A24 Cruzeiro Research Articles

Broad immunogenic spectrum of monovalent and trivalent foot-and-mouth disease virus vaccines containing O1 campos, A24 cruzeiro and A Argentina 2001 strains against circulating viral lineages in cattle and pigs

FMD remains endemic in many Asian and African countries where multiple variants of serotypes O and A, among others, currently circulate. Due to lack of cross-protection between serotypes and incomplete protection between some strains even within a serotype, an important challenge for the application of effective vaccination programs is to select highly immunogenic and widely cross-reactive vaccine strains. Adaptation of a candidate field virus for use as a vaccine can be quite complex, so that whenever possible, the use of well-established vaccine viruses could have enormous advantages. FMD vaccine strains harmonized for use in South America have shown excellent results in FMD control, not only in the region, where it is still used systematically as a preventive measure, but also more recently in some Asian countries. To gain further insight into the immunogenic spectrum of these strains, VN tests (VNT) were performed with sera from cattle and/or pigs vaccinated with monovalent (type O) or trivalent (types O and A) formulations against 122 type O and 32 type A field viruses isolated from 35 countries in Asia and Africa, belonging to different lineages. Almost all VNT titers obtained were within the expected protective level, indicating the wide immunogenic spectrum of high potency FMD vaccines formulated with O1 Campos, A24 Cruzeiro and A Argentina 2001 South American vaccine strains belonging to EURO-SA topotypes against currently active viruses from other topotypes. These in vitro results are in line with previously reported in vivo challenge tests in pigs against three A/ASIA/Sea-97 isolates and two isolates belonging to type O lineages O/SEA/Mya-98 and O/ME-SA/Ind-2001e.

Molecular detection and phylogenetic analysis of newly emerging foot-and-mouth disease virus type A, Lineage EURO-SA in Egypt in 2022

A newly emerging and exotic foot-and-mouth disease virus (FMDV) caused a recent outbreak of serotype A in Egypt in 2022, which affected cattle and water buffalo. Previous phylogenetic studies on FMDV circulating in Egypt have mainly focused on genomic regions encoding the structural proteins which determine FMDV serotype. No study has yet determined structural proteins sequences of the newly emerging Europe-South America (EURO-SA) lineage which was recently isolated from Egypt during a routine surveillance in 2022. The objective of the current study was to analyze the structural proteins of the Venezuelan type which belongs to EURO-SA. The new isolate was related to serotype A lineage Euro-South America. Phylogentic analyses have reveled that the newly isolated lineage samples were closely related to reported sequences that have been identified in Venzuela and Colombia. Analysis of structural protein sequences revealed the recent isolates belong to prototype strain A24 Cruzeiro. Notably, nucleotide sequences of the Egyptian isolate was related to Venezuelan, Brazilian, and Colombian strains with identity not exceeding 90%. The divergence which appears in the genetic identity of the Egyptian A/EURO-SA lineage from other related strains may be attributed to the absence of Euro-SA lineage sequence from Egypt. The present study is the first report on the detection of EURO-SA lineage in Egypt. The recent detection of the EURO-SA lineage samples may be explained due to imported animals from Colombia or Brazil which share geographical borders with Venezuela. The findings of the present study highlight the significance of continuous monitoring of FMDV in Egypt for newly emerging FMDVs.

Effect of Foot-and-Mouth Disease Vaccine on Pregnancy Failure in Beef Cows.

This study evaluates whether the foot-and-mouth disease (FMD) vaccination increases pregnancy failures in Bos taurus beef cows. A total of 3,379 cows were assigned to two experimental groups to receive (n = 1,722) or not receive (n = 1,657) a FMD vaccine (commercial preparation containing FMD virus, O1 Campos and A24 Cruzeiro) at different gestational age. Pregnancy diagnosis was performed by ultrasonography at vaccination time (Day 0), and the cows were classified by days of pregnancy as follows: (a) <29 days after mating (presumed pregnant cows, n = 778), (b) between 30 and 44 days of pregnancy (n = 1,100), (c) 45 and 59 days of pregnancy (n = 553), and (d) between 60 and 90 days of pregnancy (n = 948). Pregnancy failure was determined 30 days after vaccination by a second ultrasound examination. Cows that were vaccinated within 29 days after mating had a 7.8% greater pregnancy failure rate than non-vaccinated cows (44.1%, 163/370 vs. 36.3%, 148/408, respectively; P <0.05). Cows vaccinated between 30 and 44 days of gestation had a pregnancy failure rate greater than non-vaccinated cows (4.9%, 28/576 vs. 2.5%, 13/524, respectively; P <0.05). When cows received the vaccine between days 45 and 90 of gestation no differences in pregnancy failure were observed (0.8%, 6/776 vs. 1.2%, 9/725, respectively; P = NS). Body temperature and local adverse reactions to vaccine inoculation were recorded in a subset of 152 multiparous cows. Hyperthermia (>39.5°C) was detected on Day 1 or 2 in 28.0% (21/75) of vaccinated vs. 7.8% (6/77) of non-vaccinated cows (P <0.01). Local adverse reaction to the FMD vaccine inoculation increased from 0.0% (0/75) on Day 0, to 15.7% (11/75) on Day 4, and 38.7% (29/75) on Day 10 (P <0.01). On Day 30 local reaction was detected in 10.5% (34/323) and fell to 2.2% on Day 60 (7/323) post vaccination (P <0.01). In conclusion, FMD vaccine increases pregnancy failure when it is administered before 45 days of gestation, an effect that was associated with hyperthermia and local adverse reaction. No effect on pregnancy failure was found when vaccination was performed after 45 days of gestation.

Duration of Contagion of Foot-And-Mouth Disease Virus in Infected Live Pigs and Carcasses.

Data-driven modeling of incursions of high-consequence, transboundary pathogens of animals is a critical component of veterinary preparedness. However, simplifying assumptions and excessive use of proxy measures to compensate for gaps in available data may compromise modeled outcomes. The current investigation was prospectively designed to address two major gaps in current knowledge of foot-and-mouth disease virus (FMDV) pathogenesis in pigs: the end (duration) of the infectious period and the viability of FMDV in decaying carcasses. By serial exposure of sentinel groups of pigs to the same group of donor pigs infected by FMDV A24 Cruzeiro, it was demonstrated that infected pigs transmitted disease at 10 days post infection (dpi), but not at 15 dpi. Assuming a latent period of 1 day, this would result in a conservative estimate of an infectious duration of 9 days, which is considerably longer than suggested by a previous report from an experiment performed in cattle. Airborne contagion was diminished within two days of removal of infected pigs from isolation rooms. FMDV in muscle was inactivated within 7 days in carcasses stored at 4oC. By contrast, FMDV infectivity in vesicle epithelium harvested from intact carcasses stored under similar conditions remained remarkably high until the study termination at 11 weeks post mortem. The output from this study consists of experimentally determined data on contagion associated with FMDV-infected pigs. This information may be utilized to update parameterization of models used for foot-and-mouth disease outbreak simulations involving areas of substantial pig production.

Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates

Foot-and-mouth disease (FMD) is one of the most contagious veterinary viral diseases known, having economic, social and potentially devastating environmental impacts. The vaccines currently being marketed/sold around the world for disease control and prevention in bovines do not stimulate the production of antibodies having crossed reactions to different serotypes. This means that if an animal becomes infected by a serotype which has not been included in a vaccine then it will develop the disease. Synthetic peptide vaccines represent a safer option and (depending on the design) can stimulate antibodies protecting against different variants. Based on the forgoing, this work was aimed at evaluating FMDV VP1, VP2 and VP3 protein-derived, modified and chemically-synthesised peptides’ ability to induce an immune response for developing a vaccine contributing towards controlling the disease. VP1, VP2 and VP3 proteins’ conserved regions were selected for this. Peptides from these regions were chemically synthesised; binding assays were then carried out for ascertaining whether they were involved in BHK-21 cell binding. Selected peptides’ structure and location were studied. Peptides which did bind were modified and formulated with Montanide ISA 70 adjuvant; 17 animals were immunised twice with the formulation. The animals were genotyped by amplifying the BoLA-DRB3.2 gene. Blood samples were taken from 17 cattle on day 43 post-first immunisation for studying the formulation’s immunogenicity. The sera were used in ELISA, immunofluorescence, flow cytometry, immunoadsorption and seroneutralisation assays. The A24 Cruzeiro and O1 Campos virus serotypes were used for these assays. The results revealed that even though protein exposure and 3D structure might be different amongst serotypes, the antibodies so produced could inhibit virus entry to cells, thereby showing the selected peptides’ in vitro protection-inducing ability.

Evaluation of modified Vaccinia Ankara-based vaccines against foot-and-mouth disease serotype A24 in cattle

To prepare foot-and-mouth disease (FMD) recombinant vaccines in response to newly emerging FMD virus (FMDV) field strains, we evaluated Modified Vaccinia virus Ankara-Bavarian Nordic (MVA-BN®) as an FMD vaccine vector platform. The MVA-BN vector has the capacity to carry and express numerous foreign genes and thereby has the potential to encode antigens from multiple FMDV strains. Moreover, this vector has an extensive safety record in humans. All MVA-BN-FMD constructs expressed the FMDV A24 Cruzeiro P1 capsid polyprotein as antigen and the FMDV 3C protease required for processing of the polyprotein. Because the FMDV wild-type 3C protease is detrimental to mammalian cells, one of four FMDV 3C protease variants were utilized: wild-type, or one of three previously reported mutants intended to dampen protease activity (C142T, C142L) or to increase specificity and thereby reduce adverse effects (L127P). These 3C coding sequences were expressed under the control of different promoters selected to reduce 3C protease expression. Four MVA-BN-FMD constructs were evaluated in vitro for acceptable vector stability, FMDV P1 polyprotein expression, processing, and the potential for vaccine scale-up production. Two MVA-BN FMD constructs met the in vitro selection criteria to qualify for clinical studies: MVA-mBN360B (carrying a C142T mutant 3C protease and an HIV frameshift for reduced expression) and MVA-mBN386B (carrying a L127P mutant 3C protease). Both vaccines were safe in cattle and elicited low to moderate serum neutralization titers to FMDV following multiple dose administrations. Following FMDV homologous challenge, both vaccines conferred 100% protection against clinical FMD and viremia using single dose or prime-boost immunization regimens. The MVA-BN FMD vaccine platform was capable of differentiating infected from vaccinated animals (DIVA). The demonstration of the successful application of MVA-BN as an FMD vaccine vector provides a platform for further FMD vaccine development against more epidemiologically relevant FMDV strains.

Duration of protection and humoral immunity induced by an adenovirus-vectored subunit vaccine for foot-and-mouth disease (FMD) in Holstein steers

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven hooved animals that continues to cause economic disruption in both endemic countries or when introduced into a formally FMD free country. Vaccines that protect against clinical disease and virus shedding are critical to control FMD. The replication deficient human adenovirus serotype 5 (Ad5) vaccine vector expressing empty FMD virus (FMDV) capsid, AdtFMD, is a promising new vaccine platform. With no shedding or spreading of viral vector detected in field trials, this vaccine is very safe to manufacture, as there is no requirement for high containment faciitites. Here, we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9 months following a single vaccination with the commercial AdtFMD vaccine, provisionally licensed for cattle in the United States. Further, we tested the effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome and humoral immunity. Results demonstrate that a single dose vaccination in cattle with the commercial vaccine vector expressing capsid proteins of the FMDV strain A24 Cruzeiro (Adt.A24), induced protection against clinical FMD at 6 months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9 months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Anti-FMDV antibody secreting cells are detected in peripheral blood early following vaccination, but are absent after 28 days post-vaccination. Thus, the decay in antibody mediated immunity over time is likely a function of FMDV-specific antibody half-life. These data reveal the short time span of anti-FMDV antibody secreting cells (ASCs) and important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.

Recombinant mutagenic 3ABC protein and monoclonal antibody for quality-control testing in foot-and-mouth disease vaccines

To achieve the goal of performing quality control on vaccines for foot-and-mouth disease several steps were performed in this study. First, the gene that encodes the 3ABC region of the A24 Cruzeiro strain (which is used in vaccine production) was amplified. Second, to improve protein stability, the 3ABC protein was mutated at the 3Cpro catalytic site and cloned the amplification products in the pET-SUMO expression vector. In the third step, the resulting recombinant protein was tested, which the polyclonal antibody recognized, with the semi-purified viral 3ABC protein using an immunoassay test. Fourth, the muted recombinant protein was used to produce a monoclonal antibody. Of the 217 clones obtained, two of them that were particularly stable (mAb2D3 and mAb3D12) were selected to work with. One showed better results, as characterized by immunoassay (ELISA), Western blotting, spot synthesis, and sequencing methodologies; it also showed high reactivity against the 3ABC protein. This kind of monoclonal antibodies, which was considered as immunochemical inputs, have been used in industrial processes as part of quality-control procedures, to evaluate the elimination of the 3ABC protein so as to ensure regulatory approval of the vaccine. They have also been used in immunological tests to distinguish infected from vaccinated animals.

Safety profile of a replication-deficient human adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle.

The safety of a replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was evaluated in five independent safety studies. The target animal safety studies were designed in compliance with United States (U.S.) regulatory requirements (Title 9, U.S. Code of Federal Regulation [9CFR]) and international standard guidelines (VICH Topic GL-44) for veterinary live vaccines. The first three studies were conducted in a total of 22 vaccinees and demonstrated that the AdtA24 master seed virus (MSV) was safe, did not revert to virulence and was not shed or spread from vaccinees to susceptible cattle or pigs. The fourth safety study conducted in 10 lactating cows using an AdtA24 vaccine serial showed that the vaccine was completely absent from milk. The fifth safety study was conducted under typical U.S. production field conditions in 500 healthy beef and dairy cattle using two AdtA24 vaccine serials. These results demonstrated that the vaccine was safe when used per the product label recommendations. Additional data collected during these five studies confirmed that AdtA24 vaccinees developed FMDV A24 and the HAd5 vaccine vector serum neutralization antibodies that test negative in a FMDV non-structural protein antibody test, confirming AdtA24 vaccine's capability to differentiate infected from vaccinated animals (DIVA). In conclusion, results from this comprehensive set of cattle studies demonstrated the safety of the replication-deficient AdtA24 vaccine and fulfilled safety-related requirements for U.S. regulatory requirements.

Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity.

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

Transcriptomic Analysis of Persistent Infection with Foot-and-Mouth Disease Virus in Cattle Suggests Impairment of Apoptosis and Cell-Mediated Immunity in the Nasopharynx.

In order to investigate the mechanisms of persistent foot-and-mouth disease virus (FMDV) infection in cattle, transcriptome alterations associated with the FMDV carrier state were characterized using a bovine whole-transcriptome microarray. Eighteen cattle (8 vaccinated with a recombinant FMDV A vaccine, 10 non-vaccinated) were challenged with FMDV A24 Cruzeiro, and the gene expression profiles of nasopharyngeal tissues collected between 21 and 35 days after challenge were compared between 11 persistently infected carriers and 7 non-carriers. Carriers and non-carriers were further compared to 2 naïve animals that had been neither vaccinated nor challenged. At a controlled false-discovery rate of 10% and a minimum difference in expression of 50%, 648 genes were differentially expressed between FMDV carriers and non-carriers, and most (467) had higher expression in carriers. Among these, genes associated with cellular proliferation and the immune response–such as chemokines, cytokines and genes regulating T and B cells–were significantly overrepresented. Differential gene expression was significantly correlated between non-vaccinated and vaccinated animals (biological correlation +0.97), indicating a similar transcriptome profile across these groups. Genes related to prostaglandin E2 production and the induction of regulatory T cells were overexpressed in carriers. In contrast, tissues from non-carrier animals expressed higher levels of complement regulators and pro-apoptotic genes that could promote virus clearance. Based on these findings, we propose a working hypothesis for FMDV persistence in nasopharyngeal tissues of cattle, in which the virus may be maintained by an impairment of apoptosis and the local suppression of cell-mediated antiviral immunity by inducible regulatory T cells.

Protection induced by a commercial bivalent vaccine against Foot-and-Mouth Disease 2010 field virus from Ecuador

Foot-and-Mouth Disease serotype O circulated endemically in Ecuador for many years, with an upsurge occurring in 2009. This manuscript describes retrospectively in vitro and in vivo laboratory studies to predict the field effectiveness of a commercial FMD vaccine to protect against the field strain, and explains the key actions and epidemiological strategies followed by the country to control the disease.The results established that the use of a good quality oil vaccine, manufactured with strains that were isolated long ago: O1 Campos Br/58 and A24 Cruzeiro Br/55; combined with the correct epidemiological strategies, are useful to control field strains when used in periodic biannual vaccination campaigns.

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge

The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×108 to 2.1×1011 particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R2=0.97) and viremia (R2=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R2=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.

A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules.

Major histocompatibility complex (MHC) class Imolecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network-based predictions (NetMHCpan). The updated NetMHCpan server was used to predict BoLA-I binding peptides within the P1 structural polyprotein sequence of FMDV (strain A24 Cruzeiro) for Bo-LA-1*01901, BoLA-2*00801, BoLA-2*01201, and BoLA-4*02401. Peptide binding affinity and stability were determined for these BoLA-I molecules using the luminescent oxygen channeling immunoassay (LOCI) and scintillation proximity assay (SPA). The functional diversity of known BoLA alleles was predicted using theMHCcluster tool, and functional predictions for peptide motifs were compared to observed data from this and prior studies. The results of these analyses showed that BoLA alleles cluster into three distinct groups with the potential to define BBoLA supertypes.^ This streamlined approach identifies potential T cell epitopes from pathogens, such as FMDV, and provides insight into T cell immunity following infection or vaccination.

Early Detection of Foot-And-Mouth Disease Virus from Infected Cattle Using A Dry Filter Air Sampling System.

Foot-and-mouth disease (FMD) is a highly contagious livestock disease of high economic impact. Early detection of FMD virus (FMDV) is fundamental for rapid outbreak control. Air sampling collection has been demonstrated as a useful technique for detection of FMDV RNA in infected animals, related to the aerogenous nature of the virus. In the current study, air from rooms housing individual (n=17) or two groups (n=4) of cattle experimentally infected with FDMV A24 Cruzeiro of different virulence levels was sampled to assess the feasibility of applying air sampling as a non-invasive, screening tool to identify sources of FMDV infection. Detection of FMDV RNA in air was compared with first detection of clinical signs and FMDV RNA levels in serum and oral fluid. FMDV RNA was detected in room air samples 1-3days prior (seven animals) or on the same day (four animals) as the appearance of clinical signs in 11 of 12 individually housed cattle. Only in one case clinical signs preceded detection in air samples by one day. Overall, viral RNA in oral fluid or serum preceded detection in air samples by 1-2days. Six individually housed animals inoculated with attenuated strains did not show clinical signs, but virus was detected in air in one of these cases 3days prior to first detection in oral fluid. In groups of four cattle housed together, air detection always preceded appearance of clinical signs by 1-2days and coincided more often with viral shedding in oral fluid than virus in blood. These data confirm that air sampling is an effective non-invasive screening method for detecting FMDV infection in confined to enclosed spaces (e.g. auction barns, milking parlours). This technology could be a useful tool as part of a surveillance strategy during FMD prevention, control or eradication efforts.

Poly ICLC increases the potency of a replication-defective human adenovirus vectored foot-and-mouth disease vaccine

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FMDV challenge by 7 days post-vaccination. However, since relatively large amounts of Ad5-CI-A24-2B are required to induce protection this strategy could be costly for livestock production. Poly ICLC is a synthetic double stranded RNA that activates multiple innate and adaptive immune pathways. In this study, we have tested for the first time, the adjuvant effect of poly ICLC in combination with Ad5-CI-A24-2B in swine. We found that the combination resulted in a reduction of the vaccine protective dose by 80-fold. Interestingly, the lowest dose of Ad5-CI-A24-2B plus 1mg of poly ICLC protected animals against challenge even in the absence of detectable FMDV-specific neutralizing antibodies at the time of challenge.

Infection with foot-and-mouth disease virus (FMDV) induces a natural killer (NK) cell response in cattle that is lacking following vaccination.

Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46(+) cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3(+) cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV.

Validación de una técnica de RT-PCR en tiempo real para la detección del virus de fiebre aftosa y evaluación de su desempeño en la infección aguda

A specific real time reverse transcription polymerase chain reaction (RT-PCRrt) for the detection of foot-and-mouth disease virus was validated using the LightCycler thermocycler 2.0 and its reagents as recommended by the World Organization for Animal Health and was assessed for the detection of the virus in acute infection of cattle experimentally vaccinated and challenged with virus A Argentina/2001 or A24 Cruzeiro. The technique proved to be robust, showing coefficients of variation lower than 4% for different ARN extractions, days or repetitions and was able to detect up to 0,4 TCID 50%, and/or up to 100 RNA molecules. In probang samples, diagnostic sensitivity was 93.1 (95% CI 86.5–96.6) and diagnostic specificity 100 (95% CI 96.3-100). The results of the challenge in vaccinated or multivaccinated bovines showed that although there were high levels of clinical protection in the vaccinated group, FMDV could be detected in all challenged groups. However, detection was 100 times lower in immunized animals.

Repeated exposure to 5D9, an inhibitor of 3D polymerase, effectively limits the replication of foot-and-mouth disease virus in host cells

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock caused by a highly variable RNA virus (FMDV) that has seven serotypes and more than sixty subtypes. Both prophylactic and post-infection means of controlling the disease outbreak, including universally applicable vaccines and emergency response measures such as therapeutic treatments, are on high demand. In this study, we analyzed the long-term exposure outcome to a previously identified inhibitor of 3D polymerase (FMDV 3Dpol) for controlling FMDV infection and for the selection of resistance mutants. The results showed that no escape mutant viruses were isolated from FMDV A24 Cruzeiro infections in cell culture treated with gradually increasing concentrations of the antiviral compound 5D9 (4-chloro-N′-thieno, [2,3-d]pyrimidin-4-ylbenzenesulfonohydrazide) over ten passages. Biochemical and plaque assays revealed that when 5D9 was used at concentrations within a non-toxic range in cells, it drove the virus to undetectable levels at passage eight to ten. This is in contrast with observations made on parallel control (untreated) passages exhibiting fully viable and stable virus progenies. Collectively, the results demonstrated that under the experimental conditions, treatment with 5D9 does not confer a resistant phenotype and the virus is unable to evade the antiviral effect of the inhibitor. Further efforts using quantitative structure–property relationship (QSPR) based modifications of the 5D9 compound may result in the successful development of an effective in vivo antiviral drug targeting FMDV.

Examination of soluble integrin resistant mutants of foot-and-mouth disease virus

BackgroundFoot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules αvβ1, αvβ3, αvβ6, or αvβ8 by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the αvβ6 interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted αvβ6 (ssαvβ6) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction.ResultsGenetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ssαvβ6. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ssαvβ6 neutralization, relative ability to utilize αvβ6 for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for αvβ6. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ssαvβ6. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ssαvβ6 resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding.ConclusionsThese findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.