragments spanning its length were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. These fusion proteins were tested for reactivity with sera from ERAV-infected horses and with polyclonal sera from ERAV-immunized rabbits and mice. Regions at the N- and C-termini as well as the betaE-betaF and the betaG-betaH loop regions contained B cell epitopes that elicited antibodies in the natural host. GST fusion proteins of these regions also elicited antibodies following immunization of rabbits and mice, which, in general, strongly recognized native ERAV VP1 but which were non-neutralizing. It is concluded that the N-terminal region of ERAV VP1, in particular, contains strong B cell epitopes.

A plasmid DNA vaccine candidate (pCEIS) encoding two foot-and-mouth disease virus (FMDV) VP1 epitopes (amino acid residues 141-160 and 200-213) has been demonstrated to have the ability to elicit both FMDV-specific T cell proliferation and neutralizing antibody against FMD in swine. In this study, the efficiency of the pCEIS DNA vaccine when administrated by intramuscularly injection in swine was confirmed, and the immunogenicity of the pCEIS vaccine candidate was found to be enhanced through co-administration with a newly constructed plasmid (pIL2S) encoding the swine interleukin-2 (IL-2) cDNA. The expression of the pIL2S plasmid was driven by a CMV promotor provided by a pcDNA3.1 vector. Swine IL-2 cDNA was cloned by RT-PCR from swine spleen cells. The pIL2S plasmid was expressed in COS-7 cells after 24 and 96h of transfection in vitro. In an animal trial, results from T cell proliferation assay indicated that the stimulation index (SI) in response to stimulation of FMDV proteins in the swine groups injected with pCEIS plus pIL2S (SI ranging from 9.9 to 15.5) were significantly higher than that with pCEIS alone (SI ranging from 3.3 to 6.6). However, there was no significant difference in FMDV-neutralizing antibody level detected in these two swine groups. Mouse protection tests (MPTs) showed that the blood sera from immunized swine injected with either pCEIS alone or pCEIS plus pIL2S were able to protect suckling mice from FMDV challenge, with protection levels ranging from 10(1) to 10(2) lethal dose 50 (LD(50)) M. In a direct FMDV challenge, all swines immunized with either pCEIS plus pIL2S or with pCEIS alone were challenged with 50LD(50)S (50 x lethal dosage in swine) of FMDV. The animals were fully protected (100%) from the FMD viral challenge. These results suggest that co-administration of the plasmids, pCEIS and pIL2S, enhances of the immunogenicity of the pCEIS DNA vaccine candidate, and both intramuscular injection of pCEIS alone and co-administration of the vaccine candidate with pIL2S can protect the swine from direct FMD challenge.

The epidemiological role of small ruminants in foot-and-mouth disease (FMD) outbreaks has been generally neglected. Although, the disease in these species is sub-clinical in nature, their role as virus carriers represents a reservoir for further infection and spread of disease. Data on the usefulness of polyvalent FMD vaccine (FMDV) in goats is scant. Thus, the present study was undertaken to evaluate the benefits of a highly potent polyvalent FMDV in goats. In the present investigations, FMDV quadrivalent double oil emulsion (Montanide ISA 206) vaccines were tested in goats at reduced doses of 2 ml per animal (antigen payload 3.5 microg per serotype per dose). The oil adjuvant elicited superior immune response at any given period than aluminium hydroxide gel (AGS) vaccine and the rapidity of development of response was quicker. The duration of immunity also appeared to be maintained for long period. The differences in immune response between two adjuvant groups were statistically significant (P<0.05). The differences were apparent even in kinetics of immune response. Unlike cattle, goats were found to be late responders for oil-adjuvanted vaccine. Our results indicate possible universal usage of double oil emulsion vaccines for disease control programs irrespective of species of animals.

The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralization epitopes of these viruses.

Foot-and-mouth disease (FMD) is the most contagious animal virus disease of cloven-hoofed livestock and requires reliable and accurate diagnosis for the implementation of measures to control effectively its spread. Routine diagnosis of FMD is carried out at the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright by the combined use of ELISA and virus isolation in cell culture supplemented by reverse transcription polymerase chain reaction (RT-PCR) methods. These techniques require skilled personnel and dedicated laboratory facilities which are expensive. The development of a rapid and simple test for the detection of FMD virus antigen using Clearview chromatographic strip test technology for field application is described. This device detected FMD viral antigen in nasal swabs, epithelial suspensions and probangs from clinical samples submitted from the field, from animals infected experimentally and in supernatant fluids resulting from their passage in cell culture. The test system was more sensitive than ELISA for the diagnosis of all seven serotypes of FMD virus in the epithelial suspensions and nasal swabs and had equivalent sensitivity to the ELISA for the detection of contemporary virus strains in cell culture supernatant fluids. The study demonstrated the potential for this device to confirm a clinical diagnosis at the site of a suspected FMD outbreak, thereby offering the possibility of implementing control procedures more rapidly. Such pen-side diagnosis would have particular benefits in FMD emergencies, relevance to FMD control programmes which operate in endemic regions of the world such as South East Asia and for increasing disease awareness in other areas where efforts to control disease may be difficult. In each circumstance the availability of a pen-side device for diagnosis would reduce the necessity for sending routine diagnostic samples to an FMD laboratory and thereby reduce the delay in diagnosis, which can in some areas be considerable.

The recent outbreak of foot-and-mouth disease (FMD) in the United Kingdom is a stark reminder of the economic devastation that this disease can wreak. Tracing the origin of such an outbreak is an essential part of disease control. Modern molecular methods have been in place for a number of years to enable scientists to identify unambiguously the strain of virus responsible. However, tracing the precise origin of such a strain is not so straightforward because the virus can move rapidly around the world with legal and illegal trade in animals and animal products. This short review describes the virus, its control and epidemiology.

Subunit vaccination is effective in eliciting humoral responses to a variety of viral antigens, however, it has not generated persistent protective immunity to foot-and-mouth disease virus (FMDV). In this study, we observed that priming mice with a DNA plasmid encoding VP1 of the FMDV O/Taiwan/97 capsid protein followed by boosting with a VP1 peptide conjugate (P29-KLH) resulted in production of not only high titers of antibodies but also antibodies with FMDV neutralizing activities. Moreover, the mice immunized in this manner cleared the virus from their sera in FMDV challenge experiments. Mice subjected to DNA plasmid priming and P29-KLH protein boosting had relatively higher ratio of IgG2a/IgG1 than those primed and boosted with P29-KLH conjugate. Addition of an oligodeoxynucleotide (ODN) containing immunostimulatory cytosine-phosphate-guanosine (CpG) motifs to P29-KLH conjugate also induced a higher ratio of IgG2a/IgG1 and significantly higher titer of neutralizing antibodies. These results indicate that treating animals with DNA plasmids priming and FMDV antigen(s) boosting may elicit immunity to FMD and this immune response may be augmented by CpG ODN.

Foot-and-mouth disease (FMD) is one of the most dangerous diseases of cloven-hoofed animals and is a constant threat in the Middle-East and other regions throughout the world despite intensive vaccination programs. In this work, we describe the ability of FMDV expression constructs to protect pigs from FMDV challenge when used as a vaccine. The construct consists of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), the entire P1 and 2A together with 3CD sequences, all in the same reading frame. Another plasmid that was tested, carries the serotype O1 (G) VP1, Asia1 VP1 and O1 (G) 3C. Between each of the genes the 3C cleavage sequences were inserted. All constructs carried the cytomegalo virus (CMV) promoter. Using immunofluorescent and immunoblot techniques, we could show the expression and processing of viral proteins. Following the application of FMDV expression constructs into pigs skin by 'Gene Gun', pigs were partially protected from FMDV challenge.

This work focuses on the development of a potential recombinant DNA vaccine against foot-and-mouth disease virus (FMDV). Such a vaccine would have significant advantages over the conventional inactivated virus vaccine, in particular having none of the risks associated with the high security requirements for working with live virus. The principal aim of this strategy was to stimulate an antibody response to native, neutralizing epitopes of empty FMDV capsids generated in vivo. Thus, a plasmid (pcDNA3.1/P1-2A3C3D) was constructed containing FMDV cDNA sequences encoding the viral structural protein precursor P1-2A and the non-structural proteins 3C and 3D. The 3C protein was included to ensure cleavage of the P1-2A precursor to VP0, VP1 and VP3, the components of self-assembling empty capsids. The non-structural protein 3D was also included in the construct in order to provide additional stimulation of CD4(+) T cells. When swine were immunized with this plasmid, antibodies to FMDV and the 3D polymerase were synthesized. Furthermore, neutralizing antibodies were detected and, after three sequential vaccinations with DNA, some of the animals were protected against challenge with live virus. Additional experiments suggested that the antibody response to FMDV proteins was improved by the co-administration of a plasmid encoding porcine granulocyte-macrophage colony-stimulating factor. Although still not as effective as the conventional virus vaccine, the results encourage further work towards the development of a DNA vaccine against FMDV.

Foot-and-mouth disease virus (FMDV) isolate C(1)-Barcelona (or C-S30) includes four replacements within its immunodominant site (GH loop, residues 136-150 of capsid protein VP1, YTTSTRGDLAHVTAT), relative to reference strain C-S8c1 (YTASARGDLAHLTTT). Although one of the mutations in C-S30 (147Leu-->Val) is known to be detrimental for antibody recognition, reactivity of this isolate with the neutralizing monoclonal antibody (mAb) 4C4, raised against FMDV C1-Brescia (GH loop: YTASTRGDLAHLTAT), was indistinguishable from those of strains C-S8c1 or C1-Brescia. A structural interpretation for these somewhat striking findings is available, based on the observation that 15-residue peptides reproducing the C-S30 and C-S8c1 GH loops adopt very similar, quasi-circular, conformations in crystal complexes with 4C4. Nevertheless, surface plasmon resonance (SPR) kinetic analyses of the interactions between these peptides and three anti-GH loop mAbs have now revealed that the linear C-S30 peptides were less antigenic in solution than their C-S8c1 and C1-Brescia counterparts. We have, therefore, tried to modulate peptide antigenicity in solution by cyclization. Functional SPR and structural two dimensional proton nuclear magnetic resonance (2D-1H NMR) studies of both linear and cyclic peptide antigens are discussed here. Conformation seems to have an important role in peptide antigenicity, even when continuous (i.e. linear) antigenic sites are involved.

The peptide of amino acids 141-160 of VP1 protein of foot-and-mouth disease virus (FMDV) is a major B cell epitope and the peptide of amino acids 21-40 is an important T cell epitope. In this study, the DNA fragments of 141-160 and 21-40 peptide epitopes of a strain of type O FMDV was chemically synthesized and arranged into a tandem repeat 141-160 (20AA)-21-40 (20AA)-141-160 (20AA). This tandem sequence was fused to the 3' end of the heavy chain constant region gene of swine immunoglobulin G and was then cloned into mammalian expression vector pCDM8 to form a recombinant plasmid pCDM8FZ3. After pCDM8FZ3 was inoculated intramuscularly into guinea pigs, it elicited a neutralizing antibody response and a specific spleen T cell proliferative response, and 66% of the vaccinated animals were protected from viral challenge. Our study indicated that the heavy chain constant region of swine IgG can act as the carrier protein for FMDV peptide epitopes, and pC-DM8FZ3 is a potential DNA vaccine candidate to prevent FMDV infection.

Analyses on diluted sera would not measure the complete activity of the natural in-vivo serum environment used by humoral immune responses. Since these humoral defences must react rapidly, serum reactions occurring during 10 and 60 s were analysed. Primo- and multiple-vaccinated, efficiently responsive, and protected animals were differentiated. A variation in the responses, particularly the quality of response, of individual animals was also now discernible. The critical importance of antibody-antigen contact time was demonstrated, wherein natural immunoglobulins and non-immunoglobulin opsonins would influence specific antibody reactivity, through an increased avidity of reaction with antigen. Although not measured directly, the influence of the non-specific serum components would be manifest through increased specific antibody binding. By considering serum as an entity, analysis of all constituent components permits increased qualitative assessment of post-vaccination sera.

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.

Nucleotide sequence and phylogenetic analysis of the VP1 structural protein have been used extensively as diagnostic and epidemiological tools for foot and mouth disease virus (FMDV). In this report we have applied this methodology to the analysis of the VP1 coding sequence from FMDV strains isolated in Argentina during 1993-1994. The results demonstrated that the field isolates were related to the vaccine strains used at that time. However the involvement of the vaccine virus appeared to be different for outbreaks caused by FMD viruses type O or C. These data provide a database essential for determining the origin of new epizootics.

A replication-defective adenovirus 5 encoding foot-and-mouth disease virus (FMDV) capsid and 3C proteinase coding regions (Ad5-FMDV3CWT) was used to vaccinate swine. A single inoculation utilizing 1 x 10(8) plaque forming units (pfu) or an inoculation of 1 x 10(8) followed by a boost of 5 x 10(8) pfu Ad5-FMDV3CWT were tested, along with an inoculation and boost using an adenovirus encoding the FMDV capsid coding region and an inactive form of the 3C proteinase (Ad5-FMDV3CMUT). Sera collected from these animals were examined for the presence of FMDV-specific antibodies using immunoprecipitation, neutralization, and ELISA assays specific for IgM, IgG1 and IgG2. Efficacy studies were performed by placing the vaccinated swine in contact with an FMDV-infected swine and monitoring for signs of disease and changes in serum antibody levels. Ad5-FMDV3CMUT, which is unable to produce FMDV capsid structures, did not elicit FMDV-neutralizing antibodies or protect against FMD. Single inoculation with Ad5-FMDV3CWT generated FMDV-specific neutralizing antibodies, and reduced clinical signs in challenged swine, but failed to completely protect the majority of swine from FMD. Swine which received a primary vaccination with Ad5-FMDV3CWT followed by the boost at 4 weeks generated high levels of FMDV-neutralizing antibodies resulting in complete protection of five of the six swine and limited disease in the remaining animal. Increased efficacy of the two-dose regimen was associated with heightened levels of FMDV-specific IgG1 and IgG2 antibodies.

The International Vaccine Bank (IVB) based at the Institute of Animal Health (IAH) in Pirbright, United Kingdom (UK), routinely monitors the suitability of the currently held strains of foot-and-mouth disease (FMD) vaccine virus, in anticipation that vaccine may be required to control FMD outbreaks that pose a threat to member countries. Using primarily the two-dimensional micro-neutralisation test (VNT), bovine polyclonal sera raised against each of the seven current 'emergency' antigens were utilised to measure the relationship of IVB stocks to selected field isolates. The 'O' serotypes, Manisa and Lausanne, exhibited adequate levels of cross-protection against most of the type 'O' field isolates examined. A(22) Iraq 24/64 showed the broadest spectrum of reactivity against the type 'A' field isolates examined and was supplemented by A(15) Thailand 1/60. Some type 'Asia1' field isolates, particularly those from South East Asia, showed antigenic difference to the Asia1 India 8/79 vaccine strain by VNT, but in-vivo testing in the guinea pig model indicated this to be insignificant. The only 'C' serotype representative, C(1) Oberbayern, may be one of the least antigenically diverse of the current portfolio of bank antigens. Comparison of the serological and sequence data shows that despite significant genetic variation between the field isolates examined the antigens held by the IVB should still prove efficacious in the field.

An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type specific antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than -2.5Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type specifically and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples confirmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections.

A serological survey was carried out on French cattle to establish a reference pattern of residual vaccine antibodies and non-specific reactions against the foot-and-mouth disease virus 6 years after the ban on vaccination and in the absence of any foot-and-mouth disease outbreak. Most of the multi-vaccinated cattle still displayed high titres of antibodies and up to 50% of those which had received a single injection still had antibodies. Non-specific reactors were also recorded among animals born during and after 1991. Most of them displayed low titres close to the threshold. Sheep were also tested and, as for cattle, 4.6% of non-specific reactors were recorded, with titres close to the threshold for two-thirds of them. As part of these animals have been resampled and retested, sera revealed negative confirming that these animals are true non-specific reactors. Serological testing as a mean of FMD control should take these facts into account.

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Foot-and-mouth disease virus (FMDV) was the first animal virus identified. Since then, FMDV has become a model system in animal virology and a considerable amount of information on its structure, biology and vaccinology has been obtained. However, the disease that this virus produces (FMD) still constitutes one of the main animal health concerns. In this review, we have attempted to summarise the state of the knowledge in different basic and applied areas of FMDV research, with emphasis on those aspects relevant to the control of the disease.

The nature of selection on capsid genes of foot-and-mouth disease virus (FMDV) was characterized by examining the ratio of nonsynonymous to synonymous substitutions in 11 data sets of sequences obtained from six different serotypes of FMDV. Using a method of analysis that assigns each codon position to one of a number of estimated values of nonsynonymous to synonymous ratio, significant evidence of positive selection was identified in 5 data sets, operating at 1-7% of codon positions. Evidence of positive selection was identified in complete capsid sequences of serotypes A and C and in VP1 sequences of serotypes SAT 1 and 2. Sequences of serotype SAT-2 recovered from a persistently infected African buffalo also revealed evidence for positive selection. Locations of codons under positive selection coincide closely with those of antigenic sites previously identified with the use of monoclonal antibody escape mutants. The vast majority of codons are under mild to strong purifying selection. However, these results suggest that arising antigenic variants benefit from a selective advantage in their interaction with the immune system, either during the course of an infection or in transmission to individuals with previous exposure to antigen. Analysis of amino acid usage at sites under positive selection indicates that this selective advantage can be conferred by amino acid substitutions that share physicochemically similar properties.

We have analysed the influence of bovine MHC (BoLA) polymorphism on the immune response and degree of protection induced by peptide vaccines against foot-and-mouth disease (FMD) in cattle. The peptides used for animal immunisation were: A (VP1(138-156)), AT (peptide A linked to VP1(21-40)) and ACT (peptide A, linked to VP1(196-209) and VP1(21-40)). Sixteen different DRB3 types were found among the 46 cattle analysed by PCR-RFLP typing. No absolute correlation was observed, for any type, with the serum neutralising titres (SNT) values and the protection induced. However, among the most common haplotypes present, associations were observed between expression of different types with the levels of SNT and/or protection induced by peptides A and ACT. Thus, types DRB3.2*1, 3 and 7 were associated with increased levels of protection. In contrast, types DRB3.2*12 and 18 were associated non-protection, and DRB3.2*12 was also associated with low SNT titres. Overall, the results indicate that the polymorphism in BoLA class II molecules affects both the immune response and protection induced by potential FMD peptide vaccines.

Foot-and-mouth disease virus (FMDV) type OR(2)/75, grown on BHK 21 clone 13 cell monolayers, was inactivated with formalin. The virus was clarified and was either concentrated with 8% polyethylene glycol 6000 (PEG) or used in its untreated form for the preparation of oil adjuvant vaccines. The oil adjuvants used in this study were Montanide ISA 206 (which renders a water-in-oil-in-water (w/o/w) type of emulsion), Montanide ISA 57 and Montanide ISA 50V (both of which render water-in-oil (w/o) type of emulsions). The vaccines were tested on guinea pigs and calves. The results indicated that vaccines emulsified using Montanide ISA 57 elicited the best protective immune response in the animals, followed by those emulsified with Montanide ISA 50V and Montanide ISA 206. It was also found that vaccines formulated with virus concentrated using 8% polyethylene glycol (PEG) were more immunogenic than the vaccines formulated with the untreated harvest virus.

VP1 is a capsid protein of foot-and-mouth disease virus (FMDV) and contains epitopes of the virus. Plasmids encoding two VP1 epitopes (amino acid residues 141-160 and 200-213) and a host-self immunoglobulin molecule were constructed to produce a new type of FMD DNA vaccine. Two plasmids, namely, pCEIM and pCEIS, containing mouse immunoglobulin (IgG) or swine IgG were subjected to immunogenicity testing in mice and swine, respectively. In mice administrated pCEIM in the abdomen using a genegun, both FMDV-specific T-cell proliferation and neutralizing antibodies were detected. In swine immunized with pCEIS at the back of the ear, immune responses were achieved after the second administration. Swine showed a T-cell proliferative response with a stimulation index (SI) of up to 8.1 and a neutralizing antibody response that was able to protect suckling mice from 10(2) LD(50) (lethal dose 50) FMDV challenge. To compare the immunogenicity of the DNA-based vaccine candidate, versus the protein-based vaccine candidates, a second group of swine was immunized with the protein F1-scIgG, which was encoded by the plasmid pCEIS. Injection with F1-scIgG elicited a T-cell proliferative response of SI < 1.7 and a neutralizing antibody response that protected suckling mice from up to 10(5) LD(50) FMDV challenge. In the challenge test, three of three swine immunized with pCEIS were fully protected from FMDV challenge., (Copyright 2000 Academic Press.)

A foot-and-mouth disease virus (FMDV) field variant, isolate C-S30 (also named C(1)-Barcelona), is known to contain four changes within the main antigenic site A (GH loop of capsid protein VP1, residues 136-150), at least one of which (Leu147-->Val) involves a highly conserved position, critical for antibody recognition in the reference strain C-S8c1. However, immunoenzymatic analysis of FMDV C-S30 showed it was recognised by 4C4, a monoclonal antibody that specifically targets site A. This remarkable behaviour has led us to analyse the individual and combined contributions of the four mutations to the antigenicity of C-S30, by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) studies of pentadecapeptides displaying all possible combinations of the four replacements. Analysis of this family of C-S30-derived analogues shows a certain level of antibody recognition by SPR. In addition, SPR data suggest that kinetic rate constants provide an indirect measure, on the one hand, of paratope accessibility (association rate constant) and, on the other hand, of peptide fitness to the same paratope (dissociation rate constant).

Epitopes containing the residues 141aa-160aa and 200aa-213aa from foot-and-mouth disease (FMD) serotype O1K HK type FMDV VP1 were joined to a swine immunoglobulin G single heavy chain constant region (scIgG), creating a novel chimeric protein, named F1-scIgG. In this study, inoculation with F1-scIgG induced both FMD virus-neutralizing antibody response and T cell response in swine. Antisera from these F1-scIgG-inoculated swine protected suckling mice against 1000 lethal dose 50 (1000LD(50)) FMD challenge. F1-scIgG-inoculated swine were also fully protected against 50LD(50) FMD virus challenge. The present study demonstrates the clear potential for viral epitopes linked with self-Ig in novel FMD vaccine design.