FMDV Infection Research Articles

Prophylactic treatment with PEGylated bovine IFNλ3 effectively bridges the gap in vaccine-induced immunity against FMD in cattle.

Foot-and-mouth disease (FMD) is a vesicular disease of cloven-hoofed animals with devastating economic implications. The current FMD vaccine, routinely used in enzootic countries, requires at least 7 days to induce protection. However, FMD vaccination is typically not recommended for use in non-enzootic areas, underscoring the need to develop new fast-acting therapies for FMD control during outbreaks. Interferons (IFNs) are among the immune system's first line of defense against viral infections. Bovine type III IFN delivered by a replication defective adenovirus (Ad) vector has effectively blocked FMD in cattle. However, the limited duration of protection-usually only 1-3 days post-treatment (dpt)-diminishes its utility as a field therapeutic. Here, we test whether polyethylene glycosylation (PEGylation) of recombinant bovine IFNλ3 (PEGboIFNλ3) can extend the duration of IFN-induced prevention of FMDV infection in both vaccinated and unvaccinated cattle. We treated groups of heifers with PEGboIFNλ3 alone or in combination with an adenovirus-based FMD O1Manisa vaccine (Adt-O1M) at either 3 or 5 days prior to challenge with homologous wild type FMDV. We found that pre-treatment with PEGboIFNλ3 was highly effective at preventing clinical FMD when administered at either time point, with or without co-administration of Adt-O1M vaccine. PEGboIFNλ3 protein was detectable systemically for >10 days and antiviral activity for 4 days following administration. Furthermore, in combination with Adt-O1M vaccine, we observed a strong induction of FMDV-specific IFNγ+ T cell response, demonstrating its adjuvanticity when co-administered with a vaccine. Our results demonstrate the promise of this modified IFN as a pre-exposure prophylactic therapy for use in emergency outbreak scenarios.

Comparison of clinical, hematological and serum biochemical parameters between foot-and-mouth disease infected and non-infected Nigerian breeds of cattle.

Foot and mouth disease (FMD) still remains one of the major hindrances to livestock productivity in Africa as a whole and Nigeria in particular, with estimated well over 7billions losses to FMDV infection per annum in Nigeria. We therefore sought to establish and compare the clinical, hematological and biochemical indices in FMD infected and non-infected cattle. A total of 60 cattle including 40 apparently healthy and 20 FMD-infected cattle were sampled in Central Abattoir, Akinyele and Akufo Farm settlement, Ibadan, Oyo state, Nigeria respectively. Confirmatory diagnosis was done by accessing the presented pathognomonic clinical signs and the use of rapid diagnostic Antibody detection kit. Clinical parameters were recorded, and blood was drawn through the jugular vein for hematological and biochemical profiles. Standard hematological and serum biochemical techniques were used to analyzed the samples for hematological and biochemical parameters respectively. Results revealed higher clinical parameters in infected compared to the non-infected. All erythrocytic parameters were significantly lower in FMD infected cattle compared to non-infected cattle with P-values of 0.016, 0.015, 0.010, 0.001, 0.001 and 0.045 for PCV, Hb conc, RBC count, MCV, MCH and MCHC values respectively. Significant differences (P<0.05) of 0.001, 0.001, 0.001, 0.000, 0.001 and 0.001 for Lymphocyte, Neutrophil, Absolute lymphocyte, Absolute monocyte, Absolute neutrophil and platelets respectively. There were significant differences (P<0.05) of 0.009,0.001, 0.026, 0.001, 0.014, 0.001,0.001, 0.001, 0.001 and 0.001 for Total serum protein, Albumin, Globulin, Glucose, Creatinine, AST, ALT, ALP, Na+ and K+ respectively in infected cattle compared to non-infected cattle. We therefore conclude that, FMD-infected cattle showed lower clinical, hematological and serum biochemical parameters compared to uninfected cattle breeds in Nigeria.

MiR-361 and miR-34a suppress foot-and-mouth disease virus proliferation by activating immune response signaling in PK-15 cells

Foot-and-mouth disease (FMD) severely impacts cloven-hoofed live-stock production, leading to serious economic losses and international restriction on the trade of animals and animal products worldwide. MiRNAs serve key roles in viral immunity and regulation. However, the knowledge about miRNAs regulation in FMDV infection is still limited. In this study, we found that FMDV infection caused rapid cytopathic in PK-15 cell. To investigate the miRNAs’ function in FMDV infection, we performed knockdown of endogenous Dgcr8 using its specific siRNA and found that interference of Dgcr8 inhibited cellular miRNA expression and increased FMDV production, including viral capsid proteins expression, viral genome copies and virus titer, suggesting that miRNAs play an important role in FMDV infection. To obtain a full perspective on miRNA expression profiling after FMDV infection, we performed miRNA sequencing and found that FMDV infection caused inhibition of miRNA expression in PK-15 cells. Together with the target prediction result, miR-34a and miR-361 were screened for further study. Function study showed that no matter plasmid or mimics-mediated overexpression of miR-34a and miR-361 both suppressed FMDV replication, while inhibition of endogenous miR-34a and miR-361 expression using specific inhibitors significantly increased FMDV replication. Further study showed that miR-34a and miR-361 stimulated IFN-β promoter activity and activated interferon-stimulated response element (ISRE). In addition, ELISA test found that miR-361 and miR-34a increased secretion level of IFN-β and IFN-γ, which may contribute to repression of FMDV replication. This study preliminary revealed that miR-361 and miR-34a inhibited FMDV proliferation via stimulating immune response.

Main factors associated with foot-and-mouth disease virus infection during the 2001 FMD epidemic in Uruguay.

Large epidemics provide the opportunity to understand the epidemiology of diseases under the specific conditions of the affected population. Whilst foot-and-mouth disease (FMD) epidemics have been extensively studied in developed countries, epidemics in developing countries have been sparsely studied. Here we address this limitation by systematically studying the 2001 epidemic in Uruguay where a total of 2,057 farms were affected. The objective of this study was to identify the risk factors (RF) associated with infection and spread of the virus within the country. The epidemic was divided into four periods: (1) the high-risk period (HRP) which was the period between the FMD virus introduction and detection of the index case; (2) the local control measures period (LCM) which encompassed the first control measures implemented before mass vaccination was adopted; (3) the first mass vaccination, and (4) the second mass vaccination round. A stochastic model was developed to estimate the time of initial infection for each of the affected farms. Our analyses indicated that during the HRP around 242 farms were probably already infected. In this period, a higher probability of infection was associated with: (1) animal movements [OR: 1.57 (95% CI: 1.19-2.06)]; (2) farms that combined livestock with crop production [OR: 1.93 (95% CI: 1.43-2.60)]; (3) large and medium farms compared to small farms (this difference was dependent on regional herd density); (4) the geographical location. Keeping cattle only (vs farms that kept also sheep) was a significant RF during the subsequent epidemic period (LCM), and remained as RF, together with large farms, for the entire epidemic. We further explored the RF associated with FMDV infection in farms that raised cattle by fitting another model to a data subset. We found that dairy farms had a higher probability of FMDV infection than beef farms during the HRP [OR: 1.81 (95% CI: 1.12-2.83)], and remained as RF until the end of the first round of vaccination. The delay in the detection of the index case associated with unrestricted animal movements during the HRP may have contributed to this large epidemic. This study contributes to the knowledge of FMD epidemiology in extensive production systems.

The Foot-and-Mouth Disease Virus Lb Protease Cleaves Intracellular Transcription Factors STAT1 and STAT2 to Antagonize IFN-β–Induced Signaling

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, one of the most highly infectious animal viruses throughout the world. The JAK-STAT signaling pathway is a highly conserved pathway for IFN-β-induced antiviral gene expression. Previous studies have shown that FMDV can strongly suppress the innate immune response. Moreover, although STAT1 and STAT2 (STAT1/2) have been well established in JAK-STAT signaling-induced antiviral gene expression, whether FMDV proteins inhibit IFN-β-induced JAK-STAT signaling remains poorly understood. In this study, we described the Lb leader protease (Lbpro) of FMDV as a candidate for inhibiting IFN-β-induced signaling transduction via directly interacting with STAT1/2. We further showed that Lbpro colocalized with STAT1/2 to inhibit their nuclear translocation. Importantly, Lbpro cleaved STAT1/2 to inhibit IFN-β-induced signal transduction, whereas the catalytically inactive mutant of LC51A (Lbpro with cysteine substituted with alanine at amino acid residue 51) had no effect on the stability of STAT1/2 proteins. The cleavage of the STAT1/2 proteins was also determined during FMDV infection in vitro. Lbpro could cleave the residues between 252 and 502 aa for STAT1 and the site spanning residues 140 - 150 aa (QQHEIESRIL) for STAT2. The in vivo results showed that Lbpro can cleave STAT1/2 in pigs. Overall, our findings suggest that FMDV Lbpro-mediated targeting of STAT1/2 may reveal a novel mechanism for viral immune evasion.

Foot-and mouth disease and peste des petits ruminants – the role of wildlife in the epidemiology and control of diseases

Foot-and mouth disease (FMD) and peste des petits ruminants (PPR) are highly contagious and an economically devastating diseases, currently endemic to the African and Asian continents. The aim of this paper was to present the role of wildlife in the epidemiology of both diseases. There are more than 100 species of wild, feral, laboratory, or domesticated animals that have been infected naturally or experimentally with FMD or PPR viruses. Experimental infections of several African wild ruminants can result in clinical FMD. African buffalo represents the best known FMD wild host reservoir in Sub-Saharan Africa. North American mule deer were found susceptible to FMDV infection with significant mortality. Other wild ruminants such as impala can also contribute to FMDV maintenance. In Europe several deer species and the Eurasian wild boar are susceptible to FMDV. PPRV has been reported to have infected some wildlife, such as African buffalo, saiga antelope, dorcas gazelles, gemsbok, Nubian ibex and some other ungulate species. The role of wildlife in FMD and PPR epidemiology may concern wildlife as indicators, victims, bridge hosts or maintenance hosts for both diseases. In addition, they are occasionally victims of disease outbreaks, and they are often relevant for disease management as either bridge or maintenance hosts. Wildlife deserves to become a key component of future integrated surveillance and disease control strategies in an ever-changing world. However, it must be stated that efforts to control FMD and PPR in wildlife may not be successful when the diseases are endemic in livestock and may cause more harm to wildlife, human livelihoods, and domestic animals.

Assessment of multiplex PCR for detection of FMDV, BVDV, BTV, and possible coinfection with Pasteurella multocida in cattle

Multiplex polymerase chain reaction (mPCR) assay is a nucleic acid amplification method that is considered reliable and practical means for several pathogen detections in a single reaction, especially when multiple pathogens are suspected. In this study, a novel mPCR assay was validated for the detection of four notifiable diseases in cattle, including foot and mouth disease (FMD), Bovine viral diarrhea (BVD), Bluetongue (BT), and Hemorrhagic Septicemia (HS). The assay was operated in a two-step procedure. The first one was a reverse transcription of viral RNA, then mPCR of viral cDNA and bacterial DNA. The optimized mPCR was applied on blood (26) and vesicular epithelium (10) samples collected from 26 clinically infected animals from three governorates (Qalubia, Sharkia, and Gharbia). mPCR detected at least 10 pg of microbial nucleic acid extracted from the local isolates. The mPCR results showed that 22/26 (84.6%) of clinically infected animals were positively infected by single or dual infection. Mixed infection of FMDV and Pasteurella multocida was recorded in 11 animals (42.3%), while single FMDV infection was recorded in 5 animals (19.2 %). Single BVDV infection was detected in 5 animals (19. 2 %) and dual infection with FMDV in 1 animal (3.8%). Notably, BTV was not detected in any of the clinical samples. The assessed mPCR was a rapid, accurate, and sensitive test for diagnosing single and mixed infections in cattle and could be used to screen the notifiable diseases affecting cattle.

Genome Sequences of Foot-and-Mouth Disease Virus SAT2 Strains Purified from Coinfected Cape Buffalo in Kenya.

ABSTRACTFoot-and-mouth disease virus (FMDV) SAT2 sequences were acquired from Cape buffalo in Kenya in 2016, from either primary passage (n = 38) or plaque purification of dually SAT1/SAT2-infected samples (n = 61). All samples were derived from asymptomatic animals. These sequences contribute to our understanding of FMDV diversity in reservoirs and during subclinical FMDV infections.

The C3d-fused foot-and-mouth disease vaccine platform overcomes maternally-derived antibody interference by inducing a potent adaptive immunity

Vaccination prevents and controls foot-and-mouth disease (FMD). However, the current FMD vaccine remains disadvantageous since it cannot overcome maternally-derived antibody (MDA) interference in weeks-old animals, which suppress active immunity via vaccination. To address this, we developed the immune-enhancing O PA2-C3d and A22-C3d FMD vaccine strains that can stimulate receptors on the surface of B cells by inserting C3d (a B cell epitope) into the VP1 region of O PA2 (FMDV type O) and A22 (FMDV type A). We purified inactivated viral antigens from these vaccine strains and evaluated their immunogenicity and host defense against FMDV infection in mice. We also verified its efficacy in inducing an adaptive immune response and overcome MDA interference in MDA-positive (MDA(+), FMD-seropositive) and -negative (MDA(−), FMD-seronegative) pigs. These results suggest a key strategy for establishing novel FMD vaccine platform to overcome MDA interference and induce a robust adaptive immune response.

Development of a competitive chemiluminescence immunoassay using a monoclonal antibody recognizing 3B of foot-and-mouth disease virus for the rapid detection of antibodies induced by FMDV infection

BackgroundFoot-and-mouth disease (FMD) is a devastating animal disease. Anti-non-structural protein (NSP) antibody detection is very important for confirming suspected cases, evaluating the prevalence of infection, certifying animals for trade and controlling the disease.MethodsIn this study, a competitive chemiluminescence immunoassay (3B-cCLIA) was developed for the rapid detection of antibodies against NSPs in different species of livestock animals using the monoclonal antibody (mAb) 9E2 as a competitive antibody that recognizes NSP 3B.ResultsThe cut-off value (50%), diagnostic sensitivity (Dsn) (97.20%, 95.71%, and 96.15%) and diagnostic specificity (Dsp) (99.51%, 99.43%, and 98.36) of the assay were estimated by testing a panel of known-background sera from swine, cattle and sheep, respectively. The accuracy rate of the 3B-cCLIA was further validated and subsequently compared with that of two commercial diagnostic kits. The early diagnostic results showed that antibodies recognizing NSPs developed later (approximately 1–2 days) than antibodies recognizing structural proteins. Furthermore, anti-NSP antibody presence in animals vaccinated multiple times (false positives), especially cattle and sheep, was confirmed, and the false-positive rate increased with the number of vaccinations.ConclusionsThese results indicate that the 3B-cCLIA is suitable for the rapid detection of antibodies against FMDV NSP 3B in a wide range of species.

Antiviral potential of ivermectin against foot-and-mouth disease virus, serotype O, A and Asia-1

Each year, foot-and-mouth disease leads to enormous economic losses to the livestock industry. Currently, the killed whole virus is widely using to control FMD. However, vaccination is constrained by lack of or incomplete protection. Therefore, along with vaccination, we need to find the antivirals against FMD. This study was conducted to investigate the antiviral potential of ivermectin against multiple serotypes of FMDV. Initially, an MTT assay was performed on the BHK-21 cell line to determine assay ivermectin cytotoxicity. Viral inhibition assays using the non-cytotoxic concentration of ivermectin were performed to check the antiviral potential of ivermectin on different stages of virus replication. At 2.5 μM and 5 μM concentrations of ivermectin, the virus titer was reduced significantly (p < 0.001) by two to three log in all three strains of viruses at both non-toxic concentrations (2.5 and 5 μM). The virus titer in strain O control was 106.0 TCID50/0.1 mL and was reduced to 104.1 TCID50/0.1 mL at a concentration of 2.5 μM and 103.10 TCID50/0.1 mL at 5 μM concentration. In the case of strain Asia-1, the virus titer was reduced to 103.8 TCID50/0.1 mL at 2.5 μM and 103.01TCID50/0.1 mL at 5 μM concentration. The titer of strain A was reduced from 105.8 TCID50/0.1 mL to 103.9 TCID50/0.1 mL at 2.5 μM concentration and 103.1 TCID50/0.1 mL at 5 μM concentration. Moreover, the virus titer was reduced more at the replication stage as compared to attachment and entry stages. This study showed the in vitro anti-FMDV potential of ivermectin for the first time and predicted its potential use against FMDV infections.

Investigations into Foot and Mouth Disease Outbreak among Small Ruminants in Karnataka State of India

Background: Foot-and-Mouth Disease (FMD) remains a serious threat to the Indian livestock sector due to significant economic loss associated with it. Systematic vaccination of large ruminants over the years has lead to a gradual reduction in the number of disease outbreaks in India. However exposure to FMDV infection in small ruminants has been recorded during the past few years (Rout et al. 2013). Sheep and goat population have not been vaccinated so far against FMD under the FMD-Control program (FMD-CP). The present study highlighted the outbreak of FMD in small ruminants in Karnataka, India.Methods: During the period 2018-19, seven suspected FMD outbreaks among sheep population in Bellary and Tumakuru districts of Karnataka state were investigated. Tongue epithelium (oral swabs) and foot lesions (n=23) from clinically affected sheep and tissues such as heart, lung, liver, spleen, lymph nodes and kidneys from lambs during post mortem (n=67) were collected. All the samples were processed in the laboratory for the detection of FMD virus antigen by employing Serotype differentiating antigen detection ELISA and by multiplex PCR. Heart tissue samples were also collected in buffered formalin for histopathology study and processed by routine paraffin embedding technique and stained with Hematoxylin and Eosin (H and E). Serum samples from the recovered animals were collected and screened by NSP-ELISA and LPB-ELISA to check the antibody status in the affected herd. Result: A total of seven suspected outbreaks of FMD involving 688 small ruminants was investigated. The outbreak of FMD due to FMDV serotype O was confirmed by ELISA and multiplex PCR assays. Clinically, the affected adult sheep showed typical signs of FMD, while mortality in young lambs was observed without apparent signs of disease. Histologically, heart tissues from FMD affected lambs showed myocardial necrosis with marked aggregations of lymphocytes and neutrophils in the myocardium and perivascular spaces. History of FMD outbreaks in cattle and common grazing land for the livestock, as well as sheep within the reach of these villages, may be the major contributing factors for the outbreaks in sheep populations.

Antiviral Activity Of Adamantane-Pyrazole Derivatives Against Foot And Mouth Disease Virus Infection In Vivo And In Vitro With Molecular Docking Study

Foot-and-mouth disease (FMD) vaccine does not afford early effective protection until adaptive immune protection caused by the vaccination occurs. Therefore, an alternative prophylactic application of antiviral agents for inhibition of the FMD virus is needed, and this is the scope of this study. In this study, we tested nine adamantane-pyrazole derivatives that could exhibit antiviral activity against FMDV infection either in vitro through baby hamster kidney cells (BHK-21 cells) infected with FMD virus serotypes O pan Asia. Cytotoxicity Concentration 50 (CC50) activity of pyrazole derivatives (1, 2, 3, 4, 5a,b, 6a-c) were detected on BHK-21 cells and ranged between 500 to 3000µg/ml. Inhibitory Concentration 50 (IC50) on BHK-21 was achieved only for the most promising three derivatives 6a-c and exhibited an antiviral activity with a therapeutic index of 30, and that was reflected on the antiviral activity response in baby mice with different concentrations where a concentration of 50 µg/ml for pyrazole derivatives 6a and 6c compounds and 40 µg/ml for bis-tolyl pyrazole 6b that achieve 100% protection and this results was as effective as 50 µg/ml of amantadine. Specifically, diaryl pyrazole derivatives 6a-c that protected for six days following FMDV challenge. These results suggested that pyrazole derivatives 6a-c could be used as an effective antiviral agent against FMD virus infection. Molecular docking simulation of the target compounds 6a-c had good binding energy and the tested compounds recommended being an excellent 3C protease inhibitor compared to Amantadine and Ribavirin. These findings may explain the antiviral activity of the target compounds.

MicroRNA-4331-5p promotes FMDV replication through inhibiting interferon pathways in PK-15 cells

MicroRNAs play vital roles in regulating the battle between pathogens and host cells during viral challenging. MiR-4331 aggravates transmissible gastroenteritis virus (TGEV) -induced mitochondrial damage, also suppresses transcription of TGEV gene 7 via targeting cellular CDCA7. Otherwise, miR-4331-5p affects H1N1/2009 influenza A virus replication by targeting viral HA and NS. However, whether microRNA ssc-miR-4331-5p (miR-4331-5p) regulates foot and mouth virus (FMDV) replication remains unclear. To explore the role of miR-4331-5p in FMDV infection, we detected the expression level of miR-4331-5p in porcine kidney (PK-15) cells. The results showed that FMDV infection directly upregulates miR-4331-5p expression, while transfection of mimics or inhibitor of miR-4331-5p promotes or inhibits FMDV replication. Further investigation clearly showed that miR-4331-5p increases FMDV replication through inhibiting type I interferon pathways. These data demonstrate that miR-4331-5p plays an important role in regulating FMDV replication.

Foot-and-Mouth Disease Virus Capsid Protein VP1 Interacts with Host Ribosomal Protein SA To Maintain Activation of the MAPK Signal Pathway and Promote Virus Replication.

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a highly contagious, economically important viral disease. The structural protein VP1 plays significant roles during FMDV infection. Here, we identified that VP1 interacted with host ribosomal protein SA (RPSA). RPSA is a viral receptor for dengue virus and classical swine fever virus infections. However, the incubation of susceptible cells using the anti-RPSA antibodies did not block the infection of FMDV. Overexpression of porcine RPSA in the insusceptible cells could not trigger FMDV infection, suggesting that RPSA was not responsible for FMDV entry and infection. On the contrary, we found that overexpression of RPSA suppressed FMDV replication, and knockdown of RPSA enhanced FMDV replication. We further determined that FMDV infection activated the mitogen-activated protein kinase (MAPK) pathway and demonstrated that MAPK pathway activation was critically important for FMDV replication. RPSA negatively regulated MAPK pathway activation during FMDV infection and displayed an antiviral function. FMDV VP1 interacted with RPSA to abrogate the RPSA-mediated suppressive role in MAPK pathway activation. Together, our study indicated that MAPK pathway activation was required for FMDV replication and that host RPSA played a negatively regulatory role on MAPK pathway activation to suppress FMDV replication. FMDV VP1 bound to RPSA to promote viral replication by repressing RPSA-mediated function and maintaining the activation of MAPK signal pathway.IMPORTANCE Identification of virus-cell interactions is essential for making strategies to limit virus replication and refine the models of virus replication. This study demonstrated that FMDV utilized the MAPK pathway for viral replication. The host RPSA protein inhibited FMDV replication by suppressing the activation of the MAPK pathway during FMDV infection. FMDV VP1 bound to RPSA to repress the RPSA-mediated regulatory effect on MAPK pathway activation. This study revealed an important implication of the MAPK pathway for FMDV infection and identified a novel mechanism by which FMDV VP1 has evolved to interact with RPSA and maintain the activation of the MAPK pathway, elucidating new information regarding the signal reprogramming of host cells by FMDV.

Ribosomal Protein L13 Promotes IRES-Driven Translation of Foot-and-Mouth Disease Virus in a Helicase DDX3-Dependent Manner.

Internal ribosome entry site (IRES)-driven translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. In the current study, we identified the ribosomal protein L13 (RPL13) as a critical regulator of IRES-driven translation of foot-and-mouth disease virus (FMDV) but found that it is not essential for cellular global translation. RPL13 is also a determinant for translation and infection of Seneca Valley virus (SVV) and classical swine fever virus (CSFV), and this suggests that its function may also be conserved in unrelated IRES-containing viruses. We further showed that depletion of DEAD box helicase DDX3 disrupts binding of RPL13 to the FMDV IRES, whereas the reduction in RPL13 expression impairs the ability of DDX3 to promote IRES-driven translation directly. DDX3 cooperates with RPL13 to support the assembly of 80S ribosomes for optimal translation initiation of viral mRNA. Finally, we demonstrated that DDX3 affects the recruitment of the eukaryotic initiation factor eIF3 subunits e and j to the viral IRES. This work provides the first connection between DDX3 and eIF3e/j and recognition of the role of RPL13 in modulating viral IRES-dependent translation. This previously uncharacterized process may be involved in selective mRNA translation.IMPORTANCE Accumulating evidence has unveiled the roles of ribosomal proteins (RPs) belonging to the large 60S subunit in regulating selective translation of specific mRNAs. The translation specificity of the large-subunit RPs in this process is thought provoking, given the role they play canonically in catalyzing peptide bond formation. Here, we have identified the ribosomal protein L13 (RPL13) as a critical regulator of IRES-driven translation during FMDV infection. Our study supports a model whereby the FMDV IRESs recruit helicase DDX3 recognizing RPL13 to facilitate IRES-driven translation, with the assistance of eIF3e and eIF3j. A better understanding of these specific interactions surrounding IRES-mediated translation initiation could have important implications for the selective translation of viral mRNA and thus for the development of effective prevention of viral infection.

In vitro antiviral efficacy of pleconaril and ribavirin on foot-and-mouth disease virus replication.

Antiviral therapy is a promising strategy to control acute viral infections. FMDV causes an acute infection and the vaccination provides a protective immunity 7days post immunization. If the infection is uncontained, then it affects the entire herd. In such circumstances, if antiviral drug is administered the infection can be checked in a herd. Ribavirin is known to cure persistently infected BHK21 cells with FMD virus. However, there have been no systematic studies on antiviral activity of ribavirin against FMDV at different time points with the application ofELISA, PCR or real-time PCR. Pleconaril is known to inhibit enteroviruses and rhinoviruses but has not been explored on FMDV. Hence, the present study evaluates the in vitro antiviral efficacy of pleconaril and ribavirin on FMDV replication. The maximum non-toxic concentrations (MNTC) of pleconaril and ribavirin for BHK21 cells respectively were 7.81μg/50μL and 15.62μg/50μL. Thus, drug concentrations below MNTC were tested for their antiviral activity against serial tenfold diluted FMDV O, A and Asia 1 serotypes. Pleconaril did not inhibit FMDV serotype O replication at 7.5μg/50μL based on CPE inhibition assay and this was further confirmed using sandwich ELISA, PCR/real-time PCR. On the other hand, ribavirin at 15.62μg/50μL inhibited the in vitro replication of FMDV O, A and Asia 1 and the inhibition was confirmed by serotype specific sandwich ELISA, PCR and real-time PCR assays. The inhibition was directly proportional to the concentration of ribavirin. Therefore, ribavirin could be explored for its in vivo efficacy as a potential therapeutic in the prevention of early spread of FMDV infection in a herd.

Differential antibody responses to the major antigenic sites of FMD virus serotype O after primo-vaccination, multiply-vaccination and after natural exposure

Foot and mouth disease (FMD) virus serotype O is the predominant cause of FMD outbreaks in several regions of the world including India. Five independent neutralizing antigenic sites have been identified on the capsid surface of FMD virus serotype O. The relative importance of these neutralizing sites in eliciting antibody responses in the polyclonal sera collected from un-infected vaccinated (both primo and multiply-vaccinated) and naturally infected cattle populations were determined through a combination of reverse genetics and serology. The known critical amino acid residues present on the five antigenic sites of FMD virus serotype O Indian vaccine strain O IND R2/1975 were mutated through site-directed mutagenesis. The mutant viruses were rescued in cell-culture and analyzed through virus-neutralization assays along with parent virus using the polyclonal sera collected from three groups of cattle. In the polyclonal sera from primo-vaccinated cattle, significantly higher level of antibodies were directed towards antigenic site 2. In contrast, in polyclonal sera from multiply vaccinated animals, both antigenic sites 1 and 2 were equally important. In case of naturally infected animals, antibody responses were elicited against all the five antigenic sites. Although a drop in neutralization titres was observed for all the mutants, in one instance, increase in titre was noticed for a site 3 mutant. The findings from this study extend our knowledge on the antibody immunodominace following FMDV vaccination and infection, and may improve our strategies for vaccine strain selection and rational vaccine design.

Sec62 Suppresses Foot-and-Mouth Disease Virus Proliferation by Promotion of IRE1α-RIG-I Antiviral Signaling.

Foot-and-mouth disease virus (FMDV) is highly infectious and causes a major plague in animal farming. Unfolded protein response is one of the major cellular responses to pathogenic infections, which performs a crucial role in cell survival, apoptosis, and antiviral innate immune response. In this study, we showed that FMDV infection activated two unfolded protein response branches (PERK-eIF2α and ATF6 signaling) in both baby hamster kidney cells (BHK-21) and porcine kidney (PK-15) cells, whereas it suppressed the IRE1α-XBP1 signaling by decreasing IRE1α level. Further study revealed IRE1α signaling as an important antiviral innate immune mechanism against FMDV. Sec62, the transport protein, was greatly decreased at the late stages of FMDV infection. By overexpression and knockdown study, we also found that the expression of Sec62 was positively involved in the levels of IRE1α and RIG-I and subsequent activation of downstream antiviral signaling pathways in FMDV-infected PK-15 cells. Taken together, our study demonstrates that Sec62 is an important antiviral factor that upregulates IRE1α-RIG-I-dependent antiviral innate immune responses, and FMDV evades antiviral host defense mechanism by downregulating Sec62-IRE1α/RIG-I.

Foot-and-Mouth Disease Virus Antagonizes NOD2-Mediated Antiviral Effects by Inhibiting NOD2 Protein Expression.

The role of nucleotide-binding oligomerization domain 2 (NOD2) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that FMDV infection activated NOD2-mediated beta interferon (IFN-β) and nuclear factor-κB (NF-ĸB) signaling pathways. NOD2 inhibited FMDV replication in the infected cells. FMDV infection triggered NOD2 transcription, while it reduced the abundance of NOD2 protein. Our results revealed that FMDV 2B, 2C, and 3C proteinase (3Cpro) were responsible for the decrease in NOD2 protein levels. 3Cpro is a viral proteinase that can cleave multiple host proteins and limit protein synthesis. Our previous studies determined that FMDV 2B suppressed protein expression of RIG-I and LGP2. Here, we found that 3Cpro and 2B also decreased NOD2 expression. However, this is the first report that 2C induced the reduction of NOD2 protein levels. We determined that both 2B- and 2C-induced decreases in NOD2 were independent of the cleavage of host eukaryotic translation initiation factor 4 gamma (eIF4G), induction of cellular apoptosis, or proteasome, lysosome, and caspase pathways. The interactions between NOD2 and 2B or 2C were observed in the context of viral infection. The carboxyl-terminal amino acids 105 to 114 and 135 to 144 of 2B were essential for the reduction of NOD2, while the residues 105 to 114 were required for the interaction. Amino acids 116 to 260 of the carboxyl terminus of 2C were essential for the interaction, while truncated 2C mutants did not reduce NOD2. These data suggested novel antagonistic mechanisms of FMDV that were mediated by 2B, 2C, and 3Cpro proteins.IMPORTANCE NOD2 was identified as a cytoplasmic viral pattern recognition receptor in 2009. Subsequently, many viruses were reported to activate NOD2-mediated signaling pathways. This study demonstrated that FMDV infection activated NOD2-mediated IFN-β and NF-ĸB signaling pathways. Host cells have developed multiple strategies against viral infection; however, viruses have evolved many strategies to escape host defenses. FMDV has evolved multiple mechanisms to inhibit host type I IFN production. Here, we showed that NOD2 suppressed FMDV replication during viral infection. FMDV 2B, 2C, and 3Cpro decreased NOD2 protein expression by different mechanisms to promote viral replication. This study provided new insight into the immune evasion mechanisms mediated by FMDV and identified 2B, 2C, and 3Cpro as antagonistic factors for FMDV to evade host antiviral responses.