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.

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 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.

[Biological research and security institutes]. / Institutos de investigación y seguridad biológica.

The threat of using biological material for ago-bioterrorist ends has risen in recent years, which means that research and diagnostic laboratories, biological agent banks and other institutions authorised to carry out scientific activities have had to implement biosafety and biosecurity measures to counter the threat, while carrying out activities to help prevent and monitor the accidental or intentional introduction of exotic animal diseases. This article briefly sets outthe basic components of biosafety and biosecurity, as well as recommendations on organisational strategies to consider in laboratories that support agro-bioterrorist surveillance and prevention programs.

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.

Expression of foot and mouth disease virus non-structural polypeptide 3ABC induces histone H3 cleavage in BHK21 cells.

Auto-processing of the non-structural polypeptide 3ABC of foot and mouth disease virus (FMDV) expressed in Escherichia coli-BL21-DE3 was prevented by mutating either four glutamic acid residues at the 3A/3B1, 3B1/2, 3B2/3 and 3B3/3C junctions (3ABCtet) or a single cysteine residue at position 383 within the 3C domain (3ABCm). Independent expression of 3ABC and 3ABCtet genes induced expression of chaperone DnaK and degradation of ribosomal S1 protein in E. coli. They also induced cleavage of nucleosomal histone H3 when transiently expressed in BHK21 cells. 3ABCtet, 3ABCm, 3AB and 3A proteins concentrated in the perinuclear region suggesting that peptide sequences within the 3A domain specify intracellular targeting of 3ABC in BHK-21 cells. We propose that 3ABC molecules localized in the nuclear periphery are a source of protease 3C activity and are responsible for histone H3 processing during FMDV infections.

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.

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.

[The role of reference laboratories in animal health programmes in South America]. / El papel de los laboratorios de referencia en los programas de sanidad animal en América del Sur.

The contribution of the Panamerican Foot and Mouth Disease (FMD) Centre (PANAFTOSA), as an OIE (World organisation for animal health) regional reference laboratory for the diagnosis of FMD and vesicular stomatitis, and for the control of the FMD vaccine, has been of fundamental importance to the development, implementation and harmonisation of modern laboratory procedures in South America. The significance of the work conducted by PANAFTOSA is particularly obvious when one considers the two pillars on which eradication programmes are based, namely: a well-structured regional laboratory network, and the creation of a system which allows technology and new developments to be transferred to Member Countries as quickly and efficiently as possible. Over the past decade, PANAFTOSA has kept pace with the changing epidemiological situation on the continent, and with developments in the international political and economical situation. This has involved the strengthening of quality policies, and the elaboration and implementation of diagnostic tools that make for more thorough epidemiological analyses. The integration of PANAFTOSA into the network of national laboratories and its cooperation with technical and scientific institutes, universities and the private sector means that local needs can be met, thanks to the design and rapid implementation of methodological tools which are validated using internationally accepted criteria. This collaboration, which ensures harmonisation of laboratory tests and enhances the quality of national Veterinary Services, serves to promote greater equity, a prerequisite for regional eradication strategies and this in turn, helps to increase competitiveness in the region.

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.

Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens.

A highly sensitive enzyme-linked immunoelectrotransfer blot (EITB) assay, capable of detecting aphthovirus-specific antibodies to replicating virus in sera from cattle with persistent infection, was developed. The assay uses a set of purified recombinant DNA-derived nonstructural viral antigens as serologic probes in lieu of the traditionally used virus infection-associated antigen(s) partially purified from baby hamster kidney-infected cells. Sera from cattle with experimentally induced aphthovirus infection were analyzed sequentially by EITB at various postinoculation days, and the results were compared with those obtained by currently used techniques. It was established that, in all cases, EITB results remained positive at late stages of infection. At these times, results of virus infection-associated antigen-antibody determinations were negative by use of the conventional immunodiffusion in agarose gel test, and virus was recovered only occasionally from esophageal-pharyngeal fluid. Specificity of the EITB test was indicated by negative results for sera from cattle in aphthovirus-free areas, including samples from cattle infected with a variety of bovine viruses. Moreover, the test eliminated a substantial number of false-positive results (on the basis of the immunodiffusion in agarose gel assay) caused by reactivity of sera from vaccinated cattle. Use of additional nonstructural viral antigens, other than RNA polymerase, is proposed to differentiate between seropositivity resulting from vaccination or infection. This procedure may be considered to have potential applications as a sensitive, safe, rapid, and economic field test for specific diagnosis of persistent aphthovirus infection in affected animals.

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.

Control of breakdown of the polyadenylate sequence in mammalian polyribosomes: role of poly(adenylic acid)-protein interactions.

The poly(adenylic acid) [poly (A)] segment in mouse sarcoma polysomes in not hydrolyzed by snake venom exonuclease under conditions which cause extensive degradation of the poly(A) in deproteinized polysomal RNA. The protecting effect of polysomes is presumably caused by the interaction between the poly(A) sequence and the protein known to be associated with it. This protection is reduced at low KCl concentration, but addition of exogenous RNA restores the protecting effect. The poly(A) segment also becomes susceptible to exonuclease after fragmentation of the polysomes by mild ribonuclease treatment. The latter treatment releases the poly(A) in association with protein. The poly(A) sequence in polysomes in readily degraded by a cytoplasmic extract of S-180 cells. Partial purification leads to a preparation active against the poly(A) in polysomes under conditions where no fragmentation of the messenger RNA is observed. Snake venom exonuclease increases the activity of the cytoplasmic preparation against poly(A) in polysomes. The active cytoplasmic factor appears to interfere with the poly(A)-protein interaction, thus rendering the polynucleotide susceptible to degradation by exonuclease. The poly(A) sequences in polysomes and in free cytoplasmic nucleoprotein particles are hydrolyzed to the same extent. The results suggest that the poly(A) sequence is normally protected from nucleases by virtue of its association with protein. The slow reduction in poly(A) size in cytoplasmic mRNA can be accounted for by a factor capable of interfering with the poly(A)-protein interaction. The latter interaction seems also dependent on the structural integrity of the polysomes or messenger ribonucleoproteins. It is suggested that a polynucleotide segment adjacent to the poly(A) can modulate the affinity of the protein for the latter sequence, thus permitting control of poly(A) stability in individual messenger RNAs.

Comparison of the 5' and 3' untranslated genomic regions of virulent and attenuated foot-and-mouth disease viruses (strains O1 Campos and C3 Resende).

The complete 5' and 3' non-coding regions of two attenuated South American foot-and-mouth disease virus (FMDV) vaccine strains, O1C-O/E and C3R-O/E, and their corresponding virulent parental strains, O1 Campos and C3 Resende, have been cloned from polymerase chain reaction-amplified primary cDNA. Differences observed in the derived nucleotide sequences between attenuated and virulent viruses seem not to affect regulatory signal structures, supporting the theory that genetic variations, primarily in the 3' halves of the viral genomes, contribute to the attenuation phenotype of the vaccine strains. In addition, this is the first report on the complete sequence of the 5' untranslated region of a C-type aphthovirus. Approximately 10% of the nucleotides differ from the corresponding known sequences of serotypes A or O.