Engineering foot-and-mouth disease virus serotype O IND R2/1975 for one-step purification by immobilized metal affinity chromatography.

Immobilized metal affinity chromatography (IMAC) allows for the efficient protein purification via metal affinity tag such as hexa-histidine (His6) sequence. To develop a new chromatography strategy for the purification and concentration of foot-and-mouth disease virus (FMDV) particles, we inserted the His6-tag at the earlier reported site in the VP1 G-H loop of the FMD virus serotype O vaccine strain IND R2/1975. Display of the His6-tag on the capsid surface, endowed the virus with an increased affinity for immobilized nickel ions. We demonstrated that the His6-tagged FMDV could be produced to high titre and purified from the infected BHK-21 cell lysates by IMAC efficiently. Further, a 1150-fold reduction in protein contaminant level and an 8400-fold reduction in DNA contaminant level were achieved in the IMAC purification of His6-tagged FMDV. Through various functional assays it has been found that the tagged virus retains its functionality and infectivity similar to the non-tagged virus. The affinity purification of the His6-tagged FMDV may offer a feasible, alternative approach to the current methods of FMDV antigen purification, concentration and process scalability.

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.

Detection of antibodies specific for foot-and-mouth disease virus infection using indirect ELISA based on recombinant nonstructural protein 2B.

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.

The expression of IL6 and 21 in crossbred calves upregulated by inactivated trivalent FMD vaccine.

Foot and mouth disease (FMD) is an economically important disease and a whole-virus inactivated trivalent virus vaccine is the mainstay for controlling the disease in India. The protective humoral immune response to FMD vaccination is a complex, but, tightly regulated process mediated by the interplay of interleukins (IL). Based on the specific role of IL6 and 21 in adaptive immune response, we hypothesized that inactivated trivalent FMD vaccine would stimulate IL6 and 21 expression in the circulating lymphocytes. The expressions of IL6 and 21 were assayed on 0, 28, 60, 90, and 120 d post-vaccination (DPV) by quantitative PCR (qPCR) with simultaneous assessment of FMDV antibody titer by liquid phase blocking ELISA. The results revealed that the peak expression of IL6 and 21 was on DPV 28 which correlated well with the FMDV antibody titer and plummeted to the prevaccination titer level by 60 DPV. As IL21 is the final effector of antibody production as compared to IL6, we investigated the expression of IL21 in calves that had protective titer (>1.8) with the unprotected group (<1.8). Expression of IL21 on 28 DPV was numerically higher in the protected than that of the unprotected group of calves.

A new lineage of foot-and-mouth disease virus serotype O in India.

The complete nucleotide sequence of a new lineage of foot and mouth disease virus (FMDV) serotype O was determined. The lineage designated as Ind2011 first appeared during 2011 in the Southern region of India. Excluding the poly C tract and poly A tail, the genome of Ind2011 ranged from 8,169 to 8,172 nucleotides. Variation in the genome length was due to insertions/deletions in LF-UTR. The lineage had a higher sequence identity with lineage PanAsia-1 at P1 and P2 regions, and with lineage PanAsia-2 at P3 and L regions. Phylogenetically, the isolates were placed closely to both PanAsia-1 and 2 lineages, and appear to be a novel variant of the PanAsian lineage.

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.

Efficient rescue of foot-and-mouth disease virus in cultured cells transfected with RNA extracted from clinical samples.

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.

Emergence of a novel lineage genetically divergent from the predominant Ind2001 lineage of serotype O foot-and-mouth disease virus in India.

In India, emergence of Ind2001 lineage of foot-and-mouth disease virus (FMDV) serotype O was recorded in the year 2001. After causing sporadic incidences, the Ind2001 lineage that re-surged in 2008 out-competed PanAsia from the field during 2009 and continued its dominance during 2010 and 2011 as well. The lineage has diversified in due course of time, leading to two sub-lineages (Ind2001a and Ind2001b). The sub-lineage Ind2001a include isolates collected during 2001-2002 and sub-lineage Ind2001b is constituted largely by isolates collected during 2008-2012. The nucleotide substitution rate of sub-lineage Ind2001b was estimated at 6.58×10⁻³ substitutions/site/year. The most stable PanAsia lineage is restricted only to few outbreaks. During 2011, emergence of a new genetic group with >9% nucleotide divergence from rest of the lineages circulating in the country was detected and named as lineage Ind2011. Two specific amino acid substitutions at positions VP1-36 (F) and VP2-133 (T) were observed in the Ind2011 lineage. The new lineage at present is restricted only to southern states of the country. It is uncertain whether the emergence was triggered by immune pressure or due to a bottleneck in transmission or selected for higher fitness value. Six sites (4, 68, 83, 135, 138 and 209) in VP1 protein were identified to undergo episodic diversifying selection in serotype O field isolates. Both emerging and re-emerging lineages had appropriate antigenic match with currently used vaccine strain, INDR2/1975. Irrespective of genetic variability, the field isolates showed remarkable conservation at antigenically critical residues that might contribute to the observed antigenic stability. With the emergence of a new genetic group after a span of 10 years, the overall epidemiological scenario in the region is expected to change in the coming years.