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Discriminating foot-and-mouth disease virus-infected and vaccinated animals by use of beta-galactosidase allosteric biosensors.

ABSTRACT: Recombinant beta-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant beta-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant beta-galactosidases, particularly those including one copy of each of the two 3B sequences, were efficiently reactivated by sera from infected pigs. We found reaction conditions that allowed differentiation between sera of FMDV-infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals.

SUBMITTER: Sanchez-Aparicio MT

PROVIDER: S-EPMC2725531 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Discriminating foot-and-mouth disease virus-infected and vaccinated animals by use of beta-galactosidase allosteric biosensors.

Sánchez-Aparicio M Teresa MT Rosas María Flora MF Ferraz Rosa Maria RM Delgui Laura L Veloso Juan J JJ Blanco Esther E Villaverde Antonio A Sobrino Francisco F

Clinical and vaccine immunology : CVI 20090624 8

Recombinant beta-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant beta-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant beta-galactosidases, particularly those including one copy of each of ...[more]

PMID: 19553549

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Project description:In foot-and-mouth disease (FMD)-endemic countries, vaccination is commonly used to control the disease, whilst in FMD-free countries, vaccination is considered as an option, in addition to culling the infected and in contact animals. FMD vaccines are mainly comprised of inactivated virions and stimulate protective antibodies to virus structural proteins. In contrast, infection with FMD virus leads to virus replication and additional antibody responses to viral nonstructural proteins (NSP). Therefore, antibodies against NSPs are used to differentiate infection in vaccinated animals (DIVA), in order to estimate the prevalence of infection or its absence. Another advantage of NSP antibody tests is that they detect FMD infection in the field, irrespective of the serotypes of virus in circulation. In cattle, the NSP tests that target the 3ABC polyprotein provides the highest sensitivity, detecting up to 90% of vaccinated animals that become carriers after exposure to infection, with a specificity of around 99%. Due to insufficient diagnostic sensitivity and specificity, detection of a low level of infection is difficult at the population level with a high degree of confidence. The low level of non-specific responses can be overcome by retesting samples scored positive using a second confirmatory test, which should have at least comparable sensitivity to the first test. In this study, six in-house tests were developed incorporating different NSP antigens, and validated using bovine sera from naïve animals, field cases and experimentally vaccinated and/or infected animals. In addition, two (short and long incubation) new commercial NSP tests based on 3ABC competitive blocking ELISAs (ID Screen® FMD NSP Competition, IDvet, France) were validated in this study. The two commercial ELISAs had very similar sensitivities and specificities that were not improved by lengthening the incubation period. Several of the new in-house tests had performance characteristics that were nearly as good as the commercial ELISAs. Finally, the in-house tests were evaluated for use as confirmatory tests following screening with the PrioCHECK® and ID Screen® FMDV NS commercial kits, to assess the diagnostic performance produced by a multiple testing strategy. The in-house tests could be used in series (to confirm) or in parallel (to augment) with the PrioCHECK® and IDvet® FMDV NS commercial kits, in order to improve either the specificity or sensitivity of the overall test system, although this comes at the cost of a reduction in the counterpart (sensitivity/specificity) parameter.

Project description:BACKGROUND: Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cattle, pigs, sheep, goats, and many cloven-hoofed wild animals. FMDV serotypes O and Asia 1 have circulated separately in China during the last fifty years, and eliminating infected animals and vaccination are the main policies to prevent and control FMD. Antibodies to NSPs exist in infected animals, and were utilized to differentiate between infected and vaccinated animals. The reliability of detection of 3AB or 3ABC antibodies is higher than that of other NSPs. The test of 3AB is still credible because 3C protein's immunogenicity is the weakest. The 2C protein, immediately N-terminal of 3AB, was used to differentiate between infected and vaccinated animals. The use of the immunochromatographic strip is facile for clinical laboratories lacking specialized equipment and for rapid field diagnosis. RESULTS: In this study, an immunochromatographic strip with non-structural protein (NSP) 2C'3AB was developed and validated to differentiate foot-and-mouth disease infected from vaccinated animals. A part of N-terminal of 2C protein gene and whole 3AB gene were connected and prokaryotically expressed as the antigens labeled with colloidal gold was used as the detector, the 2C'3AB protein and rabbits anti-2C'3AB antibodies were blotted on the nitrocellulose(NC) membrane for the test and control lines, respectively. 387 serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial 3ABC antibody ELISA kit. The coincidence rate of pigs negative serum, pigs vaccinated serum, pigs infected serum was 100%, 97.2%, 95.0%, respectively. The coincidence rate of cattle negative serum, cattle vaccinated serum, cattle infected serum was 100%, 96.7%, 98.0%, respectively. The coincidence rate of sheep negative serum, sheep infected serum was 97.6%, 96.3%, respectively. The strip was shown to be of high specificity and sensitivity, good repeatability and stability. CONCLUSION: These data suggest that the immunochromatographic strip is a useful tool for rapid on-site diagnosing animals infected foot-and-mouth disease virus.

Project description:A time-course pathogenesis study was performed to compare and contrast primary foot-and-mouth disease virus (FMDV) infection following simulated-natural (intra-nasopharyngeal) virus exposure of cattle that were non-vaccinated or vaccinated using a recombinant adenovirus-vectored FMDV vaccine. FMDV genome and infectious virus were detected during the initial phase of infection in both categories of animals with consistent predilection for the nasopharyngeal mucosa. A rapid progression of infection with viremia and widespread dissemination of virus occurred in non-vaccinated animals whilst vaccinated cattle were protected from viremia and clinical FMD. Analysis of micro-anatomic distribution of virus during early infection by lasercapture microdissection localized FMDV RNA to follicle-associated epithelium of the nasopharyngeal mucosa in both groups of animals, with concurrent detection of viral genome in nasopharyngeal MALT follicles in vaccinated cattle only. FMDV structural and non-structural proteins were detected in epithelial cells of the nasopharyngeal mucosa by immunomicroscopy 24 hours after inoculation in both non-vaccinated and vaccinated steers. Co-localization of CD11c+/MHC II+ cells with viral protein occurred early at primary infection sites in vaccinated steers while similar host-virus interactions were observed at later time points in non-vaccinated steers. Additionally, numerous CD8+/CD3- host cells, representing presumptive natural killer cells, were observed in association with foci of primary FMDV infection in the nasopharyngeal mucosa of vaccinated steers but were absent in non-vaccinated steers. Immunomicroscopic evidence of an activated antiviral response at primary infection sites of vaccinated cattle was corroborated by a relative induction of interferon -α, -β, -γ and -λ mRNA in micro-dissected samples of nasopharyngeal mucosa. Although vaccination protected cattle from viremia and clinical FMD, there was subclinical infection of epithelial cells of the nasopharyngeal mucosa that could enable shedding and long-term persistence of infectious virus. Additionally, these data indicate different mechanisms within the immediate host response to infection between non-vaccinated and vaccinated cattle.

Dataset Information

Discriminating foot-and-mouth disease virus-infected and vaccinated animals by use of beta-galactosidase allosteric biosensors.

Publications

Discriminating foot-and-mouth disease virus-infected and vaccinated animals by use of beta-galactosidase allosteric biosensors.

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