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1.
Viruses ; 14(2)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1715771

ABSTRACT

The innate immunological response in mammals involves a diverse and complex network of many proteins. Over the last years, the tripartite motif-containing protein 5 (TRIM5) and 22 (TRIM22) have shown promise as restriction factors of a plethora of viruses that infect primates. Although there have been studies describing the evolution of these proteins in a wide range of mammals, no prior studies of the TRIM6/34/5/22 gene cluster have been performed in the Chiroptera order. Here, we provide a detailed analysis of the evolution of this gene cluster in several bat genomes. Examination of different yangochiroptera and yinpterochiroptera bat species revealed a dynamic history of gene expansion occurring in TRIM5 and TRIM22 genes. Multiple copies of TRIM5 were found in the genomes of several bats, demonstrating a very low degree of conservation in the synteny of this gene among species of the Chiroptera order. Our findings also reveal that TRIM22 is often found duplicated in yangochiroptera bat species, an evolutionary phenomenon not yet observed in any other lineages of mammals. In total, we identified 31 TRIM5 and 19 TRIM22 amino acids to be evolving under positive selection, with most of the residues being placed in the PRYSPRY domain, known to be responsible for binding to the viral capsid during restriction in the primate orthologous TRIM proteins. Altogether, our results help to shed light on the distinctive role of bats in nature as reservoirs of viruses, many of which have become threatening zoonotic diseases through virus spillover in the last decades.


Subject(s)
Chiroptera/genetics , Evolution, Molecular , Gene Duplication , Tripartite Motif Proteins/genetics , Amino Acid Sequence , Animals , Chiroptera/classification , Chiroptera/metabolism , Multigene Family , Phylogeny , Tripartite Motif Proteins/metabolism
2.
PLoS One ; 17(1): e0261344, 2022.
Article in English | MEDLINE | ID: covidwho-1602770

ABSTRACT

With more than 1400 chiropteran species identified to date, bats comprise one-fifth of all mammalian species worldwide. Many studies have associated viral zoonoses with 45 different species of bats in the EU, which cluster within 5 families of bats. For example, the Serotine bats are infected by European Bat 1 Lyssavirus throughout Europe while Myotis bats are shown infected by coronavirus, herpesvirus and paramyxovirus. Correct host species identification is important to increase our knowledge of the ecology and evolutionary pattern of bat viruses in the EU. Bat species identification is commonly determined using morphological keys. Morphological determination of bat species from bat carcasses can be limited in some cases, due to the state of decomposition or nearly indistinguishable morphological features in juvenile bats and can lead to misidentifications. The overall objective of our study was to identify insectivorous bat species using molecular biology tools with the amplification of the partial cytochrome b gene of mitochondrial DNA. Two types of samples were tested in this study, bat wing punches and bat faeces. A total of 163 bat wing punches representing 22 species, and 31 faecal pellets representing 7 species were included in the study. From the 163 bat wing punches tested, a total of 159 were genetically identified from amplification of the partial cyt b gene. All 31 faecal pellets were genetically identified based on the cyt b gene. A comparison between morphological and genetic determination showed 21 misidentifications from the 163 wing punches, representing ~12.5% of misidentifications of morphological determination compared with the genetic method, across 11 species. In addition, genetic determination allowed the identification of 24 out of 25 morphologically non-determined bat samples. Our findings demonstrate the importance of a genetic approach as an efficient and reliable method to identify bat species precisely.


Subject(s)
Chiroptera/classification , Chiroptera/genetics , DNA, Mitochondrial/analysis , Animals , Epidemiological Monitoring , Feces/chemistry , France , Rabies/veterinary , Wings, Animal/chemistry , Zoonoses
3.
Front Immunol ; 12: 807134, 2021.
Article in English | MEDLINE | ID: covidwho-1604257

ABSTRACT

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.


Subject(s)
COVID-19/immunology , Chiroptera/immunology , Immunity/immunology , Immunoglobulin Domains/immunology , Viral Proteins/immunology , Animals , Binding Sites/genetics , COVID-19/virology , Cells, Cultured , Chiroptera/genetics , Chiroptera/metabolism , Crystallography, X-Ray , Humans , Immunity/genetics , Immunoglobulin Domains/genetics , Lipopolysaccharide Receptors/immunology , Lipopolysaccharide Receptors/metabolism , Models, Molecular , Monocytes/immunology , Monocytes/metabolism , Mutation , Protein Binding , Species Specificity , Viral Proteins/classification , Viral Proteins/genetics
4.
Front Immunol ; 12: 735866, 2021.
Article in English | MEDLINE | ID: covidwho-1590052

ABSTRACT

Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this symbiotic relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this, we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFNα protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but were not associated with age, sex, or health status suggesting constitutive IFNα protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.


Subject(s)
Chiroptera/blood , Immunity, Innate , Interferon-alpha/blood , Viruses/immunology , Animals , Cell Line , Chiroptera/genetics , Chiroptera/immunology , Chiroptera/virology , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Host-Pathogen Interactions , Interferon-alpha/genetics , Species Specificity , Symbiosis , Transcription, Genetic , Viruses/pathogenicity
5.
Viruses ; 13(10)2021 10 06.
Article in English | MEDLINE | ID: covidwho-1460085

ABSTRACT

According to various estimates, only a small percentage of existing viruses have been discovered, naturally much less being represented in the genomic databases. High-throughput sequencing technologies develop rapidly, empowering large-scale screening of various biological samples for the presence of pathogen-associated nucleotide sequences, but many organisms are yet to be attributed specific loci for identification. This problem particularly impedes viral screening, due to vast heterogeneity in viral genomes. In this paper, we present a new bioinformatic pipeline, VirIdAl, for detecting and identifying viral pathogens in sequencing data. We also demonstrate the utility of the new software by applying it to viral screening of the feces of bats collected in the Moscow region, which revealed a significant variety of viruses associated with bats, insects, plants, and protozoa. The presence of alpha and beta coronavirus reads, including the MERS-like bat virus, deserves a special mention, as it once again indicates that bats are indeed reservoirs for many viral pathogens. In addition, it was shown that alignment-based methods were unable to identify the taxon for a large proportion of reads, and we additionally applied other approaches, showing that they can further reveal the presence of viral agents in sequencing data. However, the incompleteness of viral databases remains a significant problem in the studies of viral diversity, and therefore necessitates the use of combined approaches, including those based on machine learning methods.


Subject(s)
Alphacoronavirus/isolation & purification , Betacoronavirus/isolation & purification , Chiroptera/virology , Genome, Viral/genetics , Metagenome/genetics , Alphacoronavirus/classification , Alphacoronavirus/genetics , Animals , Betacoronavirus/classification , Betacoronavirus/genetics , Chiroptera/genetics , Computational Biology/methods , Feces/virology , High-Throughput Nucleotide Sequencing , Metagenomics/methods , Moscow , Phycodnaviridae/classification , Phycodnaviridae/genetics , Phycodnaviridae/isolation & purification , Sequence Analysis, DNA
6.
Viruses ; 13(10)2021 09 29.
Article in English | MEDLINE | ID: covidwho-1441884

ABSTRACT

Bats have been identified as natural reservoirs of a variety of coronaviruses. They harbor at least 19 of the 33 defined species of alpha- and betacoronaviruses. Previously, the bat coronavirus HKU10 was found in two bat species of different suborders, Rousettus leschenaultia and Hipposideros pomona, in south China. However, its geographic distribution and evolution history are not fully investigated. Here, we screened this viral species by a nested reverse transcriptase PCR in our archived samples collected over 10 years from 25 provinces of China and one province of Laos. From 8004 bat fecal samples, 26 were found to be positive for bat coronavirus HKU10 (BtCoV HKU10). New habitats of BtCoV HKU10 were found in the Yunnan, Guangxi, and Hainan Provinces of China, and Louang Namtha Province in Laos. In addition to H. pomona, BtCoV HKU10 variants were found circulating in Aselliscus stoliczkanus and Hipposideros larvatus. We sequenced full-length genomes of 17 newly discovered BtCoV HKU10 strains and compared them with previously published sequences. Our results revealed a much higher genetic diversity of BtCoV HKU10, particularly in spike genes and accessory genes. Besides the two previously reported lineages, we found six novel lineages in their new habitats, three of which were located in Yunnan province. The genotypes of these viruses are closely related to sampling locations based on polyproteins, and correlated to bat species based on spike genes. Combining phylogenetic analysis, selective pressure, and molecular-clock calculation, we demonstrated that Yunnan bats harbor a gene pool of BtCoV HKU10, with H. pomona as a natural reservoir. The cell tropism test using spike-pseudotyped lentivirus system showed that BtCoV HKU10 could enter cells from human and bat, suggesting a potential interspecies spillover. Continuous studies on these bat coronaviruses will expand our understanding of the evolution and genetic diversity of coronaviruses, and provide a prewarning of potential zoonotic diseases from bats.


Subject(s)
Alphacoronavirus/genetics , Chiroptera/virology , Alphacoronavirus/pathogenicity , Animals , Base Sequence/genetics , Biological Evolution , China , Chiroptera/genetics , Coronavirus/genetics , Coronavirus/pathogenicity , Coronavirus Infections/virology , Evolution, Molecular , Genetic Variation/genetics , Genome, Viral/genetics , Genotype , Phylogeny , Sequence Analysis, DNA/methods , Viral Proteins/genetics
7.
Science ; 374(6567): eabj3624, 2021 Oct 29.
Article in English | MEDLINE | ID: covidwho-1440797

ABSTRACT

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.


Subject(s)
2',5'-Oligoadenylate Synthetase/genetics , 2',5'-Oligoadenylate Synthetase/metabolism , COVID-19/genetics , COVID-19/physiopathology , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , SARS-CoV-2/physiology , 5' Untranslated Regions , A549 Cells , Animals , COVID-19/enzymology , COVID-19/immunology , Chiroptera/genetics , Chiroptera/virology , Coronaviridae/enzymology , Coronaviridae/genetics , Coronaviridae/physiology , Endoribonucleases/metabolism , Humans , Interferons/immunology , Isoenzymes/genetics , Isoenzymes/metabolism , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Polymorphism, Single Nucleotide , Protein Prenylation , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Retroelements , SARS-CoV-2/genetics , Severity of Illness Index , Virus Replication
8.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Article in English | MEDLINE | ID: covidwho-1428995

ABSTRACT

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.


Subject(s)
Aminohydrolases/genetics , COVID-19/genetics , Formate-Tetrahydrofolate Ligase/genetics , Methylenetetrahydrofolate Dehydrogenase (NADP)/genetics , Multienzyme Complexes/genetics , Pandemics , Aminohydrolases/antagonists & inhibitors , Animals , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Cell Line , Chiroptera/genetics , Chiroptera/virology , Formate-Tetrahydrofolate Ligase/antagonists & inhibitors , Humans , Methylenetetrahydrofolate Dehydrogenase (NADP)/antagonists & inhibitors , Minor Histocompatibility Antigens , Multienzyme Complexes/antagonists & inhibitors , RNA Viruses/genetics , SARS-CoV-2/pathogenicity , Virus Replication/genetics
9.
PLoS One ; 16(6): e0236971, 2021.
Article in English | MEDLINE | ID: covidwho-1262536

ABSTRACT

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 alpha- and 6 betacoronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance.


Subject(s)
Animals, Wild/virology , Chiroptera/virology , Coronavirus Infections/veterinary , Coronavirus/isolation & purification , Rodentia/virology , Animals , Animals, Wild/genetics , Chiroptera/genetics , Congo/epidemiology , Coronavirus/genetics , Coronavirus Infections/enzymology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Democratic Republic of the Congo/epidemiology , Environmental Monitoring/methods , Phylogeny , RNA, Viral/genetics , Rodentia/genetics
10.
J Mol Evol ; 89(6): 341-356, 2021 07.
Article in English | MEDLINE | ID: covidwho-1227833

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 is a zoonotic virus with a possible origin in bats and potential transmission to humans through an intermediate host. When zoonotic viruses jump to a new host, they undergo both mutational and natural selective pressures that result in non-synonymous and synonymous adaptive changes, necessary for efficient replication and rapid spread of diseases in new host species. The nucleotide composition and codon usage pattern of SARS-CoV-2 indicate the presence of a highly conserved, gene-specific codon usage bias. The codon usage pattern of SARS-CoV-2 is mostly antagonistic to human and bat codon usage. SARS-CoV-2 codon usage bias is mainly shaped by the natural selection, while mutational pressure plays a minor role. The time-series analysis of SARS-CoV-2 genome indicates that the virus is slowly evolving. Virus isolates from later stages of the outbreak have more biased codon usage and nucleotide composition than virus isolates from early stages of the outbreak.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , Codon Usage/genetics , Evolution, Molecular , Host-Pathogen Interactions/genetics , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Adaptation, Physiological/genetics , Animals , COVID-19/transmission , Chiroptera/genetics , Genome, Viral/genetics , Humans , Mutation , Pandemics , Principal Component Analysis , Selection, Genetic/genetics , Time Factors , Virus Replication
11.
PLoS Pathog ; 17(4): e1009501, 2021 04.
Article in English | MEDLINE | ID: covidwho-1175434

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , COVID-19/virology , Chiroptera/metabolism , SARS-CoV-2/genetics , Animals , COVID-19/genetics , Chiroptera/genetics , Host Specificity/genetics , Host Specificity/immunology , Humans , Models, Molecular , Mutation , Protein Binding/genetics , Protein Binding/physiology , Receptors, Virus/metabolism , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/metabolism
12.
Molecules ; 26(6)2021 Mar 23.
Article in English | MEDLINE | ID: covidwho-1154456

ABSTRACT

Bats are unique in their potential to serve as reservoir hosts for intracellular pathogens. Recently, the impact of COVID-19 has relegated bats from biomedical darkness to the frontline of public health as bats are the natural reservoir of many viruses, including SARS-Cov-2. Many bat genomes have been sequenced recently, and sequences coding for antimicrobial peptides are available in the public databases. Here we provide a structural analysis of genome-predicted bat cathelicidins as components of their innate immunity. A total of 32 unique protein sequences were retrieved from the NCBI database. Interestingly, some bat species contained more than one cathelicidin. We examined the conserved cysteines within the cathelin-like domain and the peptide portion of each sequence and revealed phylogenetic relationships and structural dissimilarities. The antibacterial, antifungal, and antiviral activity of peptides was examined using bioinformatic tools. The peptides were modeled and subjected to docking analysis with the region binding domain (RBD) region of the SARS-CoV-2 Spike protein. The appearance of multiple forms of cathelicidins verifies the complex microbial challenges encountered by these species. Learning more about antiviral defenses of bats and how they drive virus evolution will help scientists to investigate the function of antimicrobial peptides in these species.


Subject(s)
Cathelicidins/chemistry , Cathelicidins/pharmacology , Chiroptera/genetics , Spike Glycoprotein, Coronavirus/metabolism , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Cathelicidins/genetics , Cathelicidins/metabolism , Computational Biology/methods , Computer Simulation , Genome , Molecular Docking Simulation , Phylogeny
14.
Lancet Microbe ; 1(5): e218-e225, 2020 09.
Article in English | MEDLINE | ID: covidwho-1087372

ABSTRACT

BACKGROUND: In December, 2019, a novel zoonotic severe acute respiratory syndrome-related coronavirus emerged in China. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became pandemic within weeks and the number of human infections and severe cases is increasing. We aimed to investigate the susceptibilty of potential animal hosts and the risk of anthropozoonotic spill-over infections. METHODS: We intranasally inoculated nine fruit bats (Rousettus aegyptiacus), ferrets (Mustela putorius), pigs (Sus scrofa domesticus), and 17 chickens (Gallus gallus domesticus) with 105 TCID50 of a SARS-CoV-2 isolate per animal. Direct contact animals (n=3) were included 24 h after inoculation to test viral transmission. Animals were monitored for clinical signs and for virus shedding by nucleic acid extraction from nasal washes and rectal swabs (ferrets), oral swabs and pooled faeces samples (fruit bats), nasal and rectal swabs (pigs), or oropharyngeal and cloacal swabs (chickens) on days 2, 4, 8, 12, 16, and 21 after infection by quantitative RT-PCR (RT-qPCR). On days 4, 8, and 12, two inoculated animals (or three in the case of chickens) of each species were euthanised, and all remaining animals, including the contacts, were euthanised at day 21. All animals were subjected to autopsy and various tissues were collected for virus detection by RT-qPCR, histopathology immunohistochemistry, and in situ hybridisation. Presence of SARS-CoV-2 reactive antibodies was tested by indirect immunofluorescence assay and virus neutralisation test in samples collected before inoculation and at autopsy. FINDINGS: Pigs and chickens were not susceptible to SARS-CoV-2. All swabs, organ samples, and contact animals were negative for viral RNA, and none of the pigs or chickens seroconverted. Seven (78%) of nine fruit bats had a transient infection, with virus detectable by RT-qPCR, immunohistochemistry, and in situ hybridisation in the nasal cavity, associated with rhinitis. Viral RNA was also identified in the trachea, lung, and lung-associated lymphatic tissue in two animals euthanised at day 4. One of three contact bats became infected. More efficient virus replication but no clinical signs were observed in ferrets, with transmission to all three direct contact animals. Mild rhinitis was associated with viral antigen detection in the respiratory and olfactory epithelium. Prominent viral RNA loads of 0-104 viral genome copies per mL were detected in the upper respiratory tract of fruit bats and ferrets, and both species developed SARS-CoV-2-reactive antibodies reaching neutralising titres of up to 1/1024 after 21 days. INTERPRETATION: Pigs and chickens could not be infected intranasally by SARS-CoV-2, whereas fruit bats showed characteristics of a reservoir host. Virus replication in ferrets resembled a subclinical human infection with efficient spread. Ferrets might serve as a useful model for further studies-eg, testing vaccines or antivirals. FUNDING: German Federal Ministry of Food and Agriculture.


Subject(s)
COVID-19 , Chiroptera , Rhinitis , Animals , Antibodies, Viral , COVID-19/veterinary , Chickens/genetics , Chiroptera/genetics , Ferrets/genetics , RNA, Viral/genetics , SARS-CoV-2
15.
PLoS One ; 16(1): e0244006, 2021.
Article in English | MEDLINE | ID: covidwho-1074162

ABSTRACT

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.


Subject(s)
Alphacoronavirus/genetics , Chiroptera/genetics , Alphacoronavirus/classification , Alphacoronavirus/isolation & purification , Animals , Caves , Chiroptera/classification , Chiroptera/virology , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/metabolism , DNA, Viral/chemistry , DNA, Viral/metabolism , Far East , Feces/virology , Gene Flow , Genetic Variation , High-Throughput Nucleotide Sequencing , Monte Carlo Method , Phylogeny
16.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Article in English | MEDLINE | ID: covidwho-1066044

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2-related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19/genetics , COVID-19/metabolism , Chiroptera , SARS-CoV-2 , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/epidemiology , Chiroptera/genetics , Chiroptera/metabolism , Chiroptera/virology , HEK293 Cells , Humans , Mutation, Missense , Pandemics , Protein Binding , Protein Domains , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Species Specificity
17.
Sci Rep ; 11(1): 1702, 2021 01 18.
Article in English | MEDLINE | ID: covidwho-1035943

ABSTRACT

SARS-CoV-2 can transmit efficiently in humans, but it is less clear which other mammals are at risk of being infected. SARS-CoV-2 encodes a Spike (S) protein that binds to human ACE2 receptor to mediate cell entry. A species with a human-like ACE2 receptor could therefore be at risk of being infected by SARS-CoV-2. We compared between 132 mammalian ACE2 genes and between 17 coronavirus S proteins. We showed that while global similarities reflected by whole ACE2 gene alignments are poor predictors of high-risk mammals, local similarities at key S protein-binding sites highlight several high-risk mammals that share good ACE2 homology with human. Bats are likely reservoirs of SARS-CoV-2, but there are other high-risk mammals that share better ACE2 homologies with human. Both SARS-CoV-2 and SARS-CoV are closely related to bat coronavirus. Yet, among host-specific coronaviruses infecting high-risk mammals, key ACE2-binding sites on S proteins share highest similarities between SARS-CoV-2 and Pangolin-CoV and between SARS-CoV and Civet-CoV. These results suggest that direct coronavirus transmission from bat to human is unlikely, and that rapid adaptation of a bat SARS-like coronavirus in different high-risk intermediate hosts could have allowed it to acquire distinct high binding potential between S protein and human-like ACE2 receptors.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/classification , Angiotensin-Converting Enzyme 2/genetics , Animals , Binding Sites , COVID-19/pathology , COVID-19/transmission , COVID-19/virology , Chiroptera/genetics , Humans , Mammals , Phylogeny , Protein Binding , Risk , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/classification , Spike Glycoprotein, Coronavirus/genetics
18.
J Virol ; 94(20)2020 09 29.
Article in English | MEDLINE | ID: covidwho-1024213

ABSTRACT

The Chinese horseshoe bat (Rhinolophus sinicus), reservoir host of severe acute respiratory syndrome coronavirus (SARS-CoV), carries many bat SARS-related CoVs (SARSr-CoVs) with high genetic diversity, particularly in the spike gene. Despite these variations, some bat SARSr-CoVs can utilize the orthologs of the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), for entry. It is speculated that the interaction between bat ACE2 and SARSr-CoV spike proteins drives diversity. Here, we identified a series of R. sinicus ACE2 variants with some polymorphic sites involved in the interaction with the SARS-CoV spike protein. Pseudoviruses or SARSr-CoVs carrying different spike proteins showed different infection efficiencies in cells transiently expressing bat ACE2 variants. Consistent results were observed by binding affinity assays between SARS-CoV and SARSr-CoV spike proteins and receptor molecules from bats and humans. All tested bat SARSr-CoV spike proteins had a higher binding affinity to human ACE2 than to bat ACE2, although they showed a 10-fold lower binding affinity to human ACE2 compared with that of their SARS-CoV counterpart. Structure modeling revealed that the difference in binding affinity between spike and ACE2 might be caused by the alteration of some key residues in the interface of these two molecules. Molecular evolution analysis indicates that some key residues were under positive selection. These results suggest that the SARSr-CoV spike protein and R. sinicus ACE2 may have coevolved over time and experienced selection pressure from each other, triggering the evolutionary arms race dynamics.IMPORTANCE Evolutionary arms race dynamics shape the diversity of viruses and their receptors. Identification of key residues which are involved in interspecies transmission is important to predict potential pathogen spillover from wildlife to humans. Previously, we have identified genetically diverse SARSr-CoVs in Chinese horseshoe bats. Here, we show the highly polymorphic ACE2 in Chinese horseshoe bat populations. These ACE2 variants support SARS-CoV and SARSr-CoV infection but with different binding affinities to different spike proteins. The higher binding affinity of SARSr-CoV spike to human ACE2 suggests that these viruses have the capacity for spillover to humans. The positive selection of residues at the interface between ACE2 and SARSr-CoV spike protein suggests long-term and ongoing coevolutionary dynamics between them. Continued surveillance of this group of viruses in bats is necessary for the prevention of the next SARS-like disease.


Subject(s)
Biological Coevolution , Chiroptera/virology , SARS Virus/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2 , Animals , Binding Sites , Chiroptera/classification , Chiroptera/genetics , Coronavirus Infections/virology , Evolution, Molecular , Genetic Variation , HeLa Cells , Humans , Models, Molecular , Mutation , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Phylogeny , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , Selection, Genetic , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
19.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Article in English | MEDLINE | ID: covidwho-990135

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2-related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19/genetics , COVID-19/metabolism , Chiroptera , SARS-CoV-2 , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/epidemiology , Chiroptera/genetics , Chiroptera/metabolism , Chiroptera/virology , HEK293 Cells , Humans , Mutation, Missense , Pandemics , Protein Binding , Protein Domains , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Species Specificity
20.
Commun Biol ; 3(1): 641, 2020 10 27.
Article in English | MEDLINE | ID: covidwho-894423

ABSTRACT

The emergence of SARS-CoV-2 has caused over a million human deaths and massive global disruption. The viral infection may also represent a threat to our closest living relatives, nonhuman primates. The contact surface of the host cell receptor, ACE2, displays amino acid residues that are critical for virus recognition, and variations at these critical residues modulate infection susceptibility. Infection studies have shown that some primate species develop COVID-19-like symptoms; however, the susceptibility of most primates is unknown. Here, we show that all apes and African and Asian monkeys (catarrhines), exhibit the same set of twelve key amino acid residues as human ACE2. Monkeys in the Americas, and some tarsiers, lemurs and lorisoids, differ at critical contact residues, and protein modeling predicts that these differences should greatly reduce SARS-CoV-2 binding affinity. Other lemurs are predicted to be closer to catarrhines in their susceptibility. Our study suggests that apes and African and Asian monkeys, and some lemurs, are likely to be highly susceptible to SARS-CoV-2. Urgent actions have been undertaken to limit the exposure of great apes to humans, and similar efforts may be necessary for many other primate species.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/veterinary , Host Specificity/genetics , Pandemics/veterinary , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/veterinary , Primate Diseases/enzymology , Primates/genetics , Receptors, Virus/genetics , Amino Acid Sequence , Amino Acid Substitution , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/physiology , Biological Evolution , COVID-19 , Chiroptera/genetics , Conserved Sequence , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Genetic Predisposition to Disease , Mammals/genetics , Models, Molecular , Mutation, Missense , Peptidyl-Dipeptidase A/chemistry , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Point Mutation , Primate Diseases/virology , Protein Binding , Protein Conformation , Risk , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
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