Viral communities in vampire bats: geographical variation and ecological drivers

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

Genetic diversity, infection prevalence, and possible transmission routes of Bartonella spp. in vampire bats

Bartonella spp. are globally distributed bacteria that cause endocarditis in humans and domestic animals. Recent work has suggested bats as zoonotic reservoirs of some human Bartonella infections; however, the ecological and spatiotemporal patterns of infection in bats remain largely unknown. Here we studied the genetic diversity, prevalence of infection across seasons and years, individual risk factors, and possible transmission routes of Bartonella in populations of common vampire bats (Desmodus rotundus) in Peru and Belize, for which high infection prevalence has previously been reported. Phylogenetic analysis of the gltA gene for a subset of PCR-positive blood samples revealed sequences that were related to Bartonella described from vampire bats from Mexico, other Neotropical bat species, and streblid bat flies. Sequences associated with vampire bats clustered significantly by country but commonly spanned Central and South America, implying limited spatial structure. Stable and nonzero Bartonella prevalence between years supported endemic transmission in all sites. The odds of Bartonella infection for individual bats was unrelated to the intensity of bat flies ectoparasitism, but nearly all infected bats were infested, which precluded conclusive assessment of support for vector-borne transmission. While metagenomic sequencing found no strong evidence of Bartonella DNA in pooled bat saliva and fecal samples, we detected PCR positivity in individual saliva and feces, suggesting the potential for bacterial transmission through both direct contact (i.e., biting) and environmental (i.e., fecal) exposures. Further investigating the relative contributions of direct contact, environmental, and vector-borne transmission for bat Bartonella is an important next step to predict infection dynamics within bats and the risks of human and livestock exposures

Identification and characterization of microsatellite loci in two socially complex old world tropical babblers (Family Timaliidae)

Background: Although the highest diversity of birds occurs in tropical regions, little is known about the genetic mating systems of most tropical species. We describe microsatellite markers isolated in the chestnut-crested yuhina (Staphida everetti), endemic to the island of Borneo, and the grey-throated babbler (Stachyris nigriceps), widely distributed across Southeast Asia. Both species belong to the avian family Timaliidae and are highly social, putatively cooperatively breeding birds in which helpers attend the nests of members of their social group. We obtained DNA from individuals in social groups breeding in Kinabalu Park, Malaysian Borneo. Results: We used a shotgun sequencing approach and 454-technology to identify 36 microsatellite loci in the yuhina and 40 in the babbler. We tested 13 primer pairs in yuhinas and 20 in babblers and characterized eight polymorphic loci in 20 unrelated female yuhinas and 21 unrelated female babblers. Polymorphism at the yuhina loci ranged from 3 to 9 alleles, observed heterozygosities from 0.58 to 1.00, and expected heterozygosities from 0.64 to 0.81. Polymorphism at the babbler loci ranged from 3 to 12 alleles, observed heterozygosities from 0.14 to 0.90 and expected heterozygosities from 0.14 to 0.87. One locus in the yuhina deviated significantly from Hardy–Weinberg equilibrium. We detected nonrandom allele associations between two pairs of microsatellite loci in each species. Conclusions: Microsatellite markers will be used to describe the genetic mating system of these socially complex species and to measure genetic parentage and relatedness within social groups

European colonization, not Polynesian arrival, impacted population size and genetic diversity in the critically endangered New Zealand Kākāpō.

Island endemic species are often vulnerable to decline and extinction following human settlement, and the genetic study of historical museum specimens can be useful in understanding these processes. The kākāpō (Strigops habroptilus) is a critically endangered New Zealand parrot that was formerly widespread and abundant. It is well established that both Polynesian and European colonization of New Zealand impacted the native avifauna, but the timeframe and severity of impacts have differed depending on species. Here, we investigated the relative importance of the 2 waves of human settlement on kākāpō decline, using microsatellites and mitochondrial DNA (mtDNA) to characterize recent kākāpō genetic and demographic history. We analyzed samples from 49 contemporary individuals and 54 museum specimens dating from 1884 to 1985. Genetic diversity decreased significantly between historical and contemporary kākāpō, with a decline in mean number of microsatellite alleles from 6.15 to 3.08 and in number of mtDNA haplotypes from 17 to 3. Modeling of demographic history indicated a recent population bottleneck linked to the period of European colonization (approximately 5 generations ago) but did not support a major decline linked to Polynesian settlement. Effective population size estimates were also larger for historical than contemporary kākāpō. Our findings inform contemporary kākāpō management by indicating the timeframe and possible cause of the bottleneck, which has implications for the management of extant genetic diversity. We demonstrate the broader utility of a historical perspective in understanding causes of decline and managing extinction risk in contemporary endangered species

Complete Alphacoronavirus genome sequence from common vampire bats in Peru

Bats host diverse coronaviruses, including taxa capable of pandemic spread in humans. We report the genome of an alphacoronavirus from a neotropical bat species (Desmodus rotundus) in Peru, which contributes to our understanding of bat coronaviruses in nature

Temporal patterns of vampire bat rabies and host connectivity in Belize

In the Neotropics, vampire bats (Desmodus rotundus ) are the main reservoir host for rabies, a highly fatal encephalitis caused by viruses in the genus Lyssavirus . Although patterns of rabies virus exposure and infection have been well studied for vampire bats in South America and Mexico, exploring the ecology of vampire bat rabies in other regions is crucial for predicting risks to livestock and humans. In Belize, rabies outbreaks in livestock have increased in recent years, underscoring the need for systematic data on viral dynamics in vampire bats. In this study, we examine the first three years of a longitudinal study on the ecology of vampire bat rabies in northern Belize. Rabies seroprevalence in bats was high across years (29%–80%), suggesting active and endemic virus circulation. Across two locations, the seroprevalence time series per site were inversely related and out of phase by at least a year. Microsatellite data demonstrated historic panmixia of vampire bats, and mark–recapture detected rare but contemporary inter‐site dispersal. This degree of movement could facilitate spatial spread of rabies virus but is likely insufficient to synchronize infection dynamics, which offers one explanation for the observed phase lag in seroprevalence. More broadly, our analyses suggest frequent transmission of rabies virus within and among vampire bat roosts in northern Belize and highlight the need for future spatiotemporal, phylogenetic and ecological studies of vampire bat rabies in Central America

Detection of Trypanosoma cruzi in the saliva of diverse neotropical bats

Trypanosoma cruzi is widely reported in bats, yet transmission routes remain unclear. We present evidence from metagenomic sequence data that T. cruzi occurs in the saliva of diverse Neotropical bats. Phylogenetic analyses demonstrated that the bat‐associated T. cruzi sequences described here formed part of a bat‐specific clade, suggesting an independent transmission cycle. Our results highlight the value in repurposing metagenomic data generated for viral discovery to reveal insights into the biology of other parasites. Evaluating whether the presence of T. cruzi in the saliva of two hematophagous bat species represents an ecological route for zoonotic transmission of Chagas disease is an interesting avenue for future research

Characterizing and evaluating the zoonotic potential of novel viruses discovered in vampire bats

The contemporary surge in metagenomic sequencing has transformed knowledge of viral diversity in wildlife. However, evaluating which newly discovered viruses pose sufficient risk of infecting humans to merit detailed laboratory characterization and surveillance remains largely speculative. Machine learning algorithms have been developed to address this imbalance by ranking the relative likelihood of human infection based on viral genome sequences, but are not yet routinely applied to viruses at the time of their discovery. Here, we characterized viral genomes detected through metagenomic sequencing of feces and saliva from common vampire bats (Desmodus rotundus) and used these data as a case study in evaluating zoonotic potential using molecular sequencing data. Of 58 detected viral families, including 17 which infect mammals, the only known zoonosis detected was rabies virus; however, additional genomes were detected from the families Hepeviridae, Coronaviridae, Reoviridae, Astroviridae and Picornaviridae, all of which contain human-infecting species. In phylogenetic analyses, novel vampire bat viruses most frequently grouped with other bat viruses that are not currently known to infect humans. In agreement, machine learning models built from only phylogenetic information ranked all novel viruses similarly, yielding little insight into zoonotic potential. In contrast, genome composition-based machine learning models estimated different levels of zoonotic potential, even for closely related viruses, categorizing one out of four detected hepeviruses and two out of three picornaviruses as having high priority for further research. We highlight the value of evaluating zoonotic potential beyond ad hoc consideration of phylogeny and provide surveillance recommendations for novel viruses in a wildlife host which has frequent contact with humans and domestic animals

Using noninvasive metagenomics to characterize viral communities from wildlife

Microbial communities play an important role in organismal and ecosystem health. While high‐throughput metabarcoding has revolutionized the study of bacterial communities, generating comparable viral communities has proven elusive, particularly in wildlife samples where the diversity of viruses and limited quantities of viral nucleic acid present distinctive challenges. Metagenomic sequencing is a promising solution for studying viral communities, but the lack of standardized methods currently precludes comparisons across host taxa or localities. Here, we developed an untargeted shotgun metagenomic sequencing protocol to generate comparable viral communities from noninvasively collected faecal and oropharyngeal swabs. Using samples from common vampire bats (Desmodus rotundus), a key species for virus transmission to humans and domestic animals, we tested how different storage media, nucleic acid extraction procedures and enrichment steps affect viral community detection. Based on finding viral contamination in foetal bovine serum, we recommend storing swabs in RNAlater or another nonbiological medium. We recommend extracting nucleic acid directly from swabs rather than from supernatant or pelleted material, which had undetectable levels of viral RNA. Results from a low‐input RNA library preparation protocol suggest that ribosomal RNA depletion and light DNase treatment reduce host and bacterial nucleic acid, and improve virus detection. Finally, applying our approach to twelve pooled samples from seven localities in Peru, we showed that detected viral communities saturated at the attained sequencing depth, allowing unbiased comparisons of viral community composition. Future studies using the methods outlined here will elucidate the determinants of viral communities across host species, environments and time