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1.
Virologie ; 26(2):186, 2022.
Article in English | EMBASE | ID: covidwho-1912865

ABSTRACT

Bats are natural reservoirs for numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction of coronaviruses and bat cells is, however, sparse. There is thus a need to develop bat cellular models to understand cell tropism, viral replication and virus-induced cell responses. Here, we report the first molecular study of SARS-CoV-2 infection in chiropteran cells. We investigated the ability of primary cells from Rhinolophus and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis and Nyctalus noctula, to support SARS-CoV-2 replication. None of these cells were permissive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species including humans. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that the restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or absence of bACE2 binding in these cells. By contrast, multiple restriction factors to viral replication exist in the three N. noctula cells since hACE2 expression was not sufficient to permit infection. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication, which correlated with a potent interferon response. Together, our data highlight the existence of species-specific molecular barriers to viral replication in bat cells. Our newly developed chiropteran cellular models are useful tools to investigate the interplay between viruses belonging to the SARS-CoV- 2 lineage and their natural reservoir, including the identification of factors responsible for viral restriction.

2.
Embase; 2021.
Preprint in English | EMBASE | ID: ppcovidwho-337394

ABSTRACT

Spillover of sarbecoviruses from animals to humans has resulted in outbreaks of severe acute respiratory syndrome SARS-CoVs and the ongoing COVID-19 pandemic. Efforts to identify the origins of SARS-CoV-1 and -2 has resulted in the discovery of numerous animal sarbecoviruses - the majority of which are only distantly related to known human pathogens and do not infect human cells. The receptor binding domain (RBD) on sarbecoviruses engages receptor molecules on the host cell and mediates cell invasion. Here, we tested the receptor tropism and serological cross reactivity for RBDs from two sarbecoviruses found in Russian horseshoe bats. While these two viruses are in a viral lineage distinct from SARS-CoV-1 and -2, one virus, Khosta-2, was capable of using human ACE2 to facilitate cell entry. Viral pseudotypes with a recombinant, SARS-CoV-2 spike encoding for the Khosta 2 RBD were resistant to both SARS-CoV-2 monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2. Our findings further demonstrate that sarbecoviruses circulating in wildlife outside of Asia also pose a threat to global health and ongoing vaccine campaigns against SARS-CoV-2.

3.
Croatian Medical Journal ; 62(3):300-302, 2021.
Article in English | EMBASE | ID: covidwho-1736744
4.
International Journal of Infectious Diseases ; 116:S86, 2022.
Article in English | EMBASE | ID: covidwho-1734445

ABSTRACT

Purpose: The origin of the current COVID-19 pandemic is unknown but horseshoe bats, of the family Rhinolophidae, are natural hosts to a suite of sarbecoviruses. Global surveillance is key to monitoring potentially pathogenic viral strains and improving the capacity for surveillance across Europe will bolster our understanding of viral populations within zoonotic reservoirs. Methods & Materials: Faecal samples were collected from Lesser horseshoe bats (Rhinolophus hipposideros) in the UK during annual population monitoring surveys, stored in RNAlater and frozen prior to genomic analysis. For metagenomic analysis, the Sequence-independent Single-Primer Amplification (SISPA) method was employed and sequencing completed using Illumina Nextera and the Oxford Nanopore GridION platforms. Results: A De novo hybrid assembly utilising shorter Illumina reads with longer Nanopore reads acting as a scaffold, generated a 29kb contig named RhGB01. Mapping raw reads against RhGB01 demonstrated a combined depth of 50x across the genome. Sequence alignment exhibits genomic organisation comparable to other sarbecoviruses isolated from animal and human hosts. Within the receptor binding domain, but excluding the receptor binding motif, RhGB01 has 77% and 81% amino acid homology compared to SARS-CoV-2 and SARS respectively. Maximum likelihood phylogenies inferred from the nucleotide sequence of RNA dependent RNA polymerase, spike glycoprotein and entire coding sequence exhibit clustering with the only other fully sequenced zoonotic Sarbecovirus from Europe which was isolated from Rhinolophus blasii. The structure of the receptor binding domain of RhGB01 was predicted by homology modelling using a crystal structure of the receptor binding domain of SARS-CoV as a template. This model was selected with a Global Model Quality Estimate (GMQE) > 0.7 and Quaternary Structure Quality Estimate (QMEAN) of -2.18. Structural comparisons between the predicted receptor binding domain of RhGB01 and SARS-CoV-2 highlight structurally different regions which house hACE2 contact residues. Conclusion: Phylogenetic inference and structural modelling suggest an absence of pathogenic potential for RhGB01. However, the discovery of a novel Sarbecovirus at the western limit of Lesser horseshoe bats demonstrates their presence throughout the entire horseshoe bat distribution and indicates the need for viral surveillance systems in Western Europe.

5.
Genome Biol Evol ; 14(2)2022 02 04.
Article in English | MEDLINE | ID: covidwho-1684680

ABSTRACT

The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (members of the Sarbecovirus subgenus) have been identified, is fueling speculation on the natural origins of SARS-CoV-2. We performed a comprehensive phylogenetic study on SARS-CoV-2 and all the related bat and pangolin sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Distribution of the inferred recombination events is nonrandom with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift events in the ancestry of bat sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm that horseshoe bats, Rhinolophus, are the likely reservoir species for the SARS-CoV-2 progenitor. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and cocirculation of these viruses' ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years. We confirm that a direct proximal ancestor to SARS-CoV-2 has not yet been sampled, since the closest known relatives collected in Yunnan shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for dramatically more wildlife sampling to: 1) pinpoint the exact origins of SARS-CoV-2's animal progenitor, 2) the intermediate species that facilitated transmission from bats to humans (if there is one), and 3) survey the extent of the diversity in the related sarbecoviruses' phylogeny that present high risk for future spillovers.


Subject(s)
Chiroptera/virology , Coronavirus/genetics , Pangolins/virology , Phylogeny , Recombination, Genetic , Animals , Humans , Phylogeography
6.
Viruses ; 14(1)2022 01 09.
Article in English | MEDLINE | ID: covidwho-1611142

ABSTRACT

We found and genetically described two novel SARS-like coronaviruses in feces and oral swabs of the greater (R. ferrumequinum) and the lesser (R. hipposideros) horseshoe bats in southern regions of Russia. The viruses, named Khosta-1 and Khosta-2, together with related viruses from Bulgaria and Kenya, form a separate phylogenetic lineage. We found evidence of recombination events in the evolutionary history of Khosta-1, which involved the acquisition of the structural proteins S, E, and M, as well as the nonstructural genes ORF3, ORF6, ORF7a, and ORF7b, from a virus that is related to the Kenyan isolate BtKY72. The examination of bats by RT-PCR revealed that 62.5% of the greater horseshoe bats in one of the caves were positive for Khosta-1 virus, while its overall prevalence was 14%. The prevalence of Khosta-2 was 1.75%. Our results show that SARS-like coronaviruses circulate in horseshoe bats in the region, and we provide new data on their genetic diversity.


Subject(s)
Chiroptera/virology , SARS Virus/genetics , Animals , Base Sequence , Chiroptera/classification , Evolution, Molecular , Feces/virology , Metagenomics , Mouth/virology , Phylogeny , Prevalence , Recombination, Genetic , Russia , SARS Virus/classification , Species Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
7.
Gastroenterology ; 160(6):S-234, 2021.
Article in English | EMBASE | ID: covidwho-1593970

ABSTRACT

The ongoing COVID-19 pandemic is caused by the severe acute respiratory corona virus-2 (SARS-CoV-2) which as of right now has infected 10% of world’s population and has caused >1.5 million deaths worldwide. In addition to respiratory symptoms, COVID-19 causes nausea, vomiting and diarrhea in more than half of infected subjects. This indicates that SARS-CoV-2 not only infects the respiratory tract, but also the gastrointestinal. Bats are thought to be the original reservoir for SARS-CoV-2, since SARS-CoV-2 is 96% identical to the bat coronavirus RatG13, which was identified in horseshoe bats. However, coronaviruses fail to cause overt disease in the bats, whereas strong cytopathic effects were observed in human respiratory and gastrointestinal epithelial cells upon SARS-CoV-2 infection. The goal of our research is to compare the response of primary intestinal epithelial cells of bats and humans to SARS-CoV-2 infection in order to better understand the cellular mechanism that allow bats to harbor coronaviruses without developing disease symptoms. To study the SARS-Co-V-2 infection in bats, we have, for the first time, established organoids lines from the stomach, proximal and distal small intestine of three adult Jamaican Fruit Bats (Artibeus jamaicensis). Organoids were successfully generated from both fresh and frozen tissue and could be passaged at least 25 times and frozen and thawed with no apparent changes in growth and morphology. Microscopic analysis showed that bat gastric and intestinal organoids were composed of a simple columnar epithelium and secreted variable amounts of mucus. We also observed spontaneous development of gland and crypt structures, indicating appropriate differentiation (Fig. 1). When seeded on transwell inserts, both gastric and intestinal organoid cells consistently developed a transepithelial resistance, demonstrating intact barrier function. Using confocal microscopy, we showed that both gastric and intestinal organoids from bats expressed angiotensin I converting enzyme 2 (ACE2), a key receptor for SARS-CoV-2 entry. Our innovative experimental platform will enable us to study multiple aspects of coronavirus infection including viral evolution and determinants of spillover events in a relevant primary cell model system. Importantly, we will utilize the bat organoid model to identify nonpathogenic cellular pathways that enable tolerance to SARS-CoV-2 in the reservoir hosts for this virus, potentially informing novel treatment strategies in human COVID-19 patients.

8.
Transbound Emerg Dis ; 2021 Oct 25.
Article in English | MEDLINE | ID: covidwho-1480225

ABSTRACT

The exact origin of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and source of introduction into humans has not been established yet, though it might be originated from animals. Therefore, we conducted a study to understand the putative reservoirs, transmission dynamics, and susceptibility patterns of SARS-CoV-2 in animals. Rhinolophus bats are presumed to be natural progenitors of SARS-CoV-2-related viruses. Initially, pangolin was thought to be the source of spillover to humans, but they might be infected by human or other animal species. So, the virus spillover pathways to humans remain unknown. Human-to-animal transmission has been testified in pet, farmed, zoo and free-ranging wild animals. Infected animals can transmit the virus to other animals in natural settings like mink-to-mink and mink-to-cat transmission. Animal-to-human transmission is not a persistent pathway, while mink-to-human transmission continues to be illuminated. Multiple companions and captive wild animals were infected by an emerging alpha variant of concern (B.1.1.7 lineage) whereas Asiatic lions were infected by delta variant, (B.1.617.2). To date, multiple animal species - cat, ferrets, non-human primates, hamsters and bats - showed high susceptibility to SARS-CoV-2 in the experimental condition, while swine, poultry, cattle showed no susceptibility. The founding of SARS-CoV-2 in wild animal reservoirs can confront the control of the virus in humans and might carry a risk to the welfare and conservation of wildlife as well. We suggest vaccinating pets and captive animals to stop spillovers and spillback events. We recommend sustainable One Health surveillance at the animal-human-environmental interface to detect and prevent future epidemics and pandemics by Disease X.

9.
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
10.
Sci Bull (Beijing) ; 66(12): 1215-1227, 2021 Jun 30.
Article in English | MEDLINE | ID: covidwho-1036223

ABSTRACT

Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although the binding of RaACE2 to the receptor-binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but not mouse, ACE2 enter cells efficiently. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2s. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.

11.
Emerg Infect Dis ; 26(12): 3025-3029, 2020 12.
Article in English | MEDLINE | ID: covidwho-940169

ABSTRACT

Epidemiology of bat Betacoronavirus, subgenus Sarbecovirus is largely unknown, especially outside China. We detected a sarbecovirus phylogenetically related to severe acute respiratory syndrome coronavirus 2 from Rhinolophus cornutus bats in Japan. The sarbecovirus' spike protein specifically recognizes angiotensin-converting enzyme 2 of R. cornutus, but not humans, as an entry receptor.


Subject(s)
Betacoronavirus/genetics , Chiroptera/virology , Coronavirus Infections/veterinary , Angiotensin-Converting Enzyme 2/metabolism , Animals , Betacoronavirus/physiology , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , HEK293 Cells , Humans , Japan/epidemiology , Phylogeny , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
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