A reverse transcription-multiplex PCR strategy devised for concomitant detection and differentiation of foot and mouth disease virus serotypes O, A and Asia 1 in India.

Serotype identification occupies the central part of foot and mouth disease (FMD) diagnosis workflow and vaccination decision tree. In this study, a reverse transcription-multiplex PCR (RT-mPCR) strategy wherein three assays with unique combinations of serotype specific primers targeting the VP1 region was developed to differentiate FMD virus serotypes O, A and Asia 1 based on differential size of the PCR amplicons on agarose gel. Their diagnostic performance relative to the mPCR assay in use in India was evaluated on 169 clinical samples and 210 cell culture grown virus isolates. The relative diagnostic sensitivity was found to be 99.69%, 98.78% and 99.08% for primer combinations 1, 2 and 3, respectively. These assays proved their worth by detecting serotype in three FMD suspected specimens that went undiagnosed in the existing mPCR and also by identifying multiple serotypes in the same sample. Their detection limits varied from log10 2 to log10 4 viral RNA dilution and from 100 to 0.1 TCID50 virus depending on the serotype. The validated novel mPCR assays show promise to be included in the routine diagnostic tool-box to augment the efficiency of diagnosis of FMD virus serotypes that display extreme genetic diversity and a tendency of transboundary dispersal.

Foot-and-Mouth Disease Virus Serotype O Exhibits Phenomenal Genetic Lineage Diversity in India during 2018-2022.

In India, widespread foot-and-mouth disease (FMD) outbreaks occurred in 2021. The objective of this study was to identify genetic lineages and evaluate the antigenic relationships of FMD virus (FMDV) isolates gathered from outbreaks reported between 2019 and 2022. Our study shows that the lineages O/ME-SA/Ind2001e and the O/ME-SA/Cluster-2018 were both responsible for the FMD outbreaks on an epidemic scale during 2021. This observation is in contrast to earlier findings that suggested epidemic-scale FMD outbreaks in India are often connected to a single genetic lineage. Additionally, we report here the identification of the O/ME-SA/PanAsia-2/ANT10 sub-lineage in India for the first time, which was connected to two intermittent outbreaks in Jammu and Kashmir. The current study demonstrates that the O/ME-SA/ind2001e lineage has a strong presence outside of the Indian subcontinent. Furthermore, the O/ME-SA/Cluster-2018 was observed to have a wider geographic distribution than previously, and like the O/ME-SA/Ind2001d and O/ME-SA/Ind2001e lineages in the past, it may eventually spread outside of its geographic niche. For O/ME-SA/Ind2001e and O/ME-SA/Cluster-2018, the predicted substitution rate for the VP1 region was 6.737 × 10-3 and 8.257 × 10-3 nt substitutions per site per year, respectively. The time of the most recent common ancestor of the O/ME-SA/Ind2001e and O/ME-SA/Cluster-2018 strains suggests that the viruses possibly emerged during 2003-2011 and 2009-2017, respectively. Recent sightings of the O/ME-SA/PanAsia2/ANT10 virus in India and the O/ME-SA/Ind2001e virus in Pakistan point to possible cross-border transit of the viruses. The results of a two-dimensional viral neutralization test revealed that all of the field isolates were antigenically matched to the currently used Indian vaccine strain O INDR2/1975. These results suggest that the serotype O vaccine strain can protect against outbreaks brought on by all three circulating lineages.

Emergence of a novel genetic lineage 'A/ASIA/G-18/2019' of foot and mouth disease virus serotype A in India: A challenge to reckon with.

Foot and mouth disease (FMD) has engendered large scale socioeconomic crises on numerous occasions owing to its extreme contagiousness, transboundary nature, complicated epidemiology, negative impact on productivity, trade embargo, and need for intensive surveillance and expensive control measures. Emerging FMD virus variants have been predicted to have originated and spread from endemic Pool 2, native to South Asia, to other parts of the globe. In this study, 26 Indian serotype A isolates sampled between the year 2015 and 2022 were sequenced for the VP1 region. BLAST and maximum likelihood phylogeny suggest emergence of a novel genetic group within genotype 18, named here as 'A/ASIA/G-18/2019' lineage, that is restricted so far only to India and its eastern neighbour, Bangladesh. The lineage subsequent to its first appearance in 2019 seems to have displaced all other prevalent strains, in support of the phenomenon of 'genotype/lineage turnover'. It has diversified into two distinct sub-clusters, reflecting a phase of active evolution. The rate of evolution of the VP1 region for the Indian serotype A dataset was estimated to be 6.747 × 10-3 substitutions/site/year. India is implementing a vaccination centric FMD control programme. The novel lineage showed good antigenic match with the proposed vaccine candidate A IND 27/2011 when tested in virus neutralization test, while the existing vaccine strain A IND 40/2000 showed homology with only 31% of the isolates. Therefore, in order to combat this challenge of antigenic divergence, A IND 27/2011 could be the preferred strain in the Indian vaccine formulations.

Production and characterization of monoclonal antibodies against foot-and-mouth disease virus serotype O and development of a sandwich ELISA for virus antigen detection.

Foot-and-mouth disease (FMD) is endemic in India with a majority of outbreaks caused by FMD virus (FMDV) serotype O. In the present study a panel of eight (2F9, 2G10, 3B9, 3H5, 4C8, 4D6, 4G10 and 5B6) mouse monoclonal antibodies (MAbs) were developed against FMDV serotype O Indian vaccine strain, O/IND/R2/75 via hybridoma systems. The MAbs generated were FMDV/O specific without cross-reactivity against FMDV type A and Asia 1. All the MAbs were identified as IgG1/kappa type. Out of eight, three MAbs (3B9, 3H5 and 4G10) demonstrated virus neutralizing activity. The reactivity of all MAbs increased with heat treated (@560C) serotype O antigen compared to untreated antigen in sandwich ELISA indicating that their binding epitopes are linear. Six MAbs (except 2F9 and 4D6) reacted with recombinant P1 protein of homologous virus in an indirect ELISA among which only MAb 3B9 bound to VP1. MAb profiling of 37 serotype O field viruses isolated between the years 1962 and 2021 demonstrated antigenic similarity between field isolates and reference vaccine strain. MAbs 5B6 and 4C8 consistently reacted with all 37 isolates. In indirect immunofluorescence assay MAb 5B6 bound well with FMDV/O antigen. Finally, a sandwich ELISA was successfully developed using rabbit polyclonal anti-FMDV/O serum and MAb 5B6 for detection of FMDV/O antigen in clinical samples (n = 649). The new assay exhibited 100% and 98.89% diagnostic sensitivity and specificity respectively compared to traditional polyclonal antibody-based sandwich ELISA suggesting that the MAb-based ELISA developed here could be an effective method for detection of FMDV serotype O.

Foot-and-mouth disease status in India during the second decade of the twenty-first century (2011-2020).

Foot-and-mouth disease (FMD) is a major disease of livestock in India and causes huge economic losses. The formal FMD control program started in 2003-04 in selected districts and was gradually expanded. The present study provides a descriptive review of the FMD outbreaks, prevalent serotypes, and genetic and antigenic features of the FMD virus (FMDV) that circulated in the country between 2011 and 2020. FMD outbreaks were regularly reported in cloven-hoofed domestic livestock and wildlife, with three serotypes including O, A, and Asia1. During the study period, a total of 2226 FMD outbreaks were documented and serotypes confirmed. FMDV serotype O dominated the outbreak scenario, accounting for about 92% of all outbreaks, followed by Asia1 (5% of all outbreaks) and A (3% of all outbreaks). Two major epidemics of FMD on an unprecedented scale during the years 2013 and 2018 by serotype O were recorded. The spatial distribution of FMD was characterized by a larger number of outbreaks in the southern region of the country. In an annual-scale analysis, 2020 was the year with the lowest outbreaks, and 2013 was the year with the highest. The month-scale analysis showed that outbreaks were reported throughout the year, with the highest numbers between October and March. The emergence of three major lineages (O/ME-SA/Ind2001d, O/ME-SA/Ind2001e, and O/ME-SA/Ind2018) of serotype O was observed during the period. In the cases of serotype A and Asia1, the appearance of at least one novel lineage/genetic group, including A/G-18/non-deletion/2019 and Asia1/Group-IX, was documented. While serotype A showed the advent of antigenic variants, serotypes O and Asia1 did not show any antigenic diversity. It was noticed during the course of an outbreak that animal movement contributes significantly to disease transmission. Except for 2018, when numerous FMD outbreaks were recorded, the number of annual outbreaks reported after 2016 has been lower than in the first half of the decade, probably due to mass vaccination and COVID-19 pandemic-linked movement restrictions. Even during outbreaks, disease symptoms in ruminant populations, including cattle, were found to be less severe. Regular six-monthly immunization certainly has a positive impact on the reduction of disease burden and should be followed without fail and delay, along with intensive disease surveillance.

Impact of mass vaccination on the spatiotemporal dynamics of FMD outbreaks in India, 2008-2016.

Foot-and-mouth disease (FMD) is endemic in India, where circulation of serotypes O, A and Asia1 is frequent. Here, we provide an epidemiological assessment of the ongoing mass vaccination programs in regard to post-vaccination monitoring and outbreak occurrence. The objective of this study was assessing the contribution of mass vaccination campaigns in reducing the risk of FMD in India from 2008 to 2016 by evaluating sero-monitoring data and modelling the spatiotemporal dynamics of reported outbreaks. Through analyzing antibody titre data from >1 million animals sampled as part of pre- and post-vaccination monitoring, we show that the percent of animals with inferred immunological protection (based on ELISA) was highly variable across states but generally increased through time. In addition, the number of outbreaks in a state was negatively correlated with the percent of animals with inferred protection. We then analyzed the distribution of reported FMD outbreaks across states using a Bayesian space-time model. This approach provides better acuity to disentangle the effect of mass vaccination programs on outbreak occurrence, while accounting for other factors that contribute to spatiotemporal variability in outbreak counts, notably proximity to international borders and inherent spatiotemporal correlations in incidence. This model demonstrated a ∼50% reduction in the risk of outbreaks in states that were part of the vaccination program. In addition, after controlling for spatial autocorrelation in the data, states that had international borders experienced heightened risk of FMD outbreaks. These findings help inform risk-based control strategies for India as the country progresses towards reducing reported clinical disease.

Short communication: preliminary observations on the serum levels of HSP70 and its correlation with serum cortisol, thyroid hormones, and acute-phase protein concentration in cattle naturally infected with foot-and-mouth disease virus.

The study aimed to explore the serum levels of HSP70 and identify its possible association with serum cortisol, thyroid hormones, and acute-phase protein concentrations in cattle naturally infected with foot-and-mouth disease (FMD) virus. After the FMD outbreak in an organized dairy cattle farm in India, blood samples were obtained from clinically infected (n = 40) and apparently healthy (n = 30) animals. Samples were processed and tested by an in-house DIVA assay for confirmation of FMD infection. Serum was analyzed for HSP70, cortisol, T4, T3, haptoglobin, and serum amyloid A by enzyme-linked immunosorbent assay (ELISA). HSP70 concentrations were significantly higher in the serum of clinically infected cattle (p < 0.01) than the healthy group. To the best of our knowledge, this is the first report describing the elevated serum levels of HSP70 under infectious diseases of bovines. Cortisol (p < 0.05), haptoglobin (p < 0.001), and serum amyloid A (p < 0.05) concentrations also markedly increased in the diseased animals; however, no differences (p > 0.05) were found in T4 and T3 levels between healthy and infected cattle. Elevated HSP70 concentration correlated positively with high cortisol (p < 0.05) and haptoglobin (p < 0.001) levels suggesting an essential link between these acute events during clinical infectious phase of FMD.

Genetic characterization of foot-and-mouth disease virus serotype O isolates collected during 2014-2018 revealed dominance of O/ME-SA/Ind2001e and the emergence of a novel lineage in India.

Foot-and-mouth disease (FMD) is endemic in India with a preponderance of outbreaks caused by FMD virus (FMDV) serotype O. Out of the 11 global topotypes of serotype O, only ME-SA topotype has been reported in the country so far. Lineage O/ME-SA/Ind2001 and O/ME-SA/PanAsia are documented as the most dominant ones in terms of the number of outbreaks caused by them. To understand the distribution of topotype/lineages in India and their antigenic behaviour during the year 2014-2018, a total of 286 FMDV serotype O viral isolates were sequence determined at the VP1 region, and 109 isolates were characterized antigenically. All the isolates grouped in the ME-SA topotype, being distributed in lineage O/ME-SA/Ind2001 (within sub-lineages O/ME-SA/Ind2001d and O/ME-SA/Ind2001e), and a new group designated here as O/ME-SA/2018 cluster. The sub-lineage O/ME-SA/Ind2001e reported for the first time in India during the year 2015, replaced sub-lineage O/ME-SA/Ind2001d gradually, which was dominating since 2008. During the years 2014-2018, the sub-lineage O/ME-SA/Ind2001e was found to be the most predominant one whose mean evolutionary rate was observed to be faster than that of the sub-lineage O/ME-SA/Ind2001d. The codon sites 45 and 85 of VP1 were found to be under diversifying selection in a large proportion of trees. The common ancestor predicted for sub-lineages O/ME-SA/Ind2001e and O/ME-SA/2018 dates back to 2012 and 2016, respectively. The sustenance and spread of the new O/ME-SA/2018 cluster need to be assessed by continued surveillance. The Indian vaccine strain O/INDR2/1975 was found to provide adequate antigenic coverage to the emerging and prevalent serotype O lineages. The trait association tests showed frequent virus exchange among different states, which could be an important confounder in the region-specific assessment of effectiveness of FMD control programme.

Alternate vaccine strain selection in the wake of emerging foot-and-mouth disease virus serotype A antigenic variants in India.

'National foot-and-mouth disease (FMD) control programme' is being implemented in India and therefore predicting vaccine match is a key surveillance task. Recently, a considerable proportion of field viruses (75.6%) showed antigenic drift from the existing serotype A vaccine strain A IND 40/2000 necessitating search for an alternate strain. Here, antigenic relationship ('r1' value) of 87 field viruses with each of the 8 candidate strains was estimated by virus neutralization test. A IND 27/2011 strain emerged to be the one with the widest spectrum of antigenic coverage showing 'r1' value of more than 0.3 with 81.6% of field strains. It achieved a reasonably high titre of log10 7.5 TCID50/ml in BHK-21 suspension cell which was accompanied by positive charge gaining substitutions (E82-K and E131-K in VP2) thought to have adaptive significance. However, potency trial remains to be conducted before A IND 27/2011 finds a place in the vaccine formulation.

Diagnostic assays developed for the control of foot-and-mouth disease in India.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of livestock, primarily affecting cattle, buffalo and pigs. FMD virus serotypes O, A and Asia1 are prevalent in India and systematic efforts are on to control and eventually eradicate the disease from the country. FMD epidemiology is complex due to factors like co-circulation, extinction, emergence and re-emergence of genotypes/lineages within the three serotypes, animal movement, diverse farm practices and large number of susceptible livestock in the country. Systematic vaccination, prompt diagnosis, strict biosecurity measures, and regular monitoring of vaccinal immunity and surveillance of virus circulation are indispensible features for the effective implementation of the control measures. Availability of suitable companion diagnostic tests is very important in this endeavour. In this review, the diagnostic assays developed and validated in India and their contribution in FMD control programme is presented.

Construction and characterization of yeast two-hybrid cDNA library derived from LFBK cell line.

The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system.

Indirect ELISA using recombinant nonstructural protein 3D to detect foot and mouth disease virus infection associated antibodies.

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.

Comparative evaluation of non-structural protein-antibody detecting ELISAs for foot-and-mouth disease sero-surveillance under intensive vaccination.

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.

Diagnostic potential of recombinant nonstructural protein 3B to detect antibodies induced by foot-and-mouth disease virus infection in bovines.

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.

Truncated recombinant non-structural protein 2C-based indirect ELISA for FMD sero-surveillance.

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.

Phylogenetic analysis of Indian serotype Asia1 foot-and-mouth-disease virus isolates revealed emergence and reemergence of different genetic lineages.

Foot-and-mouth-disease virus (FMDV) serotype Asia1 causes significant number of disease outbreaks in India. Indian Asia1 virus isolates were shown to be genetically heterogeneous and of the two lineages (lineage B and lineage C) described in India, lineage C caused majority of the outbreaks. Emergence of a novel divergent lineage (lineage D) within lineage C has been described in 2001. In the present report, the complete VP1 genomic region of 41 FMDV Asia1 field isolates collected between 2003 and 2008 was sequenced. Phylogenetic analysis revealed reemergence of lineage C since 2005 following exclusive dominance of lineage D in the period between 2002 and 2004. At many positions lineage specific signature residues were identified. The antigenic relationship of the field isolates with the currently used vaccine strain IND63/72 was also determined, which reflects antigenic stability of serotype Asia1 in-spite of genetic heterogeneity.

Genetic characterization of type A foot-and-mouth disease virus 3A region in context of the reemergence of VP359-deletion lineage in India.

The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. Here we analyzed the 3A region of serotype A virus in view of the emergence of a variant group in India with an amino acid deletion at an antigenically critical position of capsid protein, VP3. The 3A region exhibited extreme variability with 38% of the amino acid positions showing substitutions and the C-terminal third (127-151) region was most flexible. Genotype inclusive grouping of type A foot-and-mouth disease virus as observed in 1D region based phylogeny was much less apparent at 3A region possibly due to independent evolution of nonstructural and structural protein coding regions. Akin to the 1D region, the VP3(59)-deletion group maintained its phylogenetic distinctness even at the 3A region and was found to be diverging with time. Twelve lineage specific signature amino acid residues, of which four were identified to be experiencing positive selection, indicates fixation of advantageous mutations in a lineage specific manner. Six positions, all located in the hypervariable C-terminal third, were identified to be under positive selection and were presumed to be imparting the virus certain advantage accounting for its adaptability to wide host spectrum and rapid dissemination. A significant change of Q(44)H was noted only in the older lineage (VIIb) of the deletion group at a position where Q(44)R mutation is associated with guinea pig adaptation. As this site has been detected to be under positive selection, such a lineage specific substitution is thought to have imparted certain temporary advantage to the virus during its possible adaptation in wild or some understudied domestic hosts and must not have seriously compromised fitness upon readaptation in bovines. A conserved hydrophobic transmembrane domain from position 59 to 76 could be predicted which possibly anchors 3A to intracellular membranes for successful interaction with RNA replication complex.

Analysis of the leader proteinase (L(pro)) region of type A foot-and-mouth disease virus with due emphasis on phylogeny and evolution of the emerging VP3(59)-deletion lineage from India.

Genotype inclusive grouping of Indian type A isolates as observed in 1D region based phylogeny was distorted at complete L(pro) region, where the VP3(59)-deletion group lineages of genotype VII clustered away from both genotypes VII and VI, confirming its uniqueness and independent evolution of L(pro) and 1D region. Akin to the 1D region, this deletion group is gradually diverging genetically even at L region forming more number of lineages and inter-lineage distance at L region is considerably more than that for 1D region. The deletion group is restricted to India only as none of the exotic sequences clustered within this group. Notably, L protein exhibited variability comparable to external capsid proteins as evident from its high d(N)/d(S) ratio (0.105), number of variable amino acid positions (41%), low Ts/Tv ratio (3.47) and alignment revealed N-terminal region, beta2 sheet and C-terminal extension to be extremely variable. Basic residues at P1, P3 and only leucine at P2 were predicted to provide an optimum autocatalytic cleavage site at L/P1 junction. All of the eight sites identified to be under positive selection revealed aa substitutions of varied physicochemical properties and at two positions lineage specific signatures were observed, which supports the contention that lineages are evolving under differential selection pressure to adapt to the varied ecological environment.

Assessment of suitability of two serotype A candidate vaccine strains for inclusion in FMD vaccine in India.

The recent type A foot and mouth disease virus field isolates recovered in India are shown to be antigenically quite divergent from the in-use vaccine strain (IND 17/82), warranting the selection of a suitable vaccine strain which can cover this diversity in antigenic spectrum. In earlier studies employing neutralization test with anti-146S rabbit sera raised against eight candidate vaccine strains, IND 81/00 and IND 40/00 belonging to genotype VII were found to offer the best antigenic coverage. In order to assess the credibility of IND 81/00 and IND 40/00 as vaccine strains, 17 recent isolates received during 2005-2006 and representative isolates from older genotypes were subjected to two-dimensional micro-neutralization assay using bovine convalescent serum (against IND 81/00 and IND 40/00) and bovine vaccinate serum (against IND 40/00). From the results it is evident that both the isolates IND 81/00 (antigenic relationship 'r-value' >0.40 with 86% of isolates) and IND 40/00 ('r-value' >0.40 with 78% of isolates) show nearly equal antigenic relatedness with the recent field viruses and hence both of these are effective vaccine candidates in present context. Though very limited in its extent, these useful data obtained with antisera raised in homologous host system are logical extension of the on going quest for the appropriate vaccine strain and circumvents species disparities in the immune recognition of epitopes.

Genotype differentiating RT-PCR and sandwich ELISA: handy tools in epidemiological investigation of foot and mouth disease.

Years of molecular epidemiological surveillance has revealed co-circulation of two antigenically divergent genotypes of foot and mouth disease virus serotype A in India. Genotype differentiating RT-PCR and sandwich ELISA were developed as fast, cost-effective and user-friendly alternatives to 1D region based phylogeny for detection and differentiation of genotype VI and VII. The RT-PCR assay targeting 1D region was found to be more sensitive and authentic in distinguishing genotypes than sandwich ELISA. These assays promise to be reliable tools in the epidemiological investigation of foot and mouth disease in the country.