Coronaviruses evolve to optimize receptor recognition in their natural hosts. A key mystery of COVID-19 is why the SARS-CoV-2 receptor-binding domain (RBD) binds human ACE2 with such high affinity despite limited time for adaptation, while some bat-derived RBDs paradoxically bind human ACE2 more strongly than bat ACE2. To investigate, we compared the RBDs of SARS-CoV-2 and BANAL-52, a related bat coronavirus, examining their interactions with ACE2 from Rhinolophus sinicus (RsBat) and from human. Structural and biochemical analyses revealed that BANAL-52 RBD is well adapted for binding to RsBat ACE2, with His498 of BANAL-52 RBD pairing favorably with His41 of RsBat ACE2. By contrast, SARS-CoV-2 RBD favors human ACE2, largely due to His34 and Met82 in human ACE2, residues that generally enhance RBD binding. These results show that receptor recognition by SARS-CoV-2 and related bat coronaviruses is consistent with established structural principles and provide structural insights relevant to the evolutionary origins of COVID-19.

The emergence of MERS-CoV, SARS-CoV-1, and SARS-CoV-2 highlights the significant public health and economic threats posed by coronaviruses. In Lao PDR, SARS-CoV-2-related bat coronaviruses capable of binding to human ACE2 receptors have been found in northern regions, but little is known about coronavirus diversity in anthropized environments like temples. This study investigated coronavirus circulation, diversity, and prevalence in bats from caves and temples in Vientiane Province, Lao PDR. A total of 648 guano samples (505 Chaerephon plicatus, 100 Hipposideros spp., 43 Taphozous spp.) were collected between December 2022 and June 2023 and screened using pan-coronavirus RT-PCR approach. The overall positivity rate was 17.28%, significantly higher in caves (18.8%) than temples (4.41%) (p = 0.003). C. plicatus showed the highest positivity rate (21.38%), followed by Hipposideros spp. 4%, while Taphozous spp. were negative. Phylogenetic analysis revealed diverse coronavirus lineages within Alphacoronavirus (80.4%) and Betacoronavirus (19.6%) genera. Although none were closely related to known human pathogens, coronaviruses of Decacovirus genus related to Chinese bat viruses and Pedacovirus genus similar to porcine epidemic diarrhea virus (PEDV) were detected. Unclassified betacoronaviruses identified were also related to viruses from C. plicatus in Thailand. This study provides valuable insights into coronavirus circulation in both natural and anthropized environments. The detection of PEDV-like viruses underlines the need for continued surveillance at the human-bat interface, where activities like guano harvesting and temple visits increase contacts. Further genomic and functional studies would enhance our understanding of their evolutionary relationships and potential for further cross-species transmission.

The zoonotic potential of bat coronaviruses, especially HKU5, is a significant issue because of their capacity to utilize human angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. This study offers structural insights into the binding kinetics of HKU5 (Bat Merbecovirus HKU5) receptor-binding domain (RBD) spike protein with human ACE2 through a multiscale computational method. This study employed structural modeling, 300-nanosecond (ns) molecular dynamics (MD) simulations, alanine-scanning mutagenesis, and computational peptide design to investigate ACE2 recognition by the HKU5 RBD and its interactions with peptides. The root mean square deviation (RMSD) investigation of HKU5-ACE2 complexes indicated that HKU5 exhibited greater flexibility than SARS-CoV-2, with RMSD values reaching a maximum of 1.2 nm. Free energy analysis, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), indicated a more robust binding affinity of HKU5 to ACE2 (ΔGTotal = -21.61 kcal/mol) in contrast to SARS-CoV-2 (ΔGTotal = -5.82 kcal/mol), implying that HKU5 binding with ACE2 had higher efficiency. Additionally, a peptide was designed from the ACE2 interface, resulting in the development of 380 single-site mutants by mutational alterations. The four most promising mutant peptides were selected for 300-nanosecond (ns) MD simulations, subsequently undergoing quantum chemical calculations (DFT) to evaluate their electronic characteristics. MM/GBSA of -37.83 kcal/mol indicated that mutant-1 exhibits the most favorable binding with HKU5, hence potentially inhibiting ACE2 interaction. Mutant-1 formed hydrogen bonds involving Glu74, Ser202, Ser204, and Asn152 residues of HKU5. Finally, QM/MM calculations on the peptide-HKU5 complexes showed the most favorable ΔE_interaction of -170.47 (Hartree) for mutant-1 peptide. These findings offer a thorough comprehension of receptor-binding dynamics and are crucial for evaluating the zoonotic risk associated with HKU5-CoV and guiding the design of receptor-targeted antiviral treatments.

SARS-CoV, MERS-CoV, and SARS-CoV-2 have posed tremendous threats to human health, highlighting the necessity of monitoring cross-species transmission of animal coronaviruses to humans. Hedgehogs infected with coronaviruses have been reported in several countries across Europe and Asia, raising concerns about the potential transmission of coronaviruses from hedgehogs to humans. In this study, we investigated coronavirus infections in hedgehogs inhabiting greenspaces in metropolitan Beijing and identified a Merbecovirus subgenus coronavirus with a prevalence rate of 30% (95% CI: 25–35%) among 317 hedgehogs. Phylogenetic analysis of 23 complete viral genome sequences revealed a monophyletic origin, showing close relatedness to Erinaceus hedgehog coronavirus HKU31 (Ea-HedCoV HKU31) with genome-wide nucleotide identities of 93.24–96.42%, and evidence of recombination with Tylonycteris bat coronavirus HKU4. These findings suggest that the increase in wildlife populations associated with urban greenspace development may pose a potential threat to human health that should not be overlooked.

229E- and NL63-like coronaviruses in phyllostomid bats, Belize.

Emerged SARS-CoV-2 intermediate hosts: The missing links from One Health perspective

SARS-like betacoronaviruses (sarbecoviruses) endemic in bats pose a significant zoonotic threat to humans. Genetic pathways associated with spillover of bat sarbecoviruses into humans are incompletely understood. We previously showed that the wild-type spike of the rhinolophid bat coronavirus SHC014-CoV has poor entry activity and uncovered two distinct genetic pathways outside the receptor-binding domain (RBD) that increased spike opening, ACE2 binding, and cell entry. Herein, we show that the widely studied bat sarbecovirus WIV1-CoV is likely a cell culture-adapted variant whose progenitor bears a spike resembling that of Rs3367-CoV, which was sequenced from the same population of rhinolophid bats as SHC014-CoV. Our findings suggest that the acquisition of a single amino-acid substitution in the ‘630-loop’ of the S1 subunit was the key spike adaptation event during the successful isolation of WIV1-CoV, and that it enhances spike opening, virus-receptor recognition, and cell entry in much the same manner as the substitutions we previously identified in SHC014-CoV using a pseudotype system. The conformational constraints on both the SHC014-CoV and Rs3367-CoV spikes could be alleviated by pre-cleaving them with trypsin, suggesting that the spike-opening substitutions arose to circumvent the lack of S1–S2 cleavage. We propose that the ‘locked-down’ nature of these spikes and their requirement for S1–S2 cleavage to engage ACE2 represent viral optimizations for a fecal-oral lifestyle and immune evasion in their natural hosts. These adaptations may be a broader property of bat sarbecoviruses than currently recognized. The acquisition of a polybasic furin cleavage site at the S1–S2 boundary is accepted as a key viral adaptation for SARS-CoV-2 emergence that overcame a host protease barrier to viral entry in the mammalian respiratory tract. Our results suggest alternative spillover scenarios in which spike-opening substitutions that promote virus-receptor binding and entry could precede, or even initially replace, substitutions that enhance spike cleavage in the zoonotic host.

SARS-like betacoronaviruses (sarbecoviruses) endemic in bats pose a significant zoonotic threat to humans. Genetic pathways associated with spillover of bat sarbecoviruses into humans are incompletely understood. We previously showed that the wild-type spike of the rhinolophid bat coronavirus SHC014-CoV has poor entry activity and uncovered two distinct genetic pathways outside the receptor-binding domain (RBD) that increased spike opening, ACE2 binding, and cell entry. Herein, we show that the widely studied bat sarbecovirus WIV1-CoV is likely a cell culture-adapted variant whose progenitor bears a spike resembling that of Rs3367-CoV, which was sequenced from the same population of rhinolophid bats as SHC014-CoV. Our findings suggest that the acquisition of a single amino-acid substitution in the '630-loop' of the S1 subunit was the key spike adaptation event during the successful isolation of WIV1-CoV, and that it enhances spike opening, virus-receptor recognition, and cell entry in much the same manner as the substitutions we previously identified in SHC014-CoV using a pseudotype system. The conformational constraints on both the SHC014-CoV and Rs3367-CoV spikes could be alleviated by pre-cleaving them with trypsin, suggesting that the spike-opening substitutions arose to circumvent the lack of S1-S2 cleavage. We propose that the 'locked-down' nature of these spikes and their requirement for S1-S2 cleavage to engage ACE2 represent viral optimizations for a fecal-oral lifestyle and immune evasion in their natural hosts. These adaptations may be a broader property of bat sarbecoviruses than currently recognized. The acquisition of a polybasic furin cleavage site at the S1-S2 boundary is accepted as a key viral adaptation for SARS-CoV-2 emergence that overcame a host protease barrier to viral entry in the mammalian respiratory tract. Our results suggest alternative spillover scenarios in which spike-opening substitutions that promote virus-receptor binding and entry could precede, or even initially replace, substitutions that enhance spike cleavage in the zoonotic host.

Detection of alphacoronaviruses in Rhinolophus ferrumequinum in Ehime, Japan.

Since the appearance of the Severe Acute Respiratory Syndrome (SARS) virus, there has been increased interest in understanding the role of bats in the maintenance and circulation of coronaviruses. This study aimed to describe the phylogenetic and evolutionary relationships and antigenic architecture of a new coronavirus detected in bats in the Department of Córdoba. In a surveillance study of pathogens of interest to public health, a bat Phyllostomus hastatus was captured. Rectal swabs samples were collected from the bats, and RNA was extracted and sequenced using NGS with MGI-G50 equipment. The results were analyzed using bioinformatics software. A contig of 28,619 nucleotides associated with the Coronaviridae family was obtained. Phylogenetic and molecular clock analyses of the ORF1ab gene revealed a novel divergent Alphacoronavirus that originated directly from an ancestral node. The analysis of the spike (S) protein and receptor-binding domain (RBD) is similar to that of humans (HCoV-229E) and porcine coronaviruses. In silico analysis suggests potential RBD interaction sites with human and pig cellular receptor aminopeptidase N. There is a possible risk of interspecies jumping of the new AlphaCoV/P. hastatus in humans and pigs. This is the first study to perform phylogenetic, evolutionary, and antigenic characterization of bat coronaviruses in Colombia.

Binding of an N protein peptide to M protein of a bat coronavirus.

Inhibiting viral entry of bat-derived coronavirus HKU5-CoV-2: Targeting spike protein S1 subunit with FDA-approved antivirals-A structural dynamics and energetics study.

BackgroundStraw-coloured fruit bats (Eidolon helvum; ) are widely distributed in Africa and are known reservoirs for viruses with zoonotic potential. These bats are widely hunted in West and Central Africa for human consumption as food source and medicine. This practice increases the potential for spillover of zoonotic disease to the human population. This study investigated the presence of neutralising antibodies against SARS-CoV-2 variants in serum samples (n = 142) from E. helvum bats captured between November 2017 and March 2019 in Makurdi, Nigeria. Faecal samples (n = 120) from the roost were collected in 2022 and screened for the presence of coronavirus RNA followed by genetic sequencing.ResultsVirus neutralisation tests revealed 7.04% of the bat sera neutralised 2019-nCoV/Italy-INMI1, while 17.57% of the bat sera neutralised a SARS-CoV-2 Omicron BA.1 isolate. Partial genome obtained by next generation sequencing identified a lineage D Betacoronavirus from one faecal sample with 98.16% nucleotide sequence identity to sequences from Eidolon helvum collected in Cameroon in 2013. Epitope analysis of the spike protein sequence from the faecal sample showed conserved antigenic determinants shared with SARS-CoV-2.ConclusionsThis study demonstrated that pre-pandemic sera collected from Eidolon helvum bats had neutralising activity against SARS-CoV-2 variants. Furthermore we detected the presence of lineage D betacoronavirus in this bat population that shared epitopes with SARS-CoV-2. This work contributes to our understanding of the complexities of coronavirus cross-reactivity. Characterizing bat coronaviruses is crucial to understand their zoonotic potential for spillover events due to bushmeat hunting practices.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-025-04938-y.

COVID-19 and long COVID are characterized by a dysregulated immune response. However, the role of macrophages during viral infection is poorly defined. Here we demonstrate that SARS-CoV-2 infection results in increased macrophage numbers and extensive formation of enlarged lipid-laden macrophages or foam cells using humanized mice, rhesus macaques and post-mortem human lung tissue. Notably, infection by other coronaviruses tested, SARS-CoV-1, MERS-CoV and two bat coronaviruses (SHC014-CoV or WIV1-CoV), did not result in macrophage proliferation or foam cell formation. Foam cells in SARS-CoV-2-infected human lung tissue display a pro-fibrotic and pro-thrombotic phenotype as they are enriched for genes associated with platelet activation and aggregation, as well as extracellular matrix organization and collagen synthesis. After viral clearance, macrophage numbers remain elevated, and lung fibrosis and thrombi persist. Importantly, we show that pre-exposure prophylaxis or early treatment with a SARS-CoV-2 antiviral, EIDD-2801, prevents increases in macrophage cell numbers and foam cell formation, and reduces fibrosis markers. These observations highlight the contribution of macrophages to lung inflammation and tissue injury leading to the pulmonary fibrosis observed in COVID-19 patients.

Zoonotic diseases are increasingly affecting global health, making it crucial to understand viral dynamics in wildlife reservoirs like bats. Bats are well-established coronavirus reservoirs, and their proximity to human habitats in France raises concerns about zoonotic transmission. This study investigates the prevalence, diversity, and phylogenetic relationships of coronaviruses in bats from the Camargue and Eastern Provence, two ecologically distinct regions. A total of 785 bat guano samples were collected non-invasively, with 154 (19.6%) testing positive for coronavirus RNA, among the highest infection rates observed in European studies. Camargue samples were entirely from Pipistrellus bats, with a prevalence of 20.1%, while Provence showed greater genus diversity and prevalence ranging from 1.3% to 35.6%. Phylogenetic analysis identified Alphacoronaviruses in four subgenera: Nyctacovirus, Myotacovirus, Decacovirus, and Minunacovirus, alongside unclassified sequences. No coronavirus related to human-infecting strains (e.g., HCoV-229E) was detected. However, the combination of high prevalence, proximity to humans, and potential for host-switching led to the investigation of coronavirus infection in bats, a topic with limited data in France. These findings highlight the ecological role of bats and the importance of continuous viral monitoring, filling a gap in knowledge about coronavirus circulation in European bat populations.

Bats are the reservoir hosts of emerging coronaviruses (CoVs) affecting human and livestock health. We assessed the diversity, evolution, and geographic distribution of two alphacoronaviruses (subgenus Rhinacovirus) with considerable potential for emergence: swine acute diarrhea syndrome coronavirus (SADS-CoV), which has caused large outbreaks in pigs in China and can infect primary human airway epithelial cells in vitro; and the related Rhinolophus bat coronavirus HKU2 (HKU2-CoV). Phylogenetic analyses of 523 rhinacovirus sequences from bats in China and Southeast Asia suggest these viruses should be reclassified into at least two distinct CoV species representing two well-supported monophyletic clades. Stronger phylogenetic clustering by sampling location than by host species suggests infrequent long-distance transmission of rhinacoviruses in southern China. Ancestral state reconstruction analysis indicates that R. sinicus/thomasi and R. affinis have played an important role in rhinacovirus evolution in southern China and that R. affinis is the likely reservoir host of SADS-CoV that spilled over into pigs. We used species distribution modeling of Rhinolophus spp. bat hosts of rhinacoviruses, combined with pig and human density data, to identify potential geographic rhinacovirus spillover risk in Southeast Asia. Areas of high pig density within suitable bat habitat exist primarily in southern China and northern Vietnam, and hotspots of the highest human density within suitable bat habitat are primarily along the southern coast of China, Java, and central Thailand. Targeted surveillance of pigs and people in these regions may facilitate the timely detection of bat CoV spillover events and mitigate the risk of future outbreaks.IMPORTANCEBats are the reservoir or ancestral hosts of important emerging coronaviruses affecting people (e.g., SARS-CoV and SARS-CoV-2) and livestock (e.g., PEDV, SADS-CoV). Here, we analyzed 523 genetic sequences of SADS-CoV that caused large-scale die-offs of pigs in China, which is known to be able to infect human cells and related HKU2-CoVs. We used this information to identify the horseshoe bat Rhinolophus affinis as the likely spillover host for the outbreak in pigs, and identified the bat species within which these viruses evolved. We then modeled the distribution of these host species and their overlap with dense human and pig populations to identify the regions where surveillance programs can help identify spillover events and prevent future outbreaks.

The zoonotic transmission of bat coronaviruses poses a threat to human health. However, the diversity of bat-borne coronaviruses remains poorly characterized in many geographical areas. Here, we recovered eight coronavirus genomes by performing a metagenomic analysis of fecal samples from hundreds of individual bats captured in Spain, a country with high bat diversity. Three of these genomes corresponded to potentially novel coronavirus species belonging to the alphacoronavirus genus. Phylogenetic analyses revealed that some of these viruses are closely related to coronaviruses previously described in bats from other countries, suggesting a shared viral reservoir worldwide. Using viral pseudotypes, we investigated the receptor usage of the identified viruses and found that one of them can use human ACE2, albeit with lower affinity than SARS-CoV-2. However, the receptor usage of the other viruses remains unknown. This study broadens our understanding of coronavirus diversity and identifies research priorities for the prevention of zoonotic viral outbreaks.

The viral entry mechanisms, which are primarily related to the spike character, play a critical role in determining zoonotic potential. Among the currently identified SC2r-CoVs, BANAL-52 and BANAL-103 exhibit spike proteins with the highest sequence similarity to SARS-CoV-2, rendering them optimal models for comparative studies on S-mediated cell entry and cross-species transmission. In this study, we systematically investigated the molecular constraints governing the functionality of BANAL spikes, with a focus on S-ACE2 interactions, S activation, S structures, and host protease utilization. Notably, we resolved the cryo-EM structure of BANAL-52 S at neutral pH and the first cryo-EM structure of BANAL-103 S, revealing distinct glycan- and lipid-mediated stabilization of inactive states. Furthermore, cross-neutralization assays demonstrated that sera of convalescents from SARS-CoV-2 inhibited BANAL pseudovirus entry with an efficiency of approximately 80%, thereby highlighting conserved antigenic epitopes and informing the development of broad-spectrum therapeutic strategies against emerging SC2r-CoVs.

Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species.

Although numerous sarbecoviruses have been identified in bats, but most lack the ability to infect human cells. Some barriers limit coronavirus zoonosis, including susceptibility to host proteases. Here, we investigated whether exogenous protease treatment can circumvent host restrictions in two severe acute respiratory syndrome (SARS)-related bat coronaviruses. We found that the spike proteins of RaTG13 and Khosta-2, which are sarbecoviruses obtained from horseshoe bats in China and Russia, respectively, facilitated the ACE2-mediated entry of pseudotyped viruses into VeroE6/TMPRSS2 cells following elastase treatment. In contrast, trypsin and thermolysin exhibited no effects. Elastase-enhanced infectivity correlated with increased fusogenicity driven by the cleavage of spike proteins. This process was TMPRSS2-dependent and was inhibited by nafamostat, a TMPRSS2 inhibitor. Additionally, mutation of residue 809 within the S2 subunit of the RaTG13 spike protein (S809D) impaired elastase-induced cleavage and infectivity. Hence, proteolytic processing of the spike protein serves as a restriction to RaTG13 and Khosta-2 infections, which can be overcome by elastase. This suggests that elastase secreted in inflamed tissues during viral infection may increase the zoonotic potential of sarbecoviruses by facilitating human cell entry.