Aniket Sanyal

Status of foot-and-mouth disease in India.

Foot-and-mouth disease (FMD) is endemic in India and causes severe economic loss. Status of FMD in the country for five fiscal years is presented. Outbreaks were more in number in 2007-2008 than 2010-2011. Three serotypes of FMD virus (O, A and Asia1) are prevalent. Serotype O was responsible for 80% of the confirmed outbreaks/cases, whereas Asia1 and A caused 12% and 8%, respectively. Geographical region-wise assessment indicated varying prevalence rate in different regions viz; 43% in Eastern region, 31.5% in Southern region, 11.6% in North-eastern region, 5% Central region, 4.4% Western region and 4% in Northern region. Highest number of outbreaks/cases was recorded in the month of September and lowest in June. Emergence and re-emergence of different genotypes/lineages within the serotypes were evident in real-time investigation carried out from time to time. Continues antigenic divergence in serotype A resulted in change in the vaccine strain in 2009. As on date, all genetic diversity within the serotypes is well tolerated by the vaccine strains. Unrestricted animal movements in the country play a major role in the spread of FMD.

Evolutionary dynamics of foot-and-mouth disease virus O/ME-SA/Ind2001 lineage

Foot-and-mouth disease (FMD) virus serotype O Ind2001 lineage within the Middle East-South Asia topotype is the major cause of recent FMD incidences in India. A sub-lineage of Ind2001 caused severe outbreaks in the southern region of the country during 2013 and also reported for the first time from Libya. In this study, we conducted a detailed evolutionary analysis of Ind2001 lineage. Phylogenetic analysis of Ind2001 lineage based on maximum likelihood method revealed two major splits and three sub-lineages. The mean nucleotide substitution rate for this lineage was calculated to be 6.338×10(-3)substitutions/site/year (s/s/y), which is similar to those of PanAsian sub-lineages. Evolutionary time scale analysis indicated that the Ind2001 lineage might have originated in 1989. The sub-lineage Ind2001d that caused 2013 outbreaks seems to be relatively more divergent genetically from other Ind2001 sub-lineages. Seven codons in the VP1 region of Ind2001 were found to be under positive selection. Four out of 24 recent Ind2001 strains tested in 2D-MNT had antigenic relationship value of <0.3 with the serotype O vaccine strain indicating intra-epidemic antigenic diversity. Amino acid substitutions found in these minor variants with reference to antigenic diversity have been discussed. The dominance of antigenically homologous strains indicates absence of vaccine immunity in the majority of the affected hosts. Taken together, the evolution of Ind2001 lineage deviates from the strict molecular clock and a typical lineage evolutionary dynamics characterized by periodic emergence and re-emergence of Ind2001 and PanAsia lineage have been observed in respect of serotype O.

Serological evidence of foot-and-mouth disease virus infection in randomly surveyed goat population of Orissa, India.

India is endemic for foot-and-mouth disease (FMD), and goats constitute the second largest susceptible population of domestic livestock. FMD surveillance and control strategies in the country largely ignore small ruminants, known to be critical in the epidemiology of the disease. Here, serological investigations were carried out to generate estimates of antibody prevalence in goats of Orissa state to both non-structural (NSP-Ab) and structural proteins (SP-Ab) of FMD. The apparent overall NSP-Ab and SP-Ab seroprevalences were 38% and 20.7%, respectively, which signifies a very high level of FMD virus circulation in the goat population despite the lack of clinical signs in this species. The apparent prevalence of NSP-Ab and SP-Ab was positively correlated in the sampling areas. Interestingly, the values found for NSP-Ab prevalence were almost consistently higher than those found for SP-Ab prevalence. This could have been attributable to either issues related to sensitivity and specificity of the test systems employed or differences in the post-infection kinetics of NSP- and SP-Ab. The pattern that emerged from SP-Ab analysis indicated goats being infected with all three prevalent serotypes (O, A and Asia 1) and reinforces the concept that non-vaccinated goats can be exploited as tracer animals for detecting serotypes involved in outbreaks. The results underscore the requirement to bring caprine species under comprehensive surveillance and vaccination campaigns to check silent amplification, excretion and transmission of the virus.

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 92.1xa0% (95xa0% CI: 89.0–94.5) and 98.1xa0% (95xa0% CI: 96.9–98.8), respectively, as compared to 97.04xa0% and 95.04xa0% 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 998xa0days postinfection in experimental calves. Substantial agreement in the test results (90.6xa0%) 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.

A positively charged lysine residue at VP2 131 position allows for the enhanced adaptability of foot-and-mouth disease virus serotype A in BHK-21 cells

Field outbreak strains of foot-and-mouth disease virus (FMDV) infect host cells through certain Arg-Gly-Asp (RGD) dependent integrin family of cellular receptors. In contrast, FMDV adapted in non-host cell cultures are reported to acquire the ability to infect cells via heparin sulphate (HS) or other unidentified cell surface molecules. It has been reported that during the serial passage of FMDV serotype A in BHK-21 cell culture, VP2 E131K (E2131K) substitution was fixed within the heparin sulphate binding site. The fixation of positively charged residue at position VP2 131 of serotype A is considered to associate with the ability to utilise alternative receptor. In this study, an infectious full-length cDNA clone for Indian FMDV vaccine strain A IND 40/2000 was constructed. Through site-directed mutagenesis on the cDNA clone, recombinant virus containing positive charged amino acid residue at position VP2 131 was rescued. The recombinant mutated virus was shown to have specific and strong affinity for HS and demonstrated an enhanced infectivity in BHK-21 cell line. The introduction of lysine residue at VP2 131 position that allows cell culture adaptation of FMDV serotype A could be exploited for the generation of vaccine seed stocks with improved growth properties in BHK-21 cell line.

Evolution of foot-and-mouth disease virus serotype A capsid coding (P1) region on a timescale of three decades in an endemic context

Three decades-long (1977-2013) evolutionary trend of the capsid coding (P1) region of foot-and-mouth disease virus (FMDV) serotype A isolated in India was analysed. The exclusive presence of genotype 18 since 2001 and the dominance of the VP3(59)-deletion group of genotype 18 was evident in the recent years. Clade 18c was found to be currently the only active one among the three clades (18a, 18b and 18c) identified in the deletion group. The rate of evolution of the Indian isolates at the capsid region was found to be 4.96×10(-3)substitutions/site/year. The timescale analysis predicted the most recent common ancestor to have existed during 1962 for Indian FMDV serotype A and around 1998 for the deletion group. The evolutionary pattern of serotype A in India appears to be homogeneous as no spatial or temporal structure was observed. Bayesian skyline plots indicate a sharp decline in the effective number of infections after 2008, which might be a result of mass vaccination or inherent loss of virus fitness. Analyses of variability at 38 known antigenically critical positions in a countrywide longitudinal data set suggested that the substitutions neither followed any specific trend nor remained fixed for a long period since frequent reversions and convergence was noticed. A maximum of 6 different amino acid residues was seen in the gene pool at any antigenically critical site over the decades, suggesting a limited combination of residues being responsible for the observed antigenic variation. Evidence of positive selection at some of the antigenically critical residues and the structurally proximal positions suggest a possible role of pre-existing immunity in the host population in driving evolution. The VP1 C-terminus neither revealed variability nor positive selection, suggesting the possibility that this stretch does not contribute to the antigenic variation and adaptation under immune selection.

Application of a recombinant capsid polyprotein (P1) expressed in a prokaryotic system to detect antibodies against foot-and-mouth disease virus serotype O

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.

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.

Substitutions accrued on Foot-and-mouth disease virus capsid during propagation in cell culture

Three lineages of serotype O of foot-and-mouth disease virus were passaged serially in BHK-21 cell culture without application of any immune pressure, to study the frequency, nature and location of the substitutions accruing on the virus capsid. The viruses showed unusual stability as only 12 substitutions were observed in 13 different regimens and the majority of the substitutions reverted back to the parental genotype very soon after their appearance. Of the 12 substitutions, a maximum of 8 were found in the VP1 region. Some substitutions (81, 147, 152, 203 and 210 in VP1 and 50 in VP3) were observed at the established antigenic sites suggesting that antigenic diversification can occur in the absence of immune selection. The viruses after serial cytolytic infection of BHK-21 cells, demonstrated an ability to infect the integrin-deficient CHO-K1 cell line suggesting an expansion in their receptor usage potential. Even after 25–50 passages in BHK-21 cell system no histidine to arginine switch was observed at the 56th residue of VP3. Amino acid sequence analysis of 141 Indian field isolates for the residues involved in heparin binding sites suggest the importance of net positive charge in the HS-binding pocket or elsewhere on the capsid for interaction with the alternative receptors and cell culture adaptation rather than acquisition of positive charge at any particular position for all serotype O strains.