High frequencies of nonviral colds and respiratory bacteria colonization among children in rural Western Uganda.

Introduction: Respiratory illness is the most common childhood disease globally, especially in developing countries. Previous studies have detected viruses in approximately 70-80% of respiratory illnesses. Methods: In a prospective cohort study of 234 young children (ages 3-11 years) and 30 adults (ages 22-51 years) in rural Western Uganda sampled monthly from May 2019 to August 2021, only 24.2% of nasopharyngeal swabs collected during symptomatic disease had viruses detectable by multiplex PCR diagnostics and metagenomic sequencing. In the remaining 75.8% of swabs from symptomatic participants, we measured detection rates of respiratory bacteria Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae by quantitative PCR. Results: 100% of children tested positive for at least one bacterial species. Detection rates were 87.2%, 96.8%, and 77.6% in children and 10.0%, 36.7%, and 13.3% for adults for H. influenzae, M. catarrhalis, and S. pneumoniae, respectively. In children, 20.8% and 70.4% were coinfected with two and three pathogens, respectively, and in adults 6.7% were coinfected with three pathogens but none were coinfected with two. Detection of any of the three pathogens was not associated with season or respiratory symptoms severity, although parsing detection status by symptoms was challenged by children experiencing symptoms in 80.3% of monthly samplings, whereas adults only reported symptoms 26.6% of the time. Pathobiont colonization in children in Western Uganda was significantly more frequent than in children living in high-income countries, including in a study of age-matched US children that utilized identical diagnostic methods. Detection rates were, however, comparable to rates in children living in other Sub-Saharan African countries. Discussion: Overall, our results demonstrate that nonviral colds contribute significantly to respiratory disease burden among children in rural Uganda and that high rates of respiratory pathobiont colonization may play a role. These conclusions have implications for respiratory health interventions in the area, such as increasing childhood immunization rates and decreasing air pollutant exposure.

Common cold viruses circulating in children threaten wild chimpanzees through asymptomatic adult carriers.

Reverse zoonotic respiratory diseases threaten great apes across Sub-Saharan Africa. Studies of wild chimpanzees have identified the causative agents of most respiratory disease outbreaks as "common cold" paediatric human pathogens, but reverse zoonotic transmission pathways have remained unclear. Between May 2019 and August 2021, we conducted a prospective cohort study of 234 children aged 3-11 years in communities bordering Kibale National Park, Uganda, and 30 adults who were forest workers and regularly entered the park. We collected 2047 respiratory symptoms surveys to quantify clinical severity and simultaneously collected 1989 nasopharyngeal swabs approximately monthly for multiplex viral diagnostics. Throughout the course of the study, we also collected 445 faecal samples from 55 wild chimpanzees living nearby in Kibale in social groups that have experienced repeated, and sometimes lethal, epidemics of human-origin respiratory viral disease. We characterized respiratory pathogens in each cohort and examined statistical associations between PCR positivity for detected pathogens and potential risk factors. Children exhibited high incidence rates of respiratory infections, whereas incidence rates in adults were far lower. COVID-19 lockdown in 2020-2021 significantly decreased respiratory disease incidence in both people and chimpanzees. Human respiratory infections peaked in June and September, corresponding to when children returned to school. Rhinovirus, which caused a 2013 outbreak that killed 10% of chimpanzees in a Kibale community, was the most prevalent human pathogen throughout the study and the only pathogen present at each monthly sampling, even during COVID-19 lockdown. Rhinovirus was also most likely to be carried asymptomatically by adults. Although we did not detect human respiratory pathogens in the chimpanzees during the cohort study, we detected human metapneumovirus in two chimpanzees from a February 2023 outbreak that were genetically similar to viruses detected in study participants in 2019. Our data suggest that respiratory pathogens circulate in children and that adults become asymptomatically infected during high-transmission times of year. These asymptomatic adults may then unknowingly carry the pathogens into forest and infect chimpanzees. This conclusion, in turn, implies that intervention strategies based on respiratory symptoms in adults are unlikely to be effective for reducing reverse zoonotic transmission of respiratory viruses to chimpanzees.

Respiratory disease patterns in rural Western Uganda, 2019-2022.

Introduction: Respiratory disease is a major cause of morbidity and mortality in the developing world, but prospective studies of temporal patterns and risk factors are rare. Methods: We studied people in rural Western Uganda, where respiratory disease is pervasive. We followed 30 adults (ages 22-51 years; 534 observations) and 234 children (ages 3-11 years; 1,513 observations) between May 2019 and July 2022 and collected monthly data on their respiratory symptoms, for a total of 2,047 case records. We examined associations between demographic and temporal factors and respiratory symptoms severity. Results: The timing of our study (before, during, and after the emergence of COVID-19) allowed us to document the effects of public health measures instituted in the region. Incidence rates of respiratory symptoms before COVID-19 lockdown were 568.4 cases per 1,000 person-months in children and 254.2 cases per 1,000 person-months in adults. These rates were 2.6 times higher than the 2019 global average for children but comparable for adults. Younger children (ages 3-6 years) had the highest frequencies and severities of respiratory symptoms. Study participants were most likely to experience symptoms in February, which is a seasonal pattern not previously documented. Incidence and severity of symptoms in children decreased markedly during COVID-19 lockdown, illustrating the broad effects of public health measures on the incidence of respiratory disease. Discussion: Our results demonstrate that patterns of respiratory disease in settings such as Western Uganda resemble patterns in developed economies in some ways (age-related factors) but not in others (increased incidence in children and seasonal pattern). Factors such as indoor air quality, health care access, timing of school trimesters, and seasonal effects (rainy/dry seasons) likely contribute to the differences observed.

Selective deforestation and exposure of African wildlife to bat-borne viruses.

Proposed mechanisms of zoonotic virus spillover often posit that wildlife transmission and amplification precede human outbreaks. Between 2006 and 2012, the palm Raphia farinifera, a rich source of dietary minerals for wildlife, was nearly extirpated from Budongo Forest, Uganda. Since then, chimpanzees, black-and-white colobus, and red duiker were observed feeding on bat guano, a behavior not previously observed. Here we show that guano consumption may be a response to dietary mineral scarcity and may expose wildlife to bat-borne viruses. Videos from 2017-2019 recorded 839 instances of guano consumption by the aforementioned species. Nutritional analysis of the guano revealed high concentrations of sodium, potassium, magnesium and phosphorus. Metagenomic analyses of the guano identified 27 eukaryotic viruses, including a novel betacoronavirus. Our findings illustrate how "upstream" drivers such as socioeconomics and resource extraction can initiate elaborate chains of causation, ultimately increasing virus spillover risk.

Novel Aquareovirus isolated from channel catfish (Ictalurus punctatus) used in mussel restoration efforts in Wisconsin.

Channel catfish (Ictalurus punctatus) are a food fish extensively reared in aquaculture facilities throughout the world and are also among the most abundant wild catfish species in North America, making them a popular target of anglers. Furthermore, channel catfish are important members of aquatic ecosystems; for example, they serve as a glochidial host for the endangered winged mapleleaf mussel (Quadrula fragosa), making them critical for conserving this species through hatchery-based restoration efforts. During a routine health inspection, a novel aquareovirus was isolated from channel catfish used in mussel propagation efforts at a fish hatchery in Wisconsin. This virus was isolated on brown bullhead cells (ATCC CCL-59) and identified through metagenomic sequencing as a novel member of the family Spinareoviridae, genus Aquareovirus. The virus genome consists of 11 segments, as is typical of the aquareoviruses, with phylogenetic relationships based on RNA-dependent RNA polymerase and major outer capsid protein amino acid sequences showing it to be most closely related to golden shiner virus (aquareovirus C) and aquareovirus C/American grass carp reovirus (aquareovirus G) respectively. The potential of the new virus, which we name genictpun virus 1 (GNIPV-1), to cause disease in channel catfish or other species remains unknown.

Five Species of Wild Freshwater Sport Fish in Wisconsin, USA, Reveal Highly Diverse Viromes.

Studies of marine fish have revealed distant relatives of viruses important to global fish and animal health, but few such studies exist for freshwater fish. To investigate whether freshwater fish also host such viruses, we characterized the viromes of five wild species of freshwater fish in Wisconsin, USA: bluegill (Lepomis macrochirus), brown trout (Salmo trutta), lake sturgeon (Acipenser fulvescens), northern pike (Esox lucius), and walleye (Sander vitreus). We analyzed 103 blood serum samples collected during a state-wide survey from 2016 to 2020 and used a metagenomic approach for virus detection to identify known and previously uncharacterized virus sequences. We then characterized viruses phylogenetically and quantified prevalence, richness, and relative abundance for each virus. Within these viromes, we identified 19 viruses from 11 viral families: Amnoonviridae, Circoviridae, Coronaviridae, Hepadnaviridae, Peribunyaviridae, Picobirnaviridae, Picornaviridae, Matonaviridae, Narnaviridae, Nudnaviridae, and Spinareoviridae, 17 of which were previously undescribed. Among these viruses was the first fish-associated coronavirus from the Gammacoronavirus genus, which was present in 11/15 (73%) of S. vitreus. These results demonstrate that, similar to marine fish, freshwater fish also harbor diverse relatives of viruses important to the health of fish and other animals, although it currently remains unknown what effect, if any, the viruses we identified may have on fish health.

Virome profiling of fig wasps (Ceratosolen spp.) reveals virus diversity spanning four realms.

We investigated the virome of agaonid fig wasps (Ceratosolen spp.) inside syconia ("fruits") of various Ficus trees fed upon by frugivores such as pteropodid bats in Sub-Saharan Africa. This virome includes representatives of viral families spanning four realms and includes near-complete genome sequences of three novel viruses and fragments of five additional potentially novel viruses evolutionarily associated with insects, fungi, plants, and vertebrates. Our study provides evidence that frugivorous animals are exposed to a plethora of viruses by coincidental consumption of fig wasps, which are obligate pollinators of figs worldwide.

VESPA: an optimized protocol for accurate metabarcoding-based characterization of vertebrate eukaryotic endosymbiont and parasite assemblages.

Protocols for characterizing taxonomic assemblages by deep sequencing of short DNA barcode regions (metabarcoding) have revolutionized our understanding of microbial communities and are standardized for bacteria, archaea, and fungi. Unfortunately, comparable methods for host-associated eukaryotes have lagged due to technical challenges. Despite 54 published studies, issues remain with primer complementarity, off-target amplification, and lack of external validation. Here, we present VESPA (Vertebrate Eukaryotic endoSymbiont and Parasite Analysis) primers and optimized metabarcoding protocol for host-associated eukaryotic community analysis. Using in silico prediction, panel PCR, engineered mock community standards, and clinical samples, we demonstrate VESPA to be more effective at resolving host-associated eukaryotic assemblages than previously published methods and to minimize off-target amplification. When applied to human and non-human primate samples, VESPA enables reconstruction of host-associated eukaryotic endosymbiont communities more accurately and at finer taxonomic resolution than microscopy. VESPA has the potential to advance basic and translational science on vertebrate eukaryotic endosymbiont communities, similar to achievements made for bacterial, archaeal, and fungal microbiomes.

Mussel mass mortality in the Clinch River, USA: metabolomics detects affected pathways and biomarkers of stress.

Biologists monitoring freshwater mussel (order Unionida) populations rely on behavioral, often subjective, signs to identify moribund ("sick") or stressed mussels, such as gaping valves and slow response to probing, and they lack clinical indicators to support a diagnosis. As part of a multi-year study to investigate causes of reoccurring mortality of pheasantshell (Ortmanniana pectorosa; synonym Actinonaias pectorosa) in the Clinch River, Virginia and Tennessee, USA, we analyzed the hemolymph metabolome of a subset of mussels from the 2018 sampling period. Mussels at the mortality sites were diagnosed in the field as affected (case) or unaffected (control) based on behavioral and physical signs. Hemolymph was collected in the field by non-lethal methods from the anterior adductor muscle for analysis. We used ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectroscopy to detect targeted and untargeted metabolites in hemolymph and compared metabolomic profiles by field assessment of clinical status. Targeted biomarker analysis found 13 metabolites associated with field assessments of clinical status. Of these, increased gamma-linolenic acid and N-methyl-l-alanine were most indicative of case mussels, while adenine and inosine were the best indicators of control mussels. Five pathways in the targeted analysis differed by clinical status; two of these, purine metabolism and glycerophospholipid metabolism, were also indicated in the untargeted analysis. In the untargeted nalysis, 22 metabolic pathways were associated with clinical status. Many of the impacted pathways in the case group were catabolic processes, such as degradation of amino acids and fatty acids. Hierarchical clustering analysis matched clinical status in 72% (18 of 25) of mussels, with control mussels more frequently (5 of 16) not matching clinical status. Our study demonstrated that metabolomic analysis of hemolymph is suitable for assessing mussel condition and complements field-based indicators of health.

Viruses of Freshwater Mussels during Mass Mortality Events in Oregon and Washington, USA.

Freshwater mussels (Unionida) are globally imperiled, in part due to largely unexplained mass mortality events (MMEs). While recent studies have begun to investigate the possibility that mussel MMEs in the Eastern USA may be caused by infectious diseases, mussels in the Western USA have received relatively little attention in this regard. We conducted a two-year epidemiologic investigation of the role of viruses in ongoing MMEs of the Western pearlshell (Margaritifera falcata) and the Western ridged mussel (Gonidea angulata) in the Chehalis River and Columbia River watersheds in the Western USA. We characterized viromes of mussel hemolymph from 5 locations in 2018 and 2020 using metagenomic methods and identified 557 viruses based on assembled contiguous sequences, most of which are novel. We also characterized the distribution and diversity of a previously identified mussel Gammarhabdovirus related to pathogenic finfish viruses. Overall, we found few consistent associations between viruses and mussel health status. Variation in mussel viromes was most strongly driven by location, with little influence from date, species, or health status, though these variables together only explained ~1/3 of variation in virome composition. Our results demonstrate that Western freshwater mussels host remarkably diverse viromes, but no single virus or combination of viruses appears to be associated with morbidity or mortality during MMEs. Our findings have implications for the conservation of imperiled freshwater mussels, including efforts to enhance natural populations through captive propagation.

Sarcina sp. as a presumptive cause of fatal acute gastric dilation and gastric emphysema in rhesus macaques.

A 4-y-old female and 3-y-old male rhesus macaque (Macaca mulatta), both housed in the same facility, died unexpectedly within 2 wk. Postmortem examination revealed severe gastric dilation in both macaques and gastric emphysema in the female macaque. Histologically, bacteria consistent with Sarcina sp. were present in both macaques within the lungs and lumen of the trachea, esophagus, and gastrointestinal (GI) tract without associated inflammation. Additionally, in the female macaque, the bacteria were found in the gastric mucosa and associated with emphysematous spaces in the gastric wall without associated inflammation. PCR and Sanger sequencing of amplicons were subsequently performed on GI contents and non-alimentary tissues from the 2 affected monkeys and on comparative samples from unaffected rhesus monkeys in the same facility and an adjacent primate facility. The cases were compared using the 2-tailed Fisher exact test (p-value at 95% confidence). PCR identified Sarcina in GI contents of both affected and unaffected monkeys (p = 0.6084) and in non-alimentary tissues of affected monkeys only (p = 0.0083). These results suggest that the presence of Sarcina sp. in non-alimentary tissues is associated with gastric distension, gas accumulation, and unexpected death in nonhuman primates.

Long-term assessment of relationships between changing environmental conditions and the physiology of southern Beaufort Sea polar bears (Ursus maritimus).

Climate change is influencing polar bear (Ursus maritimus) habitat, diet, and behavior but the effects of these changes on their physiology is not well understood. Blood-based biomarkers are used to assess the physiologic health of individuals but their usefulness for evaluating population health, especially as it relates to changing environmental conditions, has rarely been explored. We describe links between environmental conditions and physiologic functions of southern Beaufort Sea polar bears using data from blood samples collected from 1984 to 2018, a period marked by extensive environmental change. We evaluated associations between 13 physiologic biomarkers and circumpolar (Arctic oscillation index) and regional (wind patterns and ice-free days) environmental metrics and seasonal and demographic co-variates (age, sex, season, and year) known to affect polar bear ecology. We observed signs of dysregulation of water balance in polar bears following years with a lower annual Arctic oscillation index. In addition, liver enzyme values increased over time, which is suggestive of potential hepatocyte damage as the Arctic has warmed. Biomarkers of immune function increased with regional-scale wind patterns and the number of ice-free days over the Beaufort Sea continental shelf and were lower in years with a lower winter Arctic oscillation index, suggesting an increased allocation of energetic resources for immune processes under these conditions. We propose that the variation in polar bear immune and metabolic function is likely indicative of physiologic plasticity, a response that allows polar bears to remain in homeostasis even as they experience changes in nutrition and habitat in response to changing environments.

Viruses in saliva from sanctuary chimpanzees (Pan troglodytes) in Republic of Congo and Uganda.

Pathogen surveillance for great ape health monitoring has typically been performed on non-invasive samples, primarily feces, in wild apes and blood in sanctuary-housed apes. However, many important primate pathogens, including known zoonoses, are shed in saliva and transmitted via oral fluids. Using metagenomic methods, we identified viruses in saliva samples from 46 wild-born, sanctuary-housed chimpanzees at two African sanctuaries in Republic of Congo and Uganda. In total, we identified 20 viruses. All but one, an unclassified CRESS DNA virus, are classified in five families: Circoviridae, Herpesviridae, Papillomaviridae, Picobirnaviridae, and Retroviridae. Overall, viral prevalence ranged from 4.2% to 87.5%. Many of these viruses are ubiquitous in primates and known to replicate in the oral cavity (simian foamy viruses, Retroviridae; a cytomegalovirus and lymphocryptovirus; Herpesviridae; and alpha and gamma papillomaviruses, Papillomaviridae). None of the viruses identified have been shown to cause disease in chimpanzees or, to our knowledge, in humans. These data suggest that the risk of zoonotic viral disease from chimpanzee oral fluids in sanctuaries may be lower than commonly assumed.

Plasticity, Paralogy, and Pseudogenization: Rhabdoviruses of Freshwater Mussels Elucidate Mechanisms of Viral Genome Diversification and the Evolution of the Finfish-Infecting Rhabdoviral Genera.

Viruses in the family Rhabdoviridae display remarkable genomic variation and ecological diversity. This plasticity occurs despite the fact that, as negative sense RNA viruses, rhabdoviruses rarely if ever recombine. Here, we describe nonrecombinatorial evolutionary processes leading to genomic diversification in the Rhabdoviridae inferred from two novel rhabdoviruses of freshwater mussels (Mollusca: Bivalvia: Unionida). Killamcar virus 1 (KILLV-1) from a plain pocketbook (Lampsilis cardium) is closely related phylogenetically and transcriptionally to finfish-infecting viruses in the subfamily Alpharhabdovirinae. KILLV-1 offers a novel example of glycoprotein gene duplication, differing from previous examples in that the paralogs overlap. Evolutionary analyses reveal a clear pattern of relaxed selection due to subfunctionalization in rhabdoviral glycoprotein paralogs, which has not previously been described in RNA viruses. Chemarfal virus 1 (CHMFV-1) from a western pearlshell (Margaritifera falcata) is closely related phylogenetically and transcriptionally to viruses in the genus Novirhabdovirus, the sole recognized genus in the subfamily Gammarhabdovirinae, representing the first known gammarhabdovirus of a host other than finfish. The CHMFV-1 G-L noncoding region contains a nontranscribed remnant gene of precisely the same length as the NV gene of most novirhabdoviruses, offering a compelling example of pseudogenization. The unique reproductive strategy of freshwater mussels involves an obligate parasitic stage in which larvae encyst in the tissues of finfish, offering a plausible ecological mechanism for viral host-switching. IMPORTANCE Viruses in the family Rhabdoviridae infect a variety of hosts, including vertebrates, invertebrates, plants and fungi, with important consequences for health and agriculture. This study describes two newly discovered viruses of freshwater mussels from the United States. One virus from a plain pocketbook (Lampsilis cardium) is closely related to fish-infecting viruses in the subfamily Alpharhabdovirinae. The other virus from a western pearlshell (Margaritifera falcata) is closely related to viruses in the subfamily Gammarhabdovirinae, which until now were only known to infect finfish. Genome features of both viruses provide new evidence of how rhabdoviruses evolved their extraordinary variability. Freshwater mussel larvae attach to fish and feed on tissues and blood, which may explain how rhabdoviruses originally jumped between mussels and fish. The significance of this research is that it improves our understanding of rhabdovirus ecology and evolution, shedding new light on these important viruses and the diseases they cause.

Multi-Year Comparison of Community- and Species-Level West Nile Virus Antibody Prevalence in Birds from Atlanta, Georgia and Chicago, Illinois, 2005-2016.

West Nile virus (WNV) is prevalent in the United States but shows considerable variation in transmission intensity. The purpose of this study was to compare patterns of WNV seroprevalence in avian communities sampled in Atlanta, Georgia and Chicago, Illinois during a 12-year period (Atlanta 2010-2016; Chicago 2005-2012) to reveal regional patterns of zoonotic activity of WNV. WNV antibodies were measured in wild bird sera using ELISA and serum neutralization methods, and seroprevalence among species, year, and location of sampling within each city were compared using binomial-distributed generalized linear mixed-effects models. Seroprevalence was highest in year-round and summer-resident species compared with migrants regardless of region; species explained more variance in seroprevalence within each city. Northern cardinals were the species most likely to test positive for WNV in each city, whereas all other species, on average, tested positive for WNV in proportion to their sample size. Despite similar patterns of seroprevalence among species, overall seroprevalence was higher in Atlanta (13.7%) than in Chicago (5%). Location and year of sampling had minor effects, with location explaining more variation in Atlanta and year explaining more variation in Chicago. Our findings highlight the nature and magnitude of regional differences in WNV urban ecology.

Viruses in sanctuary chimpanzees across Africa.

Infectious disease is a major concern for both wild and captive primate populations. Primate sanctuaries in Africa provide critical protection to thousands of wild-born, orphan primates confiscated from the bushmeat and pet trades. However, uncertainty about the infectious agents these individuals potentially harbor has important implications for their individual care and long-term conservation strategies. We used metagenomic next-generation sequencing to identify viruses in blood samples from chimpanzees (Pan troglodytes) in three sanctuaries in West, Central, and East Africa. Our goal was to evaluate whether viruses of human origin or other "atypical" or unknown viruses might infect these chimpanzees. We identified viruses from eight families: Anelloviridae, Flaviviridae, Genomoviridae, Hepadnaviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, and Rhabdoviridae. The majority (15/26) of viruses identified were members of the family Anelloviridae and represent the genera Alphatorquevirus (torque teno viruses) and Betatorquevirus (torque teno mini viruses), which are common in chimpanzees and apathogenic. Of the remaining 11 viruses, 9 were typical constituents of the chimpanzee virome that have been identified in previous studies and are also thought to be apathogenic. One virus, a novel tibrovirus (Rhabdoviridae: Tibrovirus) is related to Bas-Congo virus, which was originally thought to be a human pathogen but is currently thought to be apathogenic, incidental, and vector-borne. The only virus associated with disease was rhinovirus C (Picornaviridae: Enterovirus) infecting one chimpanzee subsequent to an outbreak of respiratory illness at that sanctuary. Our results suggest that the blood-borne virome of African sanctuary chimpanzees does not differ appreciably from that of their wild counterparts, and that persistent infection with exogenous viruses may be less common than often assumed.

Cascading effects of climate change on recreational marine flats fishes and fisheries.

Tropical and subtropical coastal flats are shallow regions of the marine environment at the intersection of land and sea. These regions provide myriad ecological goods and services, including recreational fisheries focused on flats-inhabiting fishes such as bonefish, tarpon, and permit. The cascading effects of climate change have the potential to negatively impact coastal flats around the globe and to reduce their ecological and economic value. In this paper, we consider how the combined effects of climate change, including extremes in temperature and precipitation regimes, sea level rise, and changes in nutrient dynamics, are causing rapid and potentially permanent changes to the structure and function of tropical and subtropical flats ecosystems. We then apply the available science on recreationally targeted fishes to reveal how these changes can cascade through layers of biological organization-from individuals, to populations, to communities-and ultimately impact the coastal systems that depend on them. We identify critical gaps in knowledge related to the extent and severity of these effects, and how such gaps influence the effectiveness of conservation, management, policy, and grassroots stewardship efforts.

Viruses of Atlantic Bonefish (Albula vulpes) in Florida and the Caribbean show geographic patterns consistent with population declines.

Atlantic Bonefish (Albula vulpes) are economically important due to their popularity with recreational anglers. In the State of Florida, USA, bonefish population numbers declined by approximately 60% between the 1990s and 2015. Habitat loss, water quality impairment, chemical inputs, and other anthropogenic factors have been implicated as causes, but the role of pathogens has been largely overlooked, especially with respect to viruses. We used a metagenomic approach to identify and quantify viruses in the blood of 103 A. vulpes sampled throughout their Western Atlantic range, including populations in Florida that have experienced population declines and populations in Belize, Mexico, Puerto Rico, and The Bahamas that have remained apparently stable. We identified four viruses, all of which are members of families known to infect marine fishes (Flaviviridae, Iflaviridae, Narnaviridae, and Nodaviridae), but all of which were previously undescribed. Bonefish from Florida and Mexico had higher viral richness (numbers of distinct viruses per individual fish) than fish sampled from other areas, and bonefish from the Upper Florida Keys had the highest prevalence of viral infection (proportion of positive fish) than fish sampled from any other location. Bonefish from Florida also had markedly higher viral loads than fish sampled from any other area, both for a novel narnavirus and for all viruses combined. Bonefish viruses may be indicators of environmentally driven physiological and immunological compromise, causes of ill health, or both. Supplementary information: The online version contains supplementary material available at 10.1007/s10641-022-01306-9.

Freshwater Mussels Show Elevated Viral Richness and Intensity during a Mortality Event.

Freshwater mussels (Unionida) are among the world's most imperiled taxa, but the relationship between freshwater mussel mortality events and infectious disease is largely unstudied. We surveyed viromes of a widespread and abundant species (mucket, Actinonaias ligamentina; syn: Ortmanniana ligamentina) experiencing a mortality event of unknown etiology in the Huron River, Michigan, in 2019-2020 and compared them to viromes from mucket in a healthy population in the St. Croix River, Wisconsin and a population from the Clinch River, Virginia and Tennessee, where a mortality event was affecting the congeneric pheasantshell (Actinonaias pectorosa; syn: Ortmanniana pectorosa) population. We identified 38 viruses, most of which were associated with mussels collected during the Huron River mortality event. Viral richness and cumulative viral read depths were significantly higher in moribund mussels from the Huron River than in healthy controls from each of the three populations. Our results demonstrate significant increases in the number and intensity of viral infections for freshwater mussels experiencing mortality events, whereas individuals from healthy populations have a substantially reduced virome comprising a limited number of species at low viral read depths.

Straw-Colored Fruit Bats (Eidolon helvum) and Their Bat Flies (Cyclopodia greefi) in Nigeria Host Viruses with Multifarious Modes of Transmission.

Background: Bat flies (Diptera: Hippoboscoidea: Nycteribiidae and Streblidae) are increasingly appreciated as hosts of "bat-associated" viruses. We studied straw-colored fruit bats (Eidolon helvum) and their nycteribiid bat flies (Cyclopodia greefi) in Nigeria to investigate the role of bat flies in vectoring or maintaining viruses. Methods: We captured bats and bat flies across northern Nigeria. We used metagenomics to identify viruses in 40 paired samples (20 flies from 20 bats). We characterized viruses using genomic and phylogenetic methods, and we compared infection frequencies in bats and their bat flies. Results: In 20 bats, we detected two individuals (10%) infected with eidolon helvum parvovirus 1 (BtPAR4) (Parvoviridae; Tetraparvovirus), previously described in Ghana, and 10 bats (50%) with a novel parvovirus in the genus Amdoparvovirus (Parvoviridae). The amdoparvoviruses include Aleutian disease virus of mink and viruses of other carnivores but have not previously been identified in bats or in Africa. In 20 paired bat flies (each fly from 1 bat) all (100%) were infected with a novel virus in the genus Sigmavirus (Rhabdoviridae). The sigmaviruses include vertically transmitted viruses of dipterans. We did not detect BtPAR4 in any bat flies, and we did not detect the novel sigmavirus in any bats. However, we did detect the novel amdoparvovirus in 3 out of 20 bat flies sampled (15%), including in 2 bat flies from bats in which we did not detect this virus. Discussion: Our results show that bats and their bat flies harbor some viruses that are specific to mammals and insects, respectively, and other viruses that may transmit between bats and arthropods. Our results also greatly expand the geographic and host range of the amdoparvoviruses and suggest that some could be transmitted by arthropods. Bat flies may serve as biological vectors, mechanical vectors, or maintenance hosts for "bat-associated" viruses.