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Foot and mouth disease (FMD) virus serotype O is the predominant cause of FMD outbreaks in several regions of the world including India. Five independent neutralizing antigenic sites have been identified on the capsid surface of FMD virus serotype O. The relative importance of these neutralizing sites in eliciting antibody responses in the polyclonal sera collected from un-infected vaccinated (both primo and multiply-vaccinated) and naturally infected cattle populations were determined through a combination of reverse genetics and serology. The known critical amino acid residues present on the five antigenic sites of FMD virus serotype O Indian vaccine strain O IND R2/1975 were mutated through site-directed mutagenesis. The mutant viruses were rescued in cell-culture and analyzed through virus-neutralization assays along with parent virus using the polyclonal sera collected from three groups of cattle. In the polyclonal sera from primo-vaccinated cattle, significantly higher level of antibodies were directed towards antigenic site 2. In contrast, in polyclonal sera from multiply vaccinated animals, both antigenic sites 1 and 2 were equally important. In case of naturally infected animals, antibody responses were elicited against all the five antigenic sites. Although a drop in neutralization titres was observed for all the mutants, in one instance, increase in titre was noticed for a site 3 mutant. The findings from this study extend our knowledge on the antibody immunodominace following FMDV vaccination and infection, and may improve our strategies for vaccine strain selection and rational vaccine design., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease (FMD) is an acute, highly contagious, and economically devastating viral disease of domestic and wildlife species. For effective implementation of FMD control program, there is an imperative need for developing a rapid, sensitive, and specific diagnostics which help in the identification of serotypes involved in the outbreaks. The humoral immune response of the Camelidae is unique since in these animals 75% of circulating antibodies are constituted by heavy-chain antibodies and 25% are conventional immunoglobulin with two identical heavy chains. In the present study, we developed and characterized FMD virus-specific single-domain heavy-chain antibodies (VHHs) against inactivated whole-virus antigens of FMDV serotypes O (INDR2/1975), A (IND40/2000), and Asia 1 (IND63/1972) vaccine strains. After six rounds of panning and enrichment, these VHHs were stably expressed in Escherichia coli cells. The VHHs directed against outer capsid proteins of FMD virus were successfully utilized as the capture antibody in liquid-phase blocking ELISA (LPBE) thus replacing rabbit coating antibodies. Our study demonstrated the utility of FMD virus-specific VHHs as potential candidates in FMD research and diagnostic application.

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., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Three of the seven serotypes of foot-and-mouth disease (FMD) virus are prevailing in India. A massive vaccination campaign is on to control and eradicate the disease from the country. However, FMD vaccines provide short term immunity, hence regular assessment of antibody level in the vaccinated herds is indispensible for the success of the control programme. The antibodies are quantitatively estimated, either by virus neutralization test or by end-point dilution liquid-phase-blocking ELISA (LPBE). Millions of cattle and buffalo in the country are now systematically vaccinated, and thousands of serum samples are routinely screened in the country for estimation of herd immunity against FMDV serotypes O, A and Asia1. Testing such a large number of serum samples within limited a period of time by the conventional end point dilution method of LPBE requires lots of man power, and biological reagents. A more economical high throughput single dilution LPBE (SdLPBE) assay was optimized and validated for quantitative estimation of antibody levels against the three FMD virus serotypes. The assay was thoroughly validated against LPBE method before adopting it for country-wide use. The biological reagents used in the assay were prepared in thermo-stable form to enable transportation to the field level FMD diagnostic laboratories., (Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

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 (FMD) is a highly infectious disease of transboundary importance. Routine biannual vaccination along with surveillance activities is seen as the principal to control FMD in India. Non-structural protein (NSP) based immunoassays are the test of choice for the differentiation between infected and vaccinated population. In this study, 3D protein of FMD virus was expressed in Escherichia coli and an indirect ELISA (I-ELISA) was developed to detect 3D-antibodies in the infected bovines. 3D I-ELISA demonstrated comparable diagnostic sensitivity (97.6%) but lower specificity (80.8%) as compared to the in-house r3AB3 I-ELISA. However, the specificity values varied significantly for naïve and vaccinated samples and were observed to be 98.42% and 76.93%, respectively. A moderate degree of concordance (88.5%) was observed between the overall results of two ELISAs. 3D I-ELISA displayed a considerably lower specificity in uninfected vaccinated samples, thereby suggesting against its application for serosurveillance in intensively vaccinated population. However by virtue of its high diagnostic sensitivity and longer duration of persistence of 3D-antibody post-infection, 3D I-ELISA could be adopted for seroepidemiological investigations in regions not practicing vaccination and could be extended to susceptible species which are generally not covered by vaccination., (Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Single chain fragment variable (ScFv) antibodies specific to the nucleoprotein (NP) of avian influenza virus (AIV) were developed using a phage display system. The variable heavy (VH) and the variable light (VL) chain gene fragments were derived from spleen cells of Balb/c mouse immunized with a recombinant NP (rNP) antigen (∼63 kDa) of H5N1 influenza virus. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in a phagemid to express ScFv protein in Escherichia coli cells. The specific reactivity of the ScFv with the rNP antigen and viral antigen (H5N1) was confirmed by Western blot and ELISA. A competitive inhibition ELISA (CI-ELISA) was developed using the rNP and the anti-NP ScFv for detection of type-specific antibodies to AIV in chicken sera. The ScFv based CI-ELISA was compared with hemagglutination inhibition (HI) test and agar gel immunodiffusion (AGID) test over 850 sera. Sensitivity of the CI-ELISA was 100% with HI and AGID and specificity was 98.7% with HI and 100% with AGID., (Copyright © 2014 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.)

Detection of antibodies to nonstructural proteins (NSP) of foot-and-mouth disease virus is the preferred diagnostic method to differentiate infected from vaccinated animals. In India, an endemic region practising preventive biannual vaccination, 3AB3 indirect ELISA (r3AB3 I-ELISA) has been employed as the primary screening test for serosurveillance. However, because of the variability observed in the immune response to the NSPs, the likelihood of detecting or confirming an infected animal is increased if an antibody profile against multiple NSPs is considered for diagnosis. In this study, all three copies of NSP 3B were expressed in a prokaryotic system to develop an indirect ELISA (r3B I-ELISA). At the decided cutoff of 40 percent positivity, the diagnostic sensitivity and specificity of the r3B I-ELISA were estimated to be 92.1% (95% CI: 89.0-94.5) and 98.1% (95% CI: 96.9-98.8), respectively, as compared to 97.04% and 95.04% for r3AB3 I-ELISA. Although r3B I-ELISA displayed lower sensitivity compared to the screening assay, which could possibly be attributed to additional relevant B-cell epitopes in the carboxy-terminal half of the 3A protein, the former achieved considerably higher specificity on repeatedly vaccinated animals. NSP antibodies could be detected from 10 to as late as 998 days postinfection in experimental calves. Substantial agreement in the test results (90.6%) was found between the two ELISAs. The r3B I-ELISA, when used in conjunction with the r3AB3 I-ELISA as an integrated system, can potentially augment the efficiency and confidence of detection of infected herds against the backdrop of intensive vaccination.

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.

In this study, the Indian foot-and-mouth disease virus (FMDV) vaccine strain (A IND 40/2000) was passaged under homologous bovine convalescent serum (BCS) pressure to gain insight into the evolutionary dynamics of the antigenic sites. A considerable drop in the neutralization titres of the BCS for the isolated variants as compared to the parental population in either virus neutralization or plaque reduction neutralization test was observed. T143K substitution preceding the integrin binding 'RGD'-motif in the βG-βH loop of VP1 was found to be selected consistently and exclusively under immune pressure. By virtue of its location within an immunodominant site, sequence heterogeneity observed in the field viruses and residues already mapped in the neutralizing monoclonal antibody resistant mutants, position 143 in VP1 was predicted to be a critical residue of an important neutralizing epitope in serotype A FMDV. Using next-generation sequencing approach, the gradual overtaking of the originally dominating major variant by a minor one under a selective environment could be demonstrated. In the control passage regimen, VP2 E131K substitution was fixed within the heparan sulfate binding pocket probably as a result of adaptation to use alternative cellular receptors. But at the same time, these substitutions arising under selection forces other than immune pressure changed the antigenic behaviour of the virus inadvertently., (Copyright © 2014 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.)

Foot-and-mouth disease virus (FMDV) is constantly evolving under neutralizing antibody pressure in either naturally infected or vaccinated animals. This study was carried out to understand the dynamics of evolution of antigenic sites. Neutralizing antibody-resistant populations of three strains of FMDV serotype O (INDR2/1975, IND120/2002 and IND271/2001) were isolated by serial propagation in BHK-21 cells in the presence of sub-neutralizing level of bovine vaccinal sera (BVS). In the partial neutralization escape variants, fixation of aa substitutions were observed at critical residues of all established antigenic sites of serotype O {144 of VP1 (site 1), 45 and 48 of VP1 (site 3), 72 and 134 of VP2 (Site 2)} except site 4 and 5. In majority of the variant populations, site 3 was found to be substituted and therefore immunodominance may not be associated with a particular site, rather it appears to be a virus strain and infected host specific affair. Substitutions were also observed in proximity to the identified residues {41 and 51 (βB-βC loop), 133, 140 and 143 (βG-βH loop), 201, 204 and 209 (C terminus) of VP1, 71 and 75 (βB-βC loop), 131 (βE-αB region), 174 and 179 (βG-βH loop) and 219 (C terminus) of VP3} within antigenic sites of serotype O or other serotypes which could be significant in terms of neutralizing antibody binding and immune escape. Presence of similar residues in the Indian field viruses as selected in the variants supports the importance of these sites in antigenic diversification of serotype O FMD virus., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Polymorphism of bovine leukocyte antigen (BoLA) DRB3 gene is being intensively investigated for potential association with economically important diseases of cattle. Accordingly, we investigated the association of DRB3 Exon 2 polymorphism as evidenced by the variation in the binding pockets with variability in immune response to inactivated trivalent (O, A and Asia1) foot and mouth disease virus (FMDV) vaccine in a closed population of crossbred cattle. Antibody titer of ≥ 1.8 was set as the cut off value to distinguish the protected (≥ 1.8) and unprotected (<1.8) animals. Eleven different alleles of over 3% frequency were detected in the population. We found that DRB3 alleles 0201, 0801 and 1501 always ranked high for protective immune response whereas alleles 0701, 1103 and 1101 consistently ranked low for unprotected immune response for all the three serotypes. Rank correlation of DRB3 alleles among the three serotypes was positive, high in magnitude and statistically significant (P<0.05). Logistic regression analysis revealed that odds of protection from the vaccine were highest for all the three serotypes if allele (∗)1501 was present and strengthened the results of allele ranking. Predicted amino acid substitution in the peptide binding pockets revealed that all the important sites had high Wu-Kabat index. Similarly, specific residues in pockets were crucial for immune response to FMD vaccine. There were specific substitutions in un-protected alleles such as absence of acidic amino acids substituted by basic amino acid at β71, presence of non-polar cysteine or basic histidine at β30 and presence of polar tyrosine at β37. From the observations, we hypothesize that the substitutions lead to unique conformational changes in the protein products of the studied alleles that would associate with the protective or unprotective antibody response to FMDV vaccine. The knowledge has potential implications in future selection programs if integrated with the complete BoLA haplotype details and production traits of the herd., (Copyright © 2013 Elsevier Ltd. 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.)

The aim of this study was to develop a competitive inhibition ELISA (CI-ELISA) for detection of antibodies to bovine viral diarrhea virus (BVDV) using the helicase domain of NS3 (non-structural) protein and monoclonal antibody (MAb) against it and to estimate its sensitivity and specificity using two commercial ELISA kits as independent references. The 45-kDa helicase domain of NS3 protein of BVDV was expressed in Escherichia coli and 18MAbs were developed against it. MAb-11G8 was selected for use in CI-ELISA on the basis of maximum inhibition (90%) obtained with BVDV type 1 infected calf serum. Based on the distribution of percent inhibition of known negative sera (n=166), a cut-off value was set at 40% inhibition. In testing 914 field serum samples of cattle (810) and buffaloes (104), the CI-ELISA showed a relative specificity of 95.75% and 97.38% and sensitivity of 96% and 94.43% with Ingenesa kit and Institut Pourquier kit, respectively. This study proved that the use of helicase domain of NS3 (45-kDa) is equally good as the whole NS3 protein (80-kDa) used in commercial kits for detection of BVDV antibodies in cattle and buffaloes.

The aim of this study was to develop a more specific and sensitive competitive inhibition ELISA (CI-ELISA) than the currently used indirect ELISA for detection of antibodies to bovine immunodeficiency virus (BIV) in cattle and buffaloes. Murine monoclonal antibodies (MAbs) were generated against a recombinant capsid (CA) protein of bovine immunodeficiency virus. Of the 13 anti-CA MAbs developed, MAb-9G10 was selected for CI-ELISA based on the maximum inhibition (98%) obtained with reference BIV antibody positive serum. Based on the distribution of percent inhibition of known negative sera (n=50), a cut-off value was set at 40% inhibition. The MAb-based CI-ELISA showed much higher agreement (concordance: 95.4%) than the indirect ELISA (concordance: 77.8%) with Western blot. Out of 672 sera of cattle and buffaloes tested by CI-ELISA from four states of India, 22% (113/516) of cattle and 19% (30/156) of buffalo were sero-positive for BIV with an overall seroprevalence of 21% (143/672) in India.

A set of five neutralizing monoclonal antibodies (MAbs) to an Indian strain (IND17/77) of type A (subtype A22) foot-and-mouth disease (FMD) virus (FMDV) was used in the study. Four of the MAbs (27S, 37S, 85S, and 143S) identified a trypsin-sensitive (TS) epitope(s) and were specific for VP1, while the remaining MAb (145S) reacted with a trypsin-resistant (TR) epitope and was specific for VP3 in Western blot analysis. Both the epitopes (TS and TR) were conformation-independent in nature. Results obtained in MAb-competition enzyme-linked immunosorbent assay (ELISA), and profiling of the (MAb) neutralization-escape mutants in ELISA and cross-neutralization test revealed two overlapping TS epitopes (27S/37S and 85S/143S) on the virus. Variation at both these epitopes was observed in some field isolates of serotype A. Comparison of deduced amino acid sequence in the VP1 region (aa 140-213) between the parent virus and the mutants identified Gly148 and Arg153 as critical for the formation of both the TS epitopes. Substitution of R153 by Gly or Ser was observed in mutants with no reactivity for the MAbs 85S/143S. However, these mutants maintained partial reactivity with MAbs 27S/37S, and substitution of Gly148 by Glu eliminated both the epitopes. No amino acid substitution was observed in the VP1 region of aa 200-213. Efficient neutralization of the MAb neutralization escape mutants (MAb-resistant (MAR) mutants) by bovine vaccinate serum (BVS) indicated involvement of other epitopes on the virion surface in eliciting neutralizing antibodies following vaccination.

Neutralizable antigenic sites/epitopes of serotype Asial foot-and-mouth disease virus (strain IND63/72) were identified using monoclonal antibodies (mabs) and their neutralization-escape mutants. Relative affinity/reactivity of the mabs for viral (both native and trypsin-cleaved) and subviral antigens in enzyme-linked immunosorbent assay (ELISA) showed dominance of trypsin-sensitive and conformation-dependent neutralizable antigenic sites. Characterization of neutralization escape mutants identified at least four independent trypsin-sensitive neutralizable antigenic sites on Asial FMD virus. One site was identified by mabs B3, 1A, 24, 2A, 40 and 63, second site by mabs 34 and 81, third site by mab 72 and fourth site by mab 89. The reaction profile of the mabs with selected field isolates in ELISA identified four different neutralization epitopes within the site B3/1A/24/2A/40/63.