Diagnostic application of recombinant non-structural protein 3A to detect antibodies induced by foot-and-mouth disease virus infection

Detection of antibodies to the non-structural proteins (NSPs) of FMD virus (FMDV) is the preferred differential diagnostic method for identification of FMD-infected animals in the vaccinated population. Nevertheless, due to the observed variability in the antibody response to NSPs, the likelihood of screening or confirming the FMD infection status in animals is increased if an antibody profile to multiple NSPs is considered for diagnosis. In order to develop and evaluate an additional NSP-based diagnostic assay, in this study, the recombinant 3A protein of FMDV was expressed in Escherichia coli and used as an antigen for detection of FMD infection specific antibodies. At the fixed cut-off value of 45 percentage of positivity, the diagnostic sensitivity and specificity of 3A indirect-ELISA (I-ELISA) were found to be 95.7% and 96.3%, respectively. In FMD naturally infected cattle, about 85% of clinically infected and 75% of asymptomatic in-contact populations were found positive at 13 months post-outbreak. The 3A I-ELISA was further evaluated with the bovine serum samples collected randomly from different parts of the country. Furthermore, the performance of newly developed 3A I-ELISA was compared with the extensively used in-house r3AB3 I-ELISA, and the overall concordance in test results was found to be 93.62%. The r3A I-ELISA could be useful as a screening or confirmatory assay in the sero-surveillance of FMD in India irrespective of extensive bi-annual vaccination

Chimeric foot-and-mouth disease virus serotype O displaying a serotype Asia1 antigenic epitope at the surface

Objective: To determine whether the G-H loop of foot-and-mouth disease virus (FMDV) serotype O can function as a target structure to harbour and display serotype Asia1 antigenic epitope at the surface. Results: Using reverse genetics, FMDV serotype O IND R2/1975 displaying a FMDV serotype Asia1 B cell epitope at the capsid surface was constructed. The epitope-inserted recombinant chimeric virus was genetically stable up to ten serial passages in cell culture and exhibited growth properties similar to the parental serotype O virus. Furthermore, the surface-displayed Asia1 epitope able to react with serotype Asia1 specific antibodies in a competitive ELISA. Importantly, the recombinant chimeric virus showed neutralizing activity to both serotype O and Asia1 polyclonal antibodies. Conclusion: The capsid protein of FMDV serotype O can effectively display potent epitope of other serotypes, making this an attractive approach for the design of new generation bi-valent FMD vaccines

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Not AvailableFoot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA® cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA® card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA® card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA® card impregnated with clinical samples that had been stored at varying temperature (4–37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA® card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA® card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories.Not Availabl

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Not AvailableThe development of a negative marker vaccine against the foot-and-mouth disease virus (FMDV) will enhance the capabilities to differentiate vaccinated from infected animals and move forward in the progressive control pathway for the control of FMD. Here, we report the development of mutant FMDV of Asia1 with partial deletion of non-structural proteins 3A and 3B and characterization of their infectivity and protection response in the guinea pig model. The deleted FMDV Asia1/ IND/63/1972 mutants, pAsiaΔ3A and pAsiaΔ3A3B1 were constructed from the full-length infectious clone pAsiaWT, the viable virus was rescued, and the genetic stability of the mutants was confirmed by 20 monolayer passages in BHK21 cells. The mutant Asia1 viruses showed comparable growth pattern and infectivity with that of AsiaWT in the cell culture. However, the AsiaΔ3A3B1 virus showed smaller plaque and lower virus titer with reduced infectivity in the suckling mice. In guinea pigs, the AsiaΔ3A3B1 virus failed to induce the disease, whereas the AsiaΔ3A virus induced typical secondary lesions of FMD. Vaccination with inactivated Asia1 mutant viruses induced neutralizing antibody response that was significantly lower than that of the parent virus on day 28 post-vaccination (dpv) in guinea pigs (P < 0.05). Furthermore, challenging the vaccinated guinea pigs with the homologous vaccine strain of FMDV Asia1 conferred complete protection. It is concluded that the mutant AsiaΔ3A3B1 virus has the potential to replace the wild-type virus for use as a negative marker vaccine after assessing the vaccine worth attributes in suspension cell and protective efficacy study in cattle.Not Availabl

Genetic and antigenic variation of foot-and-mouth disease virus during persistent infection in naturally infected cattle and Asian buffalo in India

The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2substitution/site/year (s/s/y) (95% CI 1.362-2.31 x 10-2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS (ω) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMD

Detection of antibodies specific for foot-and-mouth disease virus infection using indirect ELISA based on recombinant nonstructural protein 2B

Foot-and-mouth disease (FMD) is a highly contagious viral disease of transboundary importance. In India, since the launch of the FMD control programme, there has been a substantial increase in the vaccinated bovine population. In this scenario, there is a need for additional locally developed non-structural protein (NSP)-based immnoassays for efficient identification of FMD virus (FMDV)-infected animals in the vaccinated population. The 2B NSP of FMDV, lacking the transmembrane domain (Δ2B), was expressed successfully in a prokaryotic system, and an indirect ELISA (I-ELISA) was developed and validated in this study. The diagnostic sensitivity and specificity of the Δ2B I-ELISA were found to be 95.3 % and 94.6 %, respectively. In experimentally infected cattle, the assay could consistently detect Δ2B-NSP-specific antibodies from 10 to approximately 400 days postinfection. The assay was further validated with bovine serum samples collected randomly from different parts of the country. The performance of the Δ2B I-ELISA was compared with the in-house r3AB3 I-ELISA, and the overall concordance in test results was found to be 86.49 %. The Δ2B I-ELISA could be useful as a screening or confirmatory assay in the surveillance of FMD irrespective of vaccination

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Not AvailableNucleic acid detection and virus isolation (VI) techniques are preferred for many applications in diagnostic virology, but these techniques are not enough to provide microscopic localization virus or viral antigen at cellular or at tissue level. Though the anti-FMDV fluorescent antibody technique (FAT) has been used as a tool for investigation of viral pathogenesis, they could also be useful as an ancillary diagnostic tool, particularly when necroptic tissues might be the only available samples. In this study FAT was optimised for the detection of FMDV structural proteins in frozen tissues of bovine origin. Tongue epithelium (TE) and dorsal soft palates (DSP) collected from FMDV suspected animals and later confirmed for FMDV positive by RT-LAMP and its serotype by mPCR were used. TE and DSP of apparently healthy bovine collected from slaughter house used as negative control, which was previously confirmed negative for FMDV by RT-LAMP and mPCR. Fluorescent antibody labelling of FMDV was performed in conjunction with labelling of cell markers like pancytokeratin/β-tubulin. Epithelial cells were identified with anti-pancytokeratin for DSP while β-tubulin for TE. As expected, FMDV antigen was predominantly colocalized in vesicle along with β-tubulin/pancytokeratin. Tissues (TE & DSP) from the FMDV positive animals incubated with isotype control antibodies have no corresponding FMDV specific signal. The FAT established in the current study could identify virus-positive cells within the TE and DSP. As a research tool, this technique could allow the precise localization of FMDV during various stages of infection.Not Availabl

Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142 → R or H142 → F or H142 → A substitutions resulted in non-infectious FMDV, H142 → D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A

Engineering foot-and-mouth disease virus serotype O IND R2/1975 for one-step purification by immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) allows for the efficient protein purification via metal affinity tag such as hexa-histidine (His6) sequence. To develop a new chromatography strategy for the purification and concentration of foot-and-mouth disease virus (FMDV) particles, we inserted the His6-tag at the earlier reported site in the VP1 G-H loop of the FMD virus serotype O vaccine strain IND R2/1975. Display of the His6-tag on the capsid surface, endowed the virus with an increased affinity for immobilized nickel ions. We demonstrated that the His6-tagged FMDV could be produced to high titre and purified from the infected BHK-21 cell lysates by IMAC efficiently. Further, a 1150-fold reduction in protein contaminant level and an 8400-fold reduction in DNA contaminant level were achieved in the IMAC purification of His6-tagged FMDV. Through various functional assays it has been found that the tagged virus retains its functionality and infectivity similar to the non-tagged virus. The affinity purification of the His6-tagged FMDV may offer a feasible, alternative approach to the current methods of FMDV antigen purification, concentration and process scalability

Development and Validation of a Mucosal Antibody (IgA) Test to Identify Persistent Infection with Foot-and-Mouth Disease Virus

It is well known that approximately 50% of cattle infected with foot-and-mouth disease (FMD) virus (FMDV) may become asymptomatic carrier (persistently infected) animals. Although transmission of FMDV from carrier cattle to naïve cattle has not been demonstrated experimentally, circumstantial evidence from field studies has linked FMDV-carrier cattle to cause subsequent outbreaks. Therefore, the asymptomatic carrier state complicates the control and eradication of FMD. Current serological diagnosis using tests for antibodies to the viral non-structural proteins (NSP-ELISA) are not sensitive enough to detect all carrier animals, if persistently infected after vaccination and do not distinguish between carriers and non-carriers. The specificity of the NSP ELISA may also be reduced after vaccination, in particular after multiple vaccination. FMDV-specific mucosal antibodies (IgA) are not produced in vaccinated cattle but are elevated transiently during the acute phase of infection and can be detected at a high level in cattle persistently infected with FMDV, irrespective of their vaccination status. Therefore, detection of IgA by ELISA may be considered a diagnostic alternative to RT-PCR for assessing FMDV persistent infection in ruminants in both vaccinated and unvaccinated infected populations. This study reports on the development and validation of a new mucosal IgA ELISA for the detection of carrier animals using nasal, saliva, and oro-pharyngeal fluid (OPF) samples. The diagnostic performance of the IgA ELISA using nasal samples from experimentally vaccinated and infected cattle demonstrated a high level of specificity (99%) and an improved level of sensitivity (76.5%). Furthermore, the detection of carrier animals reached 96.9% when parallel testing of samples was carried out using both the IgA-ELISA and NSP-ELISA