[Marburg virus disease in travellers]. / Maladie à virus Marburg chez les voyageurs.

Marburg virus disease (MVD) is a dreadful but exceptional disease. Formerly mainly identified in Uganda, Angola and the Democratic Republic of Congo, it has recently appeared in the Republic of Guinea, Ghana, Equatorial Guinea and Tanzania, adding West Africa to the affected regions. Humans become infected through exposure to bats Roussettus aegyptiacus or during unprotected care of infected people. Five cases are linked to travellers, the last one dates to 2008 and involved a visit to caves colonized by bats. At present, there is no specific treatment or vaccine. Despite its rarity, adventurous travelers should be aware of the risks of exposure and avoid entering places inhabited by bats.

La maladie à virus Marburg est une maladie redoutable mais exceptionnelle. Autrefois identifiée en Ouganda, Angola et République démocratique du Congo, elle a récemment fait son apparition en République de Guinée, au Ghana, en Guinée équatoriale et en Tanzanie, ajoutant l'Afrique de l'Ouest aux régions touchées. Les humains s'infectent lors d'une exposition avec les chauves-souris roussettes d'Égypte ou lors de la prise en charge sans protection de personnes infectées. Cinq cas sont liés à des voyageurs, le dernier remonte à 2008 et était associé à la visite de grottes colonisées par des roussettes d'Égypte. Actuellement, il n'existe aucun traitement spécifique ni vaccin. Malgré sa rareté, les voyageurs aventureux doivent être informés des risques d'exposition et éviter de pénétrer dans des lieux habités par des chauves-souris.

The first confirmed human case of rabies, Timor-Leste, 2024.

In March 2024, the first ever human case of rabies, following a dog bite, was detected in Timor-Leste. This paper briefly discusses the circumstances of transmission, clinical presentation, palliative care of the case and public health measures taken. Timor-Leste was previously considered rabies-free. Any person who is bitten or scratched by an animal that could potentially transmit rabies virus (especially dogs, bats, monkeys or cats) in Timor-Leste should be assessed for consideration of provision of rabies post-exposure prophylaxis.

Novel betaherpesviruses and gammaherpesviruses in bats from central China.

Herpesviruses are large double-stranded DNA viruses that cause infections in animals and humans with a characteristic of latent infectious within specific tissues. Bats are natural hosts of variety human-infecting viruses and recently have been described as hosts for herpesviruses in several countries around the world. In this study we collected 140 insectivorous bats in the neighboring urban areas of Wuhan City, Hubei Province in the central China between 2020 and 2021. Nested PCR targeting the dpol gene sequence indicated that a total of 22 individuals (15.7% of the sample) tested positive for herpesvirus with 4 strains belonging to the genus Betaherpesvirus and the remaining 18 strains classified as Gammahersvirus. Furthermore, the herpesvirus prevalence in Rhinolophus pusillus was higher at 26.3%, compared to 8.4% in Myotis davidii. The RP701 strain from R. pusillus was the predominant gammaherpesvirus strain detected in bats, accounting for 94.4% (17/18) of all strains. The variations in γ-herpesviruses genomic sequences was evident in phylogenetic tree, where RP701 strain was clustered together with ruminant γ-herpesviruses, while MD704 strain formed a distinct clade with a hedgehog γ-herpesvirus. Four betaherpesviruses exclusively identified from M. davidii, with nucleotide identities ranging from 79.7 to 82.6% compared to known betaherpesviruses. Our study provided evidence that M. davidii can sever as natural host for ß-herpesviruses, which extended the host species range. In conclusion, we found that bats from central China harbored novel ß-herpesviruses and γ-herpesviruses which were phylogenetically related to ruminant γ-herpesvirus and hedgehog γ-herpesvirus. Our study indicates that bats are natural hosts of ß- and γ-herpesviruses and further studies are needed to determine whether there is cross-species transmission of herpesviruses between bats and other animals, or humans.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Deforestation and Bovine Rabies Outbreaks in Costa Rica, 1985-2020.

In Latin America, rabies virus has persisted in a cycle between Desmodus rotundus vampire bats and cattle, potentially enhanced by deforestation. We modeled bovine rabies virus outbreaks in Costa Rica relative to land-use indicators and found spatial-temporal relationships among rabies virus outbreaks with deforestation as a predictor.

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

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

Comparative Performance in the Detection of Four Coronavirus Genera from Human, Animal, and Environmental Specimens.

Emerging coronaviruses (CoVs) are understood to cause critical human and domestic animal diseases; the spillover from wildlife reservoirs can result in mild and severe respiratory illness in humans and domestic animals and can spread more readily in these naïve hosts. A low-cost CoV molecular method that can detect a variety of CoVs from humans, animals, and environmental specimens is an initial step to ensure the early identification of known and new viruses. We examine a collection of 50 human, 46 wastewater, 28 bat, and 17 avian archived specimens using 3 published pan-CoV PCR assays called Q-, W-, and X-CoV PCR, to compare the performance of each assay against four CoV genera. X-CoV PCR can detect all four CoV genera, but Q- and W-CoV PCR failed to detect δ-CoV. In total, 21 (42.0%), 9 (18.0%), and 21 (42.0%) of 50 human specimens and 30 (65.22%), 6 (13.04%), and 27 (58.70%) of 46 wastewater specimens were detected using Q-, W-, and X-CoV PCR assays, respectively. The X-CoV PCR assay has a comparable sensitivity to Q-CoV PCR in bat CoV detection. Combining Q- and X-CoV PCR assays can increase sensitivity and avoid false negative results in the early detection of novel CoVs.

Sosuga Virus Detected in Egyptian Rousette Bats (Rousettus aegyptiacus) in Sierra Leone.

Sosuga virus (SOSV), a rare human pathogenic paramyxovirus, was first discovered in 2012 when a person became ill after working in South Sudan and Uganda. During an ecological investigation, several species of bats were sampled and tested for SOSV RNA and only one species, the Egyptian rousette bat (ERBs; Rousettus aegyptiacus), tested positive. Since that time, multiple other species have been sampled and ERBs in Uganda have continued to be the only species of bat positive for SOSV infection. Subsequent studies of ERBs with SOSV demonstrated that ERBs are a competent host for SOSV and shed this infectious virus while exhibiting only minor infection-associated pathology. Following the 2014 Ebola outbreak in West Africa, surveillance efforts focused on discovering reservoirs for zoonotic pathogens resulted in the capture and testing of many bat species. Here, SOSV RNA was detected by qRT-PCR only in ERBs captured in the Moyamba District of Sierra Leone in the central region of the country. These findings represent a substantial range extension from East Africa to West Africa for SOSV, suggesting that this paramyxovirus may occur in ERB populations throughout its sub-Saharan African range.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Virome analysis of Desmodus rotundus tissue samples from the Amazon region.

BACKGROUND: Bats are renowned for harboring a high viral diversity, their characteristics contribute to emerging infectious diseases. However, environmental and anthropic factors also play a significant role in the emergence of zoonotic viruses. Metagenomic is an important tool for investigating the virome of bats and discovering new viruses. RESULTS: Twenty-four families of virus were detected in lung samples by sequencing and bioinfomatic analysis, the largest amount of reads was focused on the Retroviridae and contigs assembled to Desmodus rotundus endogenous retrovirus, which was feasible to acquire complete sequences. The reads were also abundant for phages. CONCLUSION: This lung virome of D. rotundus contributes valuable information regarding the viral diversity found in bats, which is useful for understanding the drivers of viral cycles and their ecology in this species. The identification and taxonomic categorization of viruses hosted by bats carry epidemiological significance due to the potential for viral adaptation to other animals and humans, which can have severe repercussions for public health. Furthermore, the characterization of endogenized viruses helps to understanding the host genome and the evolution of the species.

Paving new roads toward the advancement of broad-spectrum antiviral agents.

Broad-spectrum antivirals (BSAs) have the advantageous property of being effective against a wide range of viruses with a single drug, offering a promising therapeutic solution for the largely unmet need in treating both existing and emerging viral infections. In this review, we summarize the current strategies for the development of novel BSAs, focusing on either targeting the commonalities during the replication of multiple viruses or the systemic immunity of humans. In comparison to BSAs that target viral replication, these immuno-modulatory agents possess an expanded spectrum of antiviral activity. However, antiviral immunity is a double-edged sword, and maintaining immune homeostasis ultimately dictates the health status of hosts during viral infections. Therefore, establishing an ideal goal for immuno-modulation in antiviral interventions is crucial. Herein we propose a bionic approach for immuno-modulation inspired by mimicking bats, which possess a more robust immune system for combating viral invasions, compared to humans. In addition, we discuss an empirical approach to treat diverse viral infections using traditional Chinese medicines (TCMs), mainly through bidirectional immuno-modulation to restore the disrupted homeostasis. Advancing our understanding of both the immune system of bats and the mechanisms underlying antiviral TCMs will significantly contribute to the future development of novel BSAs.

Phylogenetic relationship between rabies virus (Lyssavirus rabies) variants from two different species / Relações filogenéticas do vírus da raiva (Lyssavirus rabies) em duas diferentes espécies / Relaciones filogenéticas del virus de la rabia (Lyssavirus rabies) en dos especies diferentes

Rabies is a fatal zoonotic disease that affects several mammals. Hematophagous bats are recognized hosts of the rabies virus, and their main food source is the blood of other mammals, particularly cattle. During feeding, bats transmit the virus to cattle, which are victims of the disease, contributing to economic losses and increasing the risk of infection for humans. Based on this affinity in the rabies cycle between bats and cattle, the objective of this study was to analyze the phylogenetic relationships of rabies virus samples in cattle and bats. The G gene of the rabies virus was chosen for this study because it is directly related to the infection process. Nucleotide sequences of the viral G gene were selected from GenBank for samples obtained from infected cattle and bats. Maximum parsimony analyses were conducted using the Molecular Evolutionary Genetics Analysis software. The Maxima Parsimony tree indicated a phylogenetic relationship between the G genes of both hosts, indicating that the virus evolved from bats to cattle. Analysis of parsimoniously informative sites revealed that the viral G gene carried specific mutations in each host. Knowledge of the evolutionary relationships between the rabies virus and its hosts is critical for identifying potential new hosts and the possible routes of infection for humans.

A Raiva é uma zoonose fatal que infecta várias espécies de mamíferos. Os morcegos hematófagos são reconhecidos como hospedeiros do vírus da Raiva e sua principal fonte de alimento é o sangue de outros mamíferos, especialmente os bovinos. Quando se alimentam, os morcegos transmitem o vírus para o bovino os quais são vítimas da doença, contribuindo para perdas econômicas e riscos de infecção para humanos. Baseado nesta afinidade do ciclo da Raiva entre morcegos e bovinos, o objetivo deste estudo foi analisar as relações filogenéticas de amostras do vírus da Raiva em ambos os hospedeiros, bovinos e morcegos. O gene G do vírus da Raiva foi escolhido para esta pesquisa porque ele está diretamente relacionado ao processo de infecção. Sequências de nucleotídeos do gene G viral foram selecionadas no GenBank a partir de amostras obtidas de bovinos e morcegos infectados. Análises de Máxima Parcimônia foram conduzidas utilizando o software Molecular Evolutionary Genetics Analysis. A árvore de Máxima Parcimônia indicou uma relação filogenética entre o gene G de ambos os hospedeiros, indicando que o vírus evoluiu dos morcegos para os bovinos. A análise dos sítios parcimoniosamente informativos revelou que o gene G viral apresentou mutações específicas em cada hospedeiro. O conhecimento sobre as relações evolutivas do vírus da Raiva e seus hospedeiros é crucial para identificar nos hospedeiros potenciais e novas rotas possíveis de infecção para humanos.

La rabia es una zoonosis fatal que infecta a varias especies de mamíferos. Los murciélagos hematófagos son reconocidos como huéspedes del virus de la rabia y su principal fuente de alimentación es la sangre de otros mamíferos, especialmente del ganado. Al alimentarse, los murciélagos transmiten el virus al ganado que es víctima de la enfermedad, contribuyendo a pérdidas económicas y riesgos de infección para los humanos. Basado en esta afinidad del ciclo de la rabia entre murciélagos y ganado, el objetivo de este estudio fue analizar las relaciones filogenéticas de las muestras de virus de la rabia tanto en huéspedes, ganado y murciélagos. El gen G del virus de la rabia fue elegido para esta investigación porque está directamente relacionado con el proceso de infección. Las secuencias de nucleótidos del gen G viral se seleccionaron en GenBank a partir de muestras obtenidas de bovinos y murciélagos infectados. Los análisis de parsimonia máxima se realizaron utilizando el software Molecular Evolutionary Genetics Analysis. El árbol de Máxima Parsimônia indicó una relación filogenética entre el gen G de ambos huéspedes, indicando que el virus evolucionó de murciélagos a bovinos. El análisis de los sitios parsimoniosamente informativos reveló que el gen G viral presentaba mutaciones específicas en cada huésped. El conocimiento sobre las relaciones evolutivas del virus de la rabia y sus huéspedes es crucial para identificar huéspedes potenciales y nuevas posibles rutas de infección para humanos.

Rare studies expose bats to SARS-CoV-2.

Infecting captive species could help gauge pandemic risk of coronaviruses that have yet to infect humans.

Evolution of SARS-CoV-2 Spikes shapes their binding affinities to animal ACE2 orthologs.

IMPORTANCE: Spike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant.

Pteropus vampyrus TRIM40 Is an Interferon-Stimulated Gene That Antagonizes RIG-I-like Receptors.

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats' tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNß, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

A sensitive and simple RT-LAMP assay for sarbecovirus screening in bats.

IMPORTANCE: We report the application of a colorimetric and fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to facilitate mass screening for sarbecoviruses in bats. The assay was evaluated using a total of 838 oral and alimentary samples from bats and demonstrated comparable sensitivity and specificity to quantitative reverse transcription PCR (qRT-PCR), with a simple setup. The addition of SYTO9, a fluorescent nucleic acid stain, also allows for quantitative analysis. The scalability and simplicity of the assay are believed to contribute to improving preparedness for detecting emerging coronaviruses by applying it to field studies and surveillance.

Prevalence and public health significance of rabies virus in bats in the North Region of Cameroon.

BACKGROUND: Rabies is a zoonotic disease of all warm-blooded animals including humans. There is a paucity of data on the status of rabies in wild animals in Cameroon and the disease is endemic in the country with dogs being the main source of transmission. Bat habitats are widespread in Cameroon, but there is limited information on the prevalence of rabies in bats, and their role of as potential reservoirs of rabies virus. METHODS: A cross sectional study was carried out to estimate the prevalence and to assess risk factors of rabies virus in bats in the North Region of Cameroon. A total of 212 bats belonging to three families (Pteropodidae, Vespertilionidae and Molossidae) and 5 species were sampled in 7 localities in the North Region of Cameroon and were tested for rabies virus antigen using direct Immunofluorescence Test (IFA). RESULTS: Overall, 26.9% (57/212) of the bats collected showed an IFA positive reaction. The prevalence was significantly higher (P<0.05) in adult bats (33.3% (36/108)) compared to young individuals (20.2%; 21/104). The main risk factors identified in the study for human exposure to bats were gender (Male), religion (Christianity), localities (Babla and Lagdo), the practice of bat hunting, bat consumption, unawareness of bat rabies and cohabitation with bats in close proximity. CONCLUSION: The study revealed the first evidence of Lyssavirus in bats in Cameroon. This finding showed that bat rabies are real and represents a potential public health concern in communities with bat habitats in the North Region of Cameroon. Enhancing the level of public awareness and health education on the potential of bats as reservoirs of Lyssavirus in Cameroon as well as the integration of the "One Health" approach for effective management of animal and human rabies should be emphasized.

Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

Reservoir host immunology and life history shape virulence evolution in zoonotic viruses.

The management of future pandemic risk requires a better understanding of the mechanisms that determine the virulence of emerging zoonotic viruses. Meta-analyses suggest that the virulence of emerging zoonoses is correlated with but not completely predictable from reservoir host phylogeny, indicating that specific characteristics of reservoir host immunology and life history may drive the evolution of viral traits responsible for cross-species virulence. In particular, bats host viruses that cause higher case fatality rates upon spillover to humans than those derived from any other mammal, a phenomenon that cannot be explained by phylogenetic distance alone. In order to disentangle the fundamental drivers of these patterns, we develop a nested modeling framework that highlights mechanisms that underpin the evolution of viral traits in reservoir hosts that cause virulence following cross-species emergence. We apply this framework to generate virulence predictions for viral zoonoses derived from diverse mammalian reservoirs, recapturing trends in virus-induced human mortality rates reported in the literature. Notably, our work offers a mechanistic hypothesis to explain the extreme virulence of bat-borne zoonoses and, more generally, demonstrates how key differences in reservoir host longevity, viral tolerance, and constitutive immunity impact the evolution of viral traits that cause virulence following spillover to humans. Our theoretical framework offers a series of testable questions and predictions designed to stimulate future work comparing cross-species virulence evolution in zoonotic viruses derived from diverse mammalian hosts.

Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.