Interaction between MHC diversity and constitution, gut microbiota and Astrovirus infections in a neotropical bat.

Astroviruses (AstVs) infect numerous mammalian species including reservoirs such as bats. Peptides encoded by the genes of the highly polymorphic Major Histocompatibility Complex (MHC) form the first line of host defence against pathogens. Aside from direct involvement in mounting adaptive immune responses, MHC class II genes are hypothesized to regulate gut commensal diversity and shape the production of immune-modulatory substances by microbes, indirectly affecting host susceptibility. Despite initial empirical evidence for the link between host MHC and the microbiota, associations among these factors remain largely unknown. To fill this gap, we examined MHC allelic diversity and constitution, the gut bacterial community and abundance pattern of a wild population of a neotropical bat (Artibeus jamaicensis) challenged by AstV infections. First, we show an age-dependent relationship between the host MHC class II diversity and constitution and the gut microbiota in AstV-uninfected bats. Crucially, these associations changed in AstV-infected bats. Additionally, we identify changes in the abundance of specific bacterial taxa linked to the presence of certain MHC supertypes and AstV infection. We suggest changes in the microbiota to be either a result of AstV infection or the MHC-mediated modulation of microbial communities. The latter could subsequently affect microbe-mediated immunity and resistance against AstV infection. Our results emphasize that the reciprocal nature of host immune genetics, gut microbial diversity and pathogen infection require attention, which are particularly important given their repercussions for disease susceptibility and severity in wild animal populations with a history of zoonotic spillover and frequent human contact.

Natural co-infection of divergent hepatitis B and C virus homologues in carnivores.

In humans, co-infection of hepatitis B and C viruses (HBV, HCV) is common and aggravates disease outcome. Infection-mediated disease aggravation is poorly understood, partly due to lack of suitable animal models. Carnivores are understudied for hepatitis virus homologues. We investigated Mexican carnivores (ringtails, Bassariscus astutus) for HBV and HCV homologues. Three out of eight animals were infected with a divergent HBV termed ringtail HBV (RtHBV) at high viral loads of 5 × 109 -1.4 × 1010 copies/ml serum. Two of the RtHBV-infected animals were co-infected with a divergent hepacivirus termed ringtail hepacivirus (RtHV) at 4 × 106 -7.5 × 107 copies/ml in strain-specific qRT-PCR assays. Immunofluorescence assays relying on HBV core and RtHV NS3/4a proteins indicated that none of the animals had detectable hepadnavirus core-specific antibodies, whereas one RtHV-infected animal had concomitant RtHV-specific antibodies at 1:800 end-point titre. RtHBV and RtHV complete genomes showed typical HBV and HCV structure and length. All RtHBV genomes were identical, whereas RtHV genomes showed four amino acid substitutions located predominantly in the E1/E2-encoding genomic regions. Both RtHBV (>28% genomic nucleotide sequence distance) and RtHV (>30% partial NS3/NS5B amino acid sequence distance) formed new species within their virus families. Evolutionary analyses showed that RtHBV grouped with HBV homologues from different laurasiatherian hosts (carnivores, bats, and ungulates), whereas RtHV grouped predominantly with rodent-borne viruses. Ancestral state reconstructions showed that RtHV, but not RtHBV, likely emerged via a non-recent host switch involving rodent-borne hepacivirus ancestors. Conserved hepatitis virus infection patterns in naturally infected ringtails indicate that carnivores may be promising animal models to understand HBV/HCV co-infection.

A hepatitis B virus causes chronic infections in equids worldwide.

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 109 mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na+/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection.

At Least Seven Distinct Rotavirus Genotype Constellations in Bats with Evidence of Reassortment and Zoonotic Transmissions.

Bats host many viruses pathogenic to humans, and increasing evidence suggests that rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America, and Africa were PCR-positive for RVA, and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, which included evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite various levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses.IMPORTANCE The increased research on bat coronaviruses after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) allowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general, and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents sheds light on the vast genetic diversity of rotaviruses and also hints at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

Potential zoonotic sources of SARS-CoV-2 infections.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing coronavirus disease-2019 (COVID-19) likely has evolutionary origins in other animals than humans based on genetically related viruses existing in rhinolophid bats and pangolins. Similar to other animal coronaviruses, SARS-CoV-2 contains a functional furin cleavage site in its spike protein, which may broaden the SARS-CoV-2 host range and affect pathogenesis. Whether ongoing zoonotic infections are possible in addition to efficient human-to-human transmission remains unclear. In contrast, human-to-animal transmission can occur based on evidence provided from natural and experimental settings. Carnivores, including domestic cats, ferrets and minks, appear to be particularly susceptible to SARS-CoV-2 in contrast to poultry and other animals reared as livestock such as cattle and swine. Epidemiologic evidence supported by genomic sequencing corroborated mink-to-human transmission events in farm settings. Airborne transmission of SARS-CoV-2 between experimentally infected cats additionally substantiates the possibility of cat-to-human transmission. To evaluate the COVID-19 risk represented by domestic and farmed carnivores, experimental assessments should include surveillance and health assessment of domestic and farmed carnivores, characterization of the immune interplay between SARS-CoV-2 and carnivore coronaviruses, determination of the SARS-CoV-2 host range beyond carnivores and identification of human risk groups such as veterinarians and farm workers. Strategies to mitigate the risk of zoonotic SARS-CoV-2 infections may have to be developed in a One Health framework and non-pharmaceutical interventions may have to consider free-roaming animals and the animal farming industry.

Mammalian deltavirus without hepadnavirus coinfection in the neotropical rodent Proechimys semispinosus.

Hepatitis delta virus (HDV) is a human hepatitis-causing RNA virus, unrelated to any other taxonomic group of RNA viruses. Its occurrence as a satellite virus of hepatitis B virus (HBV) is a singular case in animal virology for which no consensus evolutionary explanation exists. Here we present a mammalian deltavirus that does not occur in humans, identified in the neotropical rodent species Proechimys semispinosus The rodent deltavirus is highly distinct, showing a common ancestor with a recently described deltavirus in snakes. Reverse genetics based on a tandem minus-strand complementary DNA genome copy under the control of a cytomegalovirus (CMV) promoter confirms autonomous genome replication in transfected cells, with initiation of replication from the upstream genome copy. In contrast to HDV, a large delta antigen is not expressed and the farnesylation motif critical for HBV interaction is absent from a genome region that might correspond to a hypothetical rodent large delta antigen. Correspondingly, there is no evidence for coinfection with an HBV-related hepadnavirus based on virus detection and serology in any deltavirus-positive animal. No other coinfecting viruses were detected by RNA sequencing studies of 120 wild-caught animals that could serve as a potential helper virus. The presence of virus in blood and pronounced detection in reproductively active males suggest horizontal transmission linked to competitive behavior. Our study establishes a nonhuman, mammalian deltavirus that occurs as a horizontally transmitted infection, is potentially cleared by immune response, is not focused in the liver, and possibly does not require helper virus coinfection.

Cross-order host switches of hepatitis C-related viruses illustrated by a novel hepacivirus from sloths.

The genealogy of the hepatitis C virus (HCV) and the genus Hepacivirus remains elusive despite numerous recently discovered animal hepaciviruses (HVs). Viruses from evolutionarily ancient mammals might elucidate the HV macro-evolutionary patterns. Here, we investigated sixty-seven two-toed and nine three-toed sloths from Costa Rica for HVs using molecular and serological tools. A novel sloth HV was detected by reverse transcription polymerase chain reaction (RT-PCR) in three-toed sloths (2/9, 22.2%; 95% confidence interval (CI), 5.3-55.7). Genomic characterization revealed typical HV features including overall polyprotein gene structure, a type 4 internal ribosomal entry site in the viral 5'-genome terminus, an A-U-rich region and X-tail structure in the viral 3'-genome terminus. Different from other animal HVs, HV seropositivity in two-toed sloths was low at 4.5 per cent (3/67; CI, 1.0-12.9), whereas the RT-PCR-positive three-toed sloths were seronegative. Limited cross-reactivity of the serological assay implied exposure of seropositive two-toed sloths to HVs of unknown origin and recent infections in RT-PCR-positive animals preceding seroconversion. Recent infections were consistent with only 9 nucleotide exchanges between the two sloth HVs, located predominantly within the E1/E2 encoding regions. Translated sequence distances of NS3 and NS5 proteins and host comparisons suggested that the sloth HV represents a novel HV species. Event- and sequence distance-based reconciliations of phylogenies of HVs and of their hosts revealed complex macro-evolutionary patterns, including both long-term evolutionary associations and host switches, most strikingly from rodents into sloths. Ancestral state reconstructions corroborated rodents as predominant sources of HV host switches during the genealogy of extant HVs. Sequence distance comparisons, partial conservation of critical amino acid residues associated with HV entry and selection pressure signatures of host genes encoding entry and antiviral protein orthologs were consistent with HV host switches between genetically divergent mammals, including the projected host switch from rodents into sloths. Structural comparison of HCV and sloth HV E2 proteins suggested conserved modes of hepaciviral entry. Our data corroborate complex macro-evolutionary patterns shaping the genus Hepacivirus, highlight that host switches are possible across highly diverse host taxa, and elucidate a prominent role of rodent hosts during the Hepacivirus genealogy.

Pathogen-associated selection on innate immunity genes (TLR4, TLR7) in a neotropical rodent in landscapes differing in anthropogenic disturbance.

Toll-like receptors (TLRs) form part of the innate immune system and can recognize structurally conserved pathogen-associated molecular pattern (PAMP) molecules. Their functional importance in the resistance to pathogens has been documented in laboratory experimental settings and in humans. TLR diversity, however, has been rarely investigated in wildlife species. How the genetic diversity of TLRs is associated with various pathogens and how it is shaped by habitat disturbance are understudied. Therefore, we investigated the role of genetic diversity in the functionally important parts of TLR4 and TLR7 genes in resistance towards gastrointestinal nematodes and Hepacivirus infection. We chose a generalist study species, the rodent Proechimys semispinosus, because it is highly abundant in three Panamanian landscapes that differ in their degree of anthropogenic modification. We detected only two TLR7 haplotypes that differed by one synonymous single-nucleotide polymorphism (SNP) position. The TLR4 variability was higher, and we detected four TLR4 haplotypes that differed at one synonymous SNP and at three amino acid positions within the leucine-rich repeat region. Only TLR4 haplotypes had different frequencies in each landscape. Using generalized linear models, we found evidence that nematode loads and virus prevalence were influenced by both specific TLR4 haplotypes and landscape. Here, the variable "landscape" served as a surrogate for the important influential ecological factors distinguishing landscapes in our study, i.e. species diversity and host population density. Individuals carrying the common TLR4_Ht1 haplotype were less intensely infected by the most abundant strongyle nematode. Individuals carrying the rare TLR4_Ht3 haplotype were all Hepacivirus-positive, where those carrying the rare haplotype TLR4_Ht4 were less often infected by Hepacivirus than individuals with other haplotypes. Our study highlights the role of TLR diversity in pathogen resistance and the importance of considering immune genetic as well as ecological factors in order to understand the effects of anthropogenic changes on wildlife health.

Sloths host Anhanga virus-related phleboviruses across large distances in time and space.

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod-borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two-toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two-toed (Choloepus hoffmanni, n = 65) and three-toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT-PCR. A clinically healthy adult Hoffmann's two-toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 107  RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non-synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro-evolutionary processes associated with immune evasion during the genealogy of sloth-associated phleboviruses. However, gene-wide mean dN/dS ratios were low at 0.02-0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth-associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely-related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV-related phleboviruses with the two extant species of two-toed sloths.

Highly diversified shrew hepatitis B viruses corroborate ancient origins and divergent infection patterns of mammalian hepadnaviruses.

Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.

Evolutionary biology of human hepatitis viruses.

Hepatitis viruses are major threats to human health. During the last decade, highly diverse viruses related to human hepatitis viruses were found in animals other than primates. Herein, we describe both surprising conservation and striking differences of the unique biological properties and infection patterns of human hepatitis viruses and their animal homologues, including transmission routes, liver tropism, oncogenesis, chronicity, pathogenesis and envelopment. We discuss the potential for translation of newly discovered hepatitis viruses into preclinical animal models for drug testing, studies on pathogenesis and vaccine development. Finally, we re-evaluate the evolutionary origins of human hepatitis viruses and discuss the past and present zoonotic potential of their animal homologues.

Host Biology and Anthropogenic Factors Affect Hepadnavirus Infection in a Neotropical Bat.

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.

Astrovirus infections induce age-dependent dysbiosis in gut microbiomes of bats.

Astroviruses (AstV) are a major cause of diarrhoea in children. Interestingly, some wildlife species, including bats, remain phenotypically asymptomatic after infection. Disease symptoms, however, may only be less visible in bats and enteric viruses may indeed perturb their gut microbial communities. Gut microbiomes represent an important driver of immune defence mechanisms but potential effects of enteric virus-host microbiome interactions are largely unexplored. Using bats as a natural model system, we show that AstV-infections affect the gut microbiome, with the strength of the effect depending on host age. The gut microbial α- and ß-diversity and the predicted microbial functional orthologs decreased in young bats but surprisingly increased in adult AstV + bats. The abundance of bacterial taxa characteristic for healthy microbiomes was strongly reduced in young AstV+ bats, possibly attributable to their immature immune system. Regardless of age, pathogen-containing genera exhibited negative interactions with several commensal taxa and increased after AstV-infection, leading to pathobiont-like shifts in the gut microbiome of all infected bats. Thus, in apparently healthy bats, AstV-infections disturb gut bacterial homeostasis, possibly increasing previously suppressed health risks by promoting co-infections. If similar processes are present in humans, the effects of enteric virus infections might have longer-term impacts extending beyond the directly observed symptoms.

Ecological drivers of Hepacivirus infection in a neotropical rodent inhabiting landscapes with various degrees of human environmental change.

Anthropogenic environmental change can impact community and population traits such as species diversity and population densities, which have been shown to influence the prevalence of viruses in wildlife reservoirs. In particular, host species resilient to changes in their natural habitat may increase in numbers, which in turn can affect the prevalence of directly transmitted viruses. We have carried out a survey of small mammal communities in three tropical landscapes differing in their degree of environmental change in Central Panama and investigated the effects of community changes on Hepacivirus prevalence. The modification of continuous habitat into partly connected or isolated habitat patches during the past century was linked to changes in species diversity and species assemblages, which was further associated with shifts in the abundance of generalist marsupial (Didelphis marsupialis, Philander opossum) and rodent (Proechimys semispinosus) species. The latter has become dominant in isolated habitat patches and was the only identified Hepacivirus host in our study system. Our analyses suggest that, in addition to the effects of host age and sex, host population density in interaction with sex ratio is a crucial predictor of infection probability. Although we found no significant relationships between species diversity per se and infection probability, the lowest prevalence detected in the landscape with the highest species diversity indicates that shifts in species assemblages (e.g. changes in the presence and abundance of marsupial predators) impact the host's intraspecific contact rates, the probability of virus transmission and, thus, the virus prevalence. Our study additionally provides important data on the influence of human-induced landscape changes on infection probability and, therefore, on virus prevalence in wildlife and emphasizes the importance of a landscape-scale approach with concomitant consideration of the complex interactions between ecological factors.

A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses.

BACKGROUND & AIMS: All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. METHODS: We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. RESULTS: We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. CONCLUSIONS: Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. LAY SUMMARY: The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.

Seasonal Fluctuations of Astrovirus, But Not Coronavirus Shedding in Bats Inhabiting Human-Modified Tropical Forests.

Emerging infectious diseases (EIDs) are considered a major threat to global health. Most EIDs appear to result from increased contact between wildlife and humans, especially when humans encroach into formerly pristine habitats. Habitat deterioration may also negatively affect the physiology and health of wildlife species, which may eventually lead to a higher susceptibility to infectious agents and/or increased shedding of the pathogens causing EIDs. Bats are known to host viruses closely related to important EIDs. Here, we tested in a paleotropical forest with ongoing logging and fragmentation, whether habitat disturbance influences the occurrence of astro- and coronaviruses in eight bat species. In contrast to our hypothesis, anthropogenic habitat disturbance was not associated with corona- and astrovirus detection rates in fecal samples. However, we found that bats infected with either astro- or coronaviruses were likely to be coinfected with the respective other virus. Additionally, we identified two more risk factors influencing astrovirus shedding. First, the detection rate of astroviruses was higher at the beginning of the rainy compared to the dry season. Second, there was a trend that individuals with a poor body condition had a higher probability of shedding astroviruses in their feces. The identification of risk factors for increased viral shedding that may potentially result in increased interspecies transmission is important to prevent viral spillovers from bats to other animals, including humans.

Differential Infection Patterns and Recent Evolutionary Origins of Equine Hepaciviruses in Donkeys.

The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV. IMPORTANCE: The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

Assay optimization for molecular detection of Zika virus.

OBJECTIVE: To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. METHODS: We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays' target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays. FINDINGS: Oligonucleotides of the published real-time RT-PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine. CONCLUSION: We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results.

Hepatitis E Virus Infection in Dromedaries, North and East Africa, United Arab Emirates, and Pakistan, 1983-2015.

A new hepatitis E virus (HEV-7) was recently found in dromedaries and 1 human from the United Arab Emirates. We screened 2,438 dromedary samples from Pakistan, the United Arab Emirates, and 4 African countries. HEV-7 is long established, diversified and geographically widespread. Dromedaries may constitute a neglected source of zoonotic HEV infections.