Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus.

Inactivated purified whole virus vaccines are used for control of foot and mouth disease (FMD). ELISAs detecting antibodies to the nonstructural proteins (NSP), a marker of infection, are primarily used to differentiate FMD virus (FMDV) infected from vaccinated animals (DIVA). However, such DIVA assays have a limitation to their specificity since residual NSPs present in the relatively impure vaccines are suspected to induce an NSP-antibody response in the repeatedly vaccinated animals. Epitope-deleted negative marker vaccine strategy seems to have an advantage over the conventional vaccines in identifying the infected animals with accuracy. NSP 3AB contains an abundance of immunodominant B-cell epitopes of diagnostic importance. This study addresses the feasibility of producing 3AB-truncated FMDV mutant as a potential negative marker vaccine candidate. An infectious cDNA clone of FMDV serotype Asia 1 strain was used to engineer an array of deletion mutations in the established antigenic domain of 3AB. The maximum length of deletion tolerated by the virus was found to be restricted to amino acid residues 87-144 in the C-terminal half of 3A protein along with deletion of the first two copies of 3B peptide. The 3AB-truncated marker virus (Asia 1 IND 491/1997Δ3A87-1443B1,2+FLAG) demonstrated infectivity titres comparable to that of the parental virus in BHK-21 (log10 7.42 TCID50/ml) and LFBK-αVß6 (log10 8.30 TCID50/ml) cell monolayer culture. The protein fragment corresponding to the viable deletion in the 3AB region was expressed in a prokaryotic system to standardize a companion assay (3A87-1533B1,2 I-ELISA) for the negative marker virus which showed reasonably high diagnostic sensitivity (96.9%) and specificity (100% for naïve and 97.1% for uninfected vaccinated samples). The marker virus and its companion ELISA designed in this study provide a basis to devise a marker vaccine strategy for FMD control.

The carboxy-terminal half of nonstructural protein 3A is not essential for foot-and-mouth disease virus replication in cultured cell lines.

In foot-and-mouth disease (FMD)-endemic parts of the globe, control is mainly implemented by preventive vaccination with an inactivated purified vaccine. ELISAs detecting antibodies to the viral nonstructural proteins (NSP) distinguish FMD virus (FMDV)-infected animals in the vaccinated population (DIVA). However, residual NSPs present in the vaccines are suspected to be a cause of occasional false positive results, and therefore, an epitope-deleted negative marker vaccine strategy is considered a more logical option. In this study, employing a serotype Asia 1 FMDV infectious cDNA clone, it is demonstrated that while large deletions differing in size and location in the carboxy-terminal half of 3A downstream of the putative hydrophobic membrane-binding domain (deletion of residues 86-110, 101-149, 81-149 and 81-153) are tolerated by the virus without affecting its infectivity in cultured cell lines, deletions in the amino-terminal half (residues 5-54, 21-50, 21-80, 55-80 and 5-149) containing the dimerization and the transmembrane domains are deleterious to its multiplication. Most importantly, the virus could dispense with the entire carboxy-terminal half of 3A (residues 81-153) including the residues involved in the formation of the 3A-3B1 cleavage junction. The rescue of a replication-competent FMDV variant carrying the largest deletion ever in 3A (residues 81-153) and the fact that the deleted region contains a series of linear B-cell epitopes inspired us to devise an indirect ELISA based on a recombinant 3A carboxy-terminal fragment and to evaluate its potential to serve as a companion diagnostic assay for differential serosurveillance if the 3A-truncated virus is used as a marker vaccine.

Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions.

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 ßE-ßF loop, VP1 C-terminus and VP3 ß-B knob) surrounding the fivefold symmetry axis (VP1 ßF-ßG loop) and at other discrete sites on the capsid (VP3 ßG-ßH loop, VP1 C-terminus, VP2 ßC strand and VP1 ßG-ßH loop). A lysine insertion in the VP1 ßF-ßG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 ßG-ßH loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 ßB-ßC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the 'RGD' motif with αv-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.

Evolution of serotype A foot-and-mouth disease virus capsid under neutralizing antibody pressure in vitro.

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 ßG-ßH 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.

RNA structure disrupting G320-T transversion within the short fragment of the 5' untranslated region prevents rescue of infectious foot-and-mouth disease virus.

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.

Immunodiagnosis of foot-and-mouth disease using mutated recombinant 3ABC polyprotein in a competitive ELISA.

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.

Recombinant non-structural polyprotein 3AB-based serodiagnostic strategy for FMD surveillance in bovines irrespective of vaccination.

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.

Phylogenetic structure of serotype A foot-and-mouth disease virus: global diversity and the Indian perspective.

Global epidemiological analysis is vital for implementing progressive regional foot-and-mouth disease control programmes. Here, we have generated VP1 region sequences for 55 Indian type A outbreak strains and have included complete VP1 sequences from 46 other countries to obtain a comprehensive global phylogeographical impression. A total of 26 regional genotypes within three continental topotypes, based on a 15% nucleotide divergence cut-off criterion, could be identified. These genotypes correlated with distinct evolutionary lineages in the maximum-likelihood phylogeny. During the last decade, ten genotypes have been in circulation the world over and it was evident that no type A strain has transgressed the continental barriers during this period. A single genotype (genotype 18) within the Asia topotype has been circulating in India with neither any incursion nor any long distance movement of virus out of the country during the last ten years, although close genetic and epidemiological links between viruses from Bhutan and India were revealed.

Multiplex PCR for rapid detection of serotype A foot-and-mouth disease virus variants with amino acid deletion at position 59 of the capsid protein VP3.

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.

Isolation of bluetongue virus serotype 1 (BTV-1) from goats and its phylogenetic relationship to other BTV-1 isolates worldwide based on full-length sequence of genome segment-2.

Eight bluetongue viruses (BTV) were isolated in BHK-21 cell culture from blood of goats suffering from peste des petits ruminants. These viruses were identified as BTV serotype 1 (BTV-1) by RT-PCR using VP2-gene-based primers coupled with sequencing of the PCR products. All of the isolates showed similar genome migration profile in 8% polyacrylamide gel electrophoresis. The genome segment-2 (seg-2) of one isolate (MKD18/India/2008) was amplified piecemeal by overlapping PCR, and the products were sequenced to obtain full-length seg-2. Phylogenetic analysis based on the seg-2 sequence revealed that MKD18 is closely related to Australian BTV-1 isolates, with 86.3-86.8% nucleotide identity. Phylogenetic analysis based on the partial sequence of seg-2 (541 bp, nucleotides 1,304-1,844) showed that the Indian BTV-1 isolates, namely, MKD18, Avikanagar, Sirsa-3 and Chennai, are very closely related to each other, with more than 99.6% nucleotide identity. Although a high degree of similarity exists, the Indian BTV-1 isolates collected over the past 25 years should be studied to demonstrate the co-existence of different VP2 antigenic profiles.