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.

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.

Phylogenetic analysis of foot-and-mouth disease virus type O re-emerging in free areas of South America.

The nucleotide sequences of the complete VP(1)-coding region of foot-and-mouth disease viruses (FMDV), type O, isolated during the recent emergencies of the disease in free areas of South America (Mato Grosso do Sul, Brazil, October 2005, and Corrientes, Argentina, February 2006), were determined. Also established were the complete VP(1)-coding sequences of viruses occurring in neighbouring locations between the years 2000 and 2003. A phylogenetic analysis was performed based on comparison with continental relevant field and vaccine strains, as well as with extra-continental representative viruses.

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.

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.

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.

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.

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.

Markers of human T lymphotropic virus type I in patients with cancer of uterine cervix in Amazon, Brazil.

Human T lymphotropic virus type I(HTLV-I) has been implicated in various human diseases. Serum samples of 390 Brazilian Amazonians with cancer of various types were tested for HTLV-I antibodies by Gelatin particle agglutination test, Enzyme linked immunosorbent assay and Western blotting. Of 134 sera from patients with cancer of uterine cervix, 4 were positive by all the methods.

HTLV-I and HTLV-II infections among HIV-1 seropositive patients in Sao Paulo, Brazil.

To estimate the presence of, and the risk factors for HTLV-I and HTLV-II infections among HIV-1 infected subjects in Sao Paulo, Brazil, a serosurvey was performed in 471 HIV-1 infected patients, including 216 intravenous drug addicts (IVDA), 229 homosexual/bisexual men, and 26 with other risk factors. Serum samples were screened for HTLV seroreactivity by ELISA; reactive samples were analyzed by Western Blot (WB), using whole HTLV-I lysate as antigen. To confirm and discriminate HTLV-I and HTLV-II infections, sera presenting any bands on WB were further analyzed by a WB containing recombinant HTLV-I and HTLV-II proteins (WB 2.

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.

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.