Ingrid E. Bergmann

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.

Summary. 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.

Expression of the aphthovirus rna polymerase gene in Escherichia coli and its use together with other bioengineered nonstructural antigens in detection of late persistent infections

A plasmid has been constructed containing the DNA sequences that direct the expression of the aphthovirus RNA-dependent RNA polymerase (virus infection-associated antigen, VIAA) in its native form. The aphthovirus polypeptide was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant protein has been purified and used in enzyme-linked immunoelectrotransfer blots to detect aphthovirus-specific antibodies in the sera of persistently infected animals. Furthermore, studies were carried out to test the hypothesis that antibodies against other nonstructural antigens appear in the sera of these animals. It was established that antibodies against polypeptides 3A and 3B can serve as complementary markers for late aphthovirus-carrier state detection. The considerable potential of this approach to detect aphthovirus-specific antibodies, when the isolation of infectious virus is not possible, was demonstrated. Negative results were obtained in animals from virus-free areas and in vaccinated cattle. This assay has the added advantage that no infectious or noninfectious virus is involved during antigen production.

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.

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

Summary. 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.

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.

Biochemical characterization of an aphthovirus type C3 strain resende attenuated for cattle by serial passages in chicken embryos

We have compared several aspects of an aphthovirus strain attenuated for cattle (C3R-O/E) with the original strain (C3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C3Res. A diminution in the rate of RNA synthesis induced by C3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.

Characterization of foot-and-mouth disease virus from outbreaks in Ecuador during 2009-2010 and cross-protection studies with the vaccine strain in use in the region.

During the years 2009 and 2010 relevant epidemic waves of foot-and-mouth disease (FMD) serotype O occurred in Ecuador, representing a great drawback for the last stages of the ongoing eradication program in South America. This study describes the molecular and antigenic characterizations of 29 isolates collected from various regions in the country and their relationship to the vaccine strain. The phylogenetic tree derived from sequences spanning the complete VP(1) protein showed that, despite the widespread origin of the viruses, they were all related among themselves and to previous isolates occurring in 2008, with around 10% difference with the vaccine strain O1/Campos. The high level of sequence conservation among different isolates in the various regions of Ecuador pointed to a common origin, suggesting animal movements as possible sources of viral spread. Monoclonal antibody profiling grouped the isolates in two major reactivity patterns which differed from that of the vaccine strain. Both profiles showed loss of reactivity with the same four MAbs, three of them with neutralizing properties. Additional sites were lost in the profile representing most of the 2010s viral samples. Levels of protective antibodies induced by the vaccine against the field strains assessed by in vitro vaccine matching studies also pointed to an increased temporal pattern of loss of a protective response. Moreover, results obtained with in vivo challenge in the protection against podal generalization test in cattle, clearly indicated lack of appropriate protection of the Ecuadorian field strains by the vaccine virus in use, which in the case of a 2010 variant was observed even after revaccination.

Evolving perception on the benefits of vaccination as a foot and mouth disease control policy: contributions of South America

Within the past decade, changes in perceptions on the benefits of vaccination as an appropriate tool to achieve complete foot and mouth disease eradication have become evident. The former negative view was derived from misconceptions, resulting mainly from the belief that vaccines are not entirely effective and that vaccination masks asymptomatic viral circulation. The advent in the 1990s of vaccination policies implemented within a strategic eradication plan in South America, and during recurrence of the disease in disease-free regions contributed towards generating more reliable and visible outcomes of vaccination programs, paving the way towards a new perception. Particularly relevant was the development and application of novel serodiagnostic approaches to assess silent viral circulation, irrespective of vaccination. The use in South America of vaccination allied to serosurveys to accompany viral clarification during eradication campaigns and after emergencies clearly established the importance of this control tool to stop the spread of viral infection. This alliance gave input to break many myths associated with the use of vaccines, including the belief that immunized carrier animals pose an epidemiologic risk.This experience launched new concepts that supported the internationally recognized status of foot and mouth disease-free regions with vaccination and the ‘vaccination to live’ policy as an alternative to ‘stamping out’.

Characterization of a type O foot-and-mouth disease virus re-emerging in the year 2011 in free areas of the Southern Cone of South America and cross-protection studies with the vaccine strain in use in the region.

Molecular, antigenic and vaccine matching studies, including protective response in vivo, were conducted with a foot-and-mouth disease type O virus isolated during the outbreak in September 2011 in San Pedro, Paraguay, country internationally recognized as free with vaccination in 1997. The phylogenetic tree derived from complete VP(1) sequences as well as monoclonal antibody profiling indicated that this isolate was related to viruses responsible for previous emergencies in free areas of the Southern Cone of South America occurring sporadically between the years 2000 and 2006. Marked differences with the vaccine strain O(1)/Campos, including the loss of reactivity with neutralizing MAbs, were recognized. Levels of protective antibodies induced by the vaccine containing the O(1)/Campos strain against the San Pedro virus and the virus responsible for the previous emergency in 2006 in the Southern Cone assessed by in vitro vaccine matching studies pointed to an insufficient protective response 30 days after vaccination (DPV), which was properly attained at 79 DPV or after revaccination. In agreement with the in vitro assessment, the in vivo challenge in the Protection against Podal Generalization test in cattle indicated appropriate protection for the San Pedro strain at 79 DPV or after revaccination. The complementary conclusions that can be derived from vaccine matching tests designed differently to fit the various objectives intended: prophylaxis, emergency vaccination or incorporation of new field strains into antigen banks, is evaluated. This is the first report of the antigenic and immunogenic characterization of the variants responsible for emergencies in the Southern Cone of South America and the putative impact of the changes on the cross protection conferred by the vaccine strain.

Heterogeneity of the polyribocytidilic acid tract in aphthovirus: changes in the size of the poly(C) of viruses recovered from persistently infected cattle

A sample of aphthovirus type C3 strain Resende carrying two polyribocytidilic acid [poly(C)] tracts was cloned in tissue culture. One clone with a poly(C)-rich tract of about 145 nucleotides long (clone 3B) and another with a poly(C)-rich tract of about 230 nucleotides long (clone 12) and a mixture of both were injected intralingually into three steers. Samples from all three animals were recovered during the acute phase of the disease, from the blood and from the feet, and at various days after inoculation from the oesophageal-pharyngeal (OP) fluids. Analysis of the viral RNAs of the positive samples by means of RNase T1 maps on one- and two-dimensional gels showed (1) changes in the electrophoretic mobility of the poly(C)-rich tracts of viruses recovered from the OP fluids at various times after infection; (2) selection of virus populations with poly(C)-rich tracts of increased size; (3) later on, changes in the patterns of oligonucleotides of persistent viruses. These variations may lead to the production of new strains with altered biological properties that may contribute to the maintenance and spread of these viruses in the field.