No abstract

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. K E Y W O R D SChiroptera, community assembly, demography, Desmodus rotundus, elevational gradient, infectious diseases, population structure, shotgun metagenomics, virome, wildlife disease | 27 BERGNER Et al.

Despite the global investment in One Health disease surveillance, it remains difficult and costly to identify and monitor the wildlife reservoirs of novel zoonotic viruses. Statistical models can guide sampling target prioritisation, but the predictions from any given model might be highly uncertain; moreover, systematic model validation is rare, and the drivers of model performance are consequently under-documented. Here, we use the bat hosts of betacoronaviruses as a case study for the data-driven process of comparing and validating predictive models of probable reservoir hosts. In early 2020, we generated an ensemble of eight statistical models that predicted host–virus associations and developed priority sampling recommendations for potential bat reservoirs of betacoronaviruses and bridge hosts for SARS-CoV-2. During a time frame of more than a year, we tracked the discovery of 47 new bat hosts of betacoronaviruses, validated the initial predictions, and dynamically updated our analytical pipeline. We found that ecological trait-based models performed well at predicting these novel hosts, whereas network methods consistently performed approximately as well or worse than expected at random. These findings illustrate the importance of ensemble modelling as a buffer against mixed-model quality and highlight the value of including host ecology in predictive models. Our revised models showed an improved performance compared with the initial ensemble, and predicted more than 400 bat species globally that could be undetected betacoronavirus hosts. We show, through systematic validation, that machine learning models can help to optimise wildlife sampling for undiscovered viruses and illustrates how such approaches are best implemented through a dynamic process of prediction, data collection, validation, and updating.

Bats (Order: Chiroptera) have been widely studied as reservoir hosts for viruses of concern for human and animal health. However, whether bats are equally competent hosts of non-viral pathogens such as bacteria remains an important open question. Here, we surveyed blood and saliva samples of vampire bats from Peru and Belize for hemotropic Mycoplasma spp. (hemoplasmas), bacteria that can cause inapparent infection or anemia in hosts. 16S rRNA gene amplification of blood showed 67% (150/223) of common vampire bats (Desmodus rotundus) were infected by hemoplasmas. Sequencing of the 16S rRNA gene amplicons revealed three novel genotypes that were phylogenetically related but not identical to hemoplasmas described from other (non-vampire) bat species, rodents, humans, and non-human primates. Hemoplasma prevalence in vampire bats was highest in non-reproductive and young individuals, did not differ by country, and was relatively stable over time (i.e., endemic). Metagenomics from pooled D. rotundus saliva from Peru detected non-hemotropic Mycoplasma species and hemoplasma genotypes phylogenetically similar to those identified in blood, providing indirect evidence for potential direct transmission of hemoplasmas through biting or social contacts. This study demonstrates vampire bats host several novel hemoplasmas and sheds light on risk factors for infection and basic transmission routes. Given the high frequency of direct contacts that arise when vampire bats feed on humans, domestic animals, and wildlife, the potential of these bacteria to be transmitted between species should be investigated in future work.

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.

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.

Hepatitis delta virus (HDV) is an unusual RNA agent that replicates using host machinery but exploits hepatitis B virus (HBV) to mobilize its spread within and between hosts. In doing so, HDV enhances the virulence of HBV. How this seemingly improbable hyperparasitic lifestyle emerged is unknown, but it underpins the likelihood that HDV and related deltaviruses may alter other host–virus interactions. Here, we show that deltaviruses diversify by transmitting between mammalian species. Among 96,695 RNA sequence datasets, deltaviruses infected bats, rodents, and an artiodactyl from the Americas but were absent from geographically overrepresented Old World representatives of each mammalian order, suggesting a relatively recent diversification within the Americas. Consistent with diversification by host shifting, both bat and rodent-infecting deltaviruses were paraphyletic, and coevolutionary modeling rejected cospeciation with mammalian hosts. In addition, a 2-y field study showed common vampire bats in Peru were infected by two divergent deltaviruses, indicating multiple introductions to a single host species. One vampire bat-associated deltavirus was detected in the saliva of up to 35% of individuals, formed phylogeographically compartmentalized clades, and infected a sympatric bat, illustrating horizontal transmission within and between species on ecological timescales. Consistent absence of HBV-like viruses in two deltavirus-infected bat species indicated acquisitions of novel viral associations during the divergence of bat and human-infecting deltaviruses. Our analyses support an American zoonotic origin of HDV and reveal prospects for future cross-species emergence of deltaviruses. Given their peculiar life history, deltavirus host shifts will have different constraints and disease outcomes compared to ordinary animal pathogens.

31 51 52 Coronaviruses are a diverse family of positive-sense, single-stranded RNA viruses, found widely 53 in mammals and birds 1 . They have a broad host range, a high mutation rate, and the largest 54 genomes of any RNA viruses, but they have also evolved mechanisms for RNA proofreading and 55repair, which help to mitigate the deleterious effects of a high recombination rate acting over a 56 large genome 2 . Consequently, coronaviruses fit the profile of viruses with high zoonotic potential. 57There are seven human coronaviruses (two in the genus Alphacoronavirus and five in 58Betacoronavirus), of which three are highly pathogenic in humans: SARS-CoV, SARS-CoV-2, and 59MERS-CoV. These three are zoonotic and widely agreed to have evolutionary origins in bats 3-6 . 60 61Our collective experience with both SARS-CoV and MERS-CoV illustrate the difficulty of tracing 62 specific animal hosts of emerging coronaviruses. During the 2002-2003 SARS epidemic, SARS-63 CoV was traced to the masked palm civet (Paguma larvata) 7 , but the ultimate origin remained 64 unknown for several years. Horseshoe bats (family Rhinolophidae: Rhinolophus) were implicated 65 as reservoir hosts in 2005, but their SARS-like viruses were not identical to circulating human 66 strains 4 . Stronger evidence from 2017 placed the most likely evolutionary origin of SARS-CoV in 67 Rhinolophus ferrumequinum or potentially R. sinicus 8 . Presently, there is even less certainty in the 68 origins of MERS-CoV, although spillover to humans occurs relatively often through contact with 69 dromedary camels (Camelus dromedarius). A virus with 100% nucleotide identity in a ~200 base 70 pair region of the polymerase gene was detected in Taphozous bats (family Emballonuridae) in 71 Saudi Arabia 9 ; however, based on spike gene similarity, other sources treat HKU4 virus from 72 Tylonycteris bats (family Vespertilionidae) in China as the closest-related bat virus 10,11 . Several 73 bat coronaviruses have shown close relation to MERS-CoV, with a surprisingly broad geographic 74 distribution from Mexico to China 12,13,14,15 . 75 76 Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome 77 coronavirus-2 (SARS-CoV-2), a novel virus with presumed evolutionary origins in bats. Although 78 the earliest cases were linked to a wildlife market, contact tracing was limited, and there has been 79 no definitive identification of the wildlife contact that resulted in spillover nor a true "index case." 80 Two bat viruses are closely related to SARS-CoV-2: RaTG13 bat CoV from Rhinolophus affinis 81 (96% identical overall), and RmYN02 bat CoV from Rhinolophus malayanus (97% identical in one 82 gene but only 61% in the receptor-binding domain and with less overall similarity) 6,16 . The 83 divergence time between these bat viruses and human SARS-CoV-2 has been estimated as 40-50 84 years 17 , suggesting that the main host(s) involved in spillover remain unknown. Evidence of viral 85 recombination in pangolins has been proposed but is unresolved 17 . S...