Dear Editor, Viruses transmitted by arthropods (arboviruses) are highly diverse,both genetically and in terms of host insect species.

Merbecoviruses comprise four viral species with remarkable genetic diversity: MERS-related coronavirus, Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, and Hedgehog coronavirus 1. However, the potential human spillover risk of animal merbecoviruses remains to be investigated. Here, we reported the discovery of HKU5-CoV lineage 2 (HKU5-CoV-2) in bats that efficiently utilize human angiotensin-converting enzyme 2 (ACE2) as a functional receptor and exhibits a broad host tropism. Cryo-EM analysis of HKU5-CoV-2 receptor-binding domain (RBD) and human ACE2 complex revealed an entirely distinct binding mode compared with other ACE2-utilizing merbecoviruses with RBD footprint largely shared with ACE2-using sarbecoviruses and NL63. Structural and functional analyses indicate that HKU5-CoV-2 has a better adaptation to human ACE2 than lineage 1 HKU5-CoV. Authentic HKU5-CoV-2 infected human ACE2-expressing cell lines and human respiratory and enteric organoids. This study reveals a distinct lineage of HKU5-CoVs in bats that efficiently use human ACE2 and underscores their potential zoonotic risk.

Bats are the natural reservoirs for a variety of emerging and re-emerging viruses. Among them, rabies virus (genus Lyssavirus, family Rhabdoviridae) is one of the first and most emblematic described in these animals. Since its first description, several new bat lyssaviruses have been regularly identified. In addition to lyssaviruses, other bat rhabdoviruses have also been discovered, including members of the genera Vesiculovirus, Ledantevirus and, more recently, Alphanemrhavirus and Tupavirus. However, the family Rhabdoviridae is one of the most abundant and diverse viral families, with 434 officially recognized species, divided into 5 subfamilies and 56 different genera. The number of rhabdoviruses associated with bats is therefore probably higher than that currently available. In this study, we first developed and validated a combined nested RT-qPCR technique (pan-rhabdo RT-nqPCR) dedicated to the broad detection of animal rhabdoviruses. After validation, this technique was used for a large retrospective screening of archival bat samples (n = 1962), including blood (n = 816), brain (n = 723) and oral swab (n = 423). These samples were collected from various bat species over a 12-year period (2007-2019) in 9 different countries in Europe and Africa. A total of 23 samples (1.2%) from bat species Miniopterus schreibersii, Rhinolophus euryale and Rhinolophus ferrumequinum tested positive for rhabdovirus infection, including 17 (2.1%) blood and 6 (1.4%) oral swab samples, all collected from bats originating from the Mediterranean region. Complete virus genome sequences were obtained by next-generation sequencing for most of the positive samples. Molecular and phylogenetic analysis of these sequences demonstrated that the virus isolates, named Mediterranean bat virus (MBV), were closely related and represented a new species, Mediterranean vesiculovirus, within the genus Vesiculovirus. MBV was more specifically related to other bat vesiculoviruses previously described from China and North America, together clustering into a distinct group of bat viruses within this genus. Interestingly, our results suggest that MBV is widespread, at least in the western part of the Mediterranean region, where it circulates in the blood of several bat species. These results expand the host range and viral diversity of bat vesiculoviruses, and pave the way for further studies to determine the transmission route and dissemination dynamics of these viruses in bat colonies, as well as to assess their potential threat to public health.

High-affinity and specific binding toward the human angiotensin-converting enzyme 2 (hACE2) receptor by severe acute respiratory syndrome coronavirus (SARS)-related coronaviruses (SARSr-CoVs) remains incompletely understood. We report cryo-electron microscopy structures of eight different S-proteins from SARSr-CoVs found across Asia, Europe, and Africa. These S-proteins all adopt tightly packed, locked, prefusion conformations. These structures enable the classification of SARSr-CoV S-proteins into three types, based on their receptor-binding motif (RBM) structures and ACE2 binding characteristics. Type-2 S-proteins often preferentially bind bat ACE2 (bACE2) over hACE2. We report a structure of a type-2 BtKY72-RBD in complex with bACE2 to understand ACE2 specificity. Structure-guided mutagenesis of BtKY72-RBD reveals that multiple synergistic mutations in four different regions of RBM are required to achieve high-affinity hACE2 binding. Similar RBM changes can also confer hACE2 binding to another type-2 BM48-31 S-protein, which is primarily non-ACE2 binding. These results provide an understanding of how high-affinity hACE2 binding may be acquired by SARSr-CoV S-proteins.

The angiotensin-converting enzyme 2 (ACE2) receptor is shared by various coronaviruses with distinct receptor-binding domain (RBD) architectures, yet our understanding of these convergent acquisition events remains elusive. Here, we report that two bat MERS-related coronaviruses (MERSr-CoVs) infecting Pipistrellus nathusii (P.nat)-MOW15-22 and PnNL2018B-use ACE2 as their receptor, with narrow ortholog specificity. Cryoelectron microscopy structures of the MOW15-22/PnNL2018B RBD-ACE2 complexes unveil an unexpected and entirely distinct binding mode, mapping >45 Å away from that of any other known ACE2-using coronaviruses. Functional profiling of ACE2 orthologs from 105 mammalian species led to the identification of host tropism determinants, including an ACE2 N432-glycosylation restricting viral recognition, and the design of a soluble P.nat ACE2 mutant with potent viral neutralizing activity. Our findings reveal convergent acquisition of ACE2 usage for merbecoviruses found in European bats, underscoring the extraordinary diversity of ACE2 recognition modes among coronaviruses and the promiscuity of this receptor.

Middle East respiratory syndrome coronavirus (MERS-CoV) and the pangolin MERS-like coronavirus MjHKU4r-CoV-1 employ dipeptidyl peptidase 4 (DPP4) as an entry receptor. MjHKU4r-CoV-1 could infect transgenic mice expressing human DPP4. To understand the mechanism of MjHKU4r-CoV-1 entry into cells, we determined the crystal structures of the receptor binding domain (RBD) of MjHKU4r-CoV-1 spike protein bound to human DPP4 (hDPP4) and Malayan pangolin DPP4 (MjDPP4), respectively. The overall hDPP4-binding mode of MjHKU4r-CoV-1 RBD is similar to that of MERS-CoV RBD. MjHKU4r-CoV-1 RBD shows higher binding affinity to hDPP4 compared to the bat MERS-like coronavirus Ty-BatCoV-HKU4. Via swapping residues between MjHKU4r-CoV-1 RBD and Ty-BatCoV-HKU4 RBD, we identified critical determinants on MjHKU4r-CoV-1 that are responsible for virus usage of hDPP4. Our study suggests that MjHKU4r-CoV-1 is more adapted to the human receptor compared to the bat HKU4 coronavirus and highlights the potential of virus emergence into the human population.

Bats are the natural reservoir hosts of some viruses, some of which may spill over to humans and cause global-scale pandemics. Different from humans, bats may coexist with high pathogenic viruses without showing symptoms of diseases. As one of the most important first defenses, bat type I IFNs (IFN-Is) were thought to play a role during this virus coexistence and thus were studied in recent years. However, there are arguments about whether bats have a contracted genome locus or constitutively expressed IFNs, mainly due to species-specific findings. We hypothesized that because of the lack of pan-bat analysis, the common characteristics of bat IFN-Is have not been revealed yet. In this study, we characterized the IFN-I locus for nine Yangochiroptera bats and three Yinpterochiroptera bats on the basis of their high-quality bat genomes. We also compared the basal expression in six bats and compared the antiviral and antiproliferative activity and the thermostability of representative Rhinolophus bat IFNs. We found a dominance of unconventional IFNω-like responses in the IFN-I system, which is unique to bats. In contrast to IFNα-dominated IFN-I loci in the majority of other mammals, bats generally have shorter IFN-I loci with more unconventional IFNω-like genes (IFNω or related IFNαω), but with fewer or even no IFNα genes. In addition, bats generally have constitutively expressed IFNs, the highest expressed of which is more likely an IFNω-like gene. Likewise, the highly expressed IFNω-like protein also demonstrated the best antiviral activity, antiproliferative activity, or thermostability, as shown in a representative Rhinolophus bat species. Overall, we revealed pan-bat unique, to our knowledge, characteristics in the IFN-I system, which provide insights into our understanding of the innate immunity that contributes to a special coexistence between bats and viruses.

The COVID-19 pandemic presents a major threat to global public health. Several lines of evidence have shown that the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), along with two other highly pathogenic coronaviruses, SARS-CoV and Middle East Respiratory Syndrome (MERS-CoV) originated from bats. To prevent and control future coronavirus outbreaks, it is necessary to investigate the interspecies infection and pathogenicity risks of animal-related coronaviruses. Currently used infection models, including in vitro cell lines and in vivo animal models, fail to fully mimic the primary infection in human tissues. Here, we employed organoid technology as a promising new model for studying emerging pathogens and their pathogenic mechanisms. We investigated the key host-virus interaction patterns of five human coronaviruses (SARS-CoV-2 original strain, Omicron BA.1, MERS-CoV, HCoV-229E, and HCoV-OC43) in different human respiratory organoids. Five indicators, including cell tropism, invasion preference, replication activity, host response and virus-induced cell death, were developed to establish a comprehensive evaluation system to predict coronavirus interspecies infection and pathogenicity risks. Using this system, we further examined the pathogenicity and interspecies infection risks of three SARS-related coronaviruses (SARSr-CoV), including WIV1 and rRsSHC014S from bats, and MpCoV-GX from pangolins. Moreover, we found that cannabidiol, a non-psychoactive plant extract, exhibits significant inhibitory effects on various coronaviruses in human lung organoid. Cannabidiol significantly enhanced interferon-stimulated gene expression but reduced levels of inflammatory cytokines. In summary, our study established a reliable comprehensive evaluation system to analyse infection and pathogenicity patterns of zoonotic coronaviruses, which could aid in prevention and control of potentially emerging coronavirus diseases.

Aging is identified as a significant risk factor for severe coronavirus disease-2019 (COVID-19), often resulting in profound lung damage and mortality. Yet, the biological relationship between aging, aging-related comorbidities, and COVID-19 remains incompletely understood. This study aimed to elucidate the age-related COVID19 pathogenesis using an Hutchinson-Gilford progeria syndrome (HGPS) mouse model, a premature aging disease model, with humanized ACE2 receptors. Pathological features were compared between young, aged, and HGPS hACE2 mice following SARS-CoV-2 challenge. We demonstrated that young mice display robust interferon response and antiviral activity, whereas this response is attenuated in aged mice. Viral infection in aged mice results in severe respiratory tract hemorrhage, likely contributing a higher mortality rate. In contrast, HGPS hACE2 mice exhibit milder disease manifestations characterized by minor immune cell infiltration and dysregulation of multiple metabolic processes. Comprehensive transcriptome analysis revealed both shared and unique gene expression dynamics among different mouse groups. Collectively, our studies evaluated the impact of SARS-CoV-2 infection on progeroid syndromes using a HGPS hACE2 mouse model, which holds promise as a useful tool for investigating COVID-19 pathogenesis in individuals with premature aging.

Developing a mucosal vaccine against SARS-CoV-2 is critical for combatting the epidemic. Here, we investigated long-term immune responses and protection against SARS-CoV-2 for the intranasal vaccination of a triple receptor-binding domain (RBD) scaffold protein (3R-NC) adjuvanted with a flagellin protein (KFD) (3R-NC + KFDi.n). In mice, the vaccination elicited RBD-specific broad-neutralizing antibody responses in both serum and mucosal sites sustained at high level over a year. This long-lasting humoral immunity was correlated with the presence of long-lived RBD-specific IgG- and IgA-producing plasma cells, alongside the Th17 and Tfh17-biased T-cell responses driven by the KFD adjuvant. Based upon these preclinical findings, an open labeled clinical trial was conducted in individuals who had been primed with the inactivated SARS-CoV-2 (IAV) vaccine. With a favorable safety profile, the 3R-NC + KFDi.n boost elicited enduring broad-neutralizing IgG in plasma and IgA in salivary secretions. To meet the challenge of frequently emerged variants, we further designed an updated triple-RBD scaffold protein with mutated RBD combinations, which can induce adaptable antibody responses to neutralize the newly emerging variants, including JN.1. Our findings highlight the potential of the KFD-adjuvanted triple-RBD scaffold protein is a promising prototype for the development of a mucosal vaccine against SARS-CoV-2 infection.

Ebola virus (EBOV) and Marburg virus (MARV), members of the Filoviridae family, are highly pathogenic and can cause hemorrhagic fevers, significantly impacting human society. Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats. However, due to requirement for maximum containment laboratories when studying infectious virus, the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems. In this study, we used RACE technology to sequence the 3′-leader and 5′-trailer of MLAV and constructed a minigenome. Similar to MARV, the transcription activities of the MLAV minigenome are independent of VP30. We further assessed the effects of polymorphisms at the 5′ end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency, probably due to alterations in the RNA secondary structure. The reporter activity upon recombination of the 3′-leaders and 5′-trailers of MLAV, MARV, and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities. Additionally, we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system. Remdesivir efficiently inhibited MLAV as well as EBOV replication. In summary, this study provided new information about bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.

This study aims to comprehensively characterize the SARS-CoV-2 BA.5 variants using K18 hACE2 transgenic mice and golden hamsters as model organisms. Previous research on SARS-CoV-2 has utilized both mouse and hamster models, leading to conflicting results concerning the virus’s lethality. In our study, the finding suggests that H11-K18 hACE2 golden hamsters closely mimic the disease progression observed in human COVID-19 cases caused by BA.5 variants, demonstrating consistent severity and symptoms comparable to severe infections.Additionally, hamsters exhibit heightened respiratory viral replication, accurately reflecting the clinical viral kinetics observed in humans. The study emphasizes the critical importance of selecting an appropriate animal model for SARS-CoV-2 research, while also providing robust support for the hypothesis that BA.5 variants contribute to fatal outcomes in COVID-19 cases. These findings highlight the pivotal role of the golden hamster model in advancing our understanding of the pathogenic mechanisms underlying SARS-CoV-2 variants, as well as in the development of targeted therapeutic strategies.Significance Statement Our research work explores groundbreaking insights that could reshape our understanding of COVID-19 and pave the way for targeted therapies. We use golden hamster models to express the possibility of different animal models could contribute to human diseases. We hope this finding could clarify some conflicts existed, and help further development of medication for COVID.### Competing Interest StatementThe authors have declared no competing interest.

Bats are the natural reservoirs of a variety of emerging or re-emerging viruses. Among them, rabies virus (genus Lyssavirus , family Rhabdoviridae ) is of the first and most iconic described in these animals. Since its first description, various new bat lyssaviruses have been regularly described. Apart from lyssaviruses, other bat rhabdoviruses have been also identified, including members of the Vesiculovirus , Ledantevirus and more recently Alphanemrhavirus and Tupavirus genera. However, the family Rhabdoviridae is one of the most abundant and diverse viral families, with 318 officially recognized species divided into 3 subfamilies and 46 different genera. Thus, the number of bat- associated rhabdoviruses is probably higher. In this study, we first developed and validated a combined nested RT-qPCR technique (pan-rhabdo RT-nqPCR) dedicated to the broad detection of animal rhabdovirus. After validation, this technique was used for a large retrospective screening of archival bat samples ( n = 1962), including blood ( n = 816), brain ( n = 723) and saliva ( n = 423). These samples were collected from various bat species over a period of 12 years (2007-2019) in 9 different countries in Europe and Africa. A total of 23 samples (1.2%) from Miniopterus schreibersii , Rhinolophus euryale and Rhinolophus ferrumequinum bat species was found positive for rhabdovirus infection, including 17 (2.1%) blood and 6 (1.4%) saliva samples, all collected from bats originated from the Mediterranean region. The complete virus genome sequences were obtained by next-generation sequencing for most of the positive samples. Molecular and phylogenetic analysis of these sequences demonstrated that these virus isolates, named Mediterranean bat virus (MBV), were closely related, and represented a new species Vesiculovirus mediterranean within the Vesiculovirus genus. MBV was more specifically related to the other bat vesiculoviruses previously described in China and North America, together clustering into a distinct group of bat viruses within this genus. Interestingly, our results suggest that MBV is widely distributed, at least in the West part of the Mediterranean region, where it can act as an arbovirus infecting and circulating in multiple bat species. These findings expand the host range and the viral diversity of bat vesiculoviruses and pave the way for further investigations to determine the route of transmission and the dynamic of diffusion of these viruses into bat colonies, as well as to evaluate their potential hazard for public health. ### Competing Interest Statement The authors have declared no competing interest.

Mice are one of the most common biological models for laboratory use. However, wild‐type mice are not susceptible to COVID‐19 infection due to the low affinity of mouse ACE2, the entry protein for SARS‐CoV‐2. Although mice with human ACE2 (hACE2) driven by Ace2 promoter reflect its tissue specificity, these animals exhibit low ACE2 expression, potentially limiting their fidelity in mimicking COVID‐19 manifestations and their utility in viral studies. Here, we created and compared hACE2 mouse models generated with different strategies. Our findings show that distinct β‐globin insertion within hACE2 cassette significantly influences its expression, with downstream placement enhancing transcription. Moreover, optimizing hACE2 codons (opt‐hACE2) improves translation efficiency in multiple tissues. Notably, opt‐hACE2 mice displayed more active immune responses and severe COVID‐19 phenotypes following SARS‐CoV‐2 challenge compared to other models. Our study demonstrates the dual regulatory role of β‐globin element in transgene transcription and suggests that opt‐hACE2 mice might serve as valuable tools for SARS‐CoV‐2 research.

As a primary target of severe acute respiratory syndrome coronavirus 2, lung exhibits heterogeneous histopathological changes following infection. However, comprehensive insight into their protein basis with spatial resolution remains deficient, which hinders further understanding of coronavirus disease 2019 (COVID-19)-related pulmonary injury. Here, we generate a region -resolved proteomic atlas of hallmark pathological pulmonary structures by integrating histological examination, laser microdissection, and ultrasensitive proteomics. Over 10,000 proteins are quantified across 71 post-mortem specimens. We identify a spectrum of pathway dysregulations in alveolar epithelium, bronchial epithelium, and blood vessels compared with non-COVID-19 controls, providing evidence for transitional -state pneumocyte hyperplasia. Additionally, our data reveal the region -specific enrichment of functional markers in bronchiole mucus plugs, pulmonary fibrosis, airspace inflammation, and alveolar type 2 cells, uncovering their distinctive features. Furthermore, we detect increased protein expression associated with viral entry and inflammatory response across multiple regions, suggesting potential therapeutic targets. Collectively, this study provides a distinct perspective for deciphering COVID-19-caused pulmonary dysfunction by spatial proteomics.

Introduction. Bats are recognized as primary natural reservoirs for alpha- and betacoronaviruses. The interspecies transmission of bat coronaviruses to other mammalian hosts, including livestock and humans, can lead to epidemics, epizootics, and global pandemics. Objective. This study aims to describe coronaviruses associated with horseshoe bats (Rhinolophus spp.) in the southern regions of the European part of Russia. Materials and methods. Fecal samples were collected from bats inhabiting caves on the southern macroslope of the Greater Caucasus (Sochi-Adler region) during 2020, 2021, and 2024. Viral genomes were detected and analyzed using high-throughput sequencing (NGS) and RT-PCR. Results. A novel alphacoronavirus, designated Kudep virus (GenBank acc. # PQ649435), was identified in R. ferrumequinum. Presumably the Kudep virus represents a novel species within the subgenus Decacovirus of the genus Alphacoronavirus. The virus Showed 72% nucleotide identity to a Cardioderma bat coronavirus from Kenya and up to 67% nucleotide identity to the YN2012 virus group found in horseshoe bats in China. RT-PCR screening revealed active circulation of both Kudep virus and the previously described SARS-like betacoronavirus Khosta-1 in the study area. Infection rates in a single R. ferrumequinum colony during autumn 2021 reached 59.2% and 70.5% for Kudep and Khosta-1, respectively. Frequent co-infections with both viruses were observed in individual bats. Conclusion. Our findings expand the understanding of the distribution of bat alphacoronaviruses and their genetic diversity. We demonstrate the presence of a persistent natural foci of two potentially zoonotic bat coronaviruses, ecologically associated with R. ferrumequinum in the southern European part of Russia.

Animal models of COVID-19 facilitate the development of vaccines and antivirals against SARS-CoV-2. The efficacy of antivirals or vaccines may differ in different animal models with varied degrees of disease. Here, we introduce a mouse model expressing human angiotensin-converting enzyme 2 (ACE2). In this model, ACE2 with the human cytokeratin 18 promoter was knocked into the Hipp11 locus of C57BL/6J mouse by CRISPR - Cas9 (K18-hACE2 KI). Upon intranasal inoculation with high (3 x 105 PFU) or low (2.5 x 102 PFU) dose of SARS-CoV-2 wildtype (WT), Delta, Omicron BA.1, or Omicron BA.2 variants, all mice showed obvious infection symptoms, including weight loss, high viral loads in the lung, and interstitial pneumonia. 100% lethality was observed in K18-hACE2 KI mice infected by variants with a delay of endpoint for Delta and BA.1, and a significantly attenuated pathogenicity was observed for BA.2. The pneumonia of infected mice was accompanied by the infiltration of neutrophils and pulmonary fibrosis in the lung. Compared with K18-hACE2 Tg mice and HFH4-hACE2 Tg mice, K18-hACE2 KI mice are more susceptible to SARS-CoV-2. In the antivirals test, REGN10933 and Remdesivir had limited antiviral efficacies in K18-hACE2 KI mice upon the challenge of SARS-CoV-2 infections, while Nirmatrelvir, monoclonal antibody 4G4, and mRNA vaccines potently protected the mice from death. Our results suggest that the K18-hACE2 KI mouse model is lethal and stable for SARS-CoV-2 infection, and is practicable and stringent to antiviral development.