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Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. In India, the disease is endemic in nature and is controlled primarily by prophylactic bi-annual mass vaccination. In this control programme, liquid-phase blocking ELISA (LPBE) is being used widely for post vaccination seromonitoring. In order to develop an alternative assay to LPBE, the recombinant capsid polyprotein (rP1) of FMD virus (FMDV) serotype O was expressed in Escherichia coli and used as an antigen for the detection of antibodies to FMDV. The capsid polyprotein of FMDV serotype O could be expressed successfully as a recombinant 6xHis-SUMO tagged protein in soluble form. In a Western blot assay, the rP1 protein reacted strongly with anti-FMDV serotype O guinea pig and bovine serum. Further, in this study, an rP1 protein-based solid phase competitive ELISA (rP1-SPCE) was developed and evaluated with a set of serum samples representing the various epidemiological situation of the country. The performance of the rP1-SPCE was compared with the in-house LPBE, and overall, an excellent agreement (kappa = 0.95) was observed between the two tests. This report demonstrates that the recombinant capsid polyprotein-based ELISA has the potential to be an easy-to-perform, safe alternative to the conventional LPBE for the quantitative detection of antibodies to FMDV serotype O., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease is a highly infectious and contagious disease of livestock animals with transboundary and economical importance. Animals in the endemic settings are regularly vaccinated in addition to intensive surveillance for control of the disease. Under intensive vaccination, detection of infected animals among the vaccinated population is essential to monitor the infection and to track down the virus movement. Sero-surveillance and retrospective disease diagnosis is performed primarily by detecting antibodies against non-structural proteins (NSPs) of FMD virus which are usually absent in the inactivated vaccine formulations. The study was conducted with an objective to compare simultaneously performance of six NSP ELISAs in detecting infected animals in the areas covered under intensive vaccination, and to assess their fit-for-purpose attribute for sero-surveillance of FMD in India. A panel of bovine serum samples consisting of samples collected from infected with FMDV, vaccinated and naive animals were constituted. In addition, samples collected at random from areas having varied FMD situation and vaccination coverage were tested simultaneously by the six NSP ELISAs to compare their performances. The four indigenous assays showed varying degrees of correlation with the two commercial kits. The study validated that, in all the groups of samples, the indigenous assays were equally sensitive and specific as the two commercial kits. Among all the six assays, PrioCheck and in-house 3ABC I-ELISAs showed maximum sensitivity for detection of infected animals, whereas 3AB3 I-ELISA and 3ABC C-ELISA showed maximum specificity. The study concluded that the in-house available assays are equally capable as the commercially available kits for differentiation of infected animals under intensive vaccination and identifies the 3AB3 I-ELISA with optimum sensitivity and specificity for the purpose of sero-surveillance in India., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, an RNA transfection was used to rescue infectious foot-and-mouth disease (FMD) virus from clinical samples in BHK-21 cell line for diagnosis of FMD. Tissue samples (n=190) were subjected to FMD virus isolation by conventional cell culture and also by RNA transfection. FMD virus was isolated from 62% of the clinical samples by RNA transfection, whereas virus was isolated only from 16% of the clinical samples in conventional cell culture method, suggesting better performance of the RNA transfection. Virus was rescued from 67% and 10% of ELISA negative but multiplex PCR positive samples by RNA transfection and conventional cell culture, respectively. The efficiency of transfection was studied on clinical samples subjected to temperature as high as 37°C and varying pH (pH 4-9). Except up to 1 week of storage at 4°C at pH 7.5, virus isolation was not possible by cell culture. Virus was rescued by transfection from samples stored at 4°C for any of the applied pH up to 4 weeks, and when stored at 37°C virus could be rescued up to 4 weeks at pH 7.5 suggesting the fitness of transfection to isolate virus from clinical samples stored under inappropriate conditions. The sequence data and antigenic relationships with the vaccine strains, between virus rescued by transfection and conventional cell culture, were comparable. The RNA transfection will help to increase the efficiency of virus isolation, diagnosis and molecular epidemiological studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A full-length cDNA clone of an Indian foot-and-mouth disease virus strain, Asia 1 IND 491/1997 was assembled downstream of the T7 promoter in the pBluescript II SK (+) vector by sequential ligation of four PCR-generated subgenomic fragments. RNA transcribed from that construct were transfected into BHK-21 and LFBK cells to rescue infectious virus. The in vitro growth kinetics, plaque morphology, infectivity titer, antigenic profile and virulence characteristics in unweaned mice infected with the recombinant virus were comparable to those infected with the parental virus. However, repeated attempts to recover viable virus from the RNA transcripts with a G320-T point mutation introduced in the short fragment of the 5' untranslated region failed. The possible destabilizing effect of such a mutation on the predicted long stem-loop structure at the 5'-end of the genome and its implications for viral genome replication are discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease (FMD) is a transboundary animal disease caused by foot-and-mouth disease virus. In India, systematic preventive vaccination using inactivated trivalent (O, A and Asia 1) vaccine is the strategy being adopted to control FMD. The use of non-structural protein (NSP)-contaminated inactivated vaccine raises concerns over differentiation of infected and vaccinated animals (DIVA) by NSP based immunoassays. However, 2C being a membrane associated protein usually remain absent in vaccine formulations and thus, anti-2C response is one of the most reliable indicator of the FMDV infection. In this study, 34 amino acids from N-terminus of 2C protein were removed to eliminate membrane-binding amphipathic helicase activity for the expression of recombinant protein in soluble form. Truncated 2C (2Ct) was utilized for development of an indirect ELISA (I-ELISA) for bovine and the developed 2Ct I-ELISA was validated using a panel constituting of serum of naïve, vaccinated and infected animals. The assay was compared with the in-house r3AB3 I-ELISA and the overall concordance was 85.31%. The diagnostic sensitivity and specificity of the 2Ct I-ELISA were 92.9% and 94.0%, respectively. The apparent prevalence of anti-2C antibodies for random bovine samples tested by the developed assay was 23.7%. The developed ELISA will help in augmenting the sensitivity of detection if used in combination with r3AB3 I-ELISA for sero-surveillance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Differentiation of infected from vaccinated animals (DIVA) is essential for effective control of foot-and-mouth disease (FMD) by vaccination. The antibody response against FMD viral non-structural proteins (NSPs) has been used widely for this purpose. Among all the NSPs, the 3ABC polyprotein has been recognized as the most appropriate indicator for DIVA. In this study, mutated full-length 3ABC polyprotein was expressed in a prokaryotic system and monoclonal antibody against the recombinant protein was developed. A competitive ELISA (C-ELISA) for DIVA was standardized for different species of livestock animals using recombinant 3ABC and monoclonal antibodies. The diagnostic sensitivity and specificity of the assay were estimated by testing a panel of known serum samples consisting of sera from naive, vaccinated and infected animals as 86.9% with 66.4-97.2 (95%) confidence interval and 97% with 89.6-99.6 (95%) confidence interval respectively at 40% inhibition cut-off. The assay was validated further by testing sera from different livestock species collected at random from different parts of the country. The assay will provide a common method for testing sera from different species of livestock and wild animals. The C-ELISA is a sensitive and specific DIVA assay for FMD and can be used as a method for FMD control programme with vaccination., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In India, the proportion of bovines vaccinated against foot-and-mouth disease (FMD) is increasing since the implementation of the Government supported 'FMD Control Programme', and non-structural protein (NSP)-based serological assays for discriminating between antibodies induced by infection or vaccination (DIVA) could be useful. The FMD virus NSP 3AB was expressed in a prokaryotic system and an indirect ELISA (r3AB(3) I-ELISA) was developed and validated as a screening assay for detecting virus in vaccinated bovines. The diagnostic sensitivity of the assay was estimated to be 96%, while the diagnostic specificity varied between the naïve and vaccinates as 99.1% and 96.4%, respectively. This assay could detect antibodies to 3AB (3AB-Ab) from 10 to as late as 900 days post-infection in cattle infected experimentally. The "in-house" assay demonstrated higher sensitivity than a commercial 3ABC ELISA kit particularly with samples obtained from the late stages of infection. Transient post-vaccinal 3AB-Ab response could be detected in one of the three commercial vaccines during the six-month vaccination regimen, which emphasizes the fact that for a DIVA-compatible diagnostic strategy to be a realistic option, all vaccines need to be quality checked for the NSP content., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In India, there has been co-circulation, extinction and emergence of genotypes/lineages within serotype A foot-and-mouth disease (FMD) virus. At present an antigenically heterogeneous, unique lineage within genotype VII dominates the field outbreaks. This genetic cluster has amino acid deletion at position 59 of VP3 (VP3(59)-deletion group), considered to be critical antigenically. The emergence of this group warrants rapid and accurate detection to facilitate early planning and implementation of an effective control policy. A rapid multiplex PCR assay was developed for detection of the dominating VP3(59)-deletion group with 100% sensitivity and specificity, even before generating sequence data and confirmatory phylogenetic analysis. This development is important for surveillance of FMD in India., (Copyright © 2010 Elsevier B.V. All rights reserved.)