Development of TaqMan Probe-Based One-Step RT-qPCR Assay Targeting 2B-NSP Coding Region for Diagnosis of Foot-and-Mouth Disease in India.

A one-step TaqMan probe-based RT-qPCR assay in the duplex format simultaneously targeting FMD Virus (FMDV) 2B NSP-coding region and 18S rRNA housekeeping gene was developed and evaluated. The duplex RT-qPCR assay specifically detected FMDV genome in both infected cell culture suspensions and a variety of clinical samples such as FMD-affected tongue/feet epithelium, oral/nasal swabs, milk and oro-pharyngeal fluids. The RT-qPCR assay was found to be highly sensitive, since the assay was 105-fold more sensitive than the traditional FMDV detecting antigen-ELISA (Ag-ELISA) and 102-fold better sensitive than both virus isolation and agarose gel-based RT-multiplex PCR. In addition, the assay could detect up to 100 copies of FMDV genome per reaction. In the epithelial samples (n = 582) collected from the FMD-affected animals, the diagnostic sensitivity was 100% (95% CI 99-100%). Similarly, all the FMDV-negative samples (n = 65) tested were confirmed negative by the new RT-qPCR assay, corresponding to 100% diagnostic specificity (95% CI = 94-100%). Further, the duplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.4 to 3.56% for FMDV-2B gene target, and from 2 to 4.12% for 18S rRNA gene target. While analyzing FMDV-infected cell culture suspension, a fairly strong positive correlation (correlation coefficient = 0.85) was observed between 2B-based RT-qPCR and WOAH-approved 5'UTR RT-qPCR assays. Therefore, the one-step RT-qPCR assay developed here with an internal control could be used for rapid, effective, and reliable detection of FMDV in pan-serotypic manner, and has the potential for routine diagnosis of FMDV in high throughput manner.

Pathology, virulence-associated gene profiling, antimicrobial susceptibility, and pathogenicity of untypeable capsular serotypes of Pasteurella multocida isolated from slaughtered pigs of India.

Pasteurella multocida is widely distributed in all pig-rearing countries, affecting the economic viability and profitability of pig production. The present research highlights the molecular characterization and pathology of untypeable capsular serotypes of P. multocida in slaughtered pigs from prominent pig-rearing states of India. The prevalence of Pasteurellosis was 27.17% by Pasteurella multocida specific Pasteurella multocida specific PCR (PM-PCR). assay, while isolation rate was 7.62%. The microscopic lesions of bronchopneumonia, tonsillitis, and the presence of bacterial antigens in immunohistochemistry confirmed P. multocida with pathologies. In capsular typing, the majority of the isolates were untypeable with prevalence of 52.15% and 43.58% in molecular and microbiological methods, respectively. All the isolates showed the uniform distribution of virulence genes such as exbB, nanB, sodC, plpB, and oma87 (100%), while the variations were observed in ptfA, hasR, ptfA, pfhA, hsf-1, and plpE genes. The untypeable isolates showed higher prevalence of hsf-1 gene as compared to others. The untypeable serotypes showed a higher degree of resistance to ampicillin, amoxicillin, and penicillin antibiotics. The mouse pathogenicity testing of untypeable capsular isolates confirmed its pathogenic potential. The higher frequency of pathogenic untypeable isolates with antibiotic resistance profile might pose a serious threat to the pigs, and therefore, preventive measures should be adopted for effective control.

One-step SYBR green-based real-time RT-PCR assay for detection of foot-and-mouth disease virus circulating in India.

Rapid, sensitive, and reliable laboratory detection of foot-and-mouth disease virus (FMDV) infection is essential for containing and controlling virus infection in any geographical area. In this report a SYBR green-based 3Dpol-specific one-step real-time RT-PCR (rRT-PCR) assay was developed for the pan-serotype detection of FMDV in India. The detection limit of the SYBR green-based rRT-PCR was 10-2 TCID50/50 µl, which is 10 times more sensitive than the traditional agarose gel electrophoresis-based RT-multiplex PCR (RT-mPCR). The standard curve exhibited a linear range across 8-log10 units of viral RNA dilution. The reproducibility and specificity of this assay were reasonably high suggesting that the 3Dpol-specific SYBR green rRT-PCR could detect FMDV genome specifically and with little run-to-run variation. The new 3Dpol-specific SYBR green rRT-PCR assay was evaluated alongside the established RT-mPCR using the archived FMDV isolates and clinical field samples from suspected FMD outbreaks. A perfect concordance was observed between the new rRT-PCR and the traditional RT-mPCR on viral RNA in the archived FMDV cell culture isolates tested. Furthermore, 73% of FMDV-suspected clinical samples were detected positive through the 3Dpol-specific SYBR green rRT-PCR, while the detection rate through the traditional RT-mPCR was 57%. Therefore, the SYBR green-based 3Dpol-specific one-step rRT-PCR could be considered as a valuable assay with higher diagnostic sensitivity to complement the routine assays that are being used for FMD virus diagnosis in India.

Infection and protection responses of deletion mutants of non-structural proteins of foot-and-mouth disease virus serotype Asia1 in guinea pigs.

The development of a negative marker vaccine against the foot-and-mouth disease virus (FMDV) will enhance the capabilities to differentiate vaccinated from infected animals and move forward in the progressive control pathway for the control of FMD. Here, we report the development of mutant FMDV of Asia1 with partial deletion of non-structural proteins 3A and 3B and characterization of their infectivity and protection response in the guinea pig model. The deleted FMDV Asia1/IND/63/1972 mutants, pAsiaΔ3A and pAsiaΔ3A3B1 were constructed from the full-length infectious clone pAsiaWT, the viable virus was rescued, and the genetic stability of the mutants was confirmed by 20 monolayer passages in BHK21 cells. The mutant Asia1 viruses showed comparable growth pattern and infectivity with that of AsiaWT in the cell culture. However, the AsiaΔ3A3B1 virus showed smaller plaque and lower virus titer with reduced infectivity in the suckling mice. In guinea pigs, the AsiaΔ3A3B1 virus failed to induce the disease, whereas the AsiaΔ3A virus induced typical secondary lesions of FMD. Vaccination with inactivated Asia1 mutant viruses induced neutralizing antibody response that was significantly lower than that of the parent virus on day 28 post-vaccination (dpv) in guinea pigs (P < 0.05). Furthermore, challenging the vaccinated guinea pigs with the homologous vaccine strain of FMDV Asia1 conferred complete protection. It is concluded that the mutant AsiaΔ3A3B1 virus has the potential to replace the wild-type virus for use as a negative marker vaccine after assessing the vaccine worth attributes in suspension cell and protective efficacy study in cattle.Key points• Deletion mutant viruses of FMDV Asia1, developed by PCR-mediated mutagenesis of NSP 3A and 3B1, were genetically stable.• The growth kinetics and antigenic relatedness of the mutant viruses were comparable with that of the wild-type virus.• Vaccination of guinea pigs with the deletion mutant viruses conferred complete protection upon challenge with the homologous virus.

Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis.

BACKGROUND: RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key pre-analytical step for SARS-CoV2 detection which often achieved using commercial RNA-extraction kits. However, due to the COVID-19 pandemic, bulk production and the supply chains for the commercial RNA-extraction kit have been seriously compromised. The shortage of commercial RNA-extraction kit is even more acute in developing country. Furthermore, use of one-off design RNA-columns can generate plastic wastes that have an environmental pollution effect. METHODS AND RESULTS: To address these issues, in this study, we used warm alkaline solution containing Triton X-100 for the complete removal of the residual SARS-CoV2 RNA from the used RNA-binding silica column. Columns regenerated using the alkaline solution have the viral RNA purification capability that is comparable to the fresh silica columns. We also demonstrated that RNA-binding silica columns can be regenerated and reused for a minimum of five-times. CONCLUSIONS: Therefore, the use of the RNA-column regeneration method may benefits several SARS-CoV2 diagnostic laboratories throughout the world by cutting down the requirement of commercial RNA-purification column.

Emergence of foot-and-mouth disease virus serotype Asia1 group IX in India.

Foot-and-mouth disease virus (FMDV) serotype Asia1 is prevalent in India and is responsible for a minor proportion of FMD outbreaks. Globally, serotype Asia1 is grouped into nine different groups (GI-IX) based on genetic analysis. In India, only Asia1/G-III and Asia1/G-VIII have been documented so far. Phylogenetic analysis of recent serotype Asia1 isolates from India revealed the emergence of Asia1/G-IX. The Asia1/G-IX lineage shares recent common ancestry with Asia1/G-VIII dating to 2016. The root state posterior probabilities of Asia1/G-VIII are inclusive and there may have been either an incursion of the virus from Bangladesh, where it was first identified, or in situ evolution of the virus within India, which is an intriguing possibility.

Immunogenicity and protective efficacy of 3A truncated negative marker foot-and-mouth disease virus serotype A vaccine.

Foot-and-mouth disease (FMD) is a highly contagious, economically significant disease of cloven-hoofed animals caused by FMD virus (FMDV) of the Picornaviridae family. Vaccination of susceptible animals with inactivated virus vaccine is the standard practice for disease control. The prophylactic use of the inactivated vaccines has reduced the disease burden in many countries endemic to FMD. In the process of implementation of the mass vaccination program and disease eradication, it is essential to differentiate infected from vaccinated animals (DIVA) where a large proportion of the animal population is vaccinated, and disease-free zones are being established, to help in sero-surveillance of the disease. In such a scenario, the use of a negative marker vaccine is beneficial to rule out false-positive results in a disease-free zone. Here we report the construction and rescue of an infectious cDNA clone for FMDV serotype A Indian vaccine strain lacking 58 amino acid residues (87-144 amino acid position) in the carboxy-terminal region of the viral 3A protein. The recombinant deletion mutant virus showed similarity in the antigenic relationship with the parental strain. Immunization of guinea pigs with the inactivated vaccine formulated using the deletion mutant virus induced potent immune response with 100% protective efficacy upon challenge with homologous virus. Further, we show that sera from the guinea pigs infected with the deletion mutant virus did not show reactivity in an indirect ELISA test targeting the deleted portion of 3A protein. We conclude that the recombinant deletion mutant virus vaccine along with the newly developed companion indirect ELISA targeting portion of FMDV 3A protein could be useful in the implementation of a precise DIVA policy in our country when we reach FMD free status with vaccination.

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O.

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID50 challenge virus.

Evidence of subclinical foot-and-mouth disease virus infection in young calves born from clinically recovered cow under natural condition.

Foot-and-mouth disease (FMD) is a highly contagious and economically important, transboundary viral disease of cloven-hoofed animals. It is known that an asymptomatic, persistent FMD virus (FMDV) infection may occur subsequent to acute or subclinical FMDV infection in adult ruminants. However, virus persistence in young calves has not been studied. In the current investigation, FMDV infection parameters were examined for calves born to FMD-clinically recovered cows (CRC), asymptomatic cows from infected herds (ASC) and cows from with no history of FMD (NHF). The study was conducted in natural condition after FMD outbreaks in two dairy herds in India. No calves described herein had any clinical signs of FMD. Six out of 12 calves born to CRC had detectable FMDV RNA in oesophageal-pharyngeal fluid consistent with asymptomatic FMDV infection. Three of the 12 calves of CRC group had seroreactivity against FMDV non-structural proteins. One calf had detectable FMDV RNA at two consecutive samplings at 2 months apart. However, infectious FMDV was not isolated from any calf in the study. None of the calves in the ASC or NHF groups had any evidence of FMDV infection. Overall, these data are consistent with earlier report on calves having been infected in utero. Further investigation of FMDV persistence in calves under controlled conditions may lead to greater understanding of the viral pathogenesis.

Antigenic variability of foot-and-mouth disease virus serotype O during serial cytolytic passage.

The emergence and disappearance of antigenic variants of foot-and-mouth disease virus (FMDV) during a field outbreak occurs periodically due to the volatile nature of its genome. In the present analysis, change in antigenic behavior of serotype O FMDV during the serial cytolytic passage in the absence of immune pressure was observed. Initially, the isolate showed a poor antigenic match (relationship value <0.3) with the serotype O vaccine strain and upon serial passage increase in relationship value was observed. Comparison of capsid sequence revealed substitution at four positions (VP3:K58 â†’ E and P158 â†’ S, VP1:E83 â†’ K and R172 â†’ Q) acquired during the serial passage. Examination of passage level and amino acid substitution revealed the critical role of position VP3-58 that was identified earlier as crucial for antigenic site IV, in the observed antigenic variability. The role of position VP3-58 was further confirmed using reverse genetics approach.

Evaluation of FTA(®) card for the rescue of infectious foot-and-mouth disease virus by chemical transfection of extracted RNA in cultured cells.

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease of transboundary importance. Inadequate storage and shipment of suspected clinical samples can compromise the ability to detect and characterise FMD virus (FMDV) in endemic countries, thereby, leading to the loss of valuable virological and epidemiological data. This study, investigates the potential of using FTA(®) cards for dry transportation of clinical samples and subsequent recovery of infectious FMDV by chemical transfection of FTA(®) card fixed RNA as an alternative to the conventional cell culture based virus isolation method. A higher proportion of infectious FMDV was rescued from clinical samples (cell culture isolates, tongue epithelial suspension and impression smears) by the FTA(®) card fixed RNA transfection method (76%) compared to the conventional cell culture based virus isolation (56%), suggesting a better performance of the current RNA transfection procedure. Furthermore, it was possible to rescue live virus by the transfection of RNA extracted from FTA(®) card impregnated with clinical samples that had been stored at varying temperature (4-37 °C) up to a period of six weeks. The VP1 sequence data and antigenic relationships with the vaccine strains, between viruses rescued by FTA(®) card fixed RNA transfection and conventional cell culture, were comparable. Therefore, these results support the use of the FTA(®) card for the economic, dry, non-hazardous transport of FMD suspected clinical samples from the site of collection to national/international reference laboratories.

Partial deletion of stem-loop 2 in the 3' untranslated region of foot-and-mouth disease virus identifies a region that is dispensable for virus replication.

The 3' untranslated region (3' UTR) of the foot-and-mouth disease virus (FMDV) genome plays an essential role in virus replication, but the properties of the 3' UTR are not completely defined. In order to determine the role of different regions of the 3' UTR in FMDV replication, we conducted site-directed mutagenesis of the 3' UTR of FMDV serotype O IND R2/1975 using a cDNA clone. Through independent serial deletions in various regions of the 3' UTR, we demonstrated that deletion of nucleotides between the stem-loop (SL) structures and in the beginning and the end regions of the SL2 structure could be lethal for FMDV replication. However, a block deletion of 20 nucleotides (nt 60 to 79) in the middle of SL2 did not affect the viability of FMDV in cultured cells. Characterisation of the deletion mutant virus (O(R2/1975-Δ3'UTR 60-79)) revealed no significant difference in growth kinetics or RNA replication ability compared to the parental virus. However, the mutant virus produced slightly larger plaques when compared to the parental virus. This is the first description of a dispensable 20-nucleotide region in SL2 of the FMDV 3' UTR.

Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142 â†’ R or H142 â†’ F or H142 â†’ A substitutions resulted in non-infectious FMDV, H142 â†’ D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A.

Diagnostic application of recombinant non-structural protein 3A to detect antibodies induced by foot-and-mouth disease virus infection.

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.

Chimeric foot-and-mouth disease virus serotype O displaying a serotype Asia1 antigenic epitope at the surface.

OBJECTIVE: To determine whether the G-H loop of foot-and-mouth disease virus (FMDV) serotype O can function as a target structure to harbour and display serotype Asia1 antigenic epitope at the surface. RESULTS: Using reverse genetics, FMDV serotype O IND R2/1975 displaying a FMDV serotype Asia1 B cell epitope at the capsid surface was constructed. The epitope-inserted recombinant chimeric virus was genetically stable up to ten serial passages in cell culture and exhibited growth properties similar to the parental serotype O virus. Furthermore, the surface-displayed Asia1 epitope able to react with serotype Asia1 specific antibodies in a competitive ELISA. Importantly, the recombinant chimeric virus showed neutralizing activity to both serotype O and Asia1 polyclonal antibodies. CONCLUSION: The capsid protein of FMDV serotype O can effectively display potent epitope of other serotypes, making this an attractive approach for the design of new generation bi-valent FMD vaccines.

Capsid coding region diversity of re-emerging lineage C foot-and-mouth disease virus serotype Asia1 from India.

Foot-and-mouth disease virus (FMDV) serotype Asia1 was first reported in India in 1951, where three major genetic lineages (B, C and D) of this serotype have been described until now. In this study, the capsid protein coding region of serotype Asia1 viruses (n = 99) from India were analyzed, giving importance to the viruses circulating since 2007. All of the isolates (n = 50) recovered during 2007-2013 were found to group within the re-emerging cluster of lineage C (designated as sublineage C(R)). The evolutionary rate of sublineage C(R) was estimated to be slightly higher than that of the serotype as a whole, and the time of the most recent common ancestor for this cluster was estimated to be approximately 2001. In comparison to the older isolates of lineage C (1993-2001), the re-emerging viruses showed variation at eight amino acid positions, including substitutions at the antigenically critical residues VP279 and VP2131. However, no direct correlation was found between sequence variations and antigenic relationships. The number of codons under positive selection and the nature of the selection pressure varied widely among the structural proteins, implying a heterogeneous pattern of evolution in serotype Asia1. While episodic diversifying selection appears to play a major role in shaping the evolution of VP1 and VP3, selection pressure acting on codons of VP2 is largely pervasive. Further, episodic positive selection appears to be responsible for the early diversification of lineage C. Recombination events identified in the structural protein coding region indicates its probable role in adaptive evolution of serotype Asia1 viruses.

Megaprimer-mediated capsid swapping for the construction of custom-engineered chimeric foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the selection of appropriate candidate vaccine strain and its adaptation in cell culture to yield high titer of virus is a cumbersome process. An attractive approach to circumvent this tedious process is to replace the capsid coding sequence of an infectious full-genome length cDNA clone of a good vaccine strain with those of appropriate field strain, to produce custom-made chimeric FMD virus (FMDV). Nevertheless, the construction of chimeric virus can be difficult if the necessary endonuclease restriction sites are unavailable or unsuitable for swapping of the capsid sequence. Here we described an efficient method based on megaprimer-mediated capsid swapping for the construction of chimeric FMDV cDNA clones. Using FMDV vaccine strain A IND 40/2000 infectious clone (pA(40/2000)) as a donor plasmid, we exchanged the capsid sequence of pA(40/2000) with that of the viruses belonging to serotypes O (n = 5), A (n = 2), and Asia 1 (n = 2), and subsequently generated infectious FMDV from their respective chimeric cDNA clones. The chimeric viruses exhibited comparable infection kinetics, plaque phenotypes, antigenic profiles, and virion stability to the parental viruses. The results from this study suggest that megaprimer-based reverse genetics technology is useful for engineering chimeric vaccine strains for use in the control and prevention of FMD in endemic countries.

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.

Marker vaccine potential of foot-and-mouth disease virus with large deletion in the non-structural proteins 3A and 3B.

Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of transboundary importance. Regular vaccination with chemically inactivated FMD vaccine is the major means of controlling the disease in endemic countries like India. However, the traditional inactivated vaccines may sometimes contain traces of FMD viral (FMDV) non-structural protein (NSP), therefore, interfering with the NSP-based serological discrimination between infected and vaccinated animals. The availability of marker vaccine for differentiating FMD infected from vaccinated animals (DIVA) would be crucial for the control and subsequent eradication of FMD in India. In this study, we constructed a negative marker FMDV serotype O virus (vaccine strain O IND R2/1975), containing dual deletions of amino acid residues 93-143 and 10-37 in the non-structural proteins 3A and 3B, respectively through reverse genetics approach. The negative marker virus exhibited similar growth kinetics and plaque morphology in cell culture as compared to the wild type virus. In addition, we also developed and evaluated an indirect ELISA (I-ELISA) targeted to the deleted 3AB NSP region (truncated 3AB) which could be used as a companion differential diagnostic assay. The diagnostic sensitivity and specificity of the truncated 3AB I-ELISA were found to be 95.5% and 96%, respectively. The results from this study suggest that the availability negative marker virus and companion diagnostic assay could open a promising new avenue for the application of DIVA compatible marker vaccine for the control of FMD in India.

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.