Monoclonal antibody based solid phase competition ELISA to detect FMDV serotype A specific antibodies.

In Foot-and-mouth disease (FMD) enzootic countries, periodic vaccination is the key tool in controlling the disease incidence. Active seromonitoring of the vaccinated population is critical to assess the impact of vaccination. Virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection. Assays like liquid phase blocking ELISA (LPBE) or solid phase competition ELISA (SPCE) are preferred as they do not require handling of live FMDV and are routinely used for seromonitoring or for vaccine potency testing; however, false positives are high in LPBE. Here we report, a monoclonal antibody (mAb) based SPCE as a potential alternate assay for antibody titration. From a panel of 12 mAbs against FMDV serotype A, two mAbs were chosen for the development of SPCE. Based on a set of 453 sera, it was demonstrated that mAb 2C4G11, mAb 6E8D11and polyclonal antibody (pAb) based SPCE had a relative sensitivity of 86.1, 86.1 and 80.3 %; and specificity of 99.6, 99.1 and 99.1 %, respectively. The correlation, repeatability, and level of agreement of the assays were high demonstrating the potential use of mAb in large scale surveillance studies and regular vaccine potency testing.

Thermostable negative-marker foot-and-mouth disease virus serotype O induces protective immunity in guinea pigs.

Foot-and-mouth disease (FMD) is a contagious viral disease of high economic importance, caused by FMD virus (FMDV), a positive-sense single-stranded RNA virus, affecting cloven-hoofed animals. Preventive vaccination using inactivated virus is in practice to control the disease in many endemic countries. While the vaccination induces antibodies mainly to structural proteins, the presence of antibodies to the non-structural proteins (NSP) is suggestive of infection, a criterion for differentiation of infected from vaccinated animals (DIVA). Also, there is a growing demand for enhancing the stability of the FMD vaccine virus capsid antigen as the strength of the immune response is proportional to the amount of intact 146S particles in the vaccine. Considering the need for a DIVA compliant stable vaccine, here we report generation and rescue of a thermostable and negative marker virus FMDV serotype O (IND/R2/1975) containing a partial deletion in non-structural protein 3A, generated by reverse genetics approach. Immunization of guinea pigs with the inactivated thermostable-negative marker virus antigen induced 91% protective immune response. Additionally, a companion competitive ELISA (cELISA) targeting the deleted 3A region was developed, which showed 92.3% sensitivity and 97% specificity, at cut-off value of 36% percent inhibition. The novel thermostable-negative marker FMDV serotype O vaccine strain and the companion cELISA could be useful in FMDV serotype O enzootic countries to benefit the FMD control program. KEY POINTS: • Thermostable foot-and-mouth disease virus serotype O with partial deletion in 3A. • Inactivated thermostable marker vaccine induced 91% protection in guinea pigs. • Companion cELISA based on deleted region in 3A could potentially facilitate DIVA.

A new blocking ELISA for detection of foot-and-mouth disease non-structural protein (NSP) antibodies in a broad host range.

Large-scale monitoring of foot-and-mouth disease (FMD) in livestock is imperative in an FMD control program. Detection of antibodies against non-structural proteins (NSP) of FMD virus (FMDV) is one of the best tools to estimate the prevalence of past infection; availability of such a well-validated test is therefore essential. Using a FMDV 3B protein-specific monoclonal antibody, we have developed a new NSP antibody blocking ELISA (10H9 bELISA) and validated it on large panels of sera from different susceptible species. The diagnostic sensitivity of the ELISA was 95% with a specificity of 98%, similar to the values found using a commercial kit (PrioCHECK FMD NS test). The 10H9 bELISA can be used in a broad range of FMD susceptible species making it a very useful tool in monitoring the foot-and-mouth disease control programs by detection of virus circulation in the vaccinated populations. KEY POINTS: • A new ELISA for detection of foot and mouth disease (FMD) antibodies. • Diagnostic sensitivity of 95% and specificity of 98%. • Tested with panels of validated sera from broad host range.

Performance characteristics of virus neutralization test (VNT) and liquid phase blocking ELISA (LPBE) and their relationship in the cattle immunized with trivalent foot and mouth disease vaccine.

Virus neutralization test (VNT) and liquid phase blocking ELISA (LPBE) are accepted tests for screening and as in vitro alternativ to challenge in FMD vaccine potency testing. To replace VNT by LPBE for the screening of cattle, the optimized tests need to be first evaluated for their diagnostic performances. To replace it with LPBE in the absence of protection data, the interrelationship between VNT and LPBE have to be established to find out LPBE cut­off titer corresponding to the currently used VNT titers. Accordingly, VNT and LPBE were carried out using known negative (n = 306) and positive samples [Serotype O (n = 43), A (n = 14) and Asia1 (n = 11)], for the initial screening. The cut­off of < 1.5 log10 LPBE was comparable with that of < 1.2 log10 VNT titer for screening. LPBE was comparable to VNT in terms of specificity, sensitivity as shown by ROC curve and least varying (coefficient of variation 7.73% in LPBE vs 24.19% in VNT). Based on linear regression model using 471 bovine sera, the predicted LPBE titers corresponding to the currently used log 10 VNT titers of 1.65, 1.5 and 1.5, were 2.24, 1.87 and 2.00 for O, A and Asia1, respectively. These LPBE titers hence can be used as cut­off titers for classifying cattle as protected or not protected until correlation based on in vivo challenge between protection and antibody titer is established.

Induction of antiviral and cell mediated immune responses significantly reduce viral load in an acute foot-and-mouth disease virus infection in cattle.

Foot-and-mouth disease virus (FMDV) causes a severe infection in ruminant animals. Here we present an in-depth transcriptional analysis of soft-palate tissue from cattle experimentally infected with FMDV. The differentially expressed genes from two Indian cattle (Bos indicus) breeds (Malnad Gidda and Hallikar) and Holstein Friesian (HF) crossbred calves, highlighted the activation of metabolic processes, mitochondrial functions and significant enrichment of innate antiviral immune response pathways in the indigenous calves. The results of RT-qPCR based validation of 12 genes was in alignment with the transcriptome data. The indigenous calves showing lesser virus load, elicited early neutralizing antibodies and IFN-γ immune responses. This study revealed that induction of potent innate antiviral response and cell mediated immunity in indigenous cattle, especially Malnad Gidda, significantly restricted FMDV replication during acute infection. These data highlighting the molecular processes associated with host-pathogen interactions, could aid in the conception of novel strategies to prevent and control FMDV infection in cattle.

Hematological and serum biochemical profile in cattle experimentally infected with foot-and-mouth disease virus.

BACKGROUND AND AIM: Foot-and-mouth disease (FMD) is an acute viral infection affecting cloven-hoofed animals causing vesicular erosions in the oral cavity and interdigital space. The present study was undertaken to ascertain the time-dependent changes in clinical, hematological, and biochemical profiles in different breeds of cattle following experimental infection. MATERIALS AND METHODS: The animals were inoculated with 1.0×104 50% bovine tongue infectious dose (BTID50) by intradermolingual route. Clinical signs were observed, and blood/serum samples were collected at different time intervals. RESULTS: The white blood cell count declined sharply on days 7-13 and recovered on day 14 post-FMD infection. Biochemical analysis of serum markers for vital organ profile revealed no marked damage. However, a significant increase in blood urea nitrogen (BUN) value indicated pre-renal azotemia. Transient hyperthyroidism was indicated by the rise in T3 and T4 that can be correlated with a decrease in triglyceride and total cholesterol levels. In the cardiac damage assessment study, a distinct breed difference was observed wherein Malnad Gidda calves showed no cardiac damage. CONCLUSION: Except thyroid profile, BUN, and creatine kinase-myocardial band, all other serum biochemical parameters showed no significant abnormalities, whereas lymphopenia is the only hematological change and it is suggested that effective ameliorative measures should be targeted mainly on the feed/water intake, thyroid gland, and the level of lymphocytes.

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.

Efficient inhibition of foot-and-mouth disease virus replication in vitro by artificial microRNA targeting 3D polymerase.

Foot-and-mouth disease (FMD) is a devastating acute viral disease of livestock with cloven hooves. Among various therapeutic control measures, RNA interference (RNAi) is one of the methods being explored to inhibit FMD virus replication and spread. The RNAi is achieved by short hairpin RNAs or artificial microRNAs (amiRNAs). Utility of amiRNAs as antiviral, targeting conserved regions of the viral genome is gaining importance. However, delivery of miRNA in vivo is still a challenge. In this study, the efficacy of amiRNAs in preventing FMD virus replication in a permissive cell culture system was investigated, by generating stable cell lines expressing amiRNAs targeting three functional regions of the FMD virus (FMDV) genome (IRES, 3B3 and 3D). The results showed that amiRNA targeting 3D polymerase is relatively more efficient. However, expression of multiple microRNAs targeting the three regions did not exhibit additive effect. The data suggest that 3D specific miRNA is a potential valid strategy in developing novel antiviral measures against FMDV infection. Keywords: artificial microRNA; foot-and-mouth disease virus; virus inhibition.

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.

Generation of acid resistant virus like particles of vaccine strains of foot-and-mouth disease virus (FMDV).

Foot-and-mouth disease (FMD) is a contagious viral disease affecting cloven hoofed livestock. Insect cell expressed virus like particles (VLPs) are potential alternative to overcome the limitations of inactivated vaccine. However, at pH < 6.5, virus particles disassociate into pentameric structure resulting in loss of antigenicity. Accordingly, we generated seven mutant VLPs containing mutations in the structural genes of FMDV vaccine strains (N17D and/or H145Y for serotypes O/IND/R2/75 and Asia1/IND/63/72; and H142D for serotype A/IND/40/00) by PCR based site directed mutagenesis. Acid resistant VLPs produced by baculovirus expression system were tested for acid stability at pH 7.5, 6.5, 6.0 and 5.5 followed by reactivity in sandwich-ELISA (s-ELISA), which revealed mutant-1 (N17D) of serotype O and Asia1 retained the antigenicity in s-ELISA even at pH 5.5 as compared to other VLPs and wild-types. Further, the 75S empty capsids obtained in sucrose density gradient, when tested in liquid phase blocking ELISA (LPBE) in comparison to cell culture antigen indicated that the VLPs were stable at acidic pH. Transmission electron microscopy of OM-1 confirmed the intact morphology of the empty VLPs. It is concluded that acid resistant VLPs could be useful for developing new generation vaccine or diagnostic for FMDV.

Baculovirus expression and purification of peste-des-petits-ruminants virus nucleocapsid protein and its application in diagnostic assay.

Peste-des-petits-ruminants (PPR) is a contagious and highly devastating disease of small ruminants. For control of endemic PPR, adequate supply of affordable and reliable diagnostics is critical for effective surveillance, along with the use of highly efficacious live vaccines that are currently available. The nucleocapsid (N) protein of PPR virus (PPRV) is an important candidate antigen for developing specific diagnostic, as it is a major viral protein being highly immunogenic and conserved among the structural proteins. In the present study, we expressed the N protein of PPRV (Sungri/96 strain), in baculovirus expression system and purified using affinity column chromatography. The recombinant protein reacted well with PPRV anti-N monoclonal antibodies and PPRV-specific polyclonal antiserum, suggesting that the expressed protein was authentic and in native form. The recombinant protein was evaluated as antigen in the diagnostic ELISA as reference positive control in place of whole virus antigen. The utility of recombinant PPRV N protein circumvents the need to use live PPRV antigen in the routinely used diagnostics targeting 'N' protein of PPRV, thus allowing large-scale field application of the test.

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.

Host serum microRNA profiling during the early stage of foot-and-mouth disease virus infection.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals, with many outbreaks in the developing world. MicroRNAs (miRNAs) are non-coding RNAs that regulate antiviral defence by post-transcriptional regulation of gene expression. In this study, the host miRNA response following FMDV infection was investigated in cattle, a natural host for FMDV. A significant alteration in serum miRNA expression was detected at early stages of infection. Compared to prior to infection, on day 2 postinfection (PI), 119 miRNAs were upregulated, of which 39 were significantly upregulated (P < 0.05). Gene target prediction and pathway enrichment analysis suggested that upregulated miRNAs target innate immune signalling pathways, suggesting a homeostasis effect, possibly to limit inappropriate immune responses. Further, for the significantly upregulated miRNAs, nine miRNA recognition elements were identified in the genome sequence of FMDV serotype O, which was used for infection. The antiviral effect of four of these miRNAs was confirmed in a cell culture system. These data demonstrate that changes in miRNA expression occur during early pathogenesis, and the identification of possible miRNA targets genes could help in elucidating molecular events involved in virus-host interaction and thus could be useful in developing therapeutic strategies.

Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle.

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×109 pfu/animal) or trivalent (5×109 pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging.

Eri silkworm (Samia ricini), a non-mulberry host system for AcMNPV mediated expression of recombinant proteins.

The baculovirus expression system (BVES) based on Autographa californica nucleopolyhedrovirus (AcMNPV) is widely used for the expression of eukaryotic proteins. Several insect cells/larvae that are permissive to AcMNPV have been routinely used as hosts to express heterologous proteins. Domesticated Eri silkworm (Samia ricini), reared in many parts of India, Japan and China, is a non-mulberry silkworm. The present study shows that the Eri silkworm larvae are susceptible to intra-haemocoelical inoculation of AcMNPV. The virus replicates in the larva, as indicated by an increased viral loads in the haemolymph upon injection of a recombinant AcMNPV carrying green fluorescent protein gene. The virus showed localized replication in different tissues including the fat body, haemocytes, tracheal matrix and in the Malphigian tubules. The larval system was successfully used to express heterologous protein, by infecting with a recombinant AcMNPV carrying the 3ABC coding sequence of foot-and-mouth disease virus (FMDV). The study shows that the Eri silkworm larva can be a potential alternative bioreactor, for scaling up of the recombinant proteins employing the baculovirus system.

Immunoreactivity and trypsin sensitivity of recombinant virus-like particles of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an important infection affecting the health and productivity of cloven-hoofed livestock. Development of improved vaccines and diagnostic reagents is being explored to facilitate the disease control. There is an emerging interest in virus-like particles (VLPs), as their constituent structural proteins are the major immunogens. The VLPs are similar to natural virus particles but lack viral nucleic acid. The objective of the present study was to express the VLPs of FMD virus (FMDV) serotype Asia-1 (IND 63/72), using baculovirus system and characterize them for antigenic structure. The VLPs expressed in insect cells showed immunoreactivity similar to inactivated cell culture FMDV. Further they possess similar sensitivity to trypsin as the inactivated cell culture FMDV, suggesting that trypsin-sensitive antigenic sites could be similarly arranged. Our findings suggest that the FMD VLPs have similar antigenic conformational feature like the wild type virus, thus supporting their utility in development of non-infectious FMD vaccines and/or diagnostic assays.

Detection of replication competent adenovirus upon serial passaging of recombinant adenovirus expressing FMDV capsid proteins.

Replication deficient human adenovirus type 5 (hAd5) is an important gene delivery vehicle and has been used in various fields of biomedical sciences such as gene therapy, cancer therapy and vaccination. Inspite of its various useful features, emergence of replication competent adenovirus (RCA) in recombinant virus stocks is a great concern. In the present study, recombinant adenovirus expressing foot-and-mouth disease virus serotype-O capsid proteins was propagated in HEK-293 cells and purified by CsCl density gradient ultracentrifugation. The virus was serially passaged in HEK-293 cells and at passage level (P) 2-4, 6, 8 and 12, tested for the presence of RCA. A vector dose of 2 × 10(8) and 1 × 10(10) TCID50 of the virus was used in cell line bioassay and PCR, respectively. Our results demonstrated that the PCR is more sensitive and rapid technique for the detection of RCA in recombinant adenovirus stocks as early as at P3, whereas the bioassay detected the RCA at P8.

A multi-species indirect ELISA for detection of non-structural protein 3ABC specific antibodies to foot-and-mouth disease virus.

Reliable diagnostic tests that are able to distinguish infected from vaccinated animals are a critical component of regional control programs for foot-and-mouth disease (FMD) in the affected countries. Non-structural protein (NSP) serology based on the 3ABC protein has been widely used for this purpose, and several kits are commercially available worldwide. This report presents the development of a 3ABC-antigen-based indirect ELISA, employing a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Recombinant 3ABC protein was expressed in insect cells and purified using affinity column chromatography. Using this protein, an indirect ELISA was developed and validated for the detection of NSP antibodies in serum samples collected from animals with different status of FMD. Diagnostic sensitivity and specificity were found to be 95.8 (95 % CI: 92.8-97.8) and 97.45 % (95 % CI: 94.8-99.0), respectively. The in-house ELISA compared well with the commercially available prioCHECK FMDV NS-FMD kit, with a high agreement between the tests, as determined by the kappa coefficient, which was 0.87. The in-house ELISA showed higher sensitivity for detecting vaccinated and subsequently infected animals, when compared to the reference test. Both of the tests were able to detect NSP antibodies as early as 7-8 days after experimental infection.

Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75.

AIM: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus (FMDV) capsid protein genes along with full-length 2B, 3B and 3C(pro) and its characterization. MATERIALS AND METHODS: FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BC(wt) and P1-2AB3BC(m)) followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BC(wt) and hAd5/P1-2AB3BC(m)). Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation. Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus (dAd5). RESULTS: The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 10(8), 10(9.5) and 10(11) TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01). CONCLUSION: Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was detected by sandwich ELISA and immunofluorescence assay.