Characterization of Foot-and-Mouth Disease Viruses in Zambia-Implications for the Epidemiology of the Disease in Southern Africa.

The livestock industry supports livelihood and nutritional security of at least 42% of people in the Southern African Development Community region. However, presence of animal diseases such as foot-and-mouth disease poses a major threat to the development of this industry. Samples collected from FMD outbreaks in Zambia during 2015-2020, comprising epithelial tissues samples (n = 47) and sera (n = 120), were analysed. FMD virus was serotyped in 26 samples, while 92 sera samples tested positive on NSP-ELISA. Phylogenetic analysis revealed notable changes in the epidemiology of FMD in Zambia, which included: (i) introduction of a novel FMDV SAT-3 (topotype II) causing FMD cases in cattle in Western Province; (ii) emergence of FMDV serotype O (topotype O/EA-2) in Central, Southern, Copperbelt, Western, Lusaka Provinces; and (iii) new outbreaks due to SAT -2 (topotypes I) in Eastern Zambia. Together, these data describe eight different epizootics that occurred in Zambia, four of which were outside the known FMD high-risk areas. This study highlights the complex epidemiology of FMD in Zambia, where the country represents an interface between East Africa (Pool 4) and Southern Africa (Pool 6). These changing viral dynamics have direct impacts on FMD vaccine selection in the SADC region.

Self-Reporting of Risk Pathways and Parameter Values for Foot-and-Mouth Disease in Slaughter Cattle from Alternative Production Systems by Kenyan and Ugandan Veterinarians.

Countries in which foot-and-mouth disease (FMD) is endemic may face bans on the export of FMD-susceptible livestock and products because of the associated risk for transmission of FMD virus. Risk assessment is an essential tool for demonstrating the fitness of one's goods for the international marketplace and for improving animal health. However, it is difficult to obtain the necessary data for such risk assessments in many countries where FMD is present. This study bridged the gaps of traditional participatory and expert elicitation approaches by partnering with veterinarians from the National Veterinary Services of Kenya (n = 13) and Uganda (n = 10) enrolled in an extended capacity-building program to systematically collect rich, local knowledge in a format appropriate for formal quantitative analysis. Participants mapped risk pathways and quantified variables that determine the risk of infection among cattle at slaughter originating from each of four beef production systems in each country. Findings highlighted that risk processes differ between management systems, that disease and sale are not always independent events, and that events on the risk pathway are influenced by the actions and motivations of value chain actors. The results provide necessary information for evaluating the risk of FMD among cattle pre-harvest in Kenya and Uganda and provide a framework for similar evaluation in other endemic settings.

Combining Multiple Assays Improves Detection and Serotyping of Foot-and-Mouth Disease Virus. A Practical Example with Field Samples from East Africa.

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012-2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVß6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.

Two Cross-Protective Antigen Sites on Foot-and-Mouth Disease Virus Serotype O Structurally Revealed by Broadly Neutralizing Antibodies from Cattle.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that infects cloven-hoofed animals. Neutralizing antibodies play critical roles in antiviral infection. Although five known antigen sites that induce neutralizing antibodies have been defined, studies on cross-protective antigen sites are still scarce. We mapped two cross-protective antigen sites using 13 bovine-derived broadly neutralizing monoclonal antibodies (bnAbs) capable of neutralizing 4 lineages within 3 topotypes of FMDV serotype O. One antigen site was formed by a novel cluster of VP3-focused epitopes recognized by bnAb C4 and C4-like antibodies. The cryo-electron microscopy (cryo-EM) structure of the FMDV-OTi (O/Tibet/99)-C4 complex showed close contact with VP3 and a novel interprotomer antigen epitope around the icosahedral 3-fold axis of the FMDV particle, which is far beyond the known antigen site 4. The key determinants of the neutralizing function of C4 and C4-like antibodies on the capsid were ßB (T65), the B-C loop (T68), the E-F loop (E131 and K134), and the H-I loop (G196), revealing a novel antigen site on VP3. The other antigen site comprised two group epitopes on VP2 recognized by 9 bnAbs (B57, B73, B77, B82, F28, F145, F150, E46, and E54), which belong to the known antigen site 2 of FMDV serotype O. Notably, bnAb C4 potently promoted FMDV RNA release in response to damage to viral particles, suggesting that the targeted epitope contains a trigger mechanism for particle disassembly. This study revealed two cross-protective antigen sites that can elicit cross-reactive neutralizing antibodies in cattle and provided new structural information for the design of a broad-spectrum molecular vaccine against FMDV serotype O. IMPORTANCE FMDV is the causative agent of foot-and-mouth disease (FMD), which is one of the most contagious and economically devastating diseases of domestic animals. The antigenic structure of FMDV serotype O is rather complicated, especially for those sites that can elicit a cross-protective neutralizing antibody response. Monoclonal neutralization antibodies provide both crucial defense components against FMDV infection and valuable tools for fine analysis of the antigenic structure. In this study, we found a cluster of novel VP3-focused epitopes using 13 bnAbs against FMDV serotype O from natural host cattle, which revealed two cross-protective antigen sites on VP2 and VP3. Antibody C4 targeting this novel epitope potently promoted viral particle disassembly and RNA release before infection, which may indicate a vulnerable region of FMDV. This study reveals new structural information about cross-protective antigen sites of FMDV serotype O, providing valuable and strong support for future research on broad-spectrum vaccines against FMD.

Phylogenetic and evolutionary analysis of foot-and-mouth disease virus A/ASIA/Sea-97 lineage.

Foot-and-mouth disease virus (FMDV) A/ASIA/Sea-97 is a predominant lineage in Southeast Asia and East Asia. However, Sea-97 lineage has not been well studied since its first outbreak in Thailand in 1997. Thus, we conducted phylogenetic and evolutionary analysis of Sea-97 using 224 VP1 sequences of FMDV A/ASIA during 1960 and 2018. Phylogenetic analysis revealed that Sea-97 lineage can be classified into five groups (G1-G5). After the emergence of G2 from G1, the genetic diversity of Sea-97 increased sharply, causing divergence into G3, G4 and G5. During this evolutionary process, Sea-97 lineage, which was initially found only in some countries in Southeast Asia, gradually spread to East Asia. The evolution rate of this lineage was estimated to be 1.2 × 10-2 substitutions/site/year and there were many differences in amino acid residues compared to vaccine strain. Substitutions at antigenically important sites may affect the efficacy of the vaccine, suggesting the need for appropriate vaccine strains. Our results could provide meaningful information to understand comprehensive characteristic of Sea-97 lineage.

Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus.

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two ß-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

Application of Heparin Affinity Chromatography to Produce a Differential Vaccine without Eliciting Antibodies against the Nonstructural Proteins of the Serotype O Foot-and-Mouth Disease Viruses.

Although polyethylene glycol (PEG) application is the most widely used method in removing nonstructural proteins (NSPs) for foot-and-mouth disease (FMD) vaccine production, some NSPs remaining in the antigen could elicit antibodies against these proteins after repeated vaccinations in livestock. Therefore, the purpose of this study was to purify the FMD virus (FMDV) via affinity chromatography using a heparin ligand to remove most proteins, including NSPs. Chromatography showed an intact virus (146S) particle recovery of 70% or more for three different strains of serotype O FMDV (two locally isolated strains and one genetically modified strain). The experimental vaccine made with antigens eluted via heparin affinity chromatography elicited virus-neutralizing antibodies against homologous viruses but did not induce antibodies against NSPs even after five immunizations in goats; this indicated that the NSPs were effectively removed from the vaccine antigen. This method can then be used to produce a higher-quality vaccine compared with PEG application in terms of the purity of the FMD vaccine. Therefore, this result would be an important groundwork for advanced FMD vaccine manufacturing in the near future.

Detection of an emerging novel sublineage Ind2001BD1 and lineage PanAsia of foot-and-mouth disease virus serotype O in cattle in Manikgonj district of Bangladesh, 2018.

Background: Foot-and-mouth disease (FMD) is an endemic disease of cloven-hoofed animals in Bangladesh and multiple outbreaks occur every year because of the FMD virus (FMDV). Aim: The aim of the present investigation was to determine the molecular characterization of the VP1 coding region of FMDV serotype O outbreak in cattle. Methods: A total of four tongue epithelial specimens were collected from clinically FMD-positive cattle during June 2018 in Manikgonj district of Bangladesh. Results: All four isolates were recorded positive for FMDV serotype O. The phylogenetic analysis showed that two isolates were clustered within an emerging novel sublineage Ind2001BD1 under lineage Ind2001 of FMDV serotype O, which was identified during 2012-2016 in Bangladesh. One isolate was clustered within the lineage PanAsia of FMDV serotype O and was closely related to an isolate identified in Nepal in 2009. The phylogenetic reconstruction revealed that all the four isolates belong to the Middle East-South Asia topotype. Conclusion: Therefore, multiple lineages of the FMDV serotype O are circulating among the cattle in the outbreak area, which make it more complex for the FMD control program in Bangladesh. A comprehensive study on the genetic characteristics of FMDV across the country is required for effective FMD prevention and control strategy.

Genetic and Antigenic Evaluation of Foot-and-mouth Disease Virus Type A in the Endemic Area of Iran within 2014-2015.

The foot-and-mouth disease virus (FMDV) with a wide variety of genomes and complicated biology is one of the infectious agents that put the lives of animals at risk. Therefore, to introduce suitable strains for vaccine production, it is essential to constantly evaluate genetic changes of circulating viruses in field. Within 2014-2015, a total of 126 clinical specimens consisting of epithelial tissue and vesicular fluid from tongue, dental pad, and hoofs suspected of FMD virus were submitted to the Reference Laboratory for FMD in Razi Vaccine and Serum Research Institute, and 86 of them were identified as FMD virus type A using sandwich Enzyme-Linked Immunosorbent Assay (ELISA). This virus was isolated from 42 samples from 16 provinces using cell culture. Firstly, the coding region that produces the main part of viral capsid was amplified by Polymerase chain reaction (PCR). This part of the genome by 800 bp length was related to the 1D gene that synthesizes the VP1 protein. The phylogenetic analysis of VP1 coding region determined two distinct genotypes with more than 15% nucleotide differences. The first cluster consisted of closely related viruses registered in the GeneBank of neighboring countries, including Afghanistan, Pakistan, and Turkey. All samples in Cluster1 were determined as relative viruses with genotype Iran-05. In-vitro serological examination indicated an antigenic relationship between Cluster 1 viruses and routine vaccine strain (A-IRN-2013). The second cluster with only two members was genetically far from earlier ones and could be considered a separate genotype. Furthermore, it was revealed that cluster 2 has not been previously reported in Iran. Genetic tracing indicated that these viruses might have been originated from circulating viruses from India. Antigenic evaluation exhibited that this group could not be cross-protected by the routine vaccinal strain (A-IRN-2013) used during the research period.

Evolution of antigenic and genetic characteristics of foot-and-mouth disease virus serotype A circulating in Thailand, 2007-2019.

Foot-and-mouth disease (FMD) is a persistent, major economic concern for livestock productivity, which is highly exacerbated by outbreaks in Thailand. FMD virus (FMDV) serotype A is more highly antigenic and genetically diverse than other serotypes, which has important implications for vaccine development as well as selection. Therefore, it is essential to continuously monitor antigenic and genetic changes of field isolates of FMDV serotype A. Here we used antisera against three vaccine strains (A/118/87, A/Sakolnakorn/97, and A/Lopburi/2012) to analyze the antigenicity of 133 field isolates of FMDV serotypes A in Thailand from 2007 to 2019. The majority of the isolates from 2007 to 2008 reacted only with the antiserum against strain A/118/87. In contrast, antigenic analysis revealed broad cross-reactivity and antigenic variations of the isolates from 2009 through 2019 against strains A/Sakolnakorn/97 and A/Lopburi/2012. These results indicate periodic changes in the antigenicity of field isolates of FMDV serotype A. Phylogenetic analysis of the VP1 region revealed that all isolates were of the Sea-97 lineage within the ASIA topotype. Analysis of the L-fragment genome sequences of 30 FMDV isolates collected throughout Thailand revealed highly variable amino acid sequences of VP1 and 3A, with the lowest average identity (94.56 %) and invariant (78.43 %) rates, respectively. The present findings indicate the importance of an active routine surveillance system incorporating antigenic and genetic analysis designated to continually update information about field isolates of FMDV serotype A. Such a system is essential for establishing and improving measures to control FMDV infections in Thailand and in neighboring Asian countries.

Sequence Analysis of Egyptian Foot-and-Mouth Disease Virus Field and Vaccine Strains: Intertypic Recombination and Evidence for Accidental Release of Virulent Virus.

In spite of annual mass vaccination programs with polyvalent inactivated vaccines, the incidence and economic impact of foot-and-mouth disease (FMD) in Egypt is high. Viruses of the A, O and SAT 2 serotypes are endemic and repeated incursions of new lineages from other countries lead to an unstable situation that makes the selection of appropriate vaccine antigens very difficult. In this study, whole genome sequencing of a 2016 serotype A isolate from Egypt revealed a recombination event with an African serotype O virus. Based on available vaccine matching data, none of the vaccines currently used in Egypt are expected to sufficiently protect against this virus or other viruses of this lineage (A/AFRICA/G-IV) circulating there since 2012. In addition to the risk of vaccine failure caused by strain mismatch, the production of inactivated FMD vaccines is dangerous if adequate biosafety cannot be maintained. Using a high-throughput sequencing protocol optimized for short nucleic acid fragments, the composition of a local inactivated vaccine was analyzed in depth. The serotype O strain identified in the vaccine was genetically identical to viruses found in recent FMD outbreaks in Egypt.

Emergence of foot-and-mouth disease virus serotype Asia1 group IX in India.

Foot-and-mouth disease virus (FMDV) serotype Asia1 is prevalent in India and is responsible for a minor proportion of FMD outbreaks. Globally, serotype Asia1 is grouped into nine different groups (GI-IX) based on genetic analysis. In India, only Asia1/G-III and Asia1/G-VIII have been documented so far. Phylogenetic analysis of recent serotype Asia1 isolates from India revealed the emergence of Asia1/G-IX. The Asia1/G-IX lineage shares recent common ancestry with Asia1/G-VIII dating to 2016. The root state posterior probabilities of Asia1/G-VIII are inclusive and there may have been either an incursion of the virus from Bangladesh, where it was first identified, or in situ evolution of the virus within India, which is an intriguing possibility.

Antigenic properties of a novel vaccine strain for type Asia1 foot-and-mouth disease in pigs.

Newly developed vaccine strains to prevent foot-and-mouth disease caused by the emerging serotype Asia1 virus were evaluated. To protect against the group (G)-VIII strain, which occurred recently, we produced an infectious cDNA clone of Asia1 Shamir cDNA (Asia1 Shamir-R). In addition, by adding a site 1 epitope of VP1 of the G-VIII lineage virus to this virus, we produced a new virus (Sham GVIII- EPI), and another virus(Sham GVIII-VP1) was replaced with that of G-VIII lineage in the VP1 region of Shamir. Test vaccines were produced using these three types of vaccine virus, and their immunogenicity and protection capabilities were evaluated in mice. Immunized mice were challenged with the Asia1 Shamir or G-VIII virus, and the results show that all the vaccines have similar protective effects. As they showed similar antigenicity, we chose the Shamir-R vaccine. Pigs maintained relatively high neutralizing antibody levels against homologous viruses of the Shamir and G-VII or G-VIII lineage three to four weeks after immunization. However, they formed relatively low levels of antibodies to G-IV and G-V viruses. In conclusion, we produced a vaccine candidate capable of protection against the G-VIII virus in the vaccine experiment for the type Asia1 serotype vaccine. This Shamir-R vaccine virus was found to protect against the viruses of the Asia1 genotype G-VII and G-VIII lineages, which occurred recently in Asia.

Comparative nucleotide sequencing of the VP1 capsid gene of recent isolates of foot-and-mouth disease virus serotype O from Egypt.

Since 2006, Egypt has been affected by eleven various foot-and-mouth disease virus (FMDV) lineages. Accordingly, the nucleotide sequences of the 1D gene and the genes encoding the external capsid protein of some isolates of serotype O (the most predominant epidemic serotype in the country) collected from 2004 to 2017 were determined. All of these viruses (including the vaccine strain) belonged to serotype O, topotype ME-SA, and lineage Sharquia-72, and their sequences were of 98.6-98.9% identical to that of strain O1/Sharquia/EGY/72 (DQ164871), and differed from cultured and clinical (D197E) virus strains. The characteristic sites on the surface of the structural proteins of the Egyptian serotype O, topotype ME-SA viruses were located at residues 138 and 198 of VP1, residue 132 of VP2, and residues 56 and 104 of VP3. Furthermore, a phylogenetic tree revealed that Sharquia-72 was the only lineage present in Egypt for many decades prior to 2007. Unfortunately, however, during the last decade, five lineages of two separate topotypes of FMDV serotype O were detected in Egypt. Lineages Sharquia-72 and PanAsia-2 belong to topotype ME-SA and show ~ 14.5 to 17.5% intra-lineage divergence. In addition, lineages Qal-13, Ism-16, and Alx-17 cluster within topotype EA-3 and show ~ 4.5 to 15% intra-lineage diversity. The predecessors of the Egyptian EA-3 viruses are likely to have been from Sudan. Finally, at least a penta- or hexavalent vaccine comprising strains representing the endemic FMDV topotypes should be implemented on a wide scale in Egypt, which could combat the incursion of new lineages.

Transmission network reconstruction for foot-and-mouth disease outbreaks incorporating farm-level covariates.

Transmission network modelling to infer 'who infected whom' in infectious disease outbreaks is a highly active area of research. Outbreaks of foot-and-mouth disease have been a key focus of transmission network models that integrate genomic and epidemiological data. The aim of this study was to extend Lau's systematic Bayesian inference framework to incorporate additional parameters representing predominant species and numbers of animals held on a farm. Lau's Bayesian Markov chain Monte Carlo algorithm was reformulated, verified and pseudo-validated on 100 simulated outbreaks populated with demographic data Japan and Australia. The modified model was then implemented on genomic and epidemiological data from the 2010 outbreak of foot-and-mouth disease in Japan, and outputs compared to those from the SCOTTI model implemented in BEAST2. The modified model achieved improvements in overall accuracy when tested on the simulated outbreaks. When implemented on the actual outbreak data from Japan, infected farms that held predominantly pigs were estimated to have five times the transmissibility of infected cattle farms and be 49% less susceptible. The farm-level incubation period was 1 day shorter than the latent period, the timing of the seeding of the outbreak in Japan was inferred, as were key linkages between clusters and features of farms involved in widespread dissemination of this outbreak. To improve accessibility the modified model has been implemented as the R package 'BORIS' for use in future outbreaks.

Complete genome sequence of a potential foot-and-mouth disease virus serotype O vaccine strain from Bangladesh.

One of the six sublineages of the dominant O/ME-SA/Ind2001 lineage of foot-and-mouth disease virus (FMDV), Ind2001BD1 has already spread throughout 14 countries, including Bangladesh. Here, we report the complete genome sequence of the potential serotype O vaccine strain BAN/TA/Dh-301/2016, which has been shown to provide protection against all the circulating serotype O viruses in Bangladesh. The viral genome is 8,211 nucleotide (nt) long with an open reading frame (ORF) of 6999 nt. The ORF is flanked by a 1098-nt-long 5'-UTR and a 114-nt-long 3'-UTR. Compared to the Indian FMDV serotype O vaccine strain O/India/R2/75 (AF204276), ten mutations were identified in the major antigenic sites of BAN/TA/Dh-301/2016 (MK088170.1).

Development and validation of a solid-phase competition ELISA based on virus-like particles of foot-and-mouth disease virus serotype A for antibody detection.

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious epidemic disease, which is controlled primarily by prophylactic vaccination and serological monitoring after vaccination. Here, we have developed a solid-phase competition ELISA (SPCE) method based on virus-like particles (VLPs) of FMDV serotype A. The use of VLPs in the SPCE assay as a replacement for inactivated FMDV provides a high level of biosafety. The SPCE showed high concordance rates when compared with the virus neutralization test and liquid-phase blocking ELISA for testing clinical serum samples and successive serological monitoring (kappa = 0.925). Thus, this SPCE is an alternative method for post-immunization detection of antibodies against FMDV serotype A, with high specificity and sensitivity.

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.

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.

Immunogenicity and protective efficacy of 3A truncated negative marker foot-and-mouth disease virus serotype A vaccine.

Foot-and-mouth disease (FMD) is a highly contagious, economically significant disease of cloven-hoofed animals caused by FMD virus (FMDV) of the Picornaviridae family. Vaccination of susceptible animals with inactivated virus vaccine is the standard practice for disease control. The prophylactic use of the inactivated vaccines has reduced the disease burden in many countries endemic to FMD. In the process of implementation of the mass vaccination program and disease eradication, it is essential to differentiate infected from vaccinated animals (DIVA) where a large proportion of the animal population is vaccinated, and disease-free zones are being established, to help in sero-surveillance of the disease. In such a scenario, the use of a negative marker vaccine is beneficial to rule out false-positive results in a disease-free zone. Here we report the construction and rescue of an infectious cDNA clone for FMDV serotype A Indian vaccine strain lacking 58 amino acid residues (87-144 amino acid position) in the carboxy-terminal region of the viral 3A protein. The recombinant deletion mutant virus showed similarity in the antigenic relationship with the parental strain. Immunization of guinea pigs with the inactivated vaccine formulated using the deletion mutant virus induced potent immune response with 100% protective efficacy upon challenge with homologous virus. Further, we show that sera from the guinea pigs infected with the deletion mutant virus did not show reactivity in an indirect ELISA test targeting the deleted portion of 3A protein. We conclude that the recombinant deletion mutant virus vaccine along with the newly developed companion indirect ELISA targeting portion of FMDV 3A protein could be useful in the implementation of a precise DIVA policy in our country when we reach FMD free status with vaccination.