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The tent-making bat hepatitis B virus (TBHBV) is a hepadnavirus closely related to human hepatitis B virus. The ecology of TBHBV is unclear. We show that it is widespread and highly diversified in Peters’ tent-making bats (Uroderma bilobatum) within Panama, while local prevalence varied significantly between sample sites, ranging from 0 to 14.3%. Females showed significantly higher prevalence than males, and pregnant females were more often acutely infected than non-reproductive ones. The distribution of TBHBV in bats was significantly affected by forest cover, with higher infection rates in areas with lower forest cover. Our data indicate that loss of natural habitat may lead to positive feedback on the biotic factors driving infection possibility. These results underline the necessity of multidisciplinary studies for a better understanding of mechanisms in pathogen–host relationships and for predictions in disease ecology. Electronic supplementary material The online version of this article (10.1007/s10393-018-1387-5) contains supplementary material, which is available to authorized users.

The apicomplexan order Haemosporida is a clade of unicellular blood parasites that infect a variety of reptilian, avian and mammalian hosts. Among them are the agents of human malaria, parasites of the genus Plasmodium, which pose a major threat to human health. Illuminating the evolutionary history of Haemosporida may help us in understanding their enormous biological diversity, as well as tracing the multiple host switches and associated acquisitions of novel life-history traits. However, the deep-level phylogenetic relationships among major haemosporidian clades have remained enigmatic because the datasets employed in phylogenetic analyses were severely limited in either gene coverage or taxon sampling. Using a PCR-based approach that employs a novel set of primers, we sequenced fragments of 21 nuclear genes from seven haemosporidian parasites of the genera Leucocytozoon, Haemoproteus, Parahaemoproteus, Polychromophilus and Plasmodium. After addition of genomic data from 25 apicomplexan species, the unreduced alignment comprised 20,580 bp from 32 species. Phylogenetic analyses were performed based on nucleotide, codon and amino acid data employing Bayesian inference, maximum likelihood and maximum parsimony. All analyses resulted in highly congruent topologies. We found consistent support for a basal position of Leucocytozoon within Haemosporida. In contrast to all previous studies, we recovered a sister group relationship between the genera Polychromophilus and Plasmodium. Within Plasmodium, the sauropsid and mammal-infecting lineages were recovered as sister clades. Support for these relationships was high in nearly all trees, revealing a novel phylogeny of Haemosporida, which is robust to the choice of the outgroup and the method of tree inference.

Litomosoides spp. (Nematoda: Filarioidea: Onchocercidae) are of great interest for understanding dynamics of zoonoses because of their similarity to human-infecting filariae (e.g., Onchocerca volvulus). We encountered Litomosoides microfilaria in blood samples from 7 Panamanian bat species. Species were identified through morphological analysis. We assessed individual traits and immune response of the host individuals as well as host species ecology. We found 8 host-specific species/morphotypes of Litomosoides microfilariae in 5 bat species, with 5 individuals showing multiple infections. Our study includes the first records of Litomosoides brasiliensis in Artibeus jamaicensis and of Litomosoides-like and Litomosa-like microfilaria in Lophostoma silvicolum. Multiple binary regression analysis showed that the presence of specific Litomosoides species was influenced by host species, sex, and reproductive status.

Crimean Congo hemorrhagic fever virus (CCHFV) is a highly virulent tick-borne pathogen that causes hemorrhagic fever in humans. The geographic range of human CCHF cases largely reflects the presence of ticks. However, highly similar CCHFV lineages occur in geographically distant regions. Tick-infested migratory birds have been suggested, but not confirmed, to contribute to the dispersal. Bats have recently been shown to carry nairoviruses distinct from CCHFV. In order to assess the presence of CCHFV in a wide range of bat species over a wide geographic range, we analyzed 1,135 sera from 16 different bat species collected in Congo, Gabon, Ghana, Germany, and Panama. Using a CCHFV glycoprotein-based indirect immunofluorescence test (IIFT), we identified reactive antibodies in 10.0% (114/1,135) of tested bats, pertaining to 12/16 tested species. Depending on the species, 3.6%–42.9% of cave-dwelling bats and 0.6%–7.1% of foliage-living bats were seropositive (two-tailed t-test, p = 0.0447 cave versus foliage). 11/30 IIFT-reactive sera from 10 different African bat species had neutralizing activity in a virus-like particle assay. Neutralization of full CCHFV was confirmed in 5 of 7 sera. Widespread infection of cave-dwelling bats may indicate a role for bats in the life cycle and geographic dispersal of CCHFV.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis in tropical and temperate climates. Tropical genotypes 1 and 2 are associated with food-borne and waterborne transmission. Zoonotic reservoirs (mainly pigs, wild boar, and deer) are considered for genotypes 3 and 4, which exist in temperate climates. In view of the association of several zoonotic viruses with bats, we analyzed 3,869 bat specimens from 85 different species and from five continents for hepevirus RNA. HEVs were detected in African, Central American, and European bats, forming a novel phylogenetic clade in the family Hepeviridae . Bat hepeviruses were highly diversified and comparable to human HEV in sequence variation. No evidence for the transmission of bat hepeviruses to humans was found in over 90,000 human blood donations and individual patient sera. Full-genome analysis of one representative virus confirmed formal classification within the family Hepeviridae . Sequence- and distance-based taxonomic evaluations suggested that bat hepeviruses constitute a distinct genus within the family Hepeviridae and that at least three other genera comprising human, rodent, and avian hepeviruses can be designated. This may imply that hepeviruses invaded mammalian hosts nonrecently and underwent speciation according to their host restrictions. Human HEV-related viruses in farmed and peridomestic animals might represent secondary acquisitions of human viruses, rather than animal precursors causally involved in the evolution of human HEV.

To the Editor: The severe acute respiratory syndrome (SARS) outbreak of 2002–03 and the subsequent implication of bats as reservoir hosts of the causative agent, a coronavirus (CoV), prompted numerous studies of bats and the viruses they harbor. A novel clade 2c betacoronavirus, termed Middle East respiratory syndrome (MERS)–CoV, was recently identified as the causative agent of a severe respiratory disease that is mainly affecting humans on the Arabian Peninsula (1). Extending on previous work (2), we described European Pipistrellus bat–derived CoVs that are closely related to MERS-CoV (3). We now report the identification of a South Africa bat derived CoV that has an even closer phylogenetic relationship with MERS-CoV. During 2011–2012, fecal pellets were collected from 62 bats representing 13 different species in the KwaZulu-Natal and Western Cape Provinces of South Africa and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA). Details about the bat sample are available in the Technical Appendix. RNA was extracted by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Screening for CoVs was done by nested reverse transcription PCR using broadly reactive oligonucleotide primers targeting a conserved region in the RNA-dependent RNA polymerase (RdRp) gene (online Technical Appendix). PCR results were positive for 5 (8%) of the 62 specimens. PCR amplicons for 4 positive specimens yielded alphacoronavirus sequences related to recently described bat alphacoronaviruses from South Africa (4). The other positive specimen, termed PML/2011, was from an adult female Neoromicia cf. zuluensis bat sampled in 2011; the specimen yielded a novel betacoronavirus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC869678","term_id":"666386896"}}KC869678). Technical Appendix Figure 1 shows the distribution of this bat species. To obtain better phylogenetic resolution, we extended the 398-nt RdRp fragment generated by the screening PCR to 816 nt, as described (5). PML/2011 differed from MERS-CoV by only 1 aa exchange (0.3%) in the translated 816-nt RdRp gene fragment. Thus, PML/2011 was much more related to MERS-CoV than any other known virus. The amino acid sequence of the next closest known relatives of MERS-CoV, from European Pipistrellus bats (3), differed from MERS-CoV by 1.8%. The amino acid sequences of viruses from Nycteris bats in Ghana (3) and the 2c prototype bat CoVs, HKU4 and HKU5, from China (6) differed by 5.5%–7.7% from MERS-CoV. The smaller 152- to 396-nt RdRp fragments of 2c bat CoVs from a Hypsugo savii bat in Spain (7), bat guano in Thailand (8), and a Nyctinomops bat in Mexico (9) showed no or only partial overlap with the 816-nt fragment generated in this study; thus, a direct comparison could not be done. However, in their respective RdRp fragments, these CoVs yielded amino acid sequence distances of 3.5%–8.0% and were thus probably more distant from MERS-CoV than the virus described here. A Bayesian phylogenetic analysis of the 816-nt RdRp sequence confirmed the close relationship between PML/2011 and MERS-CoV (Figure). Their phylogenetic relatedness was as close as that of SARS-CoV and the most closely related bat coronavirus known, Rs672 from a Rhinolophus sinicus bat (Figure). Like PML/2011 and MERS-CoV, Rs672 and SARS-CoV showed only 1 aa exchange in the translated 816-nt RdRp fragment. To confirm this relatedness, we amplified and sequenced a short 269-nt sequence encompassing the 3′-terminus of the spike gene for PML/2011 (oligonucleotide primers available upon request from the authors). A partial spike gene–based phylogeny using this sequence yielded the same topology as that using the partial RdRp sequence (Technical Appendix Figure 2). Again, PML/2011 was most closely related to MERS-CoV, showing only a 10.9% aa sequence distance in this gene, which encodes the glycoprotein responsible for CoV attachment and cellular entry. This distance was less than the 13.3% aa sequence distance between MERS-CoV and the European Pipistrellus CoVs (3) and less than the 20.5%–27.3% aa sequence distance between MERS-CoV and HKU5 and between MERS-CoV and HKU4 (6) in the same sequence fragment. Figure Partial RNA-dependent RNA polymerase (RdRp) gene phylogeny, including the novel betacoronavirus from a Neoromicia zuluensis bat in South Africa (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC869678","term_id":"666386896"}}KC869678 ... Our results further support the hypothesis that, like human CoV-229E and SARS-CoV, ancestors of MERS-CoV might exist in Old World insectivorous bats belonging to the family Vespertilionidae, to which the genera Neoromicia and Pipistrellus belong (3). Knowledge of the close relatedness of PML/2011 and MERS-CoV, which contrasts with the more distant relatedness of CoVs in bats from the Americas and Asia, enables speculations of an African origin for bat reservoir hosts of MERS-CoV ancestors. This hypothesis is limited by a global sampling bias, the small sample size, and the single clade 2c betacoronavirus detection in this study. Still, a putative transfer of MERS-CoV ancestors from Africa to the Arabian Peninsula would parallel the transfer of other viruses (e.g., the exportation of Rift Valley fever virus from East Africa, which led to a severe outbreak in Saudi Arabia in 2000) (10). Studies of Vespertilionidae bats and potential intermediate hosts (e.g., carnivores and ungulates, such as camels) are urgently needed to elucidate the emergence of MERS-CoV. Such studies should focus on the Arabian Peninsula and Africa. Technical Appendix: Description of bat sampling, screened bat species, distribution of Neoromicia zuluensis bats, and spike gene phylogeny of the 2c betacoronavirus clade. Click here to view.(326K, pdf)

SUMMARYAnthropogenic influence on ecosystems, such as habitat fragmentation, impacts species diversity and interactions. There is growing evidence that degradation of habitats favours disease and hence affects ecosystem health. The prevalence of haemoparasites in the Common Fruit Bat (Artibeus jamaicensis) in a tropical lowland forest in Panamá was studied. We assessed the relation of haemoparasite to the general condition of the animals and tested for possible association of haemoparasite prevalence to habitat fragmentation, with special focus on trypanosomes. Overall, a total of 250 A. jamaicensis sampled from fragmented sites, here man-made, forested islands in Lake Gatùn, and sites in the adjacent, continuous forest in and around the Barro Colorado Nature Monument were examined. Using microscopy and DNA-sequencing 2 dominant types of haemoparasite infections, trypanosomes and Litomosoides (Nematoda) were identified. Trypanosome prevalence was significantly higher in bats from forest fragments, than in bats captured in continuous forest. We attribute this to the loss of species richness in forest fragments and specific characteristics of the fragments favouring trypanosome transmission, in particular changes in vegetation cover. Interestingly, the effect of habitat fragmentation on the prevalence of trypanosomes as multi-host parasites could not be observed in Litomosoides which probably has a higher host specificity and might be affected less by overall diversity loss.

Bats are likely natural hosts for a range of zoonotic viruses such as Marburg, Ebola, Rabies, as well as for various Corona-and Paramyxoviruses. In 2009/10, researchers discovered RNA of two novel influenza virus subtypes - H17N10 and H18N11 - in Central and South American fruit bats. The identification of bats as possible additional reservoir for influenza A viruses raises questions about the role of this mammalian taxon in influenza A virus ecology and possible public health relevance. As molecular testing can be limited by a short time window in which the virus is present, serological testing provides information about past infections and virus spread in populations after the virus has been cleared. This study aimed at screening available sera from 100 free-ranging, frugivorous bats (Eidolon helvum) sampled in 2009/10 in Ghana, for the presence of antibodies against the complete panel of influenza A haemagglutinin (HA) types ranging from H1 to H18 by means of a protein microarray platform. This technique enables simultaneous serological testing against multiple recombinant HA-types in 5 mu l of serum. Preliminary results indicate serological evidence against avian influenza subtype H9 in about 30% of the animals screened, with low-level cross-reactivity to phylogenetically closely related subtypes H8 and H12. To our knowledge, this is the first report of serological evidence of influenza A viruses other than H17 and H18 in bats. As avian influenza subtype H9 is associated with human infections, the implications of our findings from a public health context remain to be investigated.

Nature Communications 3: Article number: 796 (2012); Published: 24 April 2012; Updated: 23 January 2014. The authors inadvertently omitted Veronika M. Cottontail and Mirjam Knornschild, who collected samples in Panama and Costa Rica, from the author list. This has now been corrected in both the PDF and HTML versions of the Article.

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV., Author Summary The hepatitis C virus (HCV) is one of the most relevant causes of liver disease and cancer in humans. The lack of a small animal models represents an important hurdle on our way to understanding, treating, and preventing hepatitis C. The investigation of small mammals could identify virus infections similar to hepatitis C in animals that can be kept in laboratories, such as rodents, and can also yield insights into the evolution of those ancestral virus lineages out of which HCV developed. Here, we investigated a worldwide sample of 4,770 rodents, 2,939 bats, 210 horses and 858 cats and dogs for HCV-related viruses. New viruses were discovered in European bank voles (Myodes glareolus) and South African four-striped mice (Rhabdomys pumilio). The disease in bank voles was studied in more detail, suggesting that infection of the liver occurs with similar symptoms to those caused by HCV in humans. These rodents might thus enable the development of new laboratory models of hepatitis C. Moreover, the phylogenetic history of those viruses provides fascinating new ideas regarding the evolution of HCV ancestors.

Bats host a broad diversity of coronaviruses (CoVs), including close relatives of human pathogens. There is only limited data on neotropical bat CoVs. We analysed faecal, blood and intestine specimens from 1562 bats sampled in Costa Rica, Panama, Ecuador and Brazil for CoVs by broad-range PCR. CoV RNA was detected in 50 bats representing nine different species, both frugivorous and insectivorous. These bat CoVs were unrelated to known human or animal pathogens, indicating an absence of recent zoonotic spill-over events. Based on RNA-dependent RNA polymerase (RdRp)-based grouping units (RGUs) as a surrogate for CoV species identification, the 50 viruses represented five different alphacoronavirus RGUs and two betacoronavirus RGUs. Closely related alphacoronaviruses were detected in Carollia perspicillata and C. brevicauda across a geographical distance exceeding 5600 km. Our study expands the knowledge on CoV diversity in neotropical bats and emphasizes the association of distinct CoVs and bat host genera.

The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149–3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

We report TcBat, a recently described genetic lineage of Trypanosoma cruzi, in fruit-eating bats Artibeus from Panama. Infections were common (11.6% prevalence), but no other T. cruzi cruzi genotypes were detected. Phylogenetic analyses show an unambiguous association with Brazilian TcBat, but raise questions about the phylogenetic placement of this genotype using the 18S rRNA gene alone. However, analyses with three concatenated genes (18S rRNA, cytb, and H2B) moderately support TcBat as sister to the discrete typing unit (DTU) TcI. We demonstrate that short fragments (>500 bp) of the 18S rRNA gene are useful for identification of DTUs of T. cruzi, and provide reliable phylogenetic signal as long as they are analyzed within a matrix with reference taxa containing additional informative genes. TcBat forms a very distinctive monophyletic group that may be recognized as an additional DTU within T. cruzi cruzi.

The large virus family Paramyxoviridae includes some of the most significant human and livestock viruses, such as measles-, distemper-, mumps-, parainfluenza-, Newcastle disease-, respiratory syncytial virus and metapneumoviruses. Here we identify an estimated 66 new paramyxoviruses in a worldwide sample of 119 bat and rodent species (9,278 individuals). Major discoveries include evidence of an origin of Hendra- and Nipah virus in Africa, identification of a bat virus conspecific with the human mumps virus, detection of close relatives of respiratory syncytial virus, mouse pneumonia- and canine distemper virus in bats, as well as direct evidence of Sendai virus in rodents. Phylogenetic reconstruction of host associations suggests a predominance of host switches from bats to other mammals and birds. Hypothesis tests in a maximum likelihood framework permit the phylogenetic placement of bats as tentative hosts at ancestral nodes to both the major Paramyxoviridae subfamilies (Paramyxovirinae and Pneumovirinae). Future attempts to predict the emergence of novel paramyxoviruses in humans and livestock will have to rely fundamentally on these data., The large virus family, Paramyxoviridae, includes several human and livestock viruses. This study, testing 119 bat and rodent species distributed globally, identifies novel putative paramyxovirus species, providing data with potential uses in predictions of the emergence of novel paramyxoviruses in humans and livestock.

Bats, a globally distributed group of mammals with high ecological importance, are increasingly recognized as natural reservoir hosts for viral agents of significance to human and animal health. In the present study, we evaluated pools of blood samples obtained from two phylogenetically distant bat families, in particular from flying foxes (Pteropodidae), Eidolon helvum in West Africa, and from two species of New World leaf-nosed fruit bats (Phyllostomidae), Artibeus jamaicensis and Artibeus lituratus in Central America. A sequence-independent virus discovery technique (VIDISCA) was used in combination with high throughput sequencing to detect two novel parvoviruses: a PARV4-like virus named Eh-BtPV-1 in Eidolon helvum from Ghana and the first member of a putative new genus in Artibeus jamaicensis from Panama (Aj-BtPV-1). Those viruses were circulating in the corresponding bat colony at rates of 7–8%. Aj-BtPV-1 was also found in Artibeus lituratus (5.5%). Both viruses were detected in the blood of infected animals at high concentrations: up to 10E8 and to 10E10 copies/ml for Aj-BtPV-1 and Eh-BtPV-1 respectively. Eh-BtPV-1 was additionally detected in all organs collected from bats (brain, lungs, liver, spleen, kidneys and intestine) and spleen and kidneys were identified as the most likely sites where viral replication takes place. Our study shows that bat parvoviruses share common ancestors with known parvoviruses of humans and livestock. We also provide evidence that a variety of Parvovirinae are able to cause active infection in bats and that they are widely distributed in these animals with different geographic origin, ecologies and climatic ranges.