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.

Production of polyclonal viperin antisera using N-terminal deleted recombinant bovine viperin.

Viperin, also known as radical S-adenosyl methionine domain-containing protein (RSAD2) is a multifunctional interferon-stimulated gene (ISG) that is activated during the viral infections. Viperin belongs to S-adenosyl methionine (SAM) superfamily of enzymes known to catalyze radical-mediated reactions and viperin inhibits a wide range of DNA and RNA viruses through its broad range of activity. The present study reports cloning and expression of bovine viperin in a bacterial expression system. PCR-based site-directed mutagenesis was carried out for deletion of N-terminal 1-70 amino acid containing amphipathic helix of viperin that interferes in protein expression and purification. The resultant truncated viperin protein was expressed in Escherichia coli, BL-21(DE3) competent cells and purified using nickel charged affinity column. The truncated 54 kDa protein was confirmed by western blot using human RSAD2 as a probe. Further, in house, hyperimmune serum was raised against the truncated viperin in the rabbit and the reactivity was confirmed by western blot using mammalian expression vector construct of viperin transfected in Baby Hamster kidney (BHK) cells and in MDBK cells infected with Foot and Mouth disease Asia I virus.

Foot-and-mouth disease virus induces PERK-mediated autophagy to suppress the antiviral interferon response.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in cloven-hoofed animals. FMDV replication-associated viral protein expression induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), in turn inducing autophagy to restore cellular homeostasis. We observed that inhibition of BiP (also known as HSPA5 and GRP78), a master regulator of ER stress and UPR, decreased FMDV infection confirming their involvement. Further, we show that the FMDV infection induces UPR mainly through the PKR-like ER kinase (PERK; also known as EIF2AK3)-mediated pathway. Knockdown of PERK and chemical inhibition of PERK activation resulted in decreased expression of FMDV proteins along with the reduction of autophagy marker protein LC3B-II [the lipidated form of LC3B (also known as MAP1LC3B)]. There are conflicting reports on the role of autophagy in FMDV multiplication. Our study systematically demonstrates that during FMDV infection, PERK-mediated UPR stimulated an increased level of endogenous LC3B-II and turnover of SQSTM1, thus confirming the activation of functional autophagy. Modulation of the UPR and autophagy by pharmacological and genetic approaches resulted in reduced numbers of viral progeny, by enhancing the antiviral interferon response. Taken together, this study underscores the prospect of exploring PERK-mediated autophagy as an antiviral target.

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.

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.

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.

Short-term effect of foot-and-mouth disease (FMD) vaccination on the milk yield in the Deoni and crossbred cows.

Biannual vaccination of the cattle with inactivated foot-and-mouth disease (FMD) vaccine is the control strategy in endemic countries. Reduction in the milk yield is one of the main reasons for poor compliance of the cattle owners to FMD vaccination. As it can adversely affect the herd immunity, the present study aimed to quantify the losses in the milk yield post-FMD vaccination. Retrospective data on the milk yield (kg) recordings, days in milk, parity, and age at vaccination of the Deoni and crossbred cows were collected from 10 days before (-10) to 10 days after (+10) FMD vaccination (dpv). Days in milk were categorized into three stages of lactation for Deoni and crossbred cows. Age (month) was categorized into four classes. Least squares means of the milk yield were generated after adjusting for year, age, parity, and stage of lactation. Based on exploratory data analysis, the corrected milk yield records from -2 to +2 dpv for 5 years comprising 614 data points on Deoni cows (n=54) and 488 data points on crossbred cows (n=55) were used for the final analysis. Because of the correlated errors on the corrected milk yield, linear mixed model ANOVA was done by fitting dpv as fixed effect and cow as random effect, and the results revealed the effect of dpv was non-significant (P>0.05) in either breed. With respect to dpv 0, a marginal reduction of 90 g in the corrected milk yield in the Deoni cow was recorded on dpv 1, while the reduction was about 360 g on dpv 0 as compared dpv -1 in the crossbred cow. It was concluded that FMD vaccination caused a transient non-significant reduction in the milk yield in the Deoni and crossbred cows.

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.

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.

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.

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

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

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.

Diagnostic potential of recombinant nonstructural protein 3B to detect antibodies induced by foot-and-mouth disease virus infection in bovines.

Detection of antibodies to nonstructural proteins (NSP) of foot-and-mouth disease virus is the preferred diagnostic method to differentiate infected from vaccinated animals. In India, an endemic region practising preventive biannual vaccination, 3AB3 indirect ELISA (r3AB3 I-ELISA) has been employed as the primary screening test for serosurveillance. However, because of the variability observed in the immune response to the NSPs, the likelihood of detecting or confirming an infected animal is increased if an antibody profile against multiple NSPs is considered for diagnosis. In this study, all three copies of NSP 3B were expressed in a prokaryotic system to develop an indirect ELISA (r3B I-ELISA). At the decided cutoff of 40 percent positivity, the diagnostic sensitivity and specificity of the r3B I-ELISA were estimated to be 92.1% (95% CI: 89.0-94.5) and 98.1% (95% CI: 96.9-98.8), respectively, as compared to 97.04% and 95.04% for r3AB3 I-ELISA. Although r3B I-ELISA displayed lower sensitivity compared to the screening assay, which could possibly be attributed to additional relevant B-cell epitopes in the carboxy-terminal half of the 3A protein, the former achieved considerably higher specificity on repeatedly vaccinated animals. NSP antibodies could be detected from 10 to as late as 998 days postinfection in experimental calves. Substantial agreement in the test results (90.6%) was found between the two ELISAs. The r3B I-ELISA, when used in conjunction with the r3AB3 I-ELISA as an integrated system, can potentially augment the efficiency and confidence of detection of infected herds against the backdrop of intensive vaccination.

In-process quality control in foot-and-mouth disease vaccine production by detection of viral non-structural proteins using chemiluminescence dot blot assay.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.

Field outbreak strains of serotype O foot-and-mouth disease virus from India with a deletion in the immunodominant ßG-ßH loop of the VP1 protein.

Foot-and-mouth disease virus serotype O accounts for around 80 % of the outbreaks in India. Although Indian serotype O isolates belongs to the ME-SA topotype, circulation of different lineages has been noted. After its emergence in the year 2001, the 'Ind2001' lineage outcompeted the PanAsia lineage in causing serotype O outbreaks in the year 2009. Three isolates had an amino acid deletion at position 139 in the VP1 coding region and grouped with the 'Ind2001' lineage. The currently used Indian vaccine strain of serotype O covers all of the field isolates antigenically.

Assessment of fitness of foot-and-mouth disease virus A IND 27/2011 as candidate vaccine strain.

Antigenic profiling of recent field outbreak strains of foot-and-mouth disease virus (FMDV) serotype A in India has revealed considerable antigenic drift from the vaccine strain, A IND 40/2000, necessitating the selection of a new strain. The complete genome sequence of A IND 27/2011 was analysed. Vaccine quality attributes of the new candidate strain including potency as an inactivated vaccine in cattle were evaluated. The capsid coding region of A IND 27/2011 showed variation at eight antigenically critical amino acid positions from that of A IND 40/2000. The strain suited well with traits required by a vaccine in terms of its adaptability to adherent and suspension cell line, its immunogenicity, and potency as an inactivated vaccine formulation in cattle. Complete protection was observed upon homologous virus challenge at 4 weeks post-vaccination. Taken together, these data demonstrate the suitability of A IND 27/2011 as an effective vaccine strain of FMDV serotype A.

A single amino acid substitution in the VP2 protein of Indian foot-and-mouth disease virus serotype O vaccine strain confers thermostability and protective immunity in cattle.

Foot-and-mouth disease (FMD) is a significant threat to animal health globally. Prophylactic vaccination using inactivated FMD virus (FMDV) antigen is being practised for the control in endemic countries. A major limitation of the current vaccine is its susceptibility to high environmental temperature causing loss of immunogenicity, thus necessitating the cold chain for maintenance of its efficacy. Hence, the FMD vaccine with thermostable virus particles will be highly useful in sustaining the integrity of whole virus particle (146S) during storage at 4°C. In this study, 12 recombinant mutants of Indian vaccine strain of FMDV serotype O (O/IND/R2/1975) were generated through reverse genetics approach and evaluated for thermostability. One of the mutant viruses, VP2_Y98F was more thermostable than other mutants and the parent FMDV. The oil-adjuvanted vaccine formulated with the inactivated VP2_Y98F mutant FMDV was stable up to 8 months when stored at 4°C and induced protective antibody response till dpv 180 after primary vaccination. It is concluded that the VP2_Y98F mutant FMDV was thermostable and has the potential to replace the parent vaccine strain.

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

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

Comparative complete genome analysis of Indian type A foot-and-mouth disease virus field isolates.

Comparative complete genome analysis of 17 serotype A Indian field isolates representing different genotypes and sub-lineages is presented in this report. Overall 79% of amino acids were invariant in the coding region. Chunk deletion of nucleotide was observed in S and L fragment of 5'-UTR. More variability which is comparable to that of capsid coding region was found in L and 3A region. Functional motifs and residues critical for virus biology were conserved most. Polyprotein cleavage sites accepted few changes. Many sites were detected to be under positive selection in L, P1, 2C, 3A, 3C, and 3D region and of which some are functionally important and antigenically critical. Genotype/lineage specific signature residues could be identified which implies evolution under different selection pressure. Transmembrane domain could be predicted in 2B, 2C, 3A, and 3C proteins in agreement with their membrane binding properties. Phylogenetic analysis at complete coding region placed the isolates in genotype IV, VI, and VII and two broad clusters comprising VP3(59)-deletion and non-deletion group within genotypes VII. The VP3(59)-deletion group has diversified genetically with time giving rise to three lineages. Incongruence in tree topology observed for different non structural protein coding region and UTRs-based phylogeny indicate suspected recombination.

Expression of foot-and-mouth disease virus non-structural protein 3A upregulates the expression of autophagy and immune response genes in vitro.

Foot-and-mouth disease (FMD) virus 3A protein regulates viral replication and virulence; thus, we generated BHK-Flp-In cell line expressing 3A protein because it can serve as helper cell line for infecting a replication defective FMDV to produce a live disabled vaccine. FMDV Asia1 3A was amplified, cloned in pcDNA vector and confirmed by sequencing. The 3A gene was subcloned in pcEF/FRT vector and transfected in BHK-Flp-In cells and transformed cells were selected by resistance to hygromycin and susceptibility to zeocin antibiotics. The BHK-Flp-In cells expressing 3A protein was designated as Flp-In3A. Western blot and immunofluorescence confirmed that Flp-In3A cells expressed FMDV3A protein. Absolute quantitation of 3A transcripts showed peak expression at 6 h in Flp-In3A cells followed by a sharp decrease and the cells showed growth retardation for 2 h post-seeding with cytoplasmic vacuolations with advancing time. Response to infection with FMDV Asia1 virus revealed smaller plaques in Flp-In3A cells. Then, we investigated the effect of FMDV3A expression on autophagy related genes by real time PCR. Most autophagy genes were upregulated by 9 h post-seeding of which, autophagosome marker LC3B-II was demonstrated by western blot. Transient expression of 3A in PK-15 cells upregulated both Th1 and Th2 genes. The study suggested that the expressed 3A protein of FMDV cannot be used for 3A trans-supplementation in helper cells; however, it acts as an endogenously processed antigen that has the potential to elicit immune response in vivo.