Integrating contact tracing and whole-genome sequencing to track the elimination of dog-mediated rabies: An observational and genomic study.

Background: Dog-mediated rabies is endemic across Africa causing thousands of human deaths annually. A One Health approach to rabies is advocated, comprising emergency post-exposure vaccination of bite victims and mass dog vaccination to break the transmission cycle. However, the impacts and cost-effectiveness of these components are difficult to disentangle. Methods: We combined contact tracing with whole-genome sequencing to track rabies transmission in the animal reservoir and spillover risk to humans from 2010 to 2020, investigating how the components of a One Health approach reduced the disease burden and eliminated rabies from Pemba Island, Tanzania. With the resulting high-resolution spatiotemporal and genomic data, we inferred transmission chains and estimated case detection. Using a decision tree model, we quantified the public health burden and evaluated the impact and cost-effectiveness of interventions over a 10-year time horizon. Results: We resolved five transmission chains co-circulating on Pemba from 2010 that were all eliminated by May 2014. During this period, rabid dogs, human rabies exposures and deaths all progressively declined following initiation and improved implementation of annual islandwide dog vaccination. We identified two introductions to Pemba in late 2016 that seeded re-emergence after dog vaccination had lapsed. The ensuing outbreak was eliminated in October 2018 through reinstated islandwide dog vaccination. While post-exposure vaccines were projected to be highly cost-effective ($256 per death averted), only dog vaccination interrupts transmission. A combined One Health approach of routine annual dog vaccination together with free post-exposure vaccines for bite victims, rapidly eliminates rabies, is highly cost-effective ($1657 per death averted) and by maintaining rabies freedom prevents over 30 families from suffering traumatic rabid dog bites annually on Pemba island. Conclusions: A One Health approach underpinned by dog vaccination is an efficient, cost-effective, equitable, and feasible approach to rabies elimination, but needs scaling up across connected populations to sustain the benefits of elimination, as seen on Pemba, and for similar progress to be achieved elsewhere. Funding: Wellcome [207569/Z/17/Z, 095787/Z/11/Z, 103270/Z/13/Z], the UBS Optimus Foundation, the Department of Health and Human Services of the National Institutes of Health [R01AI141712] and the DELTAS Africa Initiative [Afrique One-ASPIRE/DEL-15-008] comprising a donor consortium of the African Academy of Sciences (AAS), Alliance for Accelerating Excellence in Science in Africa (AESA), the New Partnership for Africa's Development Planning and Coordinating (NEPAD) Agency, Wellcome [107753/A/15/Z], Royal Society of Tropical Medicine and Hygiene Small Grant 2017 [GR000892] and the UK government. The rabies elimination demonstration project from 2010-2015 was supported by the Bill & Melinda Gates Foundation [OPP49679]. Whole-genome sequencing was partially supported from APHA by funding from the UK Department for Environment, Food and Rural Affairs (Defra), Scottish government and Welsh government under projects SEV3500 and SE0421.

Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes.

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.

Improved PCR diagnostics using up-to-date in silico validation: An F-gene RT-qPCR assay for the detection of all four lineages of peste des petits ruminants virus.

Peste des petits ruminants (PPR) is a globally significant disease of small ruminants caused by the peste des petits ruminants virus (PPRV) that is considered for eradication by 2030 by the United Nations Food and Agriculture Organisation (FAO). Critical to the eradication of PPR are accurate diagnostic assays. RT-qPCR assays targeting the nucleocapsid gene of PPRV have been successfully used for the diagnosis of PPR. We describe the development of an RT-qPCR assay targeting an alternative region (the fusion (F) gene) based on the most up-to-date PPRV sequence data. In silico analysis of the F-gene RT-qPCR assay performed using PCRv software indicated 98% sensitivity and 100% specificity against all PPRV sequences published in Genbank. The assay indicated the greatest in silico sensitivity in comparison to other previously published and recommended PPRV RT-qPCR assays. We evaluated the assay using strains representative of all 4 lineages in addition to samples obtained from naturally and experimentally-infected animals. The F-gene RT-qPCR assay showed 100% diagnostic specificity and demonstrated a limit of detection of 10 PPRV genome copies per µl. This RT-qPCR assay can be used in isolation or in conjunction with other assays for confirmation of PPR and should support the global efforts for eradication.

Validation of a diagnostic tool for the diagnosis of lumpy skin disease.

BACKGROUND: Lumpy skin disease (LSD) is caused by LSD virus which is a member of the Capripoxvirus (CaPV) genus. Although PCR provides for a rapid and sensitive diagnosis, it has limited use due to its complexity in terms of cost, time and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, specific and cost-effective method with a diagnostic accuracy similar to PCR. OBJECTIVES/HYPOTHESIS: To compare the detection rate (DR) of two LAMP assays versus PCR for the detection of CaPV. ANIMALS: This study used 105 apparently health animals (AHA) and 59 clinically sick animals (CSA). METHODS AND MATERIALS: PCR and LAMP assays (LAMP1 and LAMP 2) were compared for detection of CaPV from AHA and CSA using blood and tissue samples. The detection was confirmed by sequencing of PCR positive samples. Analytical sensitivity and specificity of LAMP assays also were assessed. RESULTS: The DR in CSA was 13.6% for PCR whereas for LAMP it was 39.0% and 25.4% for LAMP 1 and 2 methods, respectively. In AHA, the LAMP assay DR was 14.3% and 1.9% for LAMP 1 and 2, respectively. Phylogenetic tree analysis confirmed the identity of CaPV. Analytic sensitivity showed a detection limit of 8 copies/µL. The analytic specificity test showed no cross detection with other infectious agents. CONCLUSION AND CLINICAL IMPORTANCE: Good sensitivity and specificity results for LAMP assay support its application in the routine diagnosis of LSD, whereas its ability to detect LSDV in apparently healthy animals shows its usefulness in identifying populations at risk of LSD.

Landscape attributes governing local transmission of an endemic zoonosis: Rabies virus in domestic dogs.

Landscape heterogeneity plays an important role in disease spread and persistence, but quantifying landscape influences and their scale dependence is challenging. Studies have focused on how environmental features or global transport networks influence pathogen invasion and spread, but their influence on local transmission dynamics that underpin the persistence of endemic diseases remains unexplored. Bayesian phylogeographic frameworks that incorporate spatial heterogeneities are promising tools for analysing linked epidemiological, environmental and genetic data. Here, we extend these methodological approaches to decipher the relative contribution and scale-dependent effects of landscape influences on the transmission of endemic rabies virus in Serengeti district, Tanzania (area ~4,900 km2 ). Utilizing detailed epidemiological data and 152 complete viral genomes collected between 2004 and 2013, we show that the localized presence of dogs but not their density is the most important determinant of diffusion, implying that culling will be ineffective for rabies control. Rivers and roads acted as barriers and facilitators to viral spread, respectively, and vaccination impeded diffusion despite variable annual coverage. Notably, we found that landscape effects were scale-dependent: rivers were barriers and roads facilitators on larger scales, whereas the distribution of dogs was important for rabies dispersal across multiple scales. This nuanced understanding of the spatial processes that underpin rabies transmission can be exploited for targeted control at the scale where it will have the greatest impact. Moreover, this research demonstrates how current phylogeographic frameworks can be adapted to improve our understanding of endemic disease dynamics at different spatial scales.

Toward Elimination of Dog-Mediated Human Rabies: Experiences from Implementing a Large-scale Demonstration Project in Southern Tanzania.

A Rabies Elimination Demonstration Project was implemented in Tanzania from 2010 through to 2015, bringing together government ministries from the health and veterinary sectors, the World Health Organization, and national and international research institutions. Detailed data on mass dog vaccination campaigns, bite exposures, use of post-exposure prophylaxis (PEP), and human rabies deaths were collected throughout the project duration and project areas. Despite no previous experience in dog vaccination within the project areas, district veterinary officers were able to implement district-wide vaccination campaigns that, for most part, progressively increased the numbers of dogs vaccinated with each phase of the project. Bite exposures declined, particularly in the southernmost districts with the smallest dog populations, and health workers successfully transitioned from primarily intramuscular administration of PEP to intradermal administration, resulting in major cost savings. However, even with improved PEP provision, vaccine shortages still occurred in some districts. In laboratory diagnosis, there were several logistical challenges in sample handling and submission but compared to the situation before the project started, there was a moderate increase in the number of laboratory samples submitted and tested for rabies in the project areas with a decrease in the proportion of rabies-positive samples over time. The project had a major impact on public health policy and practice with the formation of a One Health Coordination Unit at the Prime Minister's Office and development of the Tanzania National Rabies Control Strategy, which lays a roadmap for elimination of rabies in Tanzania by 2030 by following the Stepwise Approach towards Rabies Elimination (SARE). Overall, the project generated many important lessons relevant to rabies prevention and control in particular and disease surveillance in general. Lessons include the need for (1) a specific unit in the government for managing disease surveillance; (2) application of innovative data collection and management approaches such as the use of mobile phones; (3) close cooperation and effective communication among all key sectors and stakeholders; and (4) flexible and adaptive programs that can incorporate new information to improve their delivery, and overcome challenges of logistics and procurement.

Elucidating the phylodynamics of endemic rabies virus in eastern Africa using whole-genome sequencing.

Many of the pathogens perceived to pose the greatest risk to humans are viral zoonoses, responsible for a range of emerging and endemic infectious diseases. Phylogeography is a useful tool to understand the processes that give rise to spatial patterns and drive dynamics in virus populations. Increasingly, whole-genome information is being used to uncover these patterns, but the limits of phylogenetic resolution that can be achieved with this are unclear. Here, whole-genome variation was used to uncover fine-scale population structure in endemic canine rabies virus circulating in Tanzania. This is the first whole-genome population study of rabies virus and the first comprehensive phylogenetic analysis of rabies virus in East Africa, providing important insights into rabies transmission in an endemic system. In addition, sub-continental scale patterns of population structure were identified using partial gene data and used to determine population structure at larger spatial scales in Africa. While rabies virus has a defined spatial structure at large scales, increasingly frequent levels of admixture were observed at regional and local levels. Discrete phylogeographic analysis revealed long-distance dispersal within Tanzania, which could be attributed to human-mediated movement, and we found evidence of multiple persistent, co-circulating lineages at a very local scale in a single district, despite on-going mass dog vaccination campaigns. This may reflect the wider endemic circulation of these lineages over several decades alongside increased admixture due to human-mediated introductions. These data indicate that successful rabies control in Tanzania could be established at a national level, since most dispersal appears to be restricted within the confines of country borders but some coordination with neighbouring countries may be required to limit transboundary movements. Evidence of complex patterns of rabies circulation within Tanzania necessitates the use of whole-genome sequencing to delineate finer scale population structure that can that can guide interventions, such as the spatial scale and design of dog vaccination campaigns and dog movement controls to achieve and maintain freedom from disease.

Molecular survey for foot-and-mouth disease virus in livestock in Tanzania, 2008-2013.

Phylogeography data are of paramount importance in studying the molecular epidemiology dynamics of foot-and-mouth disease virus (FMDV). In this study, epithelial samples and oesophageal-pharyngeal fluids were collected from 361 convalescent animals (cattle and buffaloes) in the field throughout Tanzania between 2009 and 2013. The single plex real-time RT-PCR (qRT-PCR) assay for rapid and accurate diagnosis of FMDV employing the Callahan 3DF-2, 3DF-R primers and Callahan 3DP-1 probe were used. Preparation of the samples was performed according to the OIE manual, with a Kenya O serotype obtained from the attenuated vaccine serving as a positive control and samples collected from healthy animals serving as true negatives. The results indicated that 53.49% of samples (n = 176) were positive for FMDV genome by qRT-PCR, with Ct values ranging from 14 to 32. In addition, molecular typing of the FMDV genome positive samples using serotype specific primers revealed the existence of several serotypes: serotype South Africa Territory 1 (SAT1) (34.25%, n = 60), serotype A (68.92%, n = 98), serotype O (59.20%, n = 98) and SAT2 (54.54%, n = 96). The virus protein 1 sequences analysis for 35 samples was performed and the collective results indicated: 54.28% serotype O, 25.71% serotype A, 14.28% serotype SAT1 and 2.85% serotype SAT2. Therefore in this study, both the phylogenetic trees and spatial distribution of serotypes elucidated the phylodynamics of multiple FMDV field strains in Tanzania and neighbouring countries.

Complete genome sequence of Ikoma lyssavirus.

Lyssaviruses (family Rhabdoviridae) constitute one of the most important groups of viral zoonoses globally. All lyssaviruses cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Currently available vaccines are highly protective against the predominantly circulating lyssavirus species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence for a novel lyssavirus, Ikoma lyssavirus (IKOV), isolated from an African civet in Tanzania displaying clinical signs of rabies. Genetically, this virus is the most divergent within the genus Lyssavirus. Characterization of the genome will help to improve our understanding of lyssavirus diversity and enable investigation into vaccine-induced immunity and protection.

Ikoma lyssavirus, highly divergent novel lyssavirus in an African civet.

Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.

Foot-and-mouth disease virus serotypes detected in Tanzania from 2003 to 2010: conjectured status and future prospects.

This study was conducted to investigate the presence of foot-and-mouth disease virus (FMDV) in different geographic locations of Tanzania. Epithelial tissues and fluids (n = 364) were collected from cattle exhibiting oral and foot vesicular lesions suggestive of FMD and submitted for routine FMD diagnosis. The analysis of these samples collected during the period of 2002 and 2010 was performed by serotype-specific antigen capture ELISA to determine the presence of FMDV. The results of this study indicated that 167 out of 364 (46.1%) of the samples contained FMDV antigen. Of the 167 positive samples, 37 (28.4%) were type O, 7 (4.1%) type A, 45 (21.9%) SAT 1 and 79 (45.6%) SAT 2. Two FMDV serotypes (O and SAT 2) were widely distributed throughout Tanzania whilst SAT 1 and A types were only found in the Eastern zone. Our findings suggest that serotypes A, O, SAT 1 and SAT 2 prevail in Tanzania and are associated with the recent FMD outbreaks. The lack of comprehensive animal movement records and inconsistent vaccination programmes make it difficult to determine the exact source of FMD outbreaks or to trace the transmission of the disease over time. Therefore, further collection and analysis of samples from domestic and wild animals are being undertaken to investigate the genetic and antigenic characteristics of the circulating strains, so that a rational method to control FMD in Tanzania and the neighbouring countries can be recommended.