Foot-and-mouth Disease Virus Infection Research Articles

An ELISA based on the repeated foot-and-mouth disease virus 3B epitope peptide can distinguish infected and vaccinated cattle

To develop a strategy of differentiating infected from vaccinated animals (DIVA) with foot-and-mouth disease virus (FMDV), a short (27aa) peptide containing three conserved linear B cell epitopes of the FMDV 3B nonstructural protein was designed. This novel BF peptide was synthesized using a gene splicing by overlap extension protocol with preferred codons for Escherichia coli. The resultant eight tandem repeat multimer (1, 2, 4, 6, 8, 16, 24, and 32BF) were expressed as soluble fusion proteins in E. coli. An indirect ELISA was developed based on the recombinant 8BF protein with the aim of specifically distinguishing antibodies induced by FMDV infection but not those induced by vaccination. Using the cut-off value of 0.3, the sensitivity of the assay was 96.8% and the specificities for naive and vaccinated cattle were 99.8 and 99.0%, respectively. The performance of the newly developed epitope-based ELISA was compared with three commercial NSP ELISA kits. The 8BF-ELISA appears to be a promising DIVA test for FMD control and eradication.

Detection of foot-and-mouth disease virus rna by reverse transcription loop-mediated isothermal amplification

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for foot-and-mouth disease virus (FMDV) RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus. Furthermore, the assay correctly detected 84 FMDV positive samples but not 65 FMDV negative specimens. The result indicated the potential usefulness of the technique as a simple and rapid procedure for the detection of FMDV infection.

Cattle remain immunocompetent during the acute phase of foot-and-mouth disease virus infection

Infection of cattle with foot-and-mouth disease virus (FMDV) results in the development of long-term protective antibody responses. In contrast, inactivated antigen vaccines fail to induce long-term protective immunity. Differences between susceptible species have also been observed during infection with FMDV, with cattle often developing persistent infections whilst pigs develop more severe symptoms and excrete higher levels of virus. This study examined the early immune response to FMDV in naïve cattle after in-contact challenge. Cattle exposed to FMDV were found to be viraemic and produced neutralising antibody, consistent with previous reports. In contrast to previous studies in pigs these cattle did not develop leucopenia, and the proliferative responses of peripheral blood mononuclear cells to either mitogen or third party antigen were not suppressed. Low levels of type 1 interferon and IL-10 were detected in the circulation. Taken together, these results suggest that there was no generalised immunosuppression during the acute phase of FMDV infection in cattle.

Inoculation of newborn mice with non-coding regions of foot-and-mouth disease virus RNA can induce a rapid, solid and wide-range protection against viral infection

We have recently described the ability of in vitro-transcribed RNAs, mimicking structural domains in the 5′ and 3′ non-coding regions (NCRs) of the foot-and-mouth disease virus (FMDV) genome, to trigger the innate immune response in porcine cultured cells and mice. In this work, the antiviral effect exerted in vivo by these small synthetic non-infectious RNA molecules was analyzed extensively. The susceptibility of transfected newborn Swiss mice to FMDV challenge was tested using a wide range of viral doses. The level of protection depended on the specific RNA inoculated and was dose-dependent. The RNA giving the best protection was the internal ribosome entry site (IRES), followed by the transcripts corresponding to the S fragment. The time course of resistance to FMDV of the RNA-transfected mice was studied. Our results show the efficacy of these RNAs to prevent viral infection as well as to contain ongoing FMDV infection in certain time intervals. Protection proved to be independent of the serotype of FMDV used for challenge. These results support the potential use of the FMDV NCR transcripts as both prophylactic and therapeutic molecules for new FMDV control strategies.

Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells

BackgroundshRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF) and cell enzyme linked immunosorbent assays (cell ELISA), and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells.ResultsOur results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 106 TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV.ConclusionsIαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.

Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle

Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139–149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an “expected percentage of protection” above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.

English

Dendritic cells (DCs) play an important role in inducing primary antigen-specific immune responses to viral antigens. In this study, the peripheral blood monocyte-derived (PBMC) were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. After 6 days of culture, immature monocyte-derived dendritic cells (Mo-DCs) were generated. The addition of lipopolysaccharide (LPS) during differentiation of Mo-DCs enhanced their ability to stimulate allogeneic mixed lymphocyte reaction (MLR) and alter their ability to produce cytokines. Then, we investigated the interaction between foot-and-mouth disease virus (FMDV) and porcine Mo-DCs in vitro and confirmed that the immunological phenotype and function of porcine Mo-DCs were modulated during FMDV infection. A down-regulated expression of MHC II and CD1 were observed at 48 h post FMDV infection. In addition, the infected porcine Mo-DCs exhibited ultrastructural morphological changes, FMDV-infected porcine Mo-DCs failed to stimulate T cell proliferation in vitro. Moreover, infection of porcine Mo-DCs in vitroinduced the secretion of IFN-γ and the suppressive cytokine IL-10 in porcine Mo-DCs. Results indicated that the down-regulation of MHC II and CD1 molecules and the increased secretion of the IFN-γ and IL-10 cytokines might be the mechanisms that FMDV uses to evade the host immune responses.   Key words: Dendritic cells, foot-and-mouth disease virus, MHC II, modulation, cytokines.

Detection of foot-and-mouth disease virus RNA in pharyngeal epithelium biopsy samples obtained from infected cattle: Investigation of possible sites of virus replication and persistence

Foot-and-mouth disease (FMD) is a highly contagious viral infection of significant financial importance to the export and trade of agricultural products. The occurrence of persistently infected “carriers” of FMD-virus (FMDV) in ruminant species adds further complications to disease control. There have been significant discrepancies in reports regarding the pathogenesis of FMDV infection in cattle with specific emphasis on the anatomical sites involved in early and persistent virus replication. In this study, collection of small biopsy samples from the dorsal soft palate (DSP) of live animals was used to investigate the level of FMDV RNA present at this site at sequential time points during the infection. Results were compared to measurements of virus excretion in samples of oropharyngeal fluid collected at corresponding time points. Possible sites of virus persistence were investigated through measurements of the levels of FMDV RNA in the DSP as well as mandibular and retropharyngeal lymph nodes beyond 28 days after infection. Results indicated only low levels of FMDV RNA present in samples of pharyngeal epithelia during both early and persistent phases of infection with significantly higher levels of virus detected in pharyngeal excretions. It is concluded that the targeted area for sampling within the DSP does not harbour significant levels of virus replication during acute or persistent FMDV infection in cattle. Furthermore, the DSP and the mandibular and retropharyngeal lymph nodes cannot be concluded to be principal sites for persistence of FMDV.

Chemical disinfection of high-consequence transboundary animal disease viruses on nonporous surfaces

Disinfection is a critical part of the response to transboundary animal disease virus (TADV) outbreaks by inactivating viruses on fomites to help control infection. To model the inactivation of TADV on fomites, we tested selected chemicals to inactivate Foot and Mouth Disease virus (FMDV), African Swine Fever virus (ASFV), and Classical Swine Fever virus (CSFV) dried on steel and plastic surfaces. For each of these viruses, we observed a 2 to 3 log reduction of infectivity due to drying alone. We applied a modified surface disinfection method to determine the efficacy of selected disinfectants to inactivate surface-dried high-titer stocks of these three structurally different TADV. ASFV and FMDV were susceptible to sodium hypochlorite (500 and 1000 ppm, respectively) and citric acid (1%) resulting in complete disinfection. Sodium carbonate (4%), while able to reduce FMDV infectivity by greater than 4-log units, only reduced ASFV by 3 logs. Citric acid (2%) did not totally inactivate dried CSFV, suggesting it may not be completely effective for disinfection in the field. Based on these data we recommend disinfectants be formulated with a minimum of 1000 ppm sodium hypochlorite for ASFV and CSFV disinfection, and a minimum of 1% citric acid for FMDV disinfection.

Detection of foot-and-mouth disease virus in the breath of infected cattle using a hand-held device to collect aerosols

Exhaled air of individual cattle infected experimentally with foot-and-mouth disease virus (FMDV) was sampled to assess the feasibility of a rapid, non-invasive general screening approach for identifying sources of FMDV infection. The air sampler used was a handheld prototype device employing electrostatic particle capture in a microchip chamber of 10–15μL and was shown to effectively capture a high percentage of airborne microorganisms. The particles were eluted subsequently from the chip chamber and subjected to real-time RT-PCR. Sampling exhaled air for as little as 1min allowed the detection of FMDV in cattle infected experimentally. Detection in exhaled air from individual cattle was compared to FMDV detection in serum and saliva for 3 different strains of FMDV (O1/Manisa/69, C/Oberbayern/FRG/1960 and SAT1/Zimbawe/1989). Detection of FMDV in exhaled air was possible for all strains of FMDV used for experimental infection but the period that detection was possible varied among the strains. Detection in exhaled air generally peaked on day 2–4 post infection. The perspectives of monitoring for FMDV in the breath of infected cattle are discussed in the context of real-time epidemiological contingencies.

Effective inhibition of foot-and-mouth disease virus (FMDV) replication in vitro by vector-delivered microRNAs targeting the 3D gene.

BackgroundFoot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined.ResultsFour pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV.ConclusionOur results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

RNA Structural Domains in Noncoding Regions of the Foot-and-Mouth Disease Virus Genome Trigger Innate Immunity in Porcine Cells and Mice

The induction of type I interferons (alpha/beta interferon [IFN-α/β]) in response to viral infection is a crucial step leading to the antiviral state in the host. Viruses produce double-stranded RNA (dsDNA) during their replication cycle that is sensed as nonself by host cells through different receptors. A signaling cascade then is activated to block viral replication and spread. Foot-and-mouth disease virus (FMDV) is a picornavirus that is highly sensitive to IFN, and it causes one of the world's most important animal diseases. In this study, we showed the ability of structural domains predicted to enclose stable dsRNA regions in the 5'- and 3'-noncoding regions (NCRs) of the FMDV genome to trigger an IFN-α/β response in porcine kidney cultured cells and newborn mice. These RNAs, generated by in vitro transcription, were able to stimulate IFN-β transcription and induce an antiviral state in SK-6 cells. The induction levels elicited by the different NCR RNAs were compared. Among them, the 3'NCR was identified as a potent IFN activator, and the features in this region involved in signaling have been analyzed. To address whether the FMDV NCR transcripts were able to trigger the innate immune response in vivo, Swiss suckling mice were inoculated intraperitoneally with the RNAs. All transcripts induced the innate response in transfected animals, measured as IFN-α/β protein levels, antiviral activity in sera, and reduced susceptibility to FMDV infection. Our work provides new insight into innate responses against FMDV and identifies these small noninfectious RNA molecules as potential adjuvants for vaccine improvement and antiviral strategies against picornaviruses.

Analysis of the cytotoxic T lymphocyte response of MHC-defined swine to foot-and-mouth disease virus (170.14)

Abstract Foot-and-mouth disease (FMD) is a disease of cloven-hoofed animals that remains a threat to the livestock industry. FMD virus (FMDV) causes an acute infection characterized by fever and the formation of vesicles on the feet and tongue. This picornavirus is classified into seven serotypes and the current killed-virus vaccines provide antibody-mediated protection that is serotype specific. A next-generation vaccine that relies on non-replicating recombinant human adenovirus 5 (Ad5) has been shown to induce neutralizing antibodies. To date, the role of cytotoxic T lymphocytes (CTLs) has been marginally explored. We have shown that swine vaccinated with an Ad5 vectored vaccine that targets the CTL response exhibited greater CTL killing of MHC-matched target cells compared to swine given the aforementioned Ad5 FMDV vaccine. In two trials with the T cell vaccine, the CTL response showed limited epitope specificity by class I MHC tetramer staining. Using these assays, we now show that FMDV infection also elicits a CTL response with broader epitope specificity than that induced by vaccination. These data provide a basis for evaluating whether CTLs can protect against disease following virus challenge.

Antiviral activity of bovine type III interferon against foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is one of the most serious threats to the livestock industry. Despite the availability of a vaccine, recent outbreaks in disease-free countries have demonstrated that development of novel FMD control strategies is imperative. Here we report the identification and characterization of bovine (bo) interferon lambda 3 (IFN-λ3), a member of the type III IFN family. Expression of boIFN-λ3 using a replication-defective human adenovirus type 5 vector (Ad5-boIFN-λ3) yielded a glycosylated secreted protein with antiviral activity against FMD virus (FMDV) and vesicular stomatitis virus in bovine cell culture. Inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and up-regulation of IFN stimulated gene expression in multiple tissues susceptible to FMDV infection. Our results demonstrate that the type III IFN family is conserved in bovines and boIFN-λ3 has potential for further development as a biotherapeutic candidate to inhibit FMDV or other viruses in cattle.

A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

BackgroundFoot-and-mouth disease (FMD) is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must.MethodsIn the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV) under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE), and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine.ResultsIndirect immunofluorescence assay (IFA) and indirect sandwich-ELISA (IS-ELISA) showed both recombinant baculoviruses (with or without T-cell epitopes) were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ). Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P < 0.01) and higher neutralizing antibody titer (P < 0.05). Although the inactivated vaccine produced the highest titer of neutralizing antibodies, a lower IFN-γ expression was observed compared to the two recombinant pseudotype baculoviruses.ConclusionsThese results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection.

Bovine Plasmacytoid Dendritic Cells Are the Major Source of Type I Interferon in Response to Foot-and-Mouth Disease Virus In Vitro and In Vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.

Absence of Heat Intolerance (Panting) Syndrome in Foot-and-Mouth Disease-Affected Indian Cattle (Bos indicus) is Associated with Intact Thyroid Gland Function

Foot-and-mouth disease (FMD) is a highly contagious and economically important viral disease with high morbidity and reduced productivity of affected animals. We studied the heat intolerance (HI) (panting) syndrome and the effect of FMD virus (FMDV) infection on thyroid gland function in Indian cattle (Bos indicus). Experimental infection with FMDV Asia 1 resulted in a mild form of disease with superficial lesions. Heat intolerance syndrome and its signs were not observed among the recovered animals. Subtle changes in the serum level of thyroid hormones, triiodothyronine (T₃) and thyroxine (T₄) were observed. However, there were no distinct histological changes in the thyroid gland, and FMDV antigens were not detected in the thyroid tissues. Our results thus suggest that the absence of panting syndrome in FMD-affected Bos indicus cattle may be associated with intact thyroid gland function.

Proteomics Analysis of Porcine Serum Proteins by LC-MS/MS after Foot-and-Mouth Disease Virus (FMDV) Infection

To analyze serum proteomics differences between normal and foot and mouth disease virus (FMDV)-infected piglets, an analytical method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used. Samples of venous blood were collected before and after FMDV infection and high abundance serum albumin was removed using a commercial kit. After trypsin digestion, serum samples were processed with LC-MS/MS. Proteins were identified by peptide mass fingerprinting. We found that apolipoprotein A-IV precursor, haptoglobin and probable chemoreceptor glutamine deamidase cheD appeared after FMDV infection in the same piglet. This is believed to be the first time that serum proteomics analysis by LC-MS/MS after FMDV infection has been performed, and our results may provide further information about biomarkers for early diagnosis of FMD in piglets.

The role of African buffalos (syncerus caffer) in the maintenance of foot-and-mouth disease in Uganda

BackgroundTo study the role of African buffalos (Syncerus caffer) in the maintenance of foot-and-mouth disease in Uganda, serum samples were collected from 207 African buffalos, 21 impalas (Aepyceros melampus), 1 giraffe (Giraffa camelopardalis), 1 common eland (Taurotragus oryx), 7 hartebeests (Alcelaphus buselaphus) and 5 waterbucks (Kobus ellipsiprymnus) from four major National Parks in Uganda between 2005 and 2008. Serum samples were screened to detect antibodies against foot-and-mouth disease virus (FMDV) non-structural proteins (NSP) using the Ceditest® FMDV NS ELISA. Solid Phase Blocking ELISAs (SPBE) were used to determine the serotype-specificity of antibodies against the seven serotypes of FMDV among the positive samples. Virus isolation and sequencing were undertaken to identify circulating viruses and determine relatedness between them.ResultsAmong the buffalo samples tested, 85% (95% CI = 80-90%) were positive for antibodies against FMDV non-structural proteins while one hartebeest sample out of seven (14.3%; 95% CI = -11.6-40.2%) was the only positive from 35 other wildlife samples from a variety of different species. In the buffalo, high serotype-specific antibody titres (≥ 80) were found against serotypes O (7/27 samples), SAT 1 (23/29 samples), SAT 2 (18/32 samples) and SAT 3 (16/30 samples). Among the samples titrated for antibodies against the four serotypes O, SAT 1, SAT 2 and SAT 3, 17/22 (77%; CI = 59.4-94.6%) had high titres against at least two serotypes.FMDV isolates of serotypes SAT 1 (1 sample) and SAT 2 (2 samples) were obtained from buffalo probang samples collected in Queen Elizabeth National Park (QENP) in 2007. Sequence analysis and comparison of VP1 coding sequences showed that the SAT 1 isolate belonged to topotype IV while the SAT 2 isolates belonged to different lineages within the East African topotype X.ConclusionsConsistent detection of high antibody titres in buffalos supports the view that African buffalos play an important role in the maintenance of FMDV infection within National Parks in Uganda. Both SAT 1 and SAT 2 viruses were isolated, and serological data indicate that it is also likely that FMDV serotypes O and SAT 3 may be present in the buffalo population. Detailed studies should be undertaken to define further the role of wildlife in the epidemiology of FMDV in East Africa.

Identification of a conserved linear epitope on the VP1 protein of serotype O foot-and-mouth disease virus by neutralising monoclonal antibody 8E8

Foot-and-mouth disease virus (FMDV) serotype O remains an important threat to animal husbandry worldwide, and the variability of the virus presents a major problem for FMDV vaccine design. High-affinity neutralising antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype O and protect against future pandemics. We generated a novel monoclonal antibody (MAb) 8E8 that potently neutralised infection of FMDV O/YS/CHA/05 both in vitro and in vivo. Screening of a phage-displayed random 12-peptide library revealed that MAb 8E8 bound to phages displaying a consensus motif GDLNVRT, which is highly homologous to 146GDLQVLT 152 of the FMDV VP1 protein. Given that MAb 8E8 showed reactivity with the 146GDLQVLT 152 motif, we proposed that this motif represented a linear B-cell epitope of the VP1 protein. Western blot analysis revealed that the epitope peptide could be recognised by the positive sera from serotype O FMDV-infected pigs. The 147DLQVLT 152 motif was the minimal requirement for reactivity as demonstrated by reactivity of MAb 8E8 with several truncated peptides derived from the motif. For further mapping, a set of different extended motifs derived from the minimally reactive epitope was expressed with a GST-tag and subjected to western blot. The results showed that a 10-aa peptide 145RGDLQVLTPK 154 was the minimal unit with maximal binding activity to MAb 8E8. Subsequent alanine scanning mutagenesis studies revealed that D 147, Q 149 and V 150 are crucial for MAb 8E8 binding. Furthermore, the epitope was found to be highly conserved among different topotypes of serotype O FMDV through sequence alignment analysis and detection of MAb 8E8 for affinity to some isolates collected in China. Thus, the 8E8 epitope identified here should be helpful for designing epitope-based, intra-typic, cross-protective vaccines of serotype O FMDV.