Intra-serotype SAT2 chimeric foot-and-mouth disease vaccine protects cattle against FMDV challenge

The genetic diversity of the three Southern African Territories (SAT) types of foot-and-mouth diseasevirus (FMDV) reflects high antigenic variation, and indications are that vaccines targeting each SAT-specific topotype may be needed. This has serious implications for control of FMD using vaccines as wellas the choice of strains to include in regional antigen banks. Here, we investigated an intra-serotypechimeric virus, vSAT2ZIM14-SAT2, which was engineered by replacing the surface-exposed capsid-codingregion (1B-1D/2A) of a SAT2 genome-length clone, pSAT2, with that of the field isolate, SAT2/ZIM/14/90.The chimeric FMDV produced by this technique was viable, grew to high titres and stably maintained the1B-1D/2A sequence upon passage. Chemically inactivated, oil adjuvanted vaccines of both the chimericand parental immunogens were used to vaccinate cattle. The serological response to vaccination showedthe production of strong neutralizing antibody titres that correlated with protection against homolo-gous FMDV challenge. We also predicted a good likelihood that cattle vaccinated with an intra-serotypechimeric vaccine would be protected against challenge with viruses that caused recent outbreaks insouthern Africa. These results provide support that chimeric vaccines containing the external capsid offield isolates induce protective immune responses in FMD host species similar to the parental vaccine.MSD Animal Health (previously Intervet SPAH)http://www.elsevier.com/locate/vaccine2016-06-30hb201

Spatio-temporal cluster analysis and transmission drivers for Peste des Petits Ruminants in Uganda

Peste des Petits Ruminants (PPR) is a transboundary, highly contagious, and fatal disease of small ruminants. PPR causes global annual economic losses of between USD 1.5 and 2.0 billion across more than 70 affected countries. Despite the commercial availability of effective PPR vaccines, lack of financial and technical commitment to PPR control coupled with a dearth of refined PPR risk profiling data in different endemic countries has perpetuated PPR virus transmission. In Uganda, over the past 5 years, PPR has extended from northeastern Uganda (Karamoja) with sporadic incursions in other districts /regions. To identify disease cluster hotspot trends that would facilitate the design and implementation of PPR risk-based control methods (including vaccination), we employed the space–time cube approach to identify trends in the clustering of outbreaks in neighbouring space–time cells using confirmed PPR outbreak report data (2007–2020). We also used negative binomial and logistic regression models and identified high small ruminant density, extended road length, low annual precipitation and high soil water index as the most important drivers of PPR in Uganda. The study identified (with 90–99% confidence) five PPR disease hotspot trend categories across subregions of Uganda. Diminishing hotspots were identified in the Karamoja region whereas consecutive, sporadic, new and emerging hotspots were identified in central and southwestern districts of Uganda. Inter-district and cross-border small ruminant movement facilitated by longer road stretches and animal comingling precipitate PPR outbreaks as well as PPR virus spread from its initial Karamoja focus to the central and southwestern Uganda. There is therefore urgent need to prioritize considerable vaccination coverage to obtain the required herd immunity among small ruminants in the new hotspot areas to block transmission to further emerging hotspots. Findings of this study provide a basis for more robust timing and prioritization of control measures including vaccination

Spatio-temporal cluster analysis and transmission drivers for Peste des Petits Ruminants in Uganda.

Peste des Petits Ruminants (PPR) is a transboundary, highly contagious, and fatal disease of small ruminants. PPR causes global annual economic losses of between USD 1.5-2.0 billion across more than 70 affected countries. Despite the commercial availability of effective PPR vaccines, lack of financial and technical commitment to PPR control coupled with a dearth of refined PPR risk profiling data in different endemic countries has perpetuated PPR virus transmission. In Uganda, over the past five years, PPR has extended from north-eastern Uganda (Karamoja) with sporadic incursions in other districts /regions. To identify disease cluster hotspot trends that would facilitate the design and implementation of PPR risk-based control methods (including vaccination), we employed the space-time cube approach to identify trends in the clustering of outbreaks in neighbouring space-time cells using confirmed PPR outbreak report data (2007-2020). We also used negative binomial and logistic regression models and identified high small ruminant density, extended road length, low annual precipitation and high soil water index as the most important drivers of PPR in Uganda. The study identified (with 90 - 99% confidence) five PPR disease hotspot trend categories across subregions of Uganda. Diminishing hotspots were identified in the Karamoja region whereas consecutive, sporadic, new, and emerging hotspots were identified in central and southwestern districts of Uganda. Inter-district and cross-border small ruminant movement facilitated by longer road stretches and animal comingling precipitate PPR outbreaks as well as PPR virus spread from its initial Karamoja focus to the central and south-western Uganda. There is therefore urgent need to prioritize considerable vaccination coverage to obtain the required herd immunity among small ruminants in the new hotspot areas to block transmission to further emerging hotspots. Findings of this study provide a basis for more robust timing and prioritization of control measures including vaccination. This article is protected by copyright. All rights reserved

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 lowpassage 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.S1 Table. Summary of the amino acid substitutions in the outer capsid proteins of SAT1 and SAT2 viruses resulting from cytolytic passages in BHK-21 cells.S2 Table. Virus neutralization and predicted cross-reactivity of SAT2/SAU/6/00 vaccinated cattle sera to the isolates and chimeric mutant viruses.MSD Animal Health (previously Intervet); the Wellcome Trust Translation Award; the Organization for Women in Science for the Developing World (OWSD); the Wellcome Trust; a NRF UK/ SA Researchers Link travel grant; the work of the WT Centre in Oxford is supported by the WT core award and the UK MRC.http://www.plospathogens.orgam2021BiochemistryGeneticsMicrobiology and Plant PathologyVeterinary Tropical Disease

Viruses associated with measles-like illnesses in Uganda

Objectives: In this study, we investigated the causes of measles-like illnesses (MLI) in the Uganda national surveillance programme in order to inform diagnostic assay selection and vaccination strategies. Methods: We used metagenomic next-generation sequencing (M-NGS) on the Illumina platform to identify viruses associated with MLI (defined as fever and rash in the presence of either cough, coryza or conjunctivitis) in patient samples that had tested IgM negative for measles between 2010 and 2019. Results: Viral genomes were identified in 87/271 (32%) of samples, of which 44/271 (16%) contained 12 known viral pathogens. Expected viruses included rubella, human parvovirus B19, Epstein Barr virus, human herpesvirus 6B, human cytomegalovirus, varicella zoster virus and measles virus (detected within the seronegative window-period of infection) and the blood-borne hepatitis B virus. We also detected Saffold virus, human parvovirus type 4, the human adenovirus C2 and vaccine-associated poliovirus type 1. Conclusions: The study highlights the presence of undiagnosed viruses causing MLI in Uganda, including vaccine-preventable illnesses. NGS can be used to monitor common viral infections at a population level, especially in regions where such infections are prevalent, including low and middle income countries to guide vaccination policy and optimize diagnostic assays

Genetic and phenotypic characterisation of selected african foot-and-mouth disease virus isolates

Foot-and-mouth disease (FMD) is a contagious viral disease that affects cloven hoofed animals and can be economically devastating. However, vaccination can reduce the incidence of the disease when used in conjunction with other control methods. Studies described in this thesis address three aspects beneficial to the selection of vaccine seed viruses as well as improving the design of chimeric vaccines customized for use in Africa. Molecular characterization of the non-structural proteins of African FMD viruses (FMDV) can facilitate the selection of vaccine seed strains with a fitness advantage. In addition, common mutations acquired by South African Territory (SAT) serotypes on the virus surface capsid upon cell culture adaptation may be of value to the design of recombinant constructs for chimeric viruses optimized for vaccine production. Moreover, the antigenicity of a chimeric virus vZIM14-SAT2 compared to its parental virus, SAT2/ZIM/14/90, in cattle and additionally in vaccine matching assays evaluated its compatibility for future vaccine studies. Genome comparisons of the non-structural proteins for 79 African FMDV isolates from different serotypes and topotypes, spatially and temporally dispersed, showed that critical functional motifs were conserved and variation occurred away from enzymatic active sites. The southern African FMDV clustered separately from the other African viruses on phylogenetic analysis. This increased the molecular understanding of FMDV, aiding in the selection of vaccine seed strains. The BHK-21 cell line (preferred for vaccine production) was used to serially passage eleven SAT1 and four SAT2 FMDV. This resulted in Arg or Lys mutations in the VP1 capsid protein surrounding the 5-fold axis, at positions 84-85 and 111-112 respectively. Arg at position 112 was favoured in the binding of a heparin moiety onto a SAT1 model using molecular docking simulations. This knowledge can be applied to recombinant constructs for the generation of chimeric viruses that adapt rapidly to BHK-21 cell culture for vaccine production. Vaccines derived from chimeric viruses present a good prospect in the control of FMD. The antigenic nature of an intra-serotype chimeric virus, vZIM14-SAT2, consisting of the capsid-coding region of SAT2/ZIM/14/90 in a genome-length clone pSAT2, was compared to that of its parental virus. In cattle both chimeric and parental viruses elicited similar neutralizing antibody responses and provided protection against clinical disease following homologous virus challenge. In addition, antigenic matching resulted in comparable cross-reaction results for both chimeric and parental viruses. Therefore, when used as vaccines, the antigenic profiles of chimeric viruses against SAT2 isolates did not differ from parental viruses and could be used as an alternative in vaccine production combined with the advantage of including suitable mutations for cell culture adaptation. These findings increase our knowledge on the genome structure and replication of the FMDV in BHK-21 cells. The data can be used to facilitate selection of vaccine seed viruses specific to serotype and topotype. Additionally, the information can supplement the design of recombinant constructs for chimeric vaccines for future application. Taken together, these new data broaden our options to control FMD in Africa using tailor made chimeric vaccines.Thesis (PhD)--University of Pretoria, 2015.tm2015Veterinary Tropical DiseasesPhDUnrestricte

Determination of common genetic variants within the non-structural proteins of foot-and-mouth disease viruses isolated in sub-Saharan Africa

The non-structural proteins of foot-and-mouth disease virus (FMDV) are responsible for RNA replication, proteolytic processing of the viral polyprotein precursor, folding and assembly of the structural proteins and modification of the cellular translation apparatus. Investigation of the amino acid heterogeneity of the non-structural proteins of seventy- nine FMDV isolates of SAT1, SAT2, SAT3, A and O serotypes revealed between 29 and 62% amino acid variability. The Leader protease (Lpro) and 3A proteins were the most variable whilst the RNA-dependent RNA polymerase (3Dpol) the most conserved. Phylogeny based on the non-structural protein-coding regions showed separate clusters for southern African viruses for both the Lpro and 3C protease (3Cpro) and sequences unique to this group of viruses, e.g. in the 2C and 3Cpro proteins. These groupings were unlike serotype groupings based on structural protein-coding regions. The amino acid substitutions and the nature of the naturally occurring substitutions provide insight into the functional domains and regions of the non-structural proteins that are critical for structure–function. The Lpro of southern African SAT type isolates differed from A, O and SAT isolates in northern Africa, particularly in the auto-processing region. Three-dimensional structures of the 3C protease (3Cpro) and 3Dpol showed that the observed variation does not affect the enzymatic active sites or substrate binding sites. Variation in the 3Cpro cleavage sites demonstrates broad substrate specificity.THRIP of the National Research Foundation of South Africahttp://www.elsevier.com/locate/vetmic2016-05-31hb201

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

Mentorship of the next generation of One Health workers through experiential learning: A case of students of Makerere University

Abstract Multiple zoonotic disease outbreaks occurred in Uganda over the past two decades and have needed operationalization of the One Health (OH) Approach to respond effectively. Between 2016 and 2018, the African One Health University Network (AFROHUN) supported 61 students (25 females, 36 males) to join multisectoral and multidisciplinary government national and district task force disease response teams. The goal of joining these teams was to build and strengthen the students’ disease investigation and response skills in real time using a One Health approach. Qualitative methods were used to collect student and partner responses on their field experiences. The AFROHUN project identified the experiential knowledge and skills that the students gained. Student project reports were reviewed by the joint technical teams from the university and the national/district task forces. These included training materials and disease outbreak investigation and response reports. Partnerships and collaboration between the university One Health networks and the government enabled 35 graduate and 26 undergraduate students to receive joint mentorship from the national or district task force outbreak response teams. Most participants were from degree programs in Public Health, Epidemiology, Environmental Health, Veterinary, Wildlife Sciences, and Infectious Disease Management, while few students were from social sciences. Students were mentored in seven competency-based areas of disease management: (1) biorisk management, (2) community engagement and coordination, (3) epidemiology, (4) leadership, (5) outbreak investigation and response, (6) risk communication, and (7) surveillance. In conclusion, zoonotic outbreaks provided real-life learning opportunities for students in disease outbreak investigation and response using a multidisciplinary and multisectoral approach. The identified skills can be incorporated into educational materials such as curricula and present an ideal opportunity to build partnerships for workforce development. One Health impact statement The next generation of One Health workers, those capable of working across sectors and disciplines to improve the health of animals, humans, plants, and the environment, need soft and technical skills to guarantee optimal preparedness, prevention, and response to disease outbreaks and understand the animal to human transmission dynamics of disease. In countries where emerging, re-emerging, and endemic zoonoses and hemorrhagic fevers are prevalent, it is crucial to provide these real-life or experiential training opportunities for university students, working closely in multidisciplinary teams. The “real-time” joint mentorship by the government’s multidisciplinary and multisectoral outbreak response teams, during disease outbreaks, provides an opportunity to build and strengthen student skills in biorisk management, community engagement and coordination, epidemiology, leadership, outbreak investigation and response, risk communication, and surveillance