Phylogeography of horseshoe bat sarbecoviruses in Vietnam and neighbouring countries. Implications for the origins of SARS-CoV and SARS-CoV-2.

Previous studies on horseshoe bats (Rhinolophus spp.) have described many coronaviruses related to SARS-CoV (SARSCoVr) in China and only a few coronaviruses related to SARS-CoV-2 (SARSCoV2r) in Yunnan (southern China), Cambodia, Laos and Thailand. Here, we report the results of several field missions carried out in 2017, 2021 and 2022 across Vietnam during which 1218 horseshoe bats were sampled from 19 locations. Sarbecoviruses were detected in 11% of faecal RNA extracts, with much more positives among Rhinolophus thomasi (46%). We assembled 38 Sarbecovirus genomes, including 32 SARSCoVr, four SARSCoV2r, and two recombinants of SARSCoVr and SARSCoV2r (RecSar), one showing a Spike protein very similar to SARS-CoV-2. We detected a bat co-infected with four coronaviruses, including two sarbecoviruses. Our analyses revealed that Sarbecovirus genomes evolve in Vietnam under strong geographical and host constraints. First, we found evidence for a deep separation between viruses from northern Vietnam and those from central and southern Vietnam. Second, we detected only SARSCoVr in Rhinolophus thomasi, both SARSCoVr and SARSCoV2r in Rhinolophus affinis, and only RecSar in Rhinolophus pusillus captured close to the border with China. Third, the bias in favour of Uracil in synonymous third codon positions of SARSCoVr extracted from R. thomasi showed a negative correlation with latitudes. Our results also provided support for an emergence of SARS-CoV in horseshoe bats from northern Yunnan and emergence of SARS-CoV-2 in horseshoe bats from northern Indochina subtropical forests (southern Yunnan, northern Laos and north-western Vietnam).

Cryptic hybridization between the ancient lineages of Natterer's bat (Myotis nattereri).

Studying hybrid zones that form between morphologically cryptic taxa offers valuable insights into the mechanisms of cryptic speciation and the evolution of reproductive barriers. Although hybrid zones have long been the focus of evolutionary studies, the awareness of cryptic hybrid zones increased recently due to rapidly growing evidence of biological diversity lacking obvious phenotypic differentiation. The characterization of cryptic hybrid zones with genome-wide analysis is in its early stages and offers new perspectives for studying population admixture and thus the impact of gene flow. In this study, we investigate the population genomics of the Myotis nattereri complex in one of its secondary contact zones, where a putative hybrid zone is formed between two of its cryptic lineages. By utilizing a whole-genome shotgun sequencing approach, we aim to characterize this cryptic hybrid zone in detail. Demographic analysis suggests that the cryptic lineages diverged during the Pliocene, c. 3.6 million years ago. Despite this ancient separation, the populations in the contact zone exhibit mitochondrial introgression and a considerable amount of mixing in nuclear genomes. The genomic structure of the populations corresponds to geographic locations and the genomic admixture changes along a geographic gradient. These findings suggest that there is no effective hybridization barrier between both lineages, nevertheless, their population structure is shaped by dispersal barriers. Our findings highlight how such deeply diverged cryptic lineages can still readily hybridize in secondary contact.

Environmental reservoir dynamics predict global infection patterns and population impacts for the fungal disease white-nose syndrome.

Disease outbreaks and pathogen introductions can have significant effects on host populations, and the ability of pathogens to persist in the environment can exacerbate disease impacts by fueling sustained transmission, seasonal epidemics, and repeated spillover events. While theory suggests that the presence of an environmental reservoir increases the risk of host declines and threat of extinction, the influence of reservoir dynamics on transmission and population impacts remains poorly described. Here we show that the extent of the environmental reservoir explains broad patterns of host infection and the severity of disease impacts of a virulent pathogen. We examined reservoir and host infection dynamics and the resulting impacts of Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome, in 39 species of bats at 101 sites across the globe. Lower levels of pathogen in the environment consistently corresponded to delayed infection of hosts, fewer and less severe infections, and reduced population impacts. In contrast, an extensive and persistent environmental reservoir led to early and widespread infections and severe population declines. These results suggest that continental differences in the persistence or decay of P. destructans in the environment altered infection patterns in bats and influenced whether host populations were stable or experienced severe declines from this disease. Quantifying the impact of the environmental reservoir on disease dynamics can provide specific targets for reducing pathogen levels in the environment to prevent or control future epidemics.

Detection and sequence analysis of Canine morbillivirus in multiple species of the Mustelidae family.

BACKGROUND: Canine morbillivirus (canine distemper virus, CDV) is a member of the Paramyxoviridae family. Canine distemper is a serious viral disease that affects many mammalian species, including members of the Mustelidae family. These animals have an elusive nature, which makes related virological studies extremely challenging. There is a significant knowledge gap about the evolution of their viruses and about the possible effects of these viruses to the population dynamics of the host animals. Spleen and lung tissue samples of 170 road-killed mustelids belonging to six species were collected between 1997 and 2022 throughout Hungary and tested for CDV with real-time RT-PCR. RESULTS: Three species were positive for viral RNA, 2 out of 64 Steppe polecats (Mustela eversmanii), 1 out of 36 European polecats (Mustela putorius) and 2 out of 36 stone martens (Martes foina); all 18 pine martens (Martes martes), 10 least weasels (Mustela nivalis) and 6 stoats (Mustela erminea) tested negative. The complete CDV genome was sequenced in five samples using pan-genotype CDV-specific, amplicon-based Nanopore sequencing. Based on the phylogenetic analysis, all five viral sequences were grouped to the Europe/South America 1 lineage and the distribution of one sequence among trees indicated recombination of the Hemagglutinin gene. We verified the recombination with SimPlot analysis. CONCLUSIONS: This paper provides the first CDV genome sequences from Steppe polecats and additional complete genomes from European polecats and stone martens. The infected specimens of various species originated from distinct parts of the country over a long time, indicating a wide circulation of CDV among mustelids throughout Hungary. Considering the high virulence of CDV and the presence of the virus in these animals, we highlight the importance of conservation efforts for wild mustelids. In addition, we emphasize the importance of full genomic data acquisition and analysis to better understand the evolution of the virus. Since CDV is prone to recombination, specific genomic segment analyses may provide less representative evolutionary traits than using complete genome sequences.

Eco-epidemiology of Novel Bartonella Genotypes from Parasitic Flies of Insectivorous Bats.

Bats are important zoonotic reservoirs for many pathogens worldwide. Although their highly specialized ectoparasites, bat flies (Diptera: Hippoboscoidea), can transmit Bartonella bacteria including human pathogens, their eco-epidemiology is unexplored. Here, we analyzed the prevalence and diversity of Bartonella strains sampled from 10 bat fly species from 14 European bat species. We found high prevalence of Bartonella spp. in most bat fly species with wide geographical distribution. Bat species explained most of the variance in Bartonella distribution with the highest prevalence of infected flies recorded in species living in dense groups exclusively in caves. Bat gender but not bat fly gender was also an important factor with the more mobile male bats giving more opportunity for the ectoparasites to access several host individuals. We detected high diversity of Bartonella strains (18 sequences, 7 genotypes, in 9 bat fly species) comparable with tropical assemblages of bat-bat fly association. Most genotypes are novel (15 out of 18 recorded strains have a similarity of 92-99%, with three sequences having 100% similarity to Bartonella spp. sequences deposited in GenBank) with currently unknown pathogenicity; however, 4 of these sequences are similar (up to 92% sequence similarity) to Bartonella spp. with known zoonotic potential. The high prevalence and diversity of Bartonella spp. suggests a long shared evolution of these bacteria with bat flies and bats providing excellent study targets for the eco-epidemiology of host-vector-pathogen cycles.

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

Analyses of separate and concatenated cox1 and 18S rRNA gene sequences indicate that the bat piroplasm Babesia vesperuginis is phylogenetically close to Cytauxzoon felis and the 'prototheilerid' Babesia conradae.

Babesia vesperuginis is the only piroplasm known to infect bats. Unlike most members of the genus Babesia, it is probably transmitted by a soft tick species (i.e. Argas vespertilionis). Recently, two studies have been conducted to clarify the phylogenetic status of this species, and both agreed on placing it into a basal position among Babesia sensu stricto (s.s.). However, several important groups of piroplasms were not included in the already reported phylogenetic trees of B. vesperuginis isolates. Therefore, the aim of the present study was to amplify an approx. 950-bp fragment of the cytochrome c oxidase subunit 1 (cox1) gene of B. vesperuginis from A. vespertilionis specimens, and to compare its sequences with those from other piroplasmid groups in a broader phylogenetic context. Sequence comparisons focusing on either 18S rRNA or cox1 genes, as well as phylogenetic analyses involving separate and concatenated 18S rRNA and cox1 sequences indicate that B. vesperuginis is more closely related to the phylogenetic group of Theileriidae than to Babesia s.s. In particular, B. vesperuginis clustered closest to Cytauxzoon felis and the 'prototheilerid' B. conradae. The results of this study highlight that B. vesperuginis is a unique and taxonomically important species, which should be included in future studies aimed at resolving the comprehensive phylogeny of Piroplasmida.

Metagenomic analysis of bat guano samples revealed the presence of viruses potentially carried by insects, among others by Apis mellifera in Hungary.

The predominance of dietary viruses in bat guano samples had been described recently, suggesting a new opportunity to survey the prevalence and to detect new viruses of arthropods or even plant-infecting viruses circulating locally in the ecosystem. Here we describe the diversity of viruses belonging to the order Picornavirales in Hungarian insectivorous bat guano samples. The metagenomic analysis conducted on our samples has revealed the significant predominance of aphid lethal paralysis virus (ALPV) and Big Sioux River virus (BSRV) in Hungary for the first time. Phylogenetic analysis was used to clarify the relationship to previously identified ALPV strains infecting honey bees, showing that our strain possesses a close genetic relationship with the strains that have already been described as pathogenic to honey bees. Furthermore, studies have previously confirmed the ability of these viruses to replicate in adult honey bees; however, no signs related to these viruses have been revealed yet. With the identification of two recently described possibly honey bee infecting viruses for the first time in Hungary, our results might have importance for the health conditions of Hungarian honey bee colonies in the future.

Highly divergent cyclo-like virus in a great roundleaf bat (Hipposideros armiger) in Vietnam.

Members of the viral family Circoviridae are increasingly recognized worldwide. Bats seem to be natural reservoirs or dietary-related dispensers of these viruses. Here, we report a distantly related member of the genus Cyclovirus detected in the faeces of a great roundleaf bat (Hipposideros armiger). Interestingly, the novel virus lacks a Circoviridae-specific stem-loop structure, although a Geminiviridae-like nonamer sequence was detected in the large intergenic region. Based on these differences and its phylogenetic position, we propose that our new virus represents a distant and highly divergent member of the genus Cyclovirus. However it is lacking several characteristics of members of the genus, which raises a challenge in its taxonomic classification.

Molecular investigations of the bat tick Argas vespertilionis (Ixodida: Argasidae) and Babesia vesperuginis (Apicomplexa: Piroplasmida) reflect "bat connection" between Central Europe and Central Asia.

Argas vespertilionis is a geographically widespread haematophagous ectoparasite species of bats in the Old World, with a suspected role in the transmission of Babesia vesperuginis. The aims of the present study were (1) to molecularly screen A. vespertilionis larvae (collected in Europe, Africa and Asia) for the presence of piroplasms, and (2) to analyze mitochondrial markers of A. vespertilionis larvae from Central Asia (Xinjiang Province, Northwestern China) in a phylogeographical context. Out of the 193 DNA extracts from 321 A. vespertilionis larvae, 12 contained piroplasm DNA (10 from Hungary, two from China). Sequencing showed the exclusive presence of B. vesperuginis, with 100% sequence identity between samples from Hungary and China. In addition, A. vespertilionis cytochrome oxidase c subunit 1 (cox1) and 16S rRNA gene sequences had 99.1-99.2 and 99.5-100% similarities, respectively, between Hungary and China. Accordingly, in the phylogenetic analyses A. vespertilionis from China clustered with haplotypes from Europe, and (with high support) outside the group formed by haplotypes from Southeast Asia. This is the first molecular evidence on the occurrence of B. vesperuginis in Asia. Bat ticks from hosts in Vespertilionidae contained only the DNA of B. vesperuginis (in contrast with what was reported on bat ticks from Rhinolophidae and Miniopteridae). Molecular taxonomic analyses of A. vespertilionis and B. vesperuginis suggest a genetic link of bat parasites between Central Europe and Central Asia, which is epidemiologically relevant in the context of any pathogens associated with bats.

DNA of free-living bodonids (Euglenozoa: Kinetoplastea) in bat ectoparasites: potential relevance to the evolution of parasitic trypanosomatids.

Kinetoplastids are flagellated protozoa, including principally free-living bodonids and exclusively parasitic trypanosomatids. In the most species-rich genus, Trypanosoma, more than thirty species were found to infect bats worldwide. Bat trypanosomes are also known to have played a significant role in the evolution of T. cruzi, a species with high veterinary medical significance. Although preliminary data attested the occurrence of bat trypanosomes in Hungary, these were never sought for with molecular methods. Therefore, amplification of an approx. 900-bp fragment of the 18S rRNA gene of kinetoplastids was attempted from 307 ixodid and 299 argasid ticks collected from bats, and from 207 cimicid bugs collected from or near bats in Hungary and Romania. Three samples, one per each bat ectoparasite group, were PCR positive. Sequencing revealed the presence of DNA from free-living bodonids (Bodo saltans and neobodonids), but no trypanosomes were detected. The most likely source of bodonid DNA detected here in engorged bat ectoparasites is the blood of their bat hosts. However, how bodonids were acquired by bats, can only be speculated. Bats are known to drink from freshwater bodies, i.e. the natural habitats of B. saltans and related species, allowing bats to ingest bodonids. Consequently, these results suggest that at least the DNA of bodonids might pass through the alimentary mucosa of bats into their circulation. The above findings highlight the importance of studying bats and other mammals for the occurrence of bodonids in their blood and excreta, with potential relevance to the evolution of free-living kinetoplastids towards parasitism.

Genetic characterization of a novel picornavirus detected in Miniopterus schreibersii bats.

Bats are important reservoirs of many viruses with zoonotic potential worldwide, including Europe. Among bat viruses, members of the Picornaviridae family remain a neglected group. We performed viral metagenomic analyses on Miniopterus schreibersii bat faecal samples, collected in Hungary in 2013. In the present study we report the first molecular data and genomic characterization of a novel picornavirus from the bat species M. schreibersii in Europe. Based on phylogenetic analyses, the novel bat picornaviruses unambiguously belong to the Mischivirus genus and were highly divergent from other bat-derived picornaviruses of the Sapelovirus genus. Although the Hungarian viruses were most closely related to Mischivirus A, they formed a separate monophyletic branch within the genus.

Random sampling of the Central European bat fauna reveals the existence of numerous hitherto unknown adenoviruses.

From over 1250 extant species of the order Chiroptera, 25 and 28 are known to occur in Germany and Hungary, respectively. Close to 350 samples originating from 28 bat species (17 from Germany, 27 from Hungary) were screened for the presence of adenoviruses (AdVs) using a nested PCR that targets the DNA polymerase gene of AdVs. An additional PCR was designed and applied to amplify a fragment from the gene encoding the IVa2 protein of mastadenoviruses. All German samples originated from organs of bats found moribund or dead. The Hungarian samples were excrements collected from colonies of known bat species, throat or rectal swab samples, taken from live individuals that had been captured for faunistic surveys and migration studies, as well as internal organs of dead specimens. Overall, 51 samples (14.73%) were found positive. We detected 28 seemingly novel and six previously described bat AdVs by sequencing the PCR products. The positivity rate was the highest among the guano samples of bat colonies. In phylogeny reconstructions, the AdVs detected in bats clustered roughly, but not perfectly, according to the hosts' families (Vespertilionidae, Rhinolophidae, Hipposideridae, Phyllostomidae and Pteropodidae). In a few cases, identical sequences were derived from animals of closely related species. On the other hand, some bat species proved to harbour more than one type of AdV. The high prevalence of infection and the large number of chiropteran species worldwide make us hypothesise that hundreds of different yet unknown AdV types might circulate in bats.

Thumb-pads up-a new species of thick-thumbed bat from Sumatra (Chiroptera: Vespertilionidae: Glischropus).

To date, three species of the genus Glischropus are recognized from the Indomalayan zoogeographic region-G. bucephalus from the Indochinese subregion, G. tylopus from the Sundaic subregion (Peninsular Thailand and Malaysia, Borneo, Sumatra, Moluccas) and G. javanus, restricted to Java. The investigation of the holotype and three topotype specimens of G. batjanus supported the view that the name was previously correctly regarded as the junior subjective synonym of G. tylopus. During review of material recently collected in southwestern Sumatra, Indonesia, one specimen of a yet undescribed species of Thick-thumbed bat was identified. G. aquilus n. sp. markedly differs from its congeners by its dark brown pelage, nearly black ear and tragus, and in skull proportions. The phylogenetic analysis based on cytb sequences also supports the specific distinctness of G. aquilus n. sp. Its discovery brings the count to 88 species of bats known from Sumatra.

Accelerating targeted mosquito control efforts through mobile West Nile virus detection.

BACKGROUND: Different mosquito control strategies have been implemented to mitigate or prevent mosquito-related public health situations. Modern mosquito control largely relies on multiple approaches, including targeted, specific treatments. Given this, it is becoming increasingly important to supplement these activities with rapid and mobile diagnostic capacities for mosquito-borne diseases. We aimed to create and test the applicability of a rapid diagnostic system for West Nile virus that can be used under field conditions. METHODS: In this pilot study, various types of adult mosquito traps were applied within the regular mosquito monitoring activity framework for mosquito control. Then, the captured specimens were used for the detection of West Nile virus RNA under field conditions with a portable qRT-PCR approach within 3-4 h. Then, positive samples were subjected to confirmatory RT-PCR or NGS sequencing in the laboratory to obtain genome information of the virus. We implemented phylogenetic analysis to characterize circulating strains. RESULTS: A total of 356 mosquito individuals representing 7 species were processed in 54 pools, each containing up to 20 individuals. These pools were tested for the presence of West Nile virus, and two pools tested positive, containing specimens from the Culex pipiens and Anopheles atroparvus mosquito species. As a result of subsequent sequencing, we present the complete genome of West Nile virus and Bagaza virus. CONCLUSIONS: The rapid identification of infected mosquitoes is the most important component of quick response adulticide or larvicide treatments to prevent human cases. The conceptual framework of real-time surveillance can be optimized for other pathogens and situations not only in relation to West Nile virus. We present an early warning system for mosquito-borne diseases and demonstrate its application to aid rapid-response mosquito control actions.

The calls of Vietnamese bats: a major step toward the acoustic characterization of Asian bats.

Southeast Asia includes several global biodiversity hotspots and bats account for nearly one-third of mammal species currently known in the region. While acoustic methods have become widespread in bat research, basic information is often lacking on the echolocation calls produced by Asian bat species. Since such information can aid a wide variety of research and conservation initiatives, descriptions of the calls emitted by Asian bats are fundamental. The aim of our study was to provide a standardized analysis and description of the Vietnamese bat echolocation calls. We analyzed call recordings of 87 species arranged in eight families. This constitutes 74% of the echolocating bats presently known in Vietnam and includes the first call descriptions for five taxa. Our use of an open-source software and the deposition of recordings in the ChiroVox repository will facilitate comparative studies in Asia and the information we provide represents one of the most comprehensive bioacoustic databases for Asian bats to date.

Detection and genetic characterization of circoviruses in more than 80 bat species from eight countries on four continents.

Several bat-associated circoviruses and circular rep-encoding single-stranded DNA (CRESS DNA) viruses have been described, but the exact diversity and host species of these viruses are often unknown. Our goal was to describe the diversity of bat-associated circoviruses and cirliviruses, thus, 424 bat samples from more than 80 species were collected on four continents. The samples were screened for circoviruses using PCR and the resulting amino acid sequences were subjected to phylogenetic analysis. The majority of bat strains were classified in the genus Circovirus and some strains in the genus Cyclovirus and the clades CRESS1 and CRESS3. Some strains, however, could only be classified at the taxonomic level of the order and were not classified in any of the accepted or proposed clades. In the family Circoviridae, 71 new species have been predicted. This screening of bat samples revealed a great diversity of circoviruses and cirliviruses. These studies underline the importance of the discovery and description of new cirliviruses and the need to establish new species and families in the order Cirlivirales.

Isolation and genome characterization of Lloviu virus from Italian Schreibers's bats.

Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses.

Isolation of infectious Lloviu virus from Schreiber's bats in Hungary.

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics.

ChiroVox: a public library of bat calls.

Recordings of bat echolocation and social calls are used for many research purposes from ecological studies to taxonomy. Effective use of these relies on identification of species from the recordings, but comparative recordings or detailed call descriptions to support identification are often lacking for areas with high biodiversity. The ChiroVox website (https://www.chirovox.org) was created to facilitate the sharing of bat sound recordings together with their metadata, including biodiversity data and recording circumstances. To date, more than 30 researchers have contributed over 3,900 recordings of nearly 200 species, making ChiroVox the largest open-access bat call library currently available. Each recording has a unique identifier that can be cited in publications; hence the acoustic analyses are repeatable. Most of the recordings available through the website are from bats whose species identities are confirmed, so they can be used to determine species in recordings where the bats were not captured or could not be identified. We hope that with the help of the bat researcher community, the website will grow rapidly and will serve as a solid source for bat acoustic research and monitoring.