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 contribu- tion 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 ineffec- tive for rabies control. Rivers and roads acted as barriers and facilitators to viral spread, respectively, and vaccination impeded diffusion despite variable annual cov- erage. 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

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

Antigenic and genetic characterization of a divergent African virus, Ikoma lyssavirus

In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between the genome of IKOV and those of other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spillover event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone, however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in vitro and in vivo characterization of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains and (3) poorly neutralized by sera from humans and animals immunized against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya

Cancer Gene Therapy Using Plasmid DNA: Pharmacokinetic Study of DNA Following Injection in Mice

Overview summary The present study evaluates the pharmacokinetic half-life and tissue distribution of plasmid DNA following intravenous injection in mice. This study extends the time frame of previous in vivo analyses to 6 months following i.v. injection. Injected mice exhibit no expression of the encoded gene as assayed by immunofluorescence. This represents the first systematic in vivo pharmacokinetic study of intravenously injected DNA complexed with cationic lipids, and is relevant to many gene therapy protocols utilizing direct injection of plasmid DNA plus lipids. The results provide a preliminary basis for the safe initiation of cancer immunotherapy clinical trials in which plasmid DNA is directly injected into tumors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63149/1/hum.1995.6.5-553.pd

Spatio-temporal Analysis of the Genetic Diversity of Arctic Rabies Viruses and Their Reservoir Hosts in Greenland

There has been limited knowledge on spatio-temporal epidemiology of zoonotic arctic fox rabies among countries bordering the Arctic, in particular Greenland. Previous molecular epidemiological studies have suggested the occurrence of one particular arctic rabies virus (RABV) lineage (arctic-3), but have been limited by a low number of available samples preventing in-depth high resolution phylogenetic analysis of RABVs at that time. However, an improved knowledge of the evolution, at a molecular level, of the circulating RABVs and a better understanding of the historical perspective of the disease in Greenland is necessary for better direct control measures on the island. These issues have been addressed by investigating the spatio-temporal genetic diversity of arctic RABVs and their reservoir host, the arctic fox, in Greenland using both full and partial genome sequences. Using a unique set of 79 arctic RABV full genome sequences from Greenland, Canada, USA (Alaska) and Russia obtained between 1977 and 2014, a description of the historic context in relation to the genetic diversity of currently circulating RABV in Greenland and neighboring Canadian Northern territories has been provided. The phylogenetic analysis confirmed delineation into four major arctic RABV lineages (arctic 1-4) with viruses from Greenland exclusively grouping into the circumpolar arctic-3 lineage. High resolution analysis enabled distinction of seven geographically distinct subclades (3.I - 3.VII) with two subclades containing viruses from both Greenland and Canada. By combining analysis of full length RABV genome sequences and host derived sequences encoding mitochondrial proteins obtained simultaneously from brain tissues of 49 arctic foxes, the interaction of viruses and their hosts was explored in detail. Such an approach can serve as a blueprint for analysis of infectious disease dynamics and virus-host interdependencies. The results showed a fine-scale spatial population structure in Greenland arctic foxes based on mitochondrial sequences, but provided no evidence for independent isolated evolutionary development of RABV in different arctic fox lineages. These data are invaluable to support future initiatives for arctic fox rabies control and elimination in Greenland

Genetic analysis of a rabies virus host shift event reveals within-host viral dynamics in a new host

Host shift events play an important role in epizootics as adaptation to new hosts can profoundly affect the spread of the disease and the measures needed to control it. During the late 1990s, an epizootic in Turkey resulted in a sustained maintenance of rabies virus (RABV) within the fox population. We used Bayesian inferences to investigate whole genome sequences from fox and dog brain tissues from Turkey to demonstrate that the epizootic occurred in 1997 (±1 year). Furthermore, these data indicated that the epizootic was most likely due to a host shift from locally infected domestic dogs, rather than an incursion of a novel fox or dog RABV. No evidence was observed for genetic adaptation to foxes at consensus sequence level and dN/dS analysis suggested purifying selection. Therefore, the deep sequence data were analysed to investigate the sub-viral population during a host shift event. Viral heterogeneity was measured in all RABV samples; viruses from the early period after the host shift exhibited greater sequence variation in comparison to those from the later stage, and to those not involved in the host shift event, possibly indicating a role in establishing transmission within a new host. The transient increase in variation observed in the new host species may represent virus replication within a new environment, perhaps due to increased replication within the CNS, resulting in a larger population of viruses, or due to the lack of host constraints present in the new host reservoir

Whole-genome sequencing and phylogenetic analysis of rabies viruses from Jordan

Human fatalities caused by rabies are rarely reported in Jordan; however, domestic animals are more likely to fall victim to rabies compared to wild animals, at least this is the case in Jordan due to the presence of canine rabies. In this study, twelve brain samples from domestic and wild animals suspected of being infected with rabies virus from different regions of Jordan were collected during 2019. Seven of them tested positive using the fluorescent antibody test and real-time SYBR RT-PCR assay. Five specimens were from stray dogs and two from foxes. The whole genome sequences were obtained from the positive samples. Sequence analysis showed that one dog virus from Al Quwaysimah city located in Amman governorate, was closely related to an Israeli strain belonging to a Cosmopolitan ME1a clade. The genomes of the remaining six viruses (four from dogs and two from foxes) collected from different areas of Jordan were genetically-related to each other and clustered together with sequences from Iran and Turkey; all belong to Cosmopolitan ME2 clade. These sequences were analyzed with six other Jordanian rabies virus nucleoprotein (N) gene sequences available in the public database, five of them belong to ME1a clade and one belongs to ME1b clade. Rabies virus whole genome data is scarce across the Middle East. This study provides a better understanding of the molecular epidemiology of rabies virus in the region

Experimental Lagos bat virus infection in straw-colored fruit bats: A suitable model for bat rabies in a natural reservoir species.

Rabies is a fatal neurologic disease caused by lyssavirus infection. Bats are important natural reservoir hosts of various lyssaviruses that can be transmitted to people. The epidemiology and pathogenesis of rabies in bats are poorly understood, making it difficult to prevent zoonotic transmission. To further our understanding of lyssavirus pathogenesis in a natural bat host, an experimental model using straw-colored fruit bats (Eidolon helvum) and Lagos bat virus, an endemic lyssavirus in this species, was developed. To determine the lowest viral dose resulting in 100% productive infection, bats in five groups (four bats per group) were inoculated intramuscularly with one of five doses, ranging from 100.1 to 104.1 median tissue culture infectious dose (TCID50). More bats died due to the development of rabies after the middle dose (102.1 TCID50, 4/4 bats) than after lower (101.1, 2/4; 101.1, 2/4) or higher (103.1, 2/4; 104.1, 2/4) doses of virus. In the two highest dose groups, 4/8 bats developed rabies. Of those bats that remained healthy 3/4 bats seroconverted, suggesting that high antigen loads can trigger a strong immune response that abrogates a productive infection. In contrast, in the two lowest dose groups, 3/8 bats developed rabies, 1/8 remained healthy and seroconverted and 4/8 bats remained healthy and did not seroconvert, suggesting these doses are too low to reliably induce infection. The main lesion in all clinically affected bats was meningoencephalitis associated with lyssavirus-positive neurons. Lyssavirus antigen was detected in tongue epithelium (5/11 infected bats) rather than in salivary gland epithelium (0/11), suggesting viral excretion via the tongue. Thus, intramuscular inoculation of 102.1 TCID50 of Lagos bat virus into straw-colored fruit bats is a suitable model for lyssavirus associated bat rabies in a natural reservoir host, and can help with the investigation of lyssavirus infection dynamics in bats