Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting main protease and papain-like protease.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50 ) values ranging from 1.42 to 32.7 µM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half-maximum effective concentration (EC50 ) values of 21.73 and 31.19 µM, respectively. The anti-inflammatory roles of azadirachtin and withanolide_A when assessed using HEK293T cells, were found to significantly reduce the levels of CXCL10, TNFα, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-α1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2-specific enzymes and also host immune pathways involved in virus-mediated inflammation.

Mechanistic and thermodynamic characterization of antiviral inhibitors targeting nucleocapsid N-terminal domain of SARS-CoV-2.

The nucleocapsid (N) protein of SARS-CoV-2 plays a pivotal role in encapsulating the viral genome. Developing antiviral treatments for SARS-CoV-2 is imperative due to the diminishing immunity of the available vaccines. This study targets the RNA-binding site located in the N-terminal domain (NTD) of the N-protein to identify the potential antiviral molecules against SARS-CoV-2. A structure-based repurposing approach identified the twelve high-affinity molecules from FDA-approved drugs, natural products, and the LOPAC1280 compound libraries that precisely bind to the RNA binding site within the NTD. The interaction of these potential antiviral agents with the purified NTD protein was thermodynamically characterized using isothermal titration calorimetry (ITC). A fluorescence-based plate assay to assess the RNA binding inhibitory activity of small molecules against the NTD has been employed, and the selected compounds exhibited significant RNA binding inhibition with calculated IC50 values ranging from 8.8 µM to 15.7 µM. Furthermore, the antiviral efficacy of these compounds was evaluated using in vitro cell-based assays targeting the replication of SARS-CoV-2. Remarkably, two compounds, Telmisartan and BMS-189453, displayed potential antiviral activity against SARS-CoV-2, with EC50 values of approximately 1.02 µM and 0.98 µM, and a notable selective index of >98 and > 102, respectively. This study gives valuable insight into developing therapeutic interventions against SARS-CoV-2 by targeting the N-protein, a significant effort given the global public health concern posed due to the virus re-emergence and long COVID-19 disease.

Development and evaluation of recombinant gD protein based ELISA for sero-surveillance of BoHV-1 in India.

Bovine herpes virus-1 (BoHV-1) is responsible for production losses through decreased milk yields, abortions, infertility, and trade restrictions in the bovine population. The disease is endemic in many countries including India. As the virus harbors a unique feature of latency animals once infected with the virus remain sero-positive for lifetime and can re-excrete the virus when exposed to stressful conditions. Hence, identification and culling of infected animals is only the means to minimize infection-associated losses. In this study, an economical indigenous assay for the detection of BoHV-1 specific antibodies was developed to cater to the huge bovine population of the country. The viral structural gD protein, expressed in the prokaryotic system was used for optimization of an indirect ELISA for bovines followed by statistical validation of the assay. The diagnostic sensitivity and specificity of the indirect ELISA were 82.9% and 91.3% respectively. Systematically collected serum samples representing organized, unorganized and breeding farms of India were tested with the indigenously developed assay for further validation.

A retrospective study showing a high rate of seropositivity against SARS-CoV-2 in wild felines in India.

We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of seropositivity was positively correlated with that of the incidence in humans, suggesting the occurrence of large spillover events.

LSDV126 gene based molecular assays for specific detection and characterization of emerging Lumpy Skin Disease virus.

Lumpy skin disease (LSD) is a highly infectious and economically important viral disease, which is currently emerging in the Indian subcontinent. LSD is caused by Lumpy Skin Disease Virus (LSDV) under the genus Capripoxvirus and the family Poxviridae. Since its first incursion in India in the year 2019, the virus is rapidly disseminating through different means like direct contact, fomites and mainly by blood-feeding insects. As the disease has never been reported from India or neighbouring countries, there is a lack of planning and preparatory measures in terms of diagnostics and vaccines to control the disease. In the absence of any homologous vaccine, a live attenuated heterologous goat pox vaccine (Uttarkashi strain) is now being widely used in the country for the prevention of LSDV infection. Use of live attenuated goat pox virus vaccine necessitates the availability of an assay which could specifically detect and differentiate LSDV from goat pox virus. In this study, nucleotide sequences of LSDV126 gene encoding extracellular enveloped virus protein of circulating LSDV and goat pox virus were determined and analyzed. Deletion of 27 nt tandem repeats was observed in LSDV in comparison to goat pox and LSDV vaccine viruses. The deletion region was targeted for designing primers specific to LSDV, but not goat pox virus. A novel isothermal polymerase spiral reaction (PSR) was optimized as pen side diagnostic for prompt and sensitive detection of genomic DNA of LSDV. The assay was found to be highly sensitive and specific when compared to the real-time PCR. The assay was found to be specifically detecting only LSDV but not the goat pox virus. The limit of detection was identified as 9 × 10-6 ng of positive DNA. The assay will provide a point of care tool that will be a boon for the successful control of LSD in India.

Detection of SARS-CoV-2 in a free ranging leopard (Panthera pardus fusca) in India.

We report an incidence of natural infection of SARS-CoV-2 in free-ranging Indian leopard (Panthera pardus fusca). The case was detected during routine screening. Post-mortem and laboratory examination suggested virus-induced interstitial pneumonia. Viral genome could be detected in various organs including brain, lung, spleen, and lymph nodes by real-time PCR. Whole-genome sequence analysis confirmed infection of Pango lineage B.1.617.2 of SARS-CoV-2. Till now, only Asiatic lions have been reported to be infected by SARS-CoV-2 in India. Infections in animals were detected during peak phase of pandemic and all the cases were captive with close contacts with humans, whereas the present case was observed when human cases were significantly low. No tangible evidence linked to widespread infection in the wild population and the incidence seems to be isolated case. High nucleotide sequence homology with prevailing viruses in humans suggested spillover infection to the animal. This report underlines the need for intensive screening of wild animals for keeping track of the virus evolution and development of carrier status of SARS-CoV-2 among wildlife species. Supplementary Information: The online version contains supplementary material available at 10.1007/s10344-022-01608-4.

Complete Genome Sequence of a Virulent Duck Enteritis Virus (DEV/India/IVRI-2016) Isolated from Southern India.

Molecular characterization of Indian isolates of duck enteritis virus (DEV) so far has been limited to a few selected genomic regions. Here, we report the complete genome sequence of an isolate, DEV/India/IVRI-2016, from southern India that is 158,091 bp in length.

An investigation on dissimilarity of mass flow rate and N on exergo-enviro-economic parameters for solar still of single slope type integrated with N similar PVT flat plate collectors having series connection.

This paper investigates analytically the effect of dissimilarity of mass flow rate [Formula: see text] and number of collectors (N) on exergo-enviro-economic parameters for solar still of single slope type integrated with N similar photovoltaic thermal flat plate collectors having series connection (NPVTFPC-SS) keeping water depth as 0.14 m. All four kinds of weather conditions for New Delhi have been taken for the computation of different parameters. All relevant equations obtained using energy balance equations for all components of the system have been fed to a computer code inscribed in MATLAB-2015a for computing different parameters. The computation of different relevant parameters has been performed for various values of [Formula: see text] and N while keeping water depth as constant to know the effect of variation of [Formula: see text] and N on exergo-enviro-economic parameters for NPVTFPC-SS. It has been concluded that the value of carbon credit earned, enviroeconomic and exegoeconomic parameters, and productivity diminishes with the enhancement in [Formula: see text] at given N. The optimum value of N for given value of [Formula: see text] has been found to be 10 from exergoeconomic parameter viewpoint and 6 from productivity viewpoint.

Identification and characterization of DNA aptamers specific to VP2 protein of canine parvovirus.

Canine parvovirus-2 (CPV-2) is ubiquitously distributed in dog population worldwide causing a severe and often fatal gastroenteritis. Owing to its highly contagious nature, rapid detection of CPV is crucial in effective control of the disease. Aptamers have emerged as potential alternative to antibodies as affinity reagents in diagnostic field. Present study was aimed to select and validate ssDNA aptamers specific to CPV. Systematic evolution of ligands through exponential enrichment (SELEX) method was employed for selection of CPV structural protein (VP2) specific DNA aptamers. SELEX was performed using a pool of ssDNA library comprising 30 random nucleotide region. A total of seven rounds of SELEX were performed using VP2 protein as target antigen which yielded ten aptamers (1A-10A) with distinct sequences. Target binding of all ten aptamers was assessed by dot blot and enzyme-linked oligonucleotide assay (ELONA) which revealed that 5A, 6A, 9A, and 10A were superior binders. In silico analysis of the aptamers revealed the existence of binding site on VP2 protein, and binding pattern was similar to in vitro findings. The affinity (KD) of all these four binders against CPV was evaluated by ELONA indicating relatively higher affinity of 6A and 10A than remaining two DNA sequences. Out of which, aptamer 6A displayed cross-reactivity with canine distemper virus and canine corona virus. Hence, aptamer 10A was considered as better binding sequence having high specificity and affinity for CPV. The study confirms the future utility of selected aptamers in development of a reliable diagnostic for rapid detection of CPV. KEY POINTS: • Canine parvovirus-specific ssDNA aptamers were identified with nanomolar affinity (100-150 nM). • Three aptamers displayed negligible cross-reactivity with other related viruses. • Aptamer 10A displayed high binding affinity and specificity to CPV.

Selective isolation of foot-and-mouth disease virus from coinfected samples containing more than one serotype.

The foot-and-mouth disease virus (FMDV) causes a highly infectious disease of all cloven-footed animals. The RNA genome of the virus continuously evolves, leading to the generation of new strains; this necessitates the selection of new vaccine strains to ensure complete protection. Infection with one FMDV serotype does not provide cross-protection against the other FMDV serotypes. Many of the recovered animals may become carriers of the FMDV, but they still remain susceptible to the other serotypes. Coinfection with multiple FMDV serotypes has been reported and studied to understand the virus evolution. Isolation and characterization of all the involved serotypes in the mixed infection case is essential to understand the molecular evolution of the virus. In this study, two cases of coinfection were studied by selective isolation of each of the FMDV serotypes under the cross-serotype-specific immune pressure. It was estimated that the virus present in a minimum of 10-0.92 TCID50 could be isolated from the mixed population containing other serotypes in infective doses of 100.25 TCID50 or less. All involved serotypes present in the mixed infection cases were isolated, without any cross-contamination. Virus characterization revealed that genotype 2 was of serotype A virus from a sample collected in 1995, which was last reported in 1986, indicating a possible subdued prevalence of the genetic group even after vanishing from the field.

Differential responses of chicken monocyte-derived dendritic cells infected with Salmonella Gallinarum and Salmonella Typhimurium.

Salmonella enterica serovar Gallinarum is a host-restricted bacterial pathogen that causes a serious systemic disease exclusively in birds of all ages. Salmonella enterica serovar Typhimurium is a host-generalist serovar. Dendritic cells (DCs) are key antigen-presenting cells that play an important part in Salmonella host-restriction. We evaluated the differential response of chicken blood monocyte-derived dendritic cells (chMoDCs) exposed to S. Gallinarum or S. Typhimurium. S. Typhimurium was found to be more invasive while S. Gallinarum was more cytotoxic at the early phase of infection and later showed higher resistance against chMoDCs killing. S. Typhimurium promoted relatively higher upregulation of costimulatory and other immune function genes on chMoDCs in comparison to S. Gallinarum during early phase of infection (6 h) as analyzed by real-time PCR. Both Salmonella serovars strongly upregulated the proinflammatory transcripts, however, quantum was relatively narrower with S. Gallinarum. S. Typhimurium-infected chMoDCs promoted relatively higher proliferation of naïve T-cells in comparison to S. Gallinarum as assessed by mixed lymphocyte reaction. Our findings indicated that host restriction of S. Gallinarum to chicken is linked with its profound ability to interfere the DCs function. Present findings provide a valuable roadmap for future work aimed at improved vaccine strategies against this pathogen.

Relational interaction between T-lymphocytes and SARS-CoV-2: A review.

Coronavirus disease 2019 (COVID-19) has turned out as one of the worst medical and economic misfortunes across the globe. The etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the Coronaviridae family and represents a disease manifestation from asymptomatic to severe respiratory damage. High transmissibility and contagious nature of the virus helps it to flourish in a large population. The immune system aids to retain the virus, but with accelerated cytokine secretion, it could transform into double edge sword resulting in unrestrained systemic inflammation which might become life-threatening. SARS-CoV-2 sets substantial impact on T-lymphocytes during its course of infection. The number of CD4+ T, CD8+ T, and Treg cells tend to decrease profoundly in case of severe illness. Besides, the virus modulates the CD4+ T/ CD8+ T and Treg/Th17 cells ratio and induces the functional exhaustion of T cells to make them inefficient. T cells define the pathogenesis of severe cases and provide major contributions in antiviral defense. Therefore, the apprehension of T-lymphocytes in SARS-CoV-2 infection would implicate in developing antivirals, disease control, and would broaden the way for vaccine formulation. Thus, the review depicts the significance of T-lymphocytes interaction with SARS-CoV-2. Keywords: SARS-CoV-2; COVID-19; T-lymphocytes; cytokine; inflammation; immune response.

Evaluation of ELISA and PCR in parallel to single intradermal cervical tuberculin test (SICT) for diagnosis of tuberculosis in buffaloes.

Bovine tuberculosis is an economically important disease with very high zoonotic potential. Single intradermal cervical tuberculin test (SICT) is considered a gold standard assay for the diagnosis of bovine tuberculosis. However, bovines especially buffaloes may produce a false negative result when the animal becomes cell-mediated immune (CMI) anergic in the advanced stage of the disease. In the present study, ELISA and PCR assays were successfully demonstrated to be useful in diagnosing tuberculosis especially in the CMI anergic buffaloes infected with Mycobacterium bovis. ELISA and PCR assays are able to detect 8.94% and 8.13%, respectively, more animals as positive in comparison to standard SICT assay in a selected population of 123 buffaloes. The moderate agreement between SICT and ELISA (k: 0.528; 0.249-0.807), a substantial agreement between SICT and PCR (k: 0.648; 0.364-0.931), and high agreement between ELISA and PCR (k: 0.856; 0.697-1.0) highlight that ELISA and PCR, if used in parallel with SICT, will provide better sensitivity over single assay. Reduction of false negative reactors may help in minimizing the zoonotic threat from bovine tuberculosis especially in disease endemic region where human and livestock interface is quite high.

Identification of novel interactions between host and non-structural protein 2C of foot-and-mouth disease virus.

The 2C protein of foot-and-mouth disease virus (FMDV) is reported to play a critical role in the virus replication complex and modulating the host's immune response. However, the underlying molecular intricacies of subversion of cellular machinery remains poorly understood, thus emphasizing the need to study 2C-host interactions. In this study, we identified the host proteins interacting with the 2C using yeast-two hybrid (Y2H) approach, which is one of the most recognized, high-throughput tools to study protein-protein interactions. The FMDV-2C bait was characterized for auto-activation, toxicity, and expression and was found to be suitable for mating with cDNA library. On preliminary screening a total of 32 interacting host proteins were identified which were reduced to 22 on subsequent confirmation with alternative yeast based assays. Amongst these, NMI/2C interaction has been reported earlier by Wang et al. (2012) and remaining 21 are novel interactions. The Reactome analysis has revealed the role of the identified host proteins in cellular pathways exploited by 2C during FMDV replication. We also confirmed interaction of MARCH7, an E3 ubiquitin ligase with 2C using mammalian two-hybrid system and co-immunoprecipitation. This study leads to the identification of novel 2C interacting host proteins which enhance our understanding of 2C-host interface and may provide checkpoints for development of potential therapeutics against FMDV.

Foot-and-Mouth Disease Virus: Immunobiology, Advances in Vaccines and Vaccination Strategies Addressing Vaccine Failures-An Indian Perspective.

A mass vaccination campaign in India seeks to control and eventually eradicate foot-and-mouth disease (FMD). Biosanitary measures along with FMD monitoring are being conducted along with vaccination. The implementation of the FMD control program has drastically reduced the incidence of FMD. However, cases are still reported, even in regions where vaccination is carried out regularly. Control of FMD outbreaks is difficult when the virus remains in circulation in the vaccinated population. Various FMD risk factors have been identified that are responsible for FMD in vaccinated areas. The factors are discussed along with strategies to address these challenges. The current chemically inactivated trivalent vaccine formulation containing strains of serotype O, A, and Asia 1 has limitations including thermolability and induction of only short-term immunity. Advantages and disadvantages of several new-generation alternate vaccine formulations are discussed. It is unfeasible to study every incidence of FMD in vaccinated animals/areas in such a big country as India with its huge livestock population. However, at the same time, it is absolutely necessary to identify the precise reason for vaccination failure. Failure to vaccinate is one reason for the occurrence of FMD in vaccinated areas. FMD epidemiology, emerging and re-emerging virus strains, and serological status over the past 10 years are discussed to understand the impact of vaccination and incidences of vaccination failure in India. Other factors that are important in vaccination failure that we discuss include disrupted herd immunity, health status of animals, FMD carrier status, and FMD prevalence in other species. Recommendations to boost the search of alternate vaccine formulation, strengthen the veterinary infrastructure, bolster the real-time monitoring of FMD, as well as a detailed investigation and documentation of every case of vaccination failure are provided with the goal of refining the control program.

Multiplex amplification refractory mutation system PCR (ARMS-PCR) provides convenient method for differentiation of canine parvovirus vaccine and field strains.

Vaccination with canine parvovirus-2 (CPV-2) modified live attenuated vaccine remains an effective control strategy for preventing parvovirus induced enteritis in dogs. Virus shedding is a common phenomenon few days after vaccination, possessing a diagnostic dilemma for accurate differentiation of CPV-2 vaccine and wild type field strains. Though several molecular approaches are available for differentiation, the present study focuses on a simple, rapid, cost-effective differentiating infected from vaccinated animals strategy employing ARMS-PCR for differentiation of CPV-2 vaccine and wild type field strains. The ARMS-PCR was initially validated using positive controls of recombinant plasmids, further used for screening six commercial CPV-2 vaccines and 24 archived CPV-2 positive field samples as well as to check fecal shedding of vaccine virus for 10 days post-vaccination in three vaccinated dogs. Sequencing of randomly selected CPV-2 commercial vaccine strains and archived field samples confirmed authenticity of the developed ARMS-PCR assay.

Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen.

Bovine herpesvirus-1 (BHV-1) is a major viral pathogen affecting bovines leading to various clinical manifestations and causes significant economic impediment in modern livestock production system. Rapid, accurate and sensitive detection of BHV-1 infection at frozen semen stations or at dairy herds remains a priority for control of BHV-1 spread to susceptible population. Polymerase Spiral Reaction (PSR), a novel addition in the gamut of isothermal techniques, has been successfully implemented in initial optimization for detection of BHV-1 genomic DNA and further validated in clinical samples. The developed PSR assay has been validated for detection of BHV-1 from bovine semen (n=99), a major source of transmission of BHV-1 from breeding bulls to susceptible dams in artificial insemination programs. The technique has also been used for screening of BHV-1 DNA from suspected aborted fetal tissues (n=25). The developed PSR technique is 100 fold more sensitive than conventional PCR and comparable to real-time PCR. The PSR technique has been successful in detecting 13 samples positive for BHV-1 DNA in bovine semen, 4 samples more than conventional PCR. The aborted fetal tissues were negative for presence of BHV-1 DNA. The presence of BHV-1 in bovine semen samples raises a pertinent concern for extensively screening of semen from breeding bulls before been used for artificial insemination process. PSR has all the attributes for becoming a method of choice for rapid, accurate and sensitive detection of BHV-1 DNA at frozen semen stations or at dairy herds in resource constrained settings.

Aberrant course of superficial radial nerve in the forearm: an anatomical variation and its clinical implications.

The superficial radial nerve (SRN) is the terminal pure sensory branch of the radial nerve supplying dorsal aspects of the proximal portion of the lateral three and a half digits including the lateral two-thirds of the dorsum of the hand. It transits from its submuscular position to a subcutaneous position by passing between the brachioradialis and extensor carpi radialis longus tendons around 7 to 9 cm proximal to the radial styloid. We present a case where this nerve instead of its normal forearm course, pierced the brachioradialis tendon to become subcutaneous.

The influence of foot and ankle injury patterns and treatment delays on outcomes in a tertiary hospital; a one-year prospective observation.

BACKGROUND: Ankle and foot fractures are amongst the most common injuries, and patterns may vary from primary care set up to tertiary hospitals. Severe foot injuries are projected to have significantly worse outcomes and surgical delays are thought to alter prognosis. METHODS: All patients with foot and ankle trauma were prospectively evaluated at a Tertiary trauma centre over one year. The incidence, fracture patterns, risk factors, and outcomes were evaluated, and cases were divided into simple foot injuries (FASS ≤ 3) and severe foot injuries (FASS>3). Injury mechanisms, associated injuries, and delays in treatment were evaluated, and outcomes were analyzed using Visual-Analogue Scale Foot and Ankle (VASFA), Maryland Foot Score (MFS) and Foot and ankle disability index (FADI). RESULTS: 294 Foot and Ankle injuries (51 females, 243 males) were encountered in 2919 trauma cases (incidence of 10%). 80 patients (27.2%) had simple foot injuries and 214 (72.8%) had severe foot injuries. 29 patients (9.9%) were below 18 years; most (65.3%) patients were between 18 and 45 years age. Road traffic accident was most commonest mode of injury, with ankle fractures (30.6%) the most common. Metatarsal fractures (27.9%) and calcaneal fractures (21.4%) were 2nd and 3rd most common injuries in the foot. Surgical delay averaged 1 day in both severe and simple injuries. Injury led to 32 (10.9%) below knee amputations. Outcome evaluation in 127 (91 severe, 36 simple injuries) patients showed mean Maryland foot score of 89.30 in simple injury group and 84.87 in severe injury group. Mean VASFA score was 82.87 (simple) and 81.87 in severe injury, and mean FADI score was 93.13 (simple) and 91.05 (severe injury). More detailed analysis revealed that more good scores (64.4%) were documented in severe injuries group, and more excellent scores (52.8%) in simple injuries group. CONCLUSION: Foot injuries constitute 10% of all orthopaedic trauma at tertiary hospitals; Majority of them are severe foot injuries, with 68.7% being open injuries. Surgical delay was similar in simple and severe foot and ankle injuries. Outcomes of severe injuries were similar to simple foot and ankle injuries, reflecting on the quality of care that could be administered to them when they present to tertiary hospitals.

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.