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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.

Viruses infect all forms of life and play critical roles as agents of disease, drivers of biochemical cycles and sources of genetic diversity for their hosts. Our understanding of viral diversity derives primarily from comparisons among host species, precluding insight into how intraspecific variation in host ecology affects viral communities or how predictable viral communities are across populations. Here we test spatial, demographic and environmental hypotheses explaining viral richness and community composition across populations of common vampire bats, which occur in diverse habitats of North, Central and South America. We demonstrate marked variation in viral communities that was not consistently predicted by a null model of declining community similarity with increasing spatial or genetic distances separating populations. We also find no evidence that larger bat colonies host greater viral diversity. Instead, viral diversity follows an elevational gradient, is enriched by juvenile‐biased age structure, and declines with local anthropogenic food resources as measured by livestock density. Our results establish the value of linking the modern influx of metagenomic sequence data with comparative ecology, reveal that snapshot views of viral diversity are unlikely to be representative at the species level, and affirm existing ecological theories that link host ecology not only to single pathogen dynamics but also to viral communities., https://doi.org/10.1111/mec.15306