Human and bovine respiratory syncytial viruses (HRSV and BRSV), along with pneumonia virus of mice (PVM) are the members of the genus Pneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae. Although both HRSV and BRSV have been associated with important diseases in human and livestock, there is no clearcut description of the molecular aspects of their pathogenesis. For HRSV, the lack of a suitable study model is one of the main reasons hampering the study of aspects of pathogenesis of the virus. HRSV infects a wide range of animal models, however most of the common laboratory animal models are not sufficiently permissive to study the infectivity of the virus. PVM naturally infects mice and causes a disease indistinguishable from that of HRSV in humans. Two strains of PVM have been described: strain 15 (Warwick) which is not pathogenic and strain J3666 which is highly pathogenic. The main difference between these two strains lies in the organisation of the gene encoding the attachment (G) glycoprotein. The G gene in strain J3666 has two ORFs. The larger second ORF codes for the G glycoprotein and is located downstream of the first ORF which has no known function. The strain 15 G gene also contains two ORFs but in this case both the first and main ORFs overlap each other. The aim of the project was to investigate the molecular basis for pathogenesis of PVM as a model for pneumoviruses. As a first step, the pathogenesis of PVM strain J3666 was revaluated and the effect of consecutive tissue culture passages on the pathogenicity of the virus was examined. It was shown that consecutive passages of PVM strain J3666 caused attenuation of the virus. To investigate the possible mutations causing the attenuation the majority of the virus genome was sequenced from three passage stocks where the transition from pathogenic to non-pathogenic occurred. No differences in the genome sequences for the three passage stocks were found. However, sequence analysis of individual clones of the SH and G genes of the viruses showed evidence that the stocks contained a mixed population of sequences. A robust reverse genetics system was established to rescue recombinant PVM from cDNA using co-transfection of plasmids coding for the ribonucleoprotein complex of the virus (N, L, M2-1 and P proteins) and a cDNA copy of the virus genome cloned under the control of the bacteriophage T7 RNA polymerase. Using this system, four viruses differing in their G gene organisation were generated and used to infect mice to study the effect of mutations on pathogenicity. It was shown that the viruses with the G gene of strain 15 (Warwick) lacking the first ORF manifest a modest increase in their pathogenicity compared to the non-pathogenic PVM strain 15(Warwick) parent. The recombinant viruses containing the G gene organisation of strain J3666 showed the highest level of pathogenicity. The reverse genetics system was used to study the role of the first ORF in G glycoprotein expression. Using a dicistronic minigenome construct, the effect of the presence or absence of the first ORF in both the strain 15 and strain J3666 G gene organisation was studied. It was shown that the presence of the first ORF of the G gene in the strain 15 (Warwick) suppressed the expression of the G protein, while the first ORF in the strain J3666 did not have any significant effect on G protein expression.

The emergence and spread of insecticide resistance compromises the control of mosquito borne diseases that are responsible for millions of deaths every year in tropical and subtropical areas. Mathematical modelling is a valuable tool that can be used to explore different aspects of the development and management of insecticide resistance. We have used standard population genetics theory and ecological modelling techniques for developing models to evaluate the spread of resistance in the field. We started by developing a methodology to quantify the strength of selection for resistance occurring in nature. We used data from Mexico on the mosquito Aedes aegypti and a maximum likelihood methodology to estimate the selection and dominance coefficients driving the evolution of resistance in the field. We additionally explored the impact of poor data collection, data that combine information from different locations, and the consequences of selection and dominance coefficients varying over the sampling time period. This analysis highlighted factors highly relevant to field work such as the need for frequent surveillance in discrete sentinel sites. The use of insecticidal bed nets represents the primary tool for the prevention of malaria worldwide. It is of extreme importance to maintain their efficacy against mosquitoes, which has been undermined by the development of insecticide resistance. We assed the contribution of a novel design of bed nets in delaying insecticide resistance while at the same time determining the important parameters in driving resistance in an heterogeneous environment. We showed that this new bed net can indeed contribute to the delay of the spread of resistance, but surprisingly could have the reverse effect in specific circumstances. Finally we developed a model for the vector of malaria, that considers the stage-structured nature of the mosquito life cycle and, most importantly, explicitly incorporates insecticide resistance. It can be used to understand the population dynamics of mosquitoes throughout their entire lifecycle while analysing the impact of vector control interventions, alone and in combination, and the spread of insecticide resistance that those interventions induce. We showed that targeting the larval stages has the greatest effect on the adult population followed by targeting non host-seeking female adults. According to our results, low levels of resistance can induce failure of interventions, and the rate of spread of resistance is faster when insecticides target the larval stages.

Microbial communities play important roles in organismal and ecosystem health. High throughput sequencing has revolutionized our understanding of host-associated microbial communities, but the viral component of these communities remains poorly characterized relative to microbes such as bacteria, particularly in non-human hosts. This knowledge gap has implications for global health, as viruses originating in wildlife are responsible for recent disease outbreaks in humans and domestic animals. Although studies have identified factors differentiating viral communities between species, we have little understanding of the variability of viral communities within species. Comparative studies of viral communities are therefore necessary to characterize novel taxa and to evaluate the ecological factors influencing intraspecific viral diversity and distribution. Bats are recognized as “special” reservoirs for viruses because they are associated with diverse viral communities and display deep evolutionary relationships with individual viral taxa. Common vampire bats (Desmodus rotundus) represent a particularly interesting system in which to investigate viral communities, as they are obligate blood feeders that interact ecologically with many different host species, providing opportunities for the acquisition of diverse viruses. The overall objective of this thesis was to advance our understanding of intraspecific wildlife-associated viral communities using an established field network of common vampire bat colonies across Peru. Specifically, I developed a novel method for comparative viral community studies, characterized the viral communities of vampire bats, and examined the ecological correlates of vampire bat viral diversity across Peru. Metagenomic sequencing is a promising technique for comparative studies of viral communities in wildlife, but there is a need to first develop standardized methods that can be applied to samples collected in the field. In Chapter 2 I developed a shotgun metagenomic sequencing approach to characterizing viral communities from non-invasive samples. Specifically, I optimized extraction and sequencing protocols using fecal and oropharyngeal swabs collected from common vampire bats in Peru. Two preliminary sequencing runs were performed, the results of which motivated four pilot studies in which I tested how different storage media, nucleic acid extraction procedures, and enrichment steps affect the viral community detected. Metagenomic sequencing revealed viral contamination of fetal bovine serum, a component of viral transport medium, suggesting that swabs should be stored in RNALater or another non-biological medium. Extraction and qPCR tests were performed on swabs inoculated with known concentrations of virus, which revealed that nucleic acid should be directly extracted from swabs rather than from supernatant or pelleted material. Metagenomic sequencing of paired samples was used to test enrichment by ribosomal RNA depletion and light DNAse treatment, which both reduced host and bacterial nucleic acid in samples and improved virus detection. A bioinformatic pipeline was developed specifically for processing vampire bat shotgun viral metagenomic data. Finally, the optimized protocol was applied to twelve pooled samples from seven localities in Peru, and read subsampling demonstrated that the viral communities detected were consistent at commonly attained depths of sequencing. The protocol developed in this chapter enables minimally biased comparative viral community studies in non-invasive samples collected from wildlife. Having a detailed understanding of viral diversity in key wildlife hosts is an important first step in evaluating the risk of zoonotic disease emergence, but we still lack a holistic view of viral communities in many species including vampire bats. In Chapter 3, I used the metagenomic sequencing protocol developed in Chapter 2 to thoroughly characterize viral communities in the saliva and feces of vampire bats captured across Peru. Viruses were detected from a range of natural host groups including vertebrate-associated taxa that were potentially infecting vampire bats, bacteriophages associated with gut bacteria, and plant- or insect-infecting viruses potentially acquired from the environment. There were broad differences between fecal and saliva viral communities, showing evidence of body habitat compartmentalization. Overall, results established that vampire bat viral communities differ between body habitats and suggested that, for the vertebrate-infecting families analyzed, novel viruses mostly fall within bat-specific clades, without evidence of livestock or humans acting as a major source of viral diversity in vampire bats. Interspecific differences in ecological and life history traits are known to impact viral richness in bats, but the factors structuring viral communities within bat species are less well understood. In Chapter 4, I examined the spatial, demographic and environmental correlates of intraspecific viral diversity in vampire bats. Three measures of viral diversity were calculated at the colony level: richness, a novel measure of taxonomic diversity, and community composition. Generalized linear models were then used to test the effects of broad scale and local ecological variables on saliva and fecal viral diversity. The results showed for the first time that ecological variables can influence intraspecific viral diversity. In summary, the work presented in this thesis advances our understanding of wildlife-associated viral communities in an ecologically important bat host. Future directions in comparative wildlife viral metagenomics, as discussed in Chapter 5, will include exploring the determinants of viral communities across host species, environments and time.

Infectious diseases pose a significant threat to animal and human health across the globe, with much of the burden falling on low-income countries. Despite efforts to control many of these diseases, very few have ever been eradicated. Their dynamics are often embedded in complex, heterogeneous landscapes defined by interacting population and landscape level processes. As such, landscape heterogeneity plays a key role in driving disease transmission and persistence. Incorporating landscape heterogeneity in studies of pathogen dynamics is challenging but the accessibility of data, particularly next generation sequencing data, has opened new avenues of research. Landscape epidemiology involves using an integrated approach to understand spatial patterns of disease, using methods that combine landscape genetics, ecology and epidemiology. in this thesis I use these integrative methods to determine the underlying mechanisms facilitating the spread and persistence of canine rabies virus in Tanzania. Whole genome level characterisation of rabies virus samples was achieved and used in combination with cutting-edge inference techniques to explore spatial patterns of rabies at different spatial scales. Phylogeographic patterns were able to characterise spatial scales of endemic rabies transmission in Tanzania, uncovering strong viral population structure at sub-continental levels with evidence of a more fluid dispersal dynamic at local ( less than 100km2 area) spatial scales . Within-country phylogeographic patterns revealed large regional movements within Tanzania that could be attributed to human-mediated movements and revealed the presence of multiple co-circulating lineages within a single administrative district. Finely resolved incidence data from the Serengeti District complemented with whole genome sequences enabled the exploration of local scales of transmission in more detail. By extending phylogeographic diffusion models to incorporate landscape heterogeneity I was able to uncover evidence supporting landscape predictors of rabies diffusion. While much of the spatial structure was attributable to the effects of isolation by distance, landscape predictors had discernible effects on diffusion. In particular, rivers appeared to act as a barrier to dispersal and road networks facilitated diffusion and I found evidence to support vaccination as an effective control measure for canine rabies in the Serengeti District. Importantly, I also found evidence to support vaccination as resistance to diffusion and therefore an effective control measure for dog rabies. As a complementary approach a space-time-genetic algorithm was used to determine who-infected-whom in the Serengeti District. The model explicitly accounted for the possibility of exogenous sources of infection and how to incorporate genetic data available for only a proportion of samples. Direct transmission events were estimated between 42% of observed cases and highlighted the co-circulation of two major lineages in both time and space. Direct transmission events predominantly occurred over very small distances, less than 1km, but a large proportion of cases had unobserved sources that could represent transmission from dogs in neighbouring regions or larger indirect transmission events. A future development of the model is to delineate between these possibilities to assess the true contribution of exogenous sources to the system dynamic. Ultimately these integrative models are at an early stage of development but highlight the power of genetic data to delineate fine-scale transmission patterns. The results from this thesis suggest that landscape features such as rivers could be exploited as barriers in step-wise vaccination campaigns and highlight the utility of genetic surveillance to monitor control and elimination as rabies management progresses.

The endangered population of Amur tigers (Panthera tigris altaica) in the Russian Far East (RFE) faces an increasing risk of extinction due to infection with canine distemper virus (CDV). Short-lasting CDV infections are unlikely to be maintained in small populations of species with limited connectivity like tigers, where viruses fade out as susceptible hosts are depleted. Multi-host pathogens can persist in more abundant host species that can act as reservoirs of infection for threatened populations. This study combines assessments of host demography, serology and viral phylogeny to establish the relative contribution of domestic dogs and small bodied wild mesocarnivores to the maintenance of CDV, and as sources of infection for tigers. No antibodies were detected among tigers sampled prior to 2000 (n=19), but were measured in 35.7% of tigers in subsequent years (n=56), with at least five discrete transmission events occurring in one well-monitored population. Viral sequences from three tigers and one Far Eastern leopard (P. pardus orientalis) aligned within the Arctic-like clade of CDV, and shared recent common ancestry with viruses from 22 other wild carnivores from the region. Extensive spatial mixing of wild carnivore lineages suggested long chains of transmission consistent with a maintenance population. The exposure of tigers following 2000 coincides with increases in sable (Martes zibellina) numbers and hunting pressure, which could lead to greater pathogen prevalence and potential for spill over from a wild reservoir. The ratio of humans to dogs in rural areas in the RFE are among the lowest in the world (1.73), but the overall number of dogs has been stable during the period of increased CDV exposure in tigers. The only CDV sequence obtained from dogs shared high identity with Asia-4 clade viruses from dogs in Thailand, and was distantly related to wildlife sequences from the RFE. Serum antibodies were detected in dogs in all 26 communities where households were surveyed, but seroprevalence was higher in remote, less densely populated areas, suggesting possible transmission from wildlife. Although the maintenance of CDV in Russian dogs remains unconfirmed, the strong support for a wildlife reservoir limits options for managing the impact of CDV on tiger populations. The high turnover of large and often inaccessible populations of mesocarnivores combines with limitations in vaccine safety, efficacy and delivery, to render the control of CDV in a wildlife reservoir untenable. Managing the impact of CDV on Amur tigers must therefore focus on restoring the size and integrity of remaining tiger populations to withstand future outbreaks. The safety and efficacy of vaccine products for tigers should also be investigated, for use in low coverage vaccination strategies that could enhance the long-term persistence of tiger populations.