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Use of a standardized bovine serum panel to evaluate a multiplexed nonstructural protein antibody assay for serological surveillance of foot-and-mouth disease.

ABSTRACT: Liquid array technology has previously been used to show proof of principle of a multiplexed nonstructural protein serological assay to differentiate foot-and-mouth disease virus-infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B, and 3D and the recombinant protein signature 3ABC in combination with four controls. To determine the diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed by using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, United Kingdom. This serum panel has been used to assess the performance of other singleplex enzyme-linked immunosorbent assay (ELISA)-based nonstructural protein antibody assays. The 3ABC signature in the multiplexed assay showed performance comparable to that of a commercially available nonstructural protein 3ABC ELISA (Cedi test), and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex was acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promote further assay development and optimization to generate an assay for routine use in foot-and-mouth disease serological surveillance.

SUBMITTER: Perkins J

PROVIDER: S-EPMC2168180 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Use of a standardized bovine serum panel to evaluate a multiplexed nonstructural protein antibody assay for serological surveillance of foot-and-mouth disease.

Perkins Julie J Parida Satya S Clavijo Alfonso A

Clinical and vaccine immunology : CVI 20071003 11

Liquid array technology has previously been used to show proof of principle of a multiplexed nonstructural protein serological assay to differentiate foot-and-mouth disease virus-infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B, and 3D and the recombinant protein signature 3ABC in combination with four controls. To determine the diagnostic specificity of each signature in the multiplex, the assay was evaluated against a n ...[more]

PMID: 17913861

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Project description:Despite years of biannual mass vaccination of cattle, foot-and-mouth disease (FMD) remains uncontrolled in Anatolian Turkey. To evaluate protection after mass vaccination we measured post-vaccination antibodies in a cohort of cattle (serotypes O, A and Asia-1). To obtain results reflecting typical field protection, participants were randomly sampled from across Central and Western Turkey after routine vaccination. Giving two-doses one month apart is recommended when cattle are first vaccinated against FMD. However, due to cost and logistics, this is not routinely performed in Turkey, and elsewhere. Nested within the cohort, we conducted a randomised trial comparing post-vaccination antibodies after a single-dose versus a two-dose primary vaccination course. Four to five months after vaccination, only a third of single-vaccinated cattle had antibody levels above a threshold associated with protection. A third never reached this threshold, even at peak response one month after vaccination. It was not until animals had received three vaccine doses in their lifetime, vaccinating every six months, that most (64% to 86% depending on serotype) maintained antibody levels above this threshold. By this time cattle would be >20 months old with almost half the population below this age. Consequently, many vaccinated animals will be unprotected for much of the year. Compared to a single-dose, a primary vaccination course of two-doses greatly improved the level and duration of immunity. We concluded that the FMD vaccination programme in Anatolian Turkey did not produce the high levels of immunity required. Higher potency vaccines are now used throughout Turkey, with a two-dose primary course in certain areas. Monitoring post-vaccination serology is an important component of evaluation for FMD vaccination programmes. However, consideration must be given to which antigens are present in the test, the vaccine and the field virus. Differences between these antigens affect the relationship between antibody titre and protection.

Project description:Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids that is in most FMDVs examined to date, and it plays important roles in virus replication, virulence, and host range. To better understand the role of 3A during FMDV infection, we used coimmunoprecipitation followed by mass spectrometry to identify host proteins that interact with 3A in FMDV-infected cells. Here, we report that cellular vimentin is a host binding partner for 3A. The 3A-vimentin interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pull down, and immunofluorescence assays. Alanine-scanning mutagenesis indicated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin. Using reverse genetics, we demonstrate that mutations in 3A that disrupt the interaction between 3A and vimentin are also critical for virus growth. Overexpression of vimentin significantly suppressed the replication of FMDV, whereas knockdown of vimentin significantly enhanced FMDV replication. However, chemical disruption of the vimentin network by acrylamide resulted in a significant decrease in viral yield, suggesting that an intact vimentin network is needed for FMDV replication. These results indicate that vimentin interacts with FMDV 3A and negatively regulates FMDV replication and that the vimentin-3A interaction is essential for FMDV replication. This study provides information that should be helpful for understanding the molecular mechanism of FMDV replication.IMPORTANCE Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, host range, and virulence. To further understand the role of 3A during FMDV infection, identification of host cell factors that interact with FMDV 3A is needed. Here, we found that vimentin is a direct binding partner of FMDV 3A, and manipulation of vimentin has a negative effect on virus replication. We also demonstrated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin and that the 3A-vimentin interaction is critical for viral replication since the full-length cDNA clone harboring mutations in 3A, which were disrupt 3A-vimentin reactivity, could not produce viable virus progeny. This study provides information that not only provides us a better understanding of the mechanism of FMDV replication but also helps in the development of novel antiviral strategies in the future.

Dataset Information

Use of a standardized bovine serum panel to evaluate a multiplexed nonstructural protein antibody assay for serological surveillance of foot-and-mouth disease.

Publications

Use of a standardized bovine serum panel to evaluate a multiplexed nonstructural protein antibody assay for serological surveillance of foot-and-mouth disease.

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