Efficacy of a high quality O1/Campos foot-and-mouth disease vaccine upon challenge with a heterologous Korean O Mya98 lineage virus in pigs.

In 2010 serotype O foot-and-mouth disease virus of the Mya98 lineage/SEA topotype spread into most East Asian countries. During 2010-2011 it was responsible for major outbreaks in the Republic of Korea where a monovalent O/Manisa vaccine (belonging to the ME-SA topotype) was applied to help control the outbreaks. Subsequently, all susceptible animals were vaccinated every 6 months with a vaccine containing the O/Manisa antigen. Despite vaccination, the disease re-occurred in 2014 and afterwards almost annually. This study focuses on the in vivo efficacy in pigs of a high quality monovalent commercial O1/Campos vaccine against heterologous challenge with a representative 2015 isolate from the Jincheon Province of the Republic of Korea. Initially, viral characterizations and r1 determinations were performed on six viruses recovered in that region during 2014-2015, centering on their relationship with the well characterized and widely available O1/Campos vaccine strain. Genetic and antigenic analysis indicated a close similarity among 2014-2015 Korean isolates and with the previous 2010 virus, with distinct differences with the O1/Campos strain. Virus neutralisation tests using O1/Campos cattle and pig post vaccination sera and recent Korean outbreak viruses predicted acceptable cross-protection after a single vaccination, as indicated by r1 values, and in pigs, by expectancy of protection. In agreement with the in vitro estimates, in vivo challenge with a selected field isolate indicated that O1/Campos primo vaccinated pigs were protected, resulting in a PD50 value of nearly 10. The results indicated that good quality oil vaccines containing the O1/Campos strain can successfully be used against isolates belonging to the O Mya98/SEA topotype.

Antigenic and immunogenic spectrum of foot-and-mouth disease vaccine strain O1 Campos against representative viruses of topotypes that circulated in Asia over the past decade.

Identifying vaccine strains to control outbreaks of foot-and-mouth disease virus that could spread to new regions is essential for contingency plans. This is the first report on the antigenic/immunogenic relationships of the South American O1/Campos vaccine strain with representative isolates of the three currently active Asian type O topotypes. Virus neutralization tests using O1/Campos post-vaccination sera derived from cattle and pigs predicted for both species acceptable cross-protection, even after single vaccination, established by r1 values and by expectancy of protection using monovalent or polyvalent vaccines. The results indicate that effective oil vaccines containing the O1/Campos strain can be used against Asian isolates, expanding the scope of O1/Campos strain included in vaccine banks to control emergencies caused by Asian viruses, even on single-dose vaccination, and to cover the need of effective vaccines in Asia during systematic vaccination.

Development and characterization of a bovine serum evaluation panel as a standard for immunoassays based on detection of antibodies against foot-and-mouth disease viral non-capsid proteins.

The widespread perception of the effectiveness of applying tests based on the detection of antibodies against foot-and-mouth disease (FMD) viral non-capsid proteins (NCPs) to assess virus circulation irrespective of vaccination triggered the demand for international standards to evaluate the comparative performance of the upcoming assays against the OIE Index test developed at the Pan American Foot-and-Mouth Disease Center, PAHO/WHO. To this end, a panel was developed composed of 34 cattle sera from animals with an unambiguous exposed/infected status, covering serotypes O, A and C, obtained either under experimental conditions or from the field in regions with different epidemiological situations. Reference values in the Index test and their reproducibility in other laboratories, data on stability as well as results in four other commercial kits and one in house test were obtained. The characteristics of the panel which comprise adequate preparation following international guidelines, a broad range of antibody reactivity, proper stability and the ability to assess comparative diagnostic sensitivity, make it suitable as a reference standard to evaluate if tests equivalent to the OIE Index method are used in support of FMD control programs and by trading partners, and also whether they maintain their standards of diagnostic performance.

Development of an inhibition ELISA test for the detection of non-capsid polyprotein 3ABC in viral suspensions destined for inactivated foot-and-mouth disease vaccines.

The use during the last decade of immuno-enzymatic tests based on the detection of antibodies to the non-capsid proteins (NCPs) of foot-and-mouth disease virus (FMDV) to assess viral circulation, irrespective of vaccination, supported the incorporation into the OIE code of the 'free from FMDV with vaccination' category and opened the way to a 'vaccination to live' policy. Eradication programmes in South America include systematic vaccination accompanied by large serosurveys through NCP antibody testing to ensure the absence of residual viral activity. For correct interpretation of serosurveys, a major prerequisite is that vaccines made of semi-purified preparations of inactivated virions do not contain levels of NCPs, which upon proper presentation conditions, could induce an antibody response under the conditions for field immunization. This work describes the development of an inhibition ELISA to detect NCP polyprotein 3ABC in viral suspensions destined for vaccine production as an in-process control during vaccine manufacture. Antibody responses against NCP 3ABC in vaccinated and revaccinated cattle, induced by vaccines with different purification processes and formulations, are discussed.

Comparative evaluation of six ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus.

To validate the use of serology in substantiating freedom from infection after foot-and-mouth disease (FMD) outbreaks have been controlled by measures that include vaccination, 3551 sera were tested with six assays that detect antibodies to the non-structural proteins of FMD virus. The sera came from naïve, vaccinated, infected and vaccinated-and-infected animals; two-thirds from cattle, the remainder from sheep and pigs. The assays were covariant for sensitivity, but not necessarily for specificity. A commercial kit from Cedi-diagnostics and an in-house assay from IZS-Brescia were comparable to the NCPanaftosa-screening index method described in the Diagnostic Manual of the World Animal Health Organisation. Using these three tests the specificity and sensitivity for the detection of carriers in vaccinated cattle approaches or exceeds 99% and 90%, respectively.

Non-capsid proteins to identify foot-and-mouth disease viral circulation in cattle irrespective of vaccination.

The ability of foot-and-mouth disease virus (FMDV) to establish subclinical and even persistent infection, the so called carrier state, imposes the need to reliably demonstrate absence of viral circulation, to monitor the progress of control measures, either during eradication programs or after reintroduction of virus in free areas. This demonstration becomes critical in immunized populations, because of the concern that silent viral circulation could be hidden by immunization. This concern originates from the fact that vaccination against foot-and-mouth disease (FMD) protects against clinical disease, but not necessarily against subclinical infection or establishment of the carrier state in cattle. A novel approach, developed and validated at PANAFTOSA during the 1990s, based on an immunoenzymatic system for detection of antibodies against non-capsid proteins (NCP) has proven valuable for monitoring viral circulation within and between herds, irrespective of the vaccination status. Antibodies against NCP are induced during infection but, in principle, not upon vaccination. The validation of this system led to its international recognition as the OIE index test. The fitness of this serosurvey tool to assess viral circulation in systematically vaccinated populations was demonstrated through its extensive application in most regions in South America. The experience attained in these regions supported the incorporation of the "free of FMD with vaccination" provisions into the OIE code. Likewise, it opened the way to alternatives to the "stamping out" policy. The results gave input to an old controversy related to the real epidemiological significance, if any, of carrier animals under the vaccination conditions in South America, and supported the development of recommendations and guidelines that are being implemented for serosurveys that go with control measures in vaccinated populations.

Vaccination: foot-and-mouth disease experience in South America.

Vaccination against foot-and-mouth disease (FMD) constitutes an important component of the policy for its control and eradication in South America. Considering that immunization may not impair subclinical infection, it became advisable to ally to vaccination campaigns a surveillance instrument to monitor silent viral circulation. Novel approaches for the evaluation of antibodies to FMD non-capsid proteins (NCPs), developed and validated at PANAFTOSA proved valuable for assessing viral circulation in immunized populations. The extensive and coordinated application in South America of vaccination together with this serosurvey tool indicated the effectiveness of systematic vaccination to prevent FMD spread and to restrain silent viral circulation intra- and inter- herds, and gave input to an old controversy related to the real epidemiological significance, if any, of carrier animals under the vaccination conditions in South America. The fitness of NCP tests to assess viral circulation in a population supported the incorporation into the OIE code of the "free of FMD with vaccination" category as a step prior to the recognition of the "free of FMD without vaccination" category. Likewise it released the path to allow animals, vaccinated for protective purposes during emergencies, to live for the term of their productive lives.

Vaccines and companion diagnostic tests for foot-and-mouth disease virus. An overview of the experience in South America.

Vaccination constitutes an important control policy for foot-and-mouth disease (FMD) in affected areas with advanced eradication programmes, as well as in free regions that decide to use immunization as a control measure after a recent introduction of the disease. However, considering that vaccinated animals exposed to FMD virus can establish sub-clinical infection and eventually remain persistently infected, availability of tools to identify sub-clinical infection and its silent transmission within and between herds, regardless of their vaccination state, is of utmost importance. In response to the need for new diagnostic tools to support the eradication campaigns implemented in 1988 in South America, during the past decade we have developed, validated and applied a highly sensitive and specific immuno-enzymatic system for recognition of persistence at a herd level. The system is based on the detection of antibodies against non-capsid proteins required for viral replication. These proteins, in principle, are removed from the viral suspensions destined for production of BEI inactivated vaccines. Within the validation steps, evaluation of potential induction of antibodies to non-capsid proteins caused by traces of these proteins eventually remaining in the vaccines was a major concern. This report presents a review on the experience gathered through the application of the system to various experimental and field immunization conditions. It was concluded that vaccination is not expected to induce antibody responses to non-capsid proteins that could lead to misinterpretation of serological investigations. Progress on the development of approaches towards vaccine certification to guarantee absence of interference will be discussed.

Rapid serological profiling by enzyme-linked immunosorbent assay and its use as an epidemiological indicator of foot-and-mouth disease viral activity.

Frequency distribution of reactivity levels of foot-and-mouth disease infection-specific antibodies in livestock populations was analysed. Specific antibody responses against non-capsid polyprotein 3ABC were assessed through a highly sensitive indirect enzyme-linked immonosorbent assay (I-ELISA 3ABC). A graphic display of data was designed based on three negative and three positive categories to illustrate reactivity patterns. The resulting patterns were correlated to the epidemiological status. On this basis, results of over 100,000 sera derived from cattle populations in regions with various well-documented epidemiological situations were compiled and are exemplified in this paper.Distinct distributions of antibody reactivity patterns reflecting the various epidemiological situations were attained. Whereas non-affected areas presented a rather homogenous negative pattern with very limited test-positive reactions, affected regions revealed quite heterogeneous profiles, including positive and negative categories, with distributions that varied according to the region. The use of graphic prints encompassing I-ELISA 3ABC antibody profile responses constituted an adequate epidemiological indicator of the risk of foot-and-mouth disease viral activity, providing immediate visualization for a rapid inference of the epidemiological situation of a region. Moreover, such profiles allowed for convenient follow-up of infection after a focus as a function of time and geographical spread.

Uso de una proteína recombinante del virus de la fiebre aftosa, la polimerasa 3D, en una prueba de inmunodifusión en gel de agar

En este estudio, se examinó la efectividad del uso del polipéptido 3D recombinante, obtenido en su forma nativa en una prueba de IDGA (IDGA-3D), para uso en la detección de anticuerpos específicos de infección con VFA, independientemente de la condición de vacunación. Los resultados indican que en relación a la tradicional prueba de IDGA-VIAA, la IDGA 3D ofrece, particularmente cuando se evalúan sueros de bajo título, un método más consistente, con especificidad comparable, y por lo menos la misma sensibilidad. Ninguno de los antígenos ofreció una ventaja particular con respecto a la definición de las bandas de precipitación. El reemplazo del VIAA por la proteína 3D recombinante tiene considerables atracciones, dado que proporciona un suministro ilimitado de material inocuo, económico, de fácil purificación y consistente, eliminando la presencia potencial de antígenos no específicos de células BHK o componentes de la cápside del VFA.

Improvement of a serodiagnostic strategy for foot-and-mouth disease virus surveillance in cattle under systematic vaccination: a combined system of an indirect ELISA-3ABC with an enzyme-linked immunoelectrotransfer blot assay.

Foot-and-mouth disease (FMD) recombinant non-capsideal viral antigens 3A, 3B, 2C, 3D and 3ABC were assessed individually in an indirect enzyme-linked immunosorbent assay (I-ELISA) for their ability to screen for persistent infection-specific antibodies in cattle, regardless of vaccination condition. Results of sequential serum samples from non-vaccinated animals with experimentally induced persistent infection, and their correlation with virus isolation, indicated that the polypeptides 3A, 3B and 3ABC showed the most adequate characteristics for further field studies. Reliable performance of the I-ELISA with the selected antigen 3ABC was indicated by the distinct patterns observed for the frequency distribution values of naive and true positive samples. For regularly vaccinated livestock, a clear negative profile was proved in samples from regions without recent history of FMD. In contrast, at 90 and 900 days post-outbreak, coexistence of a positive and a negative population was established. These findings indicated that, irrespective of vaccination, the test allowed a classification of the herd-disease status. A high degree of agreement was observed between I-ELISA-3ABC and EITB results for clearly reactive and non-reactive sera. For samples with reactivity values close to that of the cut-off, the EITB profiles upheld the definition of the infection condition. On this basis, screening by I-ELISA-3ABC, together with confirmation of suspect or positive samples by EITB is proposed as an adequate and accurate approach for large-scale epidemiological surveillance.

Identification of foot-and-mouth disease virus-free regions by use of a standardized enzyme-linked immunoelectrotransfer blot assay.

OBJECTIVE: To assess the potential of a highly sensitive enzyme-linked immunoelectrotransfer blot (EITB) assay to monitor persistent foot-and-mouth disease (FMD) viral activity in a livestock population. DESIGN: Cattle sera were obtained in Uruguay in 1992, 2 years after the last outbreaks of FMD. Prevalence of antibodies, as assessed by the EITB assay and by the conventional immunodiffusion in agarose gel method (virus infection-associated antigen [VIAA] test), was correlated with occurrence of FMD. SAMPLE POPULATION: A total of 2,194 serum samples were acquired from animals at different farms and were separated according to age: animals < and > 2 years old. PROCEDURE: Specific antibodies to replicating virus were detected by use of the EITB assay that utilizes a set of 5 bioengineered nonstructural antigens as serologic probes. RESULTS: EITB-positive reaction was restricted to sera from cattle in areas with the last outbreaks of FMD during 1989-1990, and to animals > 2 years old. All cattle sera from regions that were free of clinical FMD since (or prior to) 1989 were EITB negative. In contrast, use of the VIAA test yielded a rather homogeneous distribution of positive results when regions without FMD during the last 4 years preceding sample collection were compared with those affected during 1989-1990. VIAA test-positive reaction was also found in sera from animals born after the last FMD outbreak. CONCLUSIONS: The EITB assay proved to be a sensitive, specific, safe, rapid, and economic tool for monitoring the progress of FMD eradication programs, mainly because it eliminated false-positive results form the VIAA test.

Detection of foot-and-mouth disease viral sequences in various fluids and tissues during persistence of the virus in cattle.

OBJECTIVE: To assess whether foot-and-mouth disease virus (FMDV)-specific sequences could be identified in tissues from persistently virus-infected animals. DESIGN: Cattle with experimentally induced persistent FMDV infections were slaughtered at 750 days after viral exposure. Experimentally infected pigs were slaughtered at 28 days after FMDV inoculation. Postmortem specimens were asceptically removed. ANIMALS: Three bovids and 3 pigs were studied, as well as 1 control animal for each species. PROCEDURE: Various tissues were examined for the presence of FMDV-specific sequences by dot-blot hybridization assay, using a molecularly cloned FMDV cDNA corresponding to the polymerase coding region. RESULTS: The FMDV-specific genomic sequences were only detected in RNA from spleen, lung, larynx, tonsils, pancreas, liver, esophagus, and WBC of bovids. CONCLUSIONS: It was established that, at late stages of the persistent infection, when virus isolation was not possible, cattle may carry FMDV-specific sequences in different tissues. Retention of viral sequences could not be demonstrated in specimens from experimentally infected swine, 28 days after viral inoculation.

Desempeño de un ensayo inmunoenzimático de electrotransferencia rápido para la detección de bovinos expuestos al virus de la fiebre aftosa

Se evaluó la efectividad de un ensayo inmunoenzimático de electrotransferencia (EITB) para la detección de anticuerpos contra antígenos de replicación del virus de la fiebre aftosa (VFA) en bovinos expuestos al mismo. El ensayo EITB, que utiliza como sondas serológicas antígenos virales no estructurales producidos por bioingeniería y altamente purificados, se comparó con la prueba tradicional de inmunodifusión en gel de agar (prueba VIAA), que emplea un antígeno asociado a la infección viral parcialmente purificado. Se evaluó la sensibilidad y especificidad da ambas pruebas mediante el estudio de 110 sueros de bovinos naturalmente expuestos, y con comprobado estado de infección, así como de sangrías seriadas de 25 animales infectados experimentalmente, y de 1.899 sueros de animales de regiones libres de FA(1.058 de los cuales provenían de bovinos vacunados sistemáticamente). Además se realizaron estudios en 43 bovinos para evaluar la sensibilidad de estas pruebas para reconocer animales persistentemente infectados. Los resultados indicaron que, comparado con la prueba VIAA, el ensayo EITB es más sensible y específico para detectar bovinos expuestos al VFA, aun durante la infección persistente, y especialmente en regiones con vacunación sistemática. Por lo tanto, se recomienda el uso del EITB como una prueba sensible, segura, rápida y económica para la detección específica de exposición viral en el campo.

Genetic variation of foot-and-mouth disease virus during persistent infection in cattle.

Genetic variation of foot-and-mouth disease virus O1 Campos has been analyzed in consecutive isolates recovered over a one- or two-year period from four cattle with experimental persistent infection. Comparisons of RNase T1 two-dimensional maps and nucleotide sequences of the VP1-coding region revealed a continual, although irregular, increase in the fixation of mutations as the infection progressed. Most changes were not conserved in consecutive isolates. These results, together with the substantial rates of genomic variation observed between some pairs of strains recovered at close time periods, suggested the coexistence of heterogeneous populations in which variants evolve independently from each other, and predominate at irregular time intervals. Furthermore, non-related patterns of variation were observed in the four animals. Similarly, genetic diversity of representative strains from major serotype O outbreaks in endemic disease regions of southeastern Brazil and central eastern Argentina which occurred between 1958 and 1983, suggested that outbreak strains are also likely to represent fluctuations of heterogeneous populations which evolve independently from each other. The possible role of persistent infections in the introduction of variant populations in the field is discussed.

Caracterización serológica y molecular de cepas del virus de la fiebre aftosa, serotipo O, aisladas a partir de brotes ocurridos entre 1958 y 1983 en Brasil y Argentina

La caracterización genética de cepas del virus de la fiebre aftosa representativas de brotes importantes del serotipo O, ocurridos entre 1958 y 1983 en el sudeste de Brasil y el centro-este de Argentina, fue obtenida usando mapas de oligonucleótidios resistentes a T1. Los resultados obtenidos constituyen la base de un banco de datos para ser aplicado en estudios epidemiológicos.

Genetic characterization of representative foot-and-mouth disease virus strains from major serotype O outbreaks which occurred between 1958 and 1983 in southeastern Brazil and centraleastern Argentina was obtained using T1 maps. The results presented constitute the basis for a data bank to be applied in epidemiological studies.

Enfoques moleculares para el diagnóstico en laboratorio de la infección persistente con el virus de la fiebre aftosa: revisión

Se describen nuevos enfoques para mejorar los métodos de indentificación de infecciones persistentes con el virus de la fiebre aftosa (VFA). Los mismos consistieron en la detección de fragmentos genómicos especificos del VFA en muestras obtenidas de la región esofágico-faríngea por medio de técnicas de hibridación, aplicadas con o sin previa amplificación del material mediante la reacción en cadena de la polimerasa. Asimismo, para la identificación de anticuerpos específicos contra el VFA indicativos de replicación viral, se llevó a cabo el desarrollo de un método inmunoenzimático altamente sensible y específico basado en el uso como sondas serológica de antígeno no estructurales purificados del VFA, obtenidos por técnicas de ADN recombinante.

Detección de secuencias genómicas del virus de la fiebre aftosa mediante la hibridación de ácidos nucleicos: un enfoque para el diagnóstico de infección persistente

Se ha desarrollado un ensayo de hibridación capaz de detectar secuencias genómicas del virus de la fiebre aftosa (VFA) en muestras de cultivos de células infectadas o de fluidos esofágico-faríngeo (OP). El ensayo se basa en el uso de una sonda marcada de ADN complementaria a la región que codifica para la polimerasa del VFA. la sonda se obtuvo amplificando en E. coli un plásmido conteniendo el fragmento de ADN complementario (cADN) a la región de 6,3 a 7,8 kilobases del genoma del VFA. Debido al alto grado de homología de la secuencia de ácidos nucleicos en esta parte del genoma, fue posible la detección de los tres serotipos sudamericanos en un único ensayo de hibridación. Los resultados positivos obtenidos con muestras de OP de animales experimentalmente infectados extraídas a tiempos tardíos de la infección, durante los cuales la recuperación de virus era principalmente negativa, revelaron el considerable potencial de este método como complemento de los procedimientos convencionales de diagnóstico de infección persistente.