Epidemiological investigation of foot-and-mouth disease outbreaks in a Vietnamese bear rescue centre.

Foot-and-mouth disease (FMD) outbreaks affecting Asiatic black bears (Ursus thibetanus) and a Malayan sun bear (Helarctos malayanus) were previously reported in 2011 in two housing facilities at a Vietnamese bear rescue centre. In this study, demographic data of all animals housed in the centre at the time of the outbreaks (n = 79) were collected. Blood samples drawn from 23 bears at different timepoints were tested for FMDV-specific antibodies targeting using a non-structural protein (NSP) ELISA and by virus neutralisation test (VNT). The relationship between seroconversion and clinical signs was explored and epidemic curves and transmission diagrams were generated for each outbreak, where FMD cases were defined as animals showing FMD clinical signs. Outbreak-specific attack rates were 18.75 and 77.77%, with corresponding basic reproduction numbers of 1.11 and 1.92, for the first and second outbreaks, respectively. Analyses of risk factors showed that after adjusting for sex there was strong evidence for a decrease in odds of showing clinical signs per year of age. All samples collected from bears before the outbreak tested negative to NSP and VNT. All cases tested positive to VNT following onset of clinical signs and remained positive during the rest of the follow up period, while only 6 out of 17 cases tested positive to NSP after developing clinical signs. Six animals without clinical signs were tested post outbreaks; five seroconverted using VNT and three animals were seropositive using NSP ELISA. This study provides initial epidemiological parameters of FMD in captive bears, showing that FMDV is easily spread between bears in close proximity and can cause clinical and subclinical disease, both of which appear to induce rapid and long-lasting immunity.

Foot and Mouth Disease Vaccine Matching and Post-Vaccination Assessment in Abu Dhabi, United Arab Emirates.

Despite the annual vaccination of livestock against foot and mouth disease (FMD) in the United Arab Emirates (UAE), outbreaks of the disease continue to be reported. The effective control of field outbreaks by vaccination requires that the vaccines used are antigenically matched to circulating field FMD viruses. In this study, a vaccine matching analysis was performed using the two-dimensional virus neutralization test (VNT) for three field isolates belonging to the O/ME-SA/PanAsia-2/ANT-10 and O/ME-SA/SA-2018 lineages collected from different FMD outbreaks that occurred within the Abu Dhabi Emirate in 2021 affecting Arabian oryx (Oryx leucoryx), goat, and sheep. In addition, post-vaccination antibodies in sheep and goats were measured using solid-phase competitive ELISA (SPCE) for FMDV serotypes A and O at five months after a single vaccine dose and a further 28 days later after a second dose of the FMD vaccine. An analysis of vaccine matching revealed that five out of the six vaccine strains tested were antigenically matched to the UAE field isolates, with r1-values ranging between 0.32 and 0.75. These results suggest that the vaccine strains (O-3039 and O1 Manisa) included in the FMD vaccine used in the Abu Dhabi Emirate are likely to provide protection against outbreaks caused by the circulating O/ME-SA/PanAsia-2/ANT-10 and O/ME-SA/SA-2018 lineages. All critical residues at site 1 and site 3 of VP1 were conserved in all isolates, although an analysis of the VP1-encoding sequences revealed 14-16 amino acid substitutions compared to the sequence of the O1 Manisa vaccine strain. This study also reports on the results of post-vaccination monitoring where the immunization coverage rates against FMDV serotypes A and O were 47% and 69% five months after the first dose of the FMD vaccine, and they were increased to 81 and 88%, respectively, 28 days after the second dose of the vaccine. These results reinforce the importance of using a second booster dose to maximize the impact of vaccination. In conclusion, the vaccine strains currently used in Abu Dhabi are antigenically matched to circulating field isolates from two serotype O clades (O/ME-SA/PanAsia-2/ANT-10 sublineage and O/ME-SA/SA-2018 lineage). The bi-annual vaccination schedule for FMD in the Abu Dhabi Emirate has the potential to establish a sufficient herd immunity, especially when complemented by additional biosecurity measures for comprehensive FMD control. These findings are pivotal for the successful implementation of the region's vaccination-based FMD control policy, showing that high vaccination coverage and the wide-spread use of booster doses in susceptible herds is required to achieve a high level of FMDV-specific antibodies in vaccinated animals.

Selective Dorsal Rhizotomy for Spastic Cerebral Palsy: Report of 18 Cases Performed in the North of Vietnam.

OBJECTIVE: The purpose of this report is to describe a case series of children undergoing selective dorsal rhizotomy (SDR) for the treatment of spastic cerebral palsy in Vietnam. Also described is an international cooperation model to facilitate the development of a new, multidisciplinary team for the evaluation and treatment of these children. METHODS: Details of international collaboration are described, including in-person travel and virtual interactions. All cases of children younger than 18 years undergoing SDR for treatment of spastic cerebral palsy at a single center in Hanoi, Vietnam are described, including preoperative evaluation of spasticity and gait as well as results at 6 and 12 months. Results are summarized using descriptive statistics. RESULTS: Since the beginning of cooperation in training and transferring SDR techniques by experts from the United States, in the period from June 2016 to December 2022, 18 SDR surgeries were performed in Hanoi. Patients' ages ranged from 2 to 14 years; 13 were male and 5 were female. Overall, approximately 60% of nerve rootlets were cut. Modified Ashworth Scale scores at 6 and 12 months after surgery in the hip, knee, and ankle joints showed improvement from preoperative values. There were 2 recorded complications: intracranial hypotension causing subdural hemorrhage and a case of skin infection at the incision site. CONCLUSIONS: The ongoing international cooperation between Vietnamese and American physicians has helped improve the surgical treatment of spasticity in children with cerebral palsy in Hanoi, providing children with a surgical treatment option with successful outcomes.

Re-emergence of foot-and-mouth disease in the Republic of Korea caused by the O/ME-SA/Ind-2001e lineage.

The O/ME-SA/Ind-2001e foot-and-mouth disease virus (FMDV) lineage is a pandemic strain that has recently become dominant within East and Southeast Asia. During May 2023, this viral lineage spread to the Republic of Korea, where 11 outbreaks were detected on cattle and goat farms located in Cheongju and Jeungpyeong. Infected animals displayed typical FMD signs including vesicular lesions with drooling and anorexia. Molecular diagnostic testing and genetic analysis (VP1 sequencing) showed that the causative FMDVs belonged to the O/ME-SA/Ind-2001e lineage and shared the closest nucleotide identity (97.95-99.21%) to viruses that have been collected from Mongolia and South-East Asian countries. Phylogenetic analyses showed that these sequences were distinct to those collected from the previous Korean O/ME-SA/Ind-2001e lineage outbreaks in 2019, demonstrating that these cases are due to a new incursion of the virus into the country. Prompt implementation of emergency vaccination using antigenically matched serotype O vaccines (r1 value: 0.74-0.93), together with intensive active surveillance on farms surrounding the infected premises has successfully prevented further spread of FMD. These recent FMD outbreaks reinforce the importance of research to understand the risks associated with transboundary pathways in the region, in order to reduce the possibility of a further reintroduction of FMD into the Republic of Korea.

Molecular Epidemiology of Foot-and-Mouth Disease Viruses in the Emirate of Abu Dhabi, United Arab Emirates.

Foot-and-mouth disease (FMD) is an endemic disease in the United Arab Emirates (UAE) in both wild and domestic animals. Despite this, no systematic FMD outbreak investigation accompanied by molecular characterisation of FMD viruses (FMDVs) in small ruminants or cattle has been performed, and only a single report that describes sequences for FMDVs in wildlife from the Emirate has been published. In this study, FMD outbreaks that occurred in 2021 in five animal farms and one animal market in the Emirate of Abu Dhabi were investigated. Cases involved sheep, goats, and cattle, as well as Arabian oryx (Oryx leucoryx). Twelve samples were positive for FMDV via RT-qPCR, and four samples (Arabian oryx n = 1, goat n = 2, and sheep n = 1) were successfully genotyped using VP1 nucleotide sequencing. These sequences shared 88~98% identity and were classified within the serotype O, Middle East-South Asia topotype (O/ME-SA). Phylogenetic analysis revealed that the Arabian oryx isolate (UAE/2/2021) belonged to the PanAsia-2 lineage, the ANT-10 sublineage, and was closely related to the FMDVs recently detected in neighbouring countries. The FMDV isolates from goats (UAE/10/2021 and UAE/11/2021) and from sheep (UAE/14/2021) formed a monophyletic cluster within the SA-2018 lineage that contained viruses from Bangladesh, India, and Sri Lanka. This is the first study describing the circulation of the FMDV O/ME-SA/SA-2018 sublineage in the UAE. These data shed light on the epidemiology of FMD in the UAE and motivate further systematic epidemiological studies and genomic sequencing to enhance the ongoing national animal health FMD control plan.

Detection of foot-and-mouth disease viruses from the A/AFRICA/G-I genotype in the Sultanate of Oman.

Rapid identification and characterization of circulating foot-and-mouth disease virus (FMDV) strains is crucial for effective disease control. In Oman, a few serological and molecular studies have been conducted to identify the strains of FMDV responsible for the outbreaks that have been occurring within the country. In this study, 13 oral epithelial tissue samples from cattle were collected from suspected cases of FMD in Ash Sharqiyah North, Al Batinah North, Dhofar and Ad Dhakhyilia governorates of Oman between 2018 and 2021. FMDV RNA was detected in all samples by real-time RT-PCR and viruses were isolated after one- or two-blind passages in the porcine Instituto Biologico-Rim Suino-2 cell line. Antigen capture ELISA characterized all isolates as serotype A and VP1 phylogenetic analysis placed all sequences within a single clade of the G-I genotype within the A/AFRICA topotype. These sequences shared the closest nucleotide identities to viruses circulating in Bahrain in 2021 (93.5% to 99.5%) and Kenya in 2017 (93.4% to 99.1%). To the best of our knowledge, this is the first time that A/AFRICA/G-I viruses have been detected in Oman. Together with the closely related viruses detected recently in Bahrain, these findings reinforce the importance of deploying effective quarantine control measures to minimize the risks of transboundary transmission of FMD associated with the importation of cattle from East Africa.

Application of the Nagoya Protocol to veterinary pathogens: concerns for the control of foot-and-mouth disease.

The Nagoya Protocol is an international agreement adopted in 2010 (and entered into force in 2014) which governs access to genetic resources and the fair and equitable sharing of benefits from their utilisation. The agreement aims to prevent misappropriation of genetic resources and, through benefit sharing, create incentives for the conservation and sustainable use of biological diversity. While the equitable sharing of the benefits arising from the utilisation of genetic resources is a widely accepted concept, the way in which the provisions of the Nagoya Protocol are currently being implemented through national access and benefit-sharing legislation places significant logistical challenges on the control of transboundary livestock diseases such as foot-and-mouth disease (FMD). Delays to access FMD virus isolates from the field disrupt the production of new FMD vaccines and other tailored tools for research, surveillance and outbreak control. These concerns were raised within the FMD Reference Laboratory Network and were explored at a recent multistakeholder meeting hosted by the European Commission for the Control of FMD. The aim of this paper is to promote wider awareness of the Nagoya Protocol, and to highlight its impacts on the regular exchange and utilisation of biological materials collected from clinical cases which underpin FMD research activities, and work to develop new epidemiologically relevant vaccines and other diagnostic tools to control the disease.

The Immunogenicity of a Foot-and-Mouth Disease Virus Serotype O Vaccine in Commercial and Subsistence Cattle Herds in Zambia.

The recent introduction of foot-and-mouth disease (FMD) virus serotype O (O/EA-2 topotype) in Southern Africa has changed the epidemiology of the disease and vaccine requirements of the region. Commercial and subsistence cattle herds in Zambia were vaccinated with an FMD virus serotype O Manisa vaccine according to a double- or single-dose vaccination schedule. Heterologous antibody responses induced by this vaccine against a representative O/EA-2 virus from Zambia were determined. Virus neutralisation tests (VNTs) showed double-dosed cattle had a mean reciprocal log virus neutralisation titre of 2.02 (standard error [SE] = 0.16, n = 9) for commercial herds and 1.65 (SE = 0.17, n = 5) for subsistence herds 56 days after the first vaccination (dpv). Significantly lower mean titres were observed for single-dosed commercial herds (0.90, SE = 0.08, n = 9) and subsistence herds (1.15, SE = 0.18, n = 3) 56 dpv. A comparison of these results and those generated by solid-phase competitive ELISA (SPCE) tests showed a statistically significant positive correlation by Cohen's kappa coefficient. Therefore, SPCE might be used in assessing the immunogenicity of vaccines in place of VNT. Furthermore, for this vaccine and field strain, a vaccination regime employing a two-dose primary course and revaccination after 4-6 months is likely to be appropriate.

Maternally Derived Antibodies to Foot-and-Mouth Disease Virus Modulate the Antigenic Specificity of Humoral Responses in Vaccinated Cattle.

Vaccination is widely used to control foot-and-mouth disease (FMD), but maternal antibodies may interfere with the response to vaccination in calves. This study, conducted on a regularly vaccinated Malaysian dairy farm, aimed to optimise the vaccination regime by measuring the in vitro neutralising virus antibody responses of 51 calves before and after vaccination with a one or two dose vaccination regime starting at 2-7 months old. The presence of maternal antibodies was associated with poor post-vaccination antibody responses after a single dose of vaccine in calves less than 6 months old. However, a second dose of vaccine given three weeks later, improved the antibody responses in all ages of calves. This confirms the view that in regularly vaccinated farms, some combination of delay and revaccination is needed to achieve effective immunization of calves. Sera from cows and pre-vaccinated calves neutralised homologous serotype A vaccine virus more strongly than a heterologous serotype A field virus, but this pattern was reversed in some calves after vaccination. The strength of heterologous responses in calves 49 days after first vaccination correlated to the amount of transferred maternal antibody, suggesting that pre-existing antibodies could have modulated the specificity of these active antibody responses. If confirmed, such an effect by pre-existing antibodies could have wider implications for broadening the coverage of FMD vaccine responses.

Establishing a molecular toolbox of lineage-specific real-time RT-PCR assays for the characterization of foot-and-mouth disease viruses circulating in Asia.

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. This molecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability.

Comparison of vaccination schedules for foot-and-mouth disease among cattle and sheep in Mongolia.

Vaccines are a critical tool for the control strategy for foot-and-mouth disease (FMD) in Mongolia where sporadic outbreaks regularly occur. A two-dose primary vaccination course is recommended for most commercial vaccines though this can be logistically challenging to deliver among nomadic pastoralist systems which predominate in the country. Although there is evidence that very high potency vaccines can provide prolonged duration of immunity, this has not been demonstrated under field conditions using commercially available vaccines. This study compared neutralizing titres to a O/ME-SA/Panasia strain over a 6-month period following either a two-dose primary course or a single double-dose vaccination among Mongolian sheep and cattle using a 6.0 PD50 vaccine. Titers were not significantly different between groups except in sheep at six-months post vaccination when the single double-dose group had significantly lower titers. These results indicate the single double-dose regimen may be a cost-effective approach for vaccination campaigns supporting FMD control in Mongolia.

Correction: King et al. Establishing an In Vitro System to Assess How Specific Antibodies Drive the Evolution of Foot-and-Mouth Disease Virus. Viruses 2022, 14, 1820.

In the original publication [...].

Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-ß production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.

Genome Sequence of a Foot-and-Mouth Disease Virus Detected in Indonesia in 2022.

During 2022, outbreaks of foot-and-mouth disease (FMD) were reported across the islands of Indonesia, a country that had previously maintained an FMD-free (without vaccination) status since 1990. This report describes the near-complete genome sequence of a representative FMD virus collected from these cases belonging to the O/ME-SA/Ind-2001e lineage.

Subsurface mobility of land applied greenhouse nutrient feed water and environmental implications.

Greenhouse nutrient feedwater (GNF) discharge is considered a potential contributor to eutrophication issues in Lake Erie, Ontario. Land application of GNF is an accepted legislated management response to mitigate the impact of such nutrient loads. To assess the potential environmental impacts of this management practice, field infiltration experiments were conducted at four different greenhouse operations near Leamington, Ontario. Over a three-year study, GNF was applied on agricultural land adjacent to the greenhouse operations in the fall during the first year, and along with a bromide tracer in the summer and fall in Years 2 and 3, respectively. The GNF was applied at the maximum allowable rates as defined in legislation. Chemical constituents (nutrients, metals and the conservative tracer bromide) were monitored within the soil profile matrix and pore water above the water table. The results showed that, even with the GNF being applied at the highest permissible rates, the species of interest remained within the unsaturated soil zone at low concentrations over three to six months sampling intervals. The bromide tracer test demonstrated that highly mobile species could move through permeable soils to the water table depth in a potential worst-case application scenario. However, considering the low initial concentrations, long vadose zone residence time and the low mass flux, it would appear that land application of GNF, when applied in accordance with Ontario's Regulations, is a feasible and environmentally reasonable treatment option for managing GNF.

Predicting cross-protection against foot-and-mouth disease virus strains by serology after vaccination.

Serology is widely used to predict whether vaccinated individuals and populations will be protected against infectious diseases, including foot-and-mouth disease (FMD), which affects cloven-hoofed animals. Neutralising antibody titres to FMD challenge viruses correlate to protection against FMD, for vaccinated cattle that are infected with the same strain as in the vaccine (homologous protection). Similar relationships exist for cross-strain protection between different vaccine and challenge viruses, although much less data are available for these heterologous studies. Poor inter-laboratory reproducibility of the virus neutralisation test (VNT) also hampers comparisons between studies. Therefore, day-of-challenge sera (n = 180) were assembled from 13 previous FMD cross-protection experiments for serotypes O (n = 2), A (n = 10), and SAT 2 (n = 1). These were tested by VNT against the challenge viruses at the FMD FAO World Reference Laboratory (WRLFMD) and the titres were compared to challenge outcomes (protected or not). This dataset was combined with equivalent serology and protection data for 61 sera from four cross-protection experiments carried out at WRLFMD for serotypes O (n = 2), A (n = 1), and Asia 1 (n = 1). VNT results and protection outcomes were also analysed for a serotype O cross-protection experiment involving 39 cattle, where the sera were not available for retesting at WRLFMD. Three categories of association between heterologous neutralising antibody titre and heterologous protection were found (Group 1-3). The log10 reciprocal titres associated on average with 75% protection (with 95% credible limits) were: Group 1: 2.46 (2.11-2.97); Group 2: 1.67 (1.49-1.92); Group 3: 1.17 (1.06-1.30). Further cross-protection data are needed to understand the factors that underpin this variability and to develop more robust antibody thresholds. Establishing cut-off serological titres that can be used to score the adequacy of vaccine-induced immunity will facilitate the monitoring and thereby the performance of FMD vaccination in the field.

Establishing an In Vitro System to Assess How Specific Antibodies Drive the Evolution of Foot-and-Mouth Disease Virus.

Viruses can evolve to respond to immune pressures conferred by specific antibodies generated after vaccination and/or infection. In this study, an in vitro system was developed to investigate the impact of serum-neutralising antibodies upon the evolution of a foot-and-mouth disease virus (FMDV) isolate. The presence of sub-neutralising dilutions of specific antisera delayed the onset of virus-induced cytopathic effect (CPE) by up to 44 h compared to the untreated control cultures. Continued virus passage with sub-neutralising dilutions of these sera resulted in a decrease in time to complete CPE, suggesting that FMDV in these cultures adapted to escape immune pressure. These phenotypic changes were associated with three separate consensus-level non-synonymous mutations that accrued in the viral RNA-encoding amino acids at positions VP266, VP280 and VP1155, corresponding to known epitope sites. High-throughput sequencing also identified further nucleotide substitutions within the regions encoding the leader (Lpro), VP4, VP2 and VP3 proteins. While association of the later mutations with the adaptation to immune pressure must be further verified, these results highlight the multiple routes by which FMDV populations can escape neutralising antibodies and support the application of a simple in vitro approach to assess the impact of the humoral immune system on the evolution of FMDV and potentially other viruses.

Cross-Serotype Reactivity of ELISAs Used to Detect Antibodies to the Structural Proteins of Foot-and-Mouth Disease Virus.

Antibodies to the foot-and-mouth disease virus (FMDV) capsid induced by infection or vaccination can provide serotype-specific protection and be measured using virus neutralization tests and viral structural-protein (SP-)ELISAs. Separate tests are needed for each serotype, but cross-serotype reactions complicate serotyping. In this study, inter-serotypic responses were quantified for five SP-ELISA formats by testing 294 monovalent mainly bovine sera collected following infection, vaccination, or vaccination and infection with one of five serotypes of FMDV. Over half of the samples, representing all three immunization categories, scored positive for at least one heterologous serotype and some scored positive for all serotypes tested. A comparative approach to identifying the strongest reaction amongst serotypes O, A and Asia 1 improved the accuracy of serotyping to 73-100% depending on the serotype and test system, but this method will be undermined where animals have been infected and/or vaccinated with multiple FMDV serotypes. Preliminary studies with stabilized recombinant capsid antigens of serotypes O and A that do not expose internal epitopes showed reduced cross-reactivity, supporting the hypothesis that capsid integrity can affect the serotype-specificity of the SP-ELISAs. The residual cross-reactivity associated with capsid surface epitopes was consistent with the evidence of cross-serotype virus neutralization.

The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication, but essential for the production of infectious virus.

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5' untranslated region (5' UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions.

Combining a Universal Capture Ligand and Pan-Serotype Monoclonal Antibody to Develop a Pan-Serotype Lateral Flow Strip Test for Foot-and-Mouth Disease Virus Detection.

Foot-and-mouth disease virus (FMDV) causes FMD, a highly contagious disease of cloven-hoofed animals including cattle, goats, pigs and sheep. Rapid detection of FMDV is critical to limit the devastating economic losses due to FMD. Current laboratory methods for FMDV detection such as virus isolation, real-time reverse transcription PCR and antigen detection enzyme-linked immunosorbent assay (AgELISA) are labor-intensive, requiring trained personnel and specialized equipment. We present the development and validation of a pan-serotype lateral flow strip test (LFST) that uses recombinant bovine integrin αvß6 as a universal capture ligand and a pan-serotype monoclonal antibody (mAb) to detect FMDV. The LFST detected all seven FMDV serotypes, where the diagnostic sensitivity was comparable to the AgELISA, and the diagnostic specificity was 100% without cross-reactivity to other viruses causing vesicular disease in livestock. This rapid test will be useful for on-site FMDV detection, as well as in laboratories in endemic countries where laboratory resources are limited.