SAT2 Viruses Research Articles

Preliminary validation of a single-spot version of a solid-phase competition ELISA for the detection of southern African territories foot-and-mouth disease serotype exposure in goats

The objective of this study was to perform a preliminary validation of a solid-phase competition ELISA (SPCE) in goats exposed to foot-and-mouth disease virus (FMDV) southern African territories (SAT) serotypes through vaccination or experimental infection. Thirty-nine goats were vaccinated with a FMDV vaccine and 37 subsequently challenged with a SAT1 virus serotype. Blood was collected every 7 days until termination at 14 days post-challenge. Single-spot SAT1 virus serotype SPCE (ss-SPCE) was performed in duplicate at two time points and a half-titration version was performed after a variable time of long-term storage. Coefficient of variation (CV) was calculated and accuracy of the ss-SPCE was estimated relative to a half-titration SPCE log10 titer of 1.6 using mixed-effect logistic regression. Additionally, sensitivity and specificity were estimated based on serological results 14-days post-challenge and at study enrolment, respectively. Three hundred and forty-two serum samples were tested in duplicate on two non-consecutive days. The median (interquartile range (IQR)) CV for the ss-SPCE for SAT1 was 2.1% (0.5, 14.3%) and 2.5% (0.6, 12.8%) for the two testing days, respectively. Median (IQR) inter-assay (different day) CV was 10.6% (2.5, 42.5%). Specificity and sensitivity of the ss-SPCE relative to the log10 titer using a 70% percentage inhibition positive threshold were 83.4% (95% confidence interval, 77.7–87.9) and 95.8% (90.7–98.2), respectively. Specificity was estimated as 100% (92.6, 100) and sensitivity as 97.3% (87.4, 99.9) when only considering serum tested at the beginning and end of the study, respectively. The SAT1 ss-SPCE is repeatable and accurate for determining FMDV serological status in goats.

Development of reverse-transcriptase, real-time PCR assays to distinguish the Southern African Territories (SAT) serotypes 1 and 3 and topotype VII of SAT2 of Foot-and-Mouth Disease Virus.

Foot-and-Mouth Disease Virus (FMDV), the causative agent of Foot-and-Mouth Disease, is a highly feared, economically devastating transboundary pathogen. This is due to the virus' extremely contagious nature and its ability to utilize multiple transmission routes. As such, rapid and accurate diagnostic testing is imperative to the control of FMD. Identification of the FMDV serotype is necessary as it provides the foundation for appropriate vaccine selection and aids in outbreak source tracing. With the vast genetic diversity, there is a desperate need to be able to characterize FMDV without relying on prior knowledge of viral serotypes. In this study, the Neptune bioinformatics tool was used to identify genetic signatures specific to each Southern African Territories (SAT) 1, 2 and 3 genomes but exclusionary to the other circulating FMDV serotypes (A, O, Asia1, and the heterologous SAT1, SAT2 and/or SAT3). Identification of these unique genomic regions allowed the design of TaqMan-based real-time reverse transcriptase PCR (rRT-PCR) primer/probe sets for SAT1, SAT2 and SAT3 viruses. These assays were optimized using prototypic FMDV cell culture isolates using the same reagents and thermocycling conditions as the FMDV pan-serotype 3D rRT-PCR assay. Cross-reactivity was evaluated in tandem with the FMDV pan-serotype 3D rRT-PCR utilizing representative strains from FMDV serotypes A, O, Asia1, SAT1, SAT2 and SAT3. The SAT1, SAT2, and SAT3 primer/probe sets were specific for the homologous serotype and exclusionary to all others. SAT1 and SAT3 primer/probe sets were able to detect several topotypes, whereas the SAT2 assay was revealed to be specific for topotype VII. The SAT2 topotype VII specificity was possibly due to the use of sequence data deposited post-2011to design the rRT-PCR primers and probes. Each assay was tested against a panel of 99 bovine tissue samples from Nigeria, where SAT2 topotype VII viruses were correctly identified and no cross-reactivity was exhibited by the SAT1 and 3 assays. These novel SAT1, SAT3 and SAT2 topotype VII rRT-PCR assays have the potential to detect and differentiate circulating FMD SAT viruses.

Diversity of SAT2 foot-and-mouth disease virus in Sudan: implication for diagnosis and control.

Like other East African countries, Sudan experienced circulation of more than one topotype of SAT2 foot-and-mouth disease virus (FMDV). In Sudan, topotype XIII of SAT2 virus was recorded in 1977 and 2008 and topotype VII in 2007, 2010, 2013, 2014 and 2017. This work evaluated the impact of such diversity on diagnosis and control. After one or three doses of a vaccine derived from a Sudanese SAT2 virus of topotype VII originated in 2010, heterologous neutralizing antibody titres with Sudanese SAT2 viruses in 2008 were ≤ 1.2 log 10, not consistent with likely protection. Simultaneously, homologous titres were 1.65 (after one dose) or 1.95 and 2.55 log10 (after 3 doses). When r1 values between the vaccine virus and the SAT2 viruses isolated in 2008, whilst topotype XIII was circulating, were derived, values (≈ 0.00) suggested similarly poor antigenic relationship and unlikely cross protection. Concurrently, SAT2 positive field sera from Sudan in 2016 were not unvaryingly identified by virus neutralization tests (VNT) employing SAT2 viruses from 2010 and 2008. Proportions of positive sera by SAT2 virus from 2010 were always higher than those by viruses from 2008; consistent with the more frequent and recent circulation of topotype VII prior to 2016. Proportions by SAT2 virus from 2010 were 0.68 (± 0.1) in one location (n = 72), 0.39 (± 0.1) in another one (n = 94) and 0.52 (± 0.1) in the whole test group (n = 166). Corresponding values by viruses of 2008 were 0.53 (± 0.1), 0.27 (± 0.1) and 0.38 (± 0.1). In the whole test group, differences were statistically significant (p = .02339). Like post-vaccination sera, field sera (natural immunity) showed no considerable cross neutralization between topotype VII and presumably XIII; almost 45% (43/96) of SAT2 positive field sera were positive to one topotype but not to the other. Experimental and surveillance findings emphasized the implication of SAT2 diversity in Sudan. It is concluded that it is difficult to control SAT2 infection in Sudan using a monovalent vaccine. Beside a prophylactic vaccine from topotype VII, stockpiling of antigens from topotype XIII and enhanced virological surveillance with rapid genotyping and matching studies are necessary approaches. When more frequent circulation of more than one SAT2 topotype occurs, retrospective diagnosis by serological surveys could be problematic or imprecise.

A Five-Year Retrospective Study of Foot-and-Mouth Disease Outbreaks in Southern Africa, 2014 to 2018.

Foot-and-mouth disease (FMD) virus (FMDv), like other ribonucleic acid (RNA) genome viruses, has a tendency to mutate rapidly. As such, available vaccines may not confer enough cross-protection against incursion of new lineages and sublineages. This paper is a retrospective study to determine the topotypes/lineages that caused previous FMD outbreaks in 6 southern African countries and the efficacy of the current vaccines to protect cattle against them. A total of 453 bovine epithelial tissue samples from 33 FMD outbreaks that occurred in these countries from 2014 to 2018 were investigated for the presence of FMDv. The genetic diversity of the identified Southern African Type (SAT)-FMD viruses was determined by comparing sequences from outbreaks and historical prototype sequences. Of the 453 samples investigated, 176 were positive for four FMDv serotypes. Out of the 176 FMD positive cases there were 105 SAT2 samples, 32 SAT1 samples, 21 SAT3 samples, and 18 serotype O samples. Phylogenetic analysis grouped the SATs VP1 gene sequences into previously observed topotypes in southern Africa. SAT1 viruses were from topotypes I and III, SAT2 viruses belonged to topotypes I, II, III, and IV, and SAT3 viruses were of topotypes I and II. Vaccine matching studies on the field FMDv isolates produced r1-values greater than or equal to 0.3 for the three SAT serotypes. This suggests that there is no significant antigenic difference between current SAT FMD vaccine strains and the circulating SAT serotypes. Therefore, the vaccines are still fit-purpose for the control FMD in the region. The study did not identify incursion of any new lineages/topotypes of FMD into the sampled southern African countries.

Isolation and Molecular Characterization of Circulating Foot and Mouth Disease Virus in Egypt During 2018-2020

Foot and Mouth Disease (FMD) is a contagious viral disease with high economic losses and primary animal health concerns. FMDV type SAT2 is endemic in Egypt since 2012. This work aimed to characterize the circulating FMDV SAT2 strains genetically in Egypt from 2018 to 2020. A total of 209 vesicular fluids and tongue epithelium were collected from infected cattle and buffaloes in Sharkia, Ismailia, and Dakhlia provinces. All samples were examined by real-time PCR and conventional PCR for FMDV using pan- serotype and serotype-specific primers targeting the VP1 region. Out of 209 samples, 45 infected animals were positive for FMDV SAT2 virus, 29 cattle (21.5%), and 16 buffaloes (13.6%). No FMDV serotype A or O were detected. The highest prevalence of FMDV SAT2 was observed in Sharkia province with a percentage of 10% followed by Ismailia and Dakhlia with a rate of 2.9 and 0.9%, respectively. Three FMDV SAT2 positive samples represented as Sharkia 2018 and Sharkia 2019 and Ismailia 2020 were selected for sequencing and phylogenetic analysis of VP1. Sequencing and phylogenetic analysis of VP1of the three Egyptian strains demonstrated that these strains are closely related to other Egyptian strains in gene bank as Alex 2018 (MK4933346), Ismailia 2018 (MK4933341), and Menofia 2018 (MT199283) with homology ranged from 95.8 to 98.2%. Phylogenetic tree of FMDV SAT2 showed clustering of Sharkia 2018, Sharkia 2019, and Ismailia 2020 with Libya 2012 topotype VII with three amino acid substitutions at the site 24, 28, and 52.

High selectivity detection of FMDV- SAT-2 using a newly-developed electrochemical nanosensors

Foot-and-mouth disease virus serotype South-Africa territories-2 (FMDV-SAT-2) is the most fastidious known type in Aphthovirus which is subsequently reflected in the diagnosis regime. Rapid and early diagnostic actions are usually taken in response to the FMDV outbreak to prevent the dramatic spread of the disease. Virus imprinted sensor (VIP sensor) is gathering huge attention for the selective detection of pathogens. Thus, the whole virus particles of SAT-2 together with an electropolymerized film of poly(o-phenylenediamine) (PoPD) on gold-copper modified screen-printed electrode were applied to fabricate SAT-2-virus imprinted polymer (SAT-2-VIP). The SAT-2-VIPs were fully characterized using cyclic voltammetry (CV), linear sweep voltammetry (LSV), Atomic force microscopy (AFM), Scanning electron microscope (SEM), and Fourier transform Infra-Red (FTIR) spectroscopy. Excellent selective binding affinity towards the targeted virus particle was achieved with limits of detection and quantification of 0.1 ng/mL and 0.4 ng/mL, respectively. In terms of viral interference, the sensor did not show cross-reactivity towards other animal viruses including FMDV serotype A, O, or even SAT-2 subtype Libya and the un-related virus Lumpy skin disease virus (LSDV). This high selectivity provides a sensible platform with 70 folds more sensitivity than the reference RT-PCR as revealed from the application of SAT-2-VIP sensor for rapid analysis of clinical samples with no need for treatment or equipped labs. Thus, as diagnostic and surveillance technologies, on-site point of care diagnostics for SAT-2 virus are supported.

Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses.

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the βD-βE loop and 110-112 in the βF-βG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.

Diagnostic and Epitope Mapping Potential of Single-Chain Antibody Fragments Against Foot-and-Mouth Disease Virus Serotypes A, SAT1, and SAT3.

Foot-and-mouth disease (FMD) affects cloven-hoofed domestic and wildlife animals and an outbreak can cause severe losses in milk production, reduction in meat production and death amongst young animals. Several parts of Asia, most of Africa, and the Middle East remain endemic, thus emphasis on improved FMD vaccines, diagnostic assays, and control measures are key research areas. FMD virus (FMDV) populations are quasispecies, which pose serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent neutralizing epitopes to elicit an adequate immune response. Further investigation of the residues that comprise the antigenic determinants of the virus will allow the identification of mutations in outbreak strains that potentially lessen the efficacy of a vaccine. Additionally, of utmost importance in endemic regions, is the accurate diagnosis of FMDV infection for the control and eradication of the disease. To this end, a phage display library was explored to identify FMDV epitopes for recombinant vaccines and for the generation of reagents for improved diagnostic FMD enzyme-linked immunosorbent assays (ELISAs). A naïve semi-synthetic chicken single chain variable fragment (scFv) phage display library i.e., the Nkuku® library was used for bio-panning against FMD Southern-African Territories (SAT) 1, SAT3, and serotype A viruses. Biopanning yielded one unique scFv against SAT1, two for SAT3, and nine for A22. SAT1 and SAT3 specific scFvs were exploited as capturing and detecting reagents to develop an improved diagnostic ELISA for FMDV. The SAT1 soluble scFv showed potential as a detecting reagent in the liquid phase blocking ELISA (LPBE) as it reacted specifically with a panel of SAT1 viruses, albeit with different ELISA absorbance signals. The SAT1svFv1 had little or no change on its paratope when coated on polystyrene plates whilst the SAT3scFv's paratope may have changed. SAT1 and SAT3 soluble scFvs did not neutralize the SAT1 and SAT3 viruses; however, three of the nine A22 binders i.e., A22scFv1, A22scFv2, and A22scFv8 were able to neutralize A22 virus. Following the generation of virus escape mutants through successive virus passage under scFv pressure, FMDV epitopes were postulated i.e., RGD+3 and +4 positions respectively, proving the epitope mapping potential of scFvs.

The role of African buffalo in the epidemiology of foot-and-mouth disease in sympatric cattle and buffalo populations in Kenya.

Quantitative knowledge on the contribution of African buffalo to the epidemiology of foot-and-mouth disease virus (FMDV) in East Africa is lacking, and this information is essential for the design of control programs in the region. The objective of this study was to investigate the epidemiology of FMDV in buffalo, including the role of buffalo in the circulation of FMDV in livestock populations. We collected blood and oropharyngeal fluids from 92 wild buffalo and 98 sympatric cattle in central Kenya and sequenced the virus' VP1 coding region. We show that FMDV has a high seroprevalence in buffalo (~77%) and targeted cattle (~93%). In addition, we recovered 80 FMDV sequences from buffalo, all of which were serotype SAT1 and SAT2, and four serotype O and A sequences from sympatric cattle. Notably, six individual buffalo were co-infected with both SAT1 and SAT2. Amongst sympatric buffalo and cattle, the fact that no SAT1 or 2 sequences were found in cattle suggests that transmission of FMDV from buffalo to sympatric cattle is rare. Similarly, there was no evidence that serotype O and A sequences found in cattle were transmitted to buffalo. However, viruses from FMDV outbreaks in cattle elsewhere in Kenya were closely related to SAT1 and SAT2 viruses found in buffalo in this study, suggesting that FMDV in cattle and buffalo do not constitute independently evolving populations. We also show that fine-scale geographic features, such as rivers, influence the circulation of FMDV in buffalo and that social segregation amongst sympatric herds may limit between-herd transmission. These results significantly advance our understanding of the ecology and molecular epidemiology of FMDV at wildlife-livestock interfaces in East Africa and will help to inform the design of control and surveillance strategies for this disease in the region.

Efficacy of a foot-and-mouth disease vaccine against a heterologous SAT1 virus challenge in goats

Goats are susceptible to infection with foot-and-mouth disease virus (FMDV), but their role in the epidemiology of the disease and response to vaccination is poorly understood. In southern Africa, FMDV serotypes Southern African Territories (SAT) 1, 2 and 3 are known to be endemic. In this study, we evaluated the efficacy of a pentavalent FMD vaccine in goats against heterologous challenge with a pool of field SAT1 FMDV. Forty FMD sero-negative goats (6–12 months of age) of mixed sexes were randomly allocated to one of five treatment groups: full cattle dose (2 ml), 1/3rd (0.67 ml), 1/6th (0.33 ml), 1/12th (0.16 ml) or unvaccinated placebo control. Goats were vaccinated with an inactivated pentavalent FMD vaccine containing serotypes SAT1, SAT2 and SAT3 on day 0 and revaccinated at day 20 post vaccination. Thereafter, thirty-four goats were challenged by tongue inoculation at day 41 post-vaccination using 104.57 50% tissue culture infective dose (TCID50) FMDV SAT1 pool. Animals were examined daily and clinical signs were scored. Rectal temperatures were measured daily, with temperatures ≥40 °C defined as fever. Clinical specimens (nasal, oral and rectal swabs) were collected on days 0, 2, 4 and 6 post challenge. Viral shedding was determined using reverse-transcriptase real-time PCR. None of the goats vaccinated with the full cattle dose developed secondary lesions. All vaccinated groups had lower temperatures compared to the unvaccinated controls (P < 0.001). Based on RT-PCR results, goats in the unvaccinated control group shed more virus compared to all groups except for 1/12th (P < 0.05), while goats in the full dose group shed less virus than goats in the 1/12th and the unvaccinated control group (P < 0.05). The results suggest that the 1/3rd (0.67 ml) dose of the vaccine is sufficient to reduce viral shedding after heterologous challenge with a FMDV SAT1 pool.

Pathogenesis, biophysical stability and phenotypic variance of SAT2 foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals, which severely decreases livestock productivity. FMD virus (FMDV), the causative agent, initiates infection by interaction with integrin cellular receptors on pharyngeal epithelium cells, causing clinical signs one to four days after transmission to a susceptible host. However, some Southern African Territories (SAT) viruses have been reported to cause mild or subclinical infections that may go undiagnosed in field conditions and are likely to be more common than previously expected. The studies presented here demonstrate that not all SAT2 viruses are equally virulent in cattle. The two SAT2 viruses, ZIM/5/83 and ZIM/7/83, were both highly attenuated in cattle, as evidenced by the mild clinical signs observed after needle challenge, while two incongruent SAT2 viruses showed significantly different clinical signs in challenged cattle. We then explored the ability of the SAT2 viruses to infect different cell types with defined receptors that are utilised by FMDV and found differences in their ability to lyse cells in culture and to compete in a controlled cell culture environment. The population sequence variation between ZIM/5/83 and ZIM/7/83 revealed multiple sites of single nucleotide variants of low frequency between the predominant virus populations, as could be expected from the genome of an RNA virus. An assessment of the biophysical stability of SAT2 virions during acidification indicated that the SAT2 virus EGY/09/12 was more resilient to acidification than the ZIM/5/83 and ZIM/7/83 viruses; however, whether this difference relates to differences in virulence in vivo is unclear. This study is a consolidated view of the key findings of SAT2 viruses studied over a 14-year period involving many different experiments.

Characterization of SAT2 foot-and-mouth disease 2013/2014 outbreak viruses at the wildlife-livestock interface in South Africa.

The Southern African Territories (SAT)-type foot-and-mouth disease viruses (FMDV) are endemic to the greater Kruger National Park (KNP) area in South Africa, where they are maintained through persistent infections in African buffalo. The occurrence of FMDV within the Greater KNP area constitutes a continual threat to the livestock industry. To expand on knowledge of FMDV diversity, the genetic and antigenic relatedness of SAT2-type viruses isolated from cattle during a FMD outbreak in Mpumalanga Province in 2013 and 2014 were investigated. Cattle from twelve diptanks tested positive on polymerase chain reaction (PCR), and molecular epidemiological relationships of the viruses were determined by VP1 sequencing. Phylogenetic analysis of the SAT2 viruses from the FMD outbreak in Mpumalanga in 2013/2014 revealed their genetic relatedness to other SAT2 isolates from topotype I (South Africa, Zimbabwe and Mozambique), albeit genetically distinct from previous South African outbreak viruses (2011 and 2012) from the same topotype. The fifteen SAT2 field isolates clustered into a novel genotype with ≥98.7% nucleotide identity. High neutralization antibody titres were observed for four 2013/2014 outbreak viruses tested against the SAT2 reference antisera representative of viruses isolated from cattle and buffalo from South Africa (topotype I) and Zimbabwe (topotype II). Comparison of the antigenic relationship (r1 values) of the outbreak viruses with reference antisera indicated a good vaccine match with 90% of r1 values>0.3. The r1 values for the 2013/2014 outbreak viruses were 0.4 and above for the three South African vaccine/reference strains. These results confirm the presence of genetic and antigenic variability in SAT2 viruses and suggest the emergence of new variants at the wildlife-livestock interface in South Africa. Continuous characterization of field viruses should be performed to identify new virus strains as epidemiological surveillance to improve vaccination efforts.

Impact of foot-and-mouth-disease on goat behaviour after experimental infection with serotype SAT1 virus

Infectious diseases and parasitic infestations can cause a set of non-specific clinical signs, such as increased body temperature and resting, and a decrease in food intake. These physiological and behavioural changes have an adaptive function facilitating defences against the pathogen and to support immune functions. These so-called’ sickness behaviours’ can also be used as an early detection tool for disease. Foot-and-mouth disease (FMD) still causes great economic losses in endemic countries, especially to smallholder farmers. The aim of this study was to determine if behavioural changes in goats can be used as an early indicator of FMD virus (FMDV) infection. The efficacy of a Southern African Territories (SAT) FMD vaccine was studied on forty South African indigenous goats. Changes in daily activities (resting, feeding, walking), as well as social behaviours (social resting, social feeding, dominance behaviours) were recorded and then compared over time and between clinically affected and unaffected goats. Pedometers were used to estimate average daily steps and to compare between groups of study animals.Eleven goats developed clinical signs of FMD, as well as non-FMD related sicknesses during the course of the study. Overall walking and resting behaviours were not significantly affected by the presence of FMD related clinical signs (p > 0.05). However, during the time of FMDV infection, social resting increased significantly (p < 0.001). Although goats developed FMD lesions on lips and tongues, percentage of time feeding was not affected (p = 0.762), suggesting that the study goats did not perceive the oral lesions as an important disturbance. Similarly, the number of steps did not consistently decrease in the presence of FMD-associated foot lesions. When affected by non-FMD related sicknesses, animals did not have an overall reduction in the time spent feeding (p = 0.867). However, goats affected with non-FMD conditions reduced the amount of social feeding (p = 0.002), potentially avoiding energetically costly competition at the feeding points. Overall, goats affected with FMD did not show more sickness behaviour, suggesting that FMDV infection in goats might not lead to obvious and therefore, easily detectable behavioural changes. This might have implications for farmers and animal health personnel, as individual goats infected with FMDV might be undetected within a flock due to the absence of obvious sickness behaviours, and the virus can therefore be spread more easily between herds through animal movements.

Evaluation of novel inactivated vaccines for the SAT 1, SAT 2 and SAT 3 serotypes of foot-and-mouth disease in pigs

BackgroundThe foot-and-mouth disease (FMD) virus is classified into seven serotypes, of which the South African types have South African Territories (SAT)1, SAT2, and SAT3 that are prevalent in Africa. Especially SAT2 have spread to Arabian Peninsula and the Palestinian Autonomous Territories. Of these viruses, the incidence of SAT2 is the highest. It is important to prepare for the spread of the virus to other continents, even though most FMD viruses are bovine-derived. In particular, due to the high breeding density of pigs in Asia, more attention is usually paid to the immunity and protection of pigs than cattle. For this reason, this study investigated the immunity and protection of pigs against the SAT viruses.MethodsSpecific vaccines were developed for SAT1, SAT2, and SAT3 serotypes. These vaccine viruses were designed to be distinguished from the wild-type strain. An immunogenicity test was conducted using these vaccines in both cattle (n = 5/group) and pigs (n = 20/group).ResultsHigh virus-neutralizing titer of antibodies (> 1:100) was induced in only 2 weeks after the immunization of cattle with the individual vaccine for SAT1, SAT2 or SAT3, and a clear immune response was induced after the second immunization in pigs. When the vaccinated pigs (n = 4–5/group) were challenged by the homologous wild-type virus strain 4 weeks after immunization, all the pigs were protected from the challenge.ConclusionsThis study confirmed that these vaccines can be used against SAT1, SAT2, and SAT3 viruses in cattle and pigs. The vaccine strains developed in this study are expected to be used as vaccines that can protect against FMD in the event of a future FMD outbreak in pigs in consideration of the situation in Asia.

Persistent Infection of African Buffalo (Syncerus caffer) with Foot-and-Mouth Disease Virus: Limited Viral Evolution and No Evidence of Antibody Neutralization Escape.

African buffaloes (Syncerus caffer) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centers of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centers, probang samples (oropharyngeal scrapings), and tonsil swabs to determine if sufficient virus variation is generated during persistence for immune escape. Most sequencing reads of the VP1 coding region of the SAT1 virus inoculum clustered around 2 subpopulations differing by 22 single-nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions, depending on the day postinfection (dpi). There was a significant difference between the populations of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral populations in samples from individual animals, germinal centers, and epithelial tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to postmortem showed less within-sample variability of VP1 than that of tissue sample sequences analyzed at the same time. Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralize the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study, there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo.IMPORTANCE Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven-hoofed animals and is recognized as the most important constraint to international trade in animals and animal products. African buffaloes (Syncerus caffer) are efficient carriers of FMDV, and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low-frequency sequence variants over time, there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants.

A novel method for performing antigenic vaccine matching for foot-and-mouth disease in absence of the homologous virus

Foot-and-mouth-disease (FMD) is a highly contagious transboundary animal disease that has negative consequences on regional and international trade. Vaccination is an important approach for FMD control and an essential consideration is the degree of cross-protection conferred by the vaccine against currently circulating field viruses. The objective of this study was to evaluate a new vaccine matching technique that does not require knowledge concerning the homologous vaccine virus. As a proof of concept, the vaccine-match was assessed for 41 FMD field viruses isolated from southern Africa over a 25-year period.A diverse group of 20 SAT1 and 21 SAT2 FMDV isolates collected from cattle and wildlife during 1991–2015 were selected for this study. Virus neutralization tests were performed against two sets of pooled sera for each serotype: vaccinated cattle sera (4–16 weeks post-vaccination) and convalescent cattle sera (3 weeks post-experimental challenge). Novel r1-values were calculated as the ratio of the titre of the vaccinated sera to the titre for convalescent cattle sera. A validation r1-value was calculated based on an assumption concerning the true homologous vaccine virus. There was a strong positive correlation between r1-values for the novel and the validation methods for SAT1 viruses (Spearman’s rho = 0.84, P < 0.01) and a very strong correlation for SAT2 viruses (Spearman’s rho = 0.90, P < 0.01). In addition, there was moderate to good agreement between the novel and validation methods for both serotypes based on a r1-value cut-off of 0.3, which is presumed to represent a good vaccine-match. The agreement between methods using prevalence-adjusted and bias-adjusted kappa (PABAK) was 0.67 and 0.84 for SAT1 and SAT2 viruses, respectively.The new r1-value method provides a feasible, alternative vaccine matching approach that could benefit FMD control in southern Africa.

Phylogeographical and cross‐species transmission dynamics of SAT1 and SAT2 foot‐and‐mouth disease virus in Eastern Africa

Understanding the dynamics of foot-and-mouth disease virus (FMDV), an endemic and economically constraining disease, is critical in designing control programmes in Africa. This study investigates the evolutionary epidemiology of SAT1 and SAT2 FMDV in Eastern Africa, as well as between cattle and wild African buffalo. Bayesian phylodynamic models were used to analyse SAT1 and SAT2 VP1 gene segments collected between 1975 and 2016, focusing on the SAT1 and SAT2 viruses currently circulating in Eastern Africa. The root state posterior probabilities inferred from our analyses suggest Zimbabwe as the ancestral location for SAT1 currently circulating in Eastern Africa (p=0.67). For the SAT2 clade, Kenya is inferred to be the ancestral location for introduction of the virus into other countries in Eastern Africa (p=0.72). Salient (Bayes factor >10) viral dispersal routes were inferred from Tanzania to Kenya, and from Kenya to Uganda for SAT1 and SAT2, respectively. Results suggest that cattle are the source of the SAT1 and SAT2 clades currently circulating in Eastern Africa. In addition, our results suggest that the majority of SAT1 and SAT2 in livestock come from other livestock rather than wildlife, with limited evidence that buffalo serve as reservoirs for cattle. Insights from the present study highlight the role of cattle movements and anthropogenic activities in shaping the evolutionary history of SAT1 and SAT2 in Eastern Africa. While the results may be affected by inherent limitations of imperfect surveillance, our analysis elucidates the dynamics between host species in this region, which is key to guiding disease intervention activities.

Inherent biophysical stability of foot-and-mouth disease SAT1, SAT2 and SAT3 viruses

Foot-and-mouth disease (FMD) virus (FMDV) isolates show variation in their ability to withstand an increase in temperature. The FMDV is surprisingly thermolabile, even though this virus is probably subjected to a strong extracellular selective pressure by heat in hot climate regions where FMD is prevalent. The three SAT serotypes, with their particularly low biophysical stability also only yield vaccines of low protective capacity, even with multiple booster vaccinations. The aim of the study was to determine the inherent biophysical stability of field SAT isolates. To characterise the biophysical stability of 20 SAT viruses from Southern Africa, the thermofluor assay was used to monitor capsid dissociation by the release of the RNA genome under a range of temperature, pH and ionic conditions.The SAT2 and SAT3 viruses had a similar range of thermostability of 48–54 °C. However, the SAT1 viruses had a wider range of thermostability with an 8 °C difference but with many viruses being unstable at 43–46 °C. The thermostable A-serotype A24 control virus had the highest thermostability of 55 °C with some SAT2 and SAT3 viruses of similar thermostability. There was a 10 °C difference between the most unstable SAT virus (SAT1/TAN/2/99) and the highly stable A24 control virus. SAT1 viruses were generally more stable compared to SAT2 and SAT3 viruses at the pH range of 6.7–9.1.The effect of ionic buffers on capsid stability showed that SAT1 and SAT2 viruses had an increased stability of 2–9 °C and 2–6 °C, respectively, with the addition of 1 M NaCl. This is in contrast to the SAT3 viruses, which did not show improved stabilisation after addition of 1 M or 0.5 M NaCl buffers. Some buffers showed differing results dependent on the virus tested, highlighting the need to test SAT viruses with different solutions to establish the most stabilising option for storage of each virus. This study confirms for the first time that more stable SAT field viruses are present in the southern Africa region. This could facilitate the selection of the most stable circulating field strains, for adaptation to cultured BHK-21 cells or manipulation by reverse genetics and targeted mutation to produce improved vaccine master seed viruses.

Molecular Epidemiology of Foot-and-Mouth Disease Virus in the Context of Transboundary Animal Movement in the Far North Region of Cameroon.

Transboundary movement of animals is an important mechanism for foot-and-mouth disease virus (FMDV) spread in endemic regions, such as Cameroon. Several transboundary animal trade routes cross the Far North Region of Cameroon, and cattle moved on foot along these routes often come in contact with native (sedentary and transhumant) herds. The purpose of this study was to investigate the role of transboundary trade cattle in the epidemiology of FMDV in the Far North Region of Cameroon. A total of 582 oropharyngeal fluid (OPF) samples were collected from asymptomatic transboundary trade cattle at official border check points and 57 vesicle epithelial tissues were collected from clinically affected native cattle in the Far North Region of Cameroon during 2010–2014. Viral protein 1 (VP1) coding sequences were obtained from 6 OPF samples from transboundary cattle (4 serotype O, 2 serotype SAT2) and 19 epithelial tissue samples from native cattle (7 serotype O, 3 serotype SAT2, 9 serotype A). FMDV serotype O viruses belonged to two topotypes (East Africa-3 and West Africa), and phylogenetic analyses suggested a pattern of continuous transmission in the region. Serotype SAT2 viruses belonged to a single topotype (VII), and phylogenetic analysis suggested a pattern of repeated introductions of different SAT2 lineages in the region. Serotype A viruses belonged to topotype AFRICA/G-IV, and the pattern of transmission was unclear. Spearman rank correlation analysis of VP1 coding sequences obtained in this study from transboundary and native cattle showed a positive correlation between genetic distance and time for serotype O (ρ = 0.71, p = 0.003) and between genetic distance and geographic distance for serotype SAT2 (ρ = 0.54, p = 0.1). These data suggest that transboundary trade cattle participate in the transmission of FMDV in the Far North Region of Cameroon, however the dynamics and direction of transmission could not be determined in this study. Results of this study contribute to the understanding of transboundary FMDV epidemiology in Central Africa and will help to inform control programs in Cameroon and in the region.

Chimeric O1K foot-and-mouth disease virus with SAT2 outer capsid as an FMD vaccine candidate

Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth disease (FMD). FMD outbreaks have severe economic impact due to production losses and associated control measures. FMDV is found as seven distinct serotypes, but there are numerous subtypes within each serotype, and effective vaccines must match the subtypes circulating in the field. In addition, the O and Southern African Territories (SAT) serotypes, are relatively more thermolabile and their viral capsids readily dissociate into non-immunogenic pentameric subunits, which can compromise the effectiveness of FMD vaccines. Here we report the construction of a chimeric clone between the SAT2 and O serotypes, designed to have SAT2 antigenicity. Characterisation of the chimeric virus showed growth kinetics equal to that of the wild type SAT2 virus with better thermostability, attributable to changes in the VP4 structural protein. Sequence and structural analyses confirmed that no changes from SAT2 were present elsewhere in the capsid as a consequence of the VP4 changes. Following exposure to an elevated temperature the thermostable SAT2-O1K chimera induced higher neutralizing-antibody titres in comparison to wild type SAT2 virus.