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.

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. Three of these were from non-transfused patients. DNA was extracted from 2 of 4 seropositive sera that gave strong reactions and were analyzed by PCR-SSCP for HTLV-I sequences. One was positive for all HTLV-I genes tested while the other one was positive for LTR and tax and negative for gag. In view of a possible pathway of the virus by sexual contact, the involvement of HTLV-I in cervical cancer warrants further studies.

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.

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.

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.

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.

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.

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 2.3), and by enzyme immunoassays using synthetic peptides specific for envelope proteins (Synth-EIA). In 22 cases, cell samples were available for polymerase chain reaction (PCR) studies. On WB, 114 sera were reactive and, of these, 37 and 25 were concordantly positive on both WB 2.3 and Synth-EIA procedures for HTLV-I and HTLV-II specific antibodies, respectively; 37 specimens were negative on both assays, and 15 gave discordant or indeterminate results. PCR findings confirmed concordant results obtained in the discriminatory serological assays. The prevalence rates of HTLV-I and HTLV-II infections were 15.3% and 11.1% in IVDA, and 0.9% and 0.4% in homosexual/bisexual men, respectively. No case of HTLV-I/HTLV-II co-infection was found.

PIP: HTLV-I is associated with adult T-cell leukemia/lymphoma and a neurological disorder known as HTLV-I-associated myelopathy or tropical spastic paraparesis. HTLV-II was initially isolated from subjects with a T-cell variant of hairy cell leukemia, but its etiological role in that or other diseases is unclear. HTLV infections, like HIV, are transmitted sexually, via blood transfusion and contaminated needles, and from mother to infant. Many reports indicate that HTLVs are present in the same populations at risk for HIV-1, and the cofactorial role of HTLVs in AIDS progression has been suggested by in vitro studies and epidemiological data. The authors report findings from a serosurvey conducted among 216 HIV-seropositive male and female intravenous drug users (IVDU), 229 HIV-seropositive homosexual and bisexual men, and 7 HIV-seropositive men and women who had had multiple transfusions, and 19 HIV-seropositive heterosexual men with multiple partners to estimate the presence of and the risk factors for HTLV-I and HTLV-II infections among HIV-1 infected individuals in Sao Paulo, Brazil. 70.9% of the subjects were classified according to CDC criteria as having AIDS. ELISA, Western blot, and polymerase chain reaction methods were used. The prevalence rates of HTLV-I and HTLV-II infections were 15.3% and 11.1% in IVDUs, and 0.9% and 0.4% in homosexual and bisexual men, respectively. No case of HTLV-I/HTLV-II co-infection was observed.