Rhinolophus Bats Research Articles

Unconventional IFNω-like Genes Dominate the Type I IFN Locus and the Constitutive Antiviral Responses in Bats.

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.

SARS CoV 2 (COVID-19) Diagnosis in Wildlife Animals using Rt-Polymerase Chain Reaction (PCR) in Dubai Safari Park

Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses. SARS-CoV-2 is a beta coronavirus, a genus that includes several coronaviruses (SARS-CoV, MERS-CoV, bat SARS-like CoV, and others) isolated from humans, bats, camels, civets, and other animals. Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is the pathogenic agent that causes the disease COVID-19. SARS-CoV-2 is thought to have emerged from an animal source, most likely a bat, and subsequently spread to humans. While genetically closely related viruses have been isolated from Rhinolophus bats, the exact source of SARS-CoV-2 and its route of introduction into the human population have not been established. Monitoring animal infections is imperative to better understanding their epidemiological significance for animal health, biodiversity, and human health. According to evidence from risk assessments, epidemiological investigations, and experimental studies, animals play no significant role in the spread of SARS-CoV-2, which is sustained by human-to-human transmission. The possibility of Coronavirus testing by veterinary labs was considered after the Iowa State veterinary lab discovered the COVID-19 virus has a similar DNA testing process to porcine epidemic diarrhea virus (PED). PED is another form of coronavirus that killed many piglets in 2013 and was unresponsive to treatment. Several vet labs optimized their equipment and processes to test for PED, helping them determine that older pigs could recover and develop immunity against the virus. Those same labs are still set up for coronavirus testing. We in Dubai Safari Park, particularly within the laboratory Veterinary Hospital, are conducting RT-PCR analysis to test diverse animal species including non-human primates, Carnivores, small mammals, and Ungulates. Even though none of our animals show any respiratory signs, we have conducted this study to ensure that our animal collection is healthy and free of SARS-COV-2.

Probiotics, An Approach towards Modern Medication

Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses. SARS-CoV-2 is a beta coronavirus, a genus that includes several coronaviruses (SARS-CoV, MERS-CoV, bat SARS-like CoV, and others) isolated from humans, bats, camels, civets, and other animals. Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is the pathogenic agent that causes the disease COVID-19. SARS-CoV-2 is thought to have emerged from an animal source, most likely a bat, and subsequently spread to humans. While genetically closely related viruses have been isolated from Rhinolophus bats, the exact source of SARS-CoV-2 and its route of introduction into the human population have not been established. Monitoring animal infections is imperative to better understanding their epidemiological significance for animal health, biodiversity, and human health. According to evidence from risk assessments, epidemiological investigations, and experimental studies, animals play no significant role in the spread of SARS-CoV-2, which is sustained by human-to-human transmission. The possibility of Coronavirus testing by veterinary labs was considered after the Iowa State veterinary lab discovered the COVID-19 virus has a similar DNA testing process to porcine epidemic diarrhea virus (PED). PED is another form of coronavirus that killed many piglets in 2013 and was unresponsive to treatment. Several vet labs optimized their equipment and processes to test for PED, helping them determine that older pigs could recover and develop immunity against the virus. Those same labs are still set up for coronavirus testing. We in Dubai Safari Park, particularly within the laboratory Veterinary Hospital, are conducting RT-PCR analysis to test diverse animal species including non-human primates, Carnivores, small mammals, and Ungulates. Even though none of our animals show any respiratory signs, we have conducted this study to ensure that our animal collection is healthy and free of SARS-COV-2.

Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus

Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S)glycoproteins remain uncharacterized. Here, we show that the African Rhinolophus bat clade 3 sarbecovirus PRD-0038S has a broad angiotensin-converting enzyme 2 (ACE2) usage and that receptor-binding domain (RBD) mutations further expand receptor promiscuity and enable human ACE2 utilization. We determine a cryo-EM structure of the PRD-0038 RBD bound to Rhinolophus alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038S using cryo-EM and monoclonal antibody reactivity reveals its distinct antigenicity relative to SARS-CoV-2 and identifies PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038S vaccination elicits greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared with SARS-CoV-2S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.

A fast and accurate identification model for Rhinolophus bats based on fine-grained information

Bats are a crucial component within ecosystems, providing valuable ecosystem services such as pollination and pest control. In practical conservation efforts, the classification and identification of bats are essential in order to develop effective conservation management programs for bats and their habitats. Traditionally, the identification of bats has been a manual and time-consuming process. With the development of artificial intelligence technology, the accuracy and speed of identification work of such fine-grained images as bats identification can be greatly improved. Bats identification relies on the fine features of their beaks and faces, so mining the fine-grained information in images is crucial to improve the accuracy of bats identification. This paper presents a deep learning-based model designed for the rapid and precise identification of common horseshoe bats (Chiroptera: Rhinolophidae: Rhinolophus) from Southern China. The model was developed by utilizing a comprehensive dataset of 883 high-resolution images of seven distinct Rhinolophus species which were collected during surveys conducted between 2010 and 2022. An improved EfficientNet model with an attention mechanism module is architected to mine the fine-grained appearance of these Rhinolophus. The performance of the model beat other classical models, including SqueezeNet, AlexNet, VGG16_BN, ShuffleNetV2, GoogleNet, ResNet50 and EfficientNet_B0, according to the predicting precision, recall, accuracy, F1-score. Our model achieved the highest identification accuracy of 94.22% and an F1-score of 0.948 with low computational complexity. Heat maps obtained with Grad-CAM show that our model meets the identification criteria of the morphology of Rhinolophus. Our study highlights the potential of artificial intelligence technology for the identification of small mammals, and facilitating fast species identification in the future.

Determination of the factors responsible for the tropism of SARS-CoV-2-related bat coronaviruses to Rhinolophus bat ACE2.

The efficiency of infection receptor use is the first step in determining the species tropism of viruses. After the coronavirus disease 2019 pandemic, a number of SARS-CoV-2-related coronaviruses (SC2r-CoVs) were identified in Rhinolophus bats, and some of them can use human angiotensin converting enzyme 2 (ACE2) for the infection receptor without acquiring additional mutations. This means that the potential of certain SC2r-CoVs to cause spillover from bats to humans is "off-the-shelf." However, both SC2r-CoVs and Rhinolophus bat species are highly diversified, and the host tropism of SC2r-CoVs remains unclear. Here, we focus on two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and determine how the tropism of SC2r-CoVs to Rhinolophus bat ACE2 is determined at the amino acid resolution level.

Khosta: A Genetic and Structural Point of View of the Forgotten Virus

Bats are well-known to be natural reservoirs of various zoonotic coronaviruses, which have caused outbreaks of severe acute respiratory syndrome (SARS) and the COVID-19 pandemic in 2002 and 2019, respectively. In late 2020, two new Sarbecoviruses were found in Russia, isolated in Rhinolophus bats, i.e., Khosta-1 in R. ferrumequinum and Khosta-2 in R. hipposideros. The potential danger associated with these new species of Sarbecovirus is that Khosta-2 has been found to interact with the same entry receptor as SARS-CoV-2. Our multidisciplinary approach in this study demonstrates that Khosta-1 and -2 currently appear to be not dangerous with low risk of spillover, as confirmed by prevalence data and by phylogenomic reconstruction. In addition, the interaction between Khosta-1 and -2 with ACE2 appears weak, and furin cleavage sites are absent. While the possibility of a spillover event cannot be entirely excluded, it is currently highly unlikely. This research further emphasizes the importance of assessing the zoonotic potential of widely distributed batborne CoV in order to monitor changes in genomic composition of viruses and prevent spillover events (if any).

Coronaviruses Are Abundant and Genetically Diverse in West and Central African Bats, including Viruses Closely Related to Human Coronaviruses.

Bats are at the origin of human coronaviruses, either directly or via an intermediate host. We tested swabs from 4597 bats (897 from the Democratic Republic of Congo (DRC), 2191 from Cameroon and 1509 from Guinea) with a broadly reactive PCR in the RdRp region. Coronaviruses were detected in 903 (19.6%) bats and in all species, with more than 25 individuals tested. The highest prevalence was observed in Eidolon helvum (239/733; 39.9%) and Rhinolophus sp. (306/899; 34.1%), followed by Hipposideros sp. (61/291; 20.9%). Frugivorous bats were predominantly infected with beta coronaviruses from the Nobecovirus subgenus (93.8%), in which at least 6 species/genus-specific subclades were observed. In contrast, insectivorous bats were infected with beta-coronaviruses from different subgenera (Nobecovirus (8.5%), Hibecovirus (32.8%), Merbecovirus (0.5%) and Sarbecovirus (57.6%)) and with a high diversity of alpha-coronaviruses. Overall, our study shows a high prevalence and genetic diversity of coronaviruses in bats and illustrates that Rhinolophus bats in Africa are infected at high levels with the Sarbecovirus subgenus, to which SARS-CoV-2 belongs. It is important to characterize in more detail the different coronavirus lineages from bats for their potential to infect human cells, their evolution and to study frequency and modes of contact between humans and bats in Africa.

Frequency of SARS-CoV-2 infection in dogs and cats: Results of a retrospective serological survey in Šumadija District, Serbia

It has long been known that coronaviruses cause various infectious diseases in animals. Although SARS-CoV-2 is genetically related to viruses isolated from Rhinolophus bats, the exact origin, mode of transmission, and how the human species has become the epidemiological reservoir of the virus have not yet been established with certainty. Although the main route of transmission is human-to-human, there are considerable numbers of reported cases of infection in animal species, predominantly among pet animals. The aim of this retrospective study was to assess SARS-CoV-2 seropositivity in dogs and cats during the COVID-19 pandemic in Šumadija District, Serbia. We used serology to identify household contacts of pet animals with infected pet owners and the degree of association. The study presented in this paper is also the first study of this type in Serbia. The results of a retrospective serosurvey, which was conducted in dogs and cats with different exposure risk factors, were analyzed to find the possible modes of transmission between humans and animals. The relative frequency of SARS-CoV-2 infection in dogs was 1.45% bounded with a 95% confidence interval (CI) of 0.0007–7.73%, while in cats, it was 5.56% (95% CI: 0.77–4.13%). The relative frequency of SARS-CoV-2 infection in pet owners was 11% (95% CI: 6.25–18.63%). In pets that were in close contact with COVID-19 positive owners, the seropositivity was found to be 9%. Out of a total of five stray dogs and cats tested, seropositivity was observed in two animals. Detected SARS-CoV-2 infection in pets shows that these animals are susceptible to infection and that the most common means of virus transmission to pets is through contact with diseased owners. However, the presence of infection in stray dogs and cats is not clear and needs further research.

Myotis fimbriatus Virome, a Window to Virus Diversity and Evolution in the Genus Myotis.

Significant efforts have been made to characterize viral diversity in bats from China. Many of these studies were prospective and focused mainly on Rhinolophus bats that could be related to zoonotic events. However, other species of bats that are part of ecosystems identified as virus diversity hotspots have not been studied in-depth. We analyzed the virome of a group of Myotis fimbriatus bats collected from the Yunnan Province during 2020. The virome of M. fimbriatus revealed the presence of families of pathogenic viruses such as Coronavirus, Astrovirus, Mastadenovirus, and Picornavirus, among others. The viral sequences identified in M. fimbriatus were characterized by significant divergence from other known viral sequences of bat origin. Complex phylogenetic landscapes implying a tendency of co-specificity and relationships with viruses from other mammals characterize these groups. The most prevalent and abundant virus in M. fimbriatus individuals was an alphacoronavirus. The genome of this virus shows evidence of recombination and is likely the product of ancestral host-switch. The close phylogenetic and ecological relationship of some species of the Myotis genus in China may have played an important role in the emergence of this alphacoronavirus.

A standardized instrument quantifying risk factors associated with bi-directional transmission of SARS-CoV-2 and other zoonotic pathogens: The COVID-19 human-animal interactions survey (CHAIS)

Similar to many zoonotic pathogens which transmit from animals to humans, SARS-CoV-2 (CoV-2), the virus responsible for the COVID-19 pandemic, most likely originated in Rhinolophus bats before spreading among humans globally. Early into the pandemic, reports of CoV-2 diagnoses in animals from various countries emerged. While most CoV-2 positive animals were confirmed to have been in close contact with CoV-2 positive humans, there has been a paucity of published evidence to-date describing risk factors associated with CoV-2 transmission among humans and animals. The COVID-19 Human-Animal Interactions Survey (CHAIS) was developed to provide a standardized instrument describing human-animal interactions during the pandemic and to evaluate behavioral, spatiotemporal, and biological risk factors associated with bi-directional zoonotic transmission of CoV-2 within shared environments, predominantly households with limited information about human-wildlife or human-livestock interactions. CHAIS measures four broad domains of transmission risk: 1) risk and intensity of infection in human hosts, 2) spatial characteristics of shared environments, 3) behaviors and human-animal interactions, and 4) susceptible animal subpopulations. Following the development of CHAIS, with a One Health approach, a multidisciplinary group of experts (n = 20) was invited to review and provide feedback on the survey for content validity. Expert feedback was incorporated into two final survey formats—an extended version and an abridged version for which specific core questions addressing zoonotic and reverse zoonotic transmission were identified. Both versions are modularized, with each section having the capacity to serve as independent instruments, allowing researchers to customize the survey based on context and research-specific needs. Further adaptations for studies seeking to investigate other zoonotic pathogens with similar routes of transmission (i.e. respiratory, direct contact) are also possible. The CHAIS instrument is a standardized human-animal interaction survey developed to provide important data on risk factors that guide transmission of CoV-2, and other similar pathogens, among humans and animals.

Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains.

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV) and SARS-CoV-2 enter cells via engagement of angiotensin converting enzyme-2 (ACE2), while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clades 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from Rhinolophus alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

Variation in synonymous nucleotide composition among genomes of sarbecoviruses and consequences for the origin of COVID-19

The subgenus Sarbecovirus includes two human viruses, SARS-CoV and SARS-CoV-2, respectively responsible for the SARS epidemic and COVID-19 pandemic, as well as many bat viruses and two pangolin viruses.Here, the synonymous nucleotide composition (SNC) of Sarbecovirus genomes was analysed by examining third codon-positions, dinucleotides, and degenerate codons. The results show evidence for the eight following groups: (i) SARS-CoV related coronaviruses (SCoVrC including many bat viruses from China), (ii) SARS-CoV-2 related coronaviruses (SCoV2rC; including five bat viruses from Cambodia, Thailand and Yunnan), (iii) pangolin sarbecoviruses, (iv) three bat sarbecoviruses showing evidence of recombination between SCoVrC and SCoV2rC genomes, (v) two highly divergent bat sarbecoviruses from Yunnan, (vi) the bat sarbecovirus from Japan, (vii) the bat sarbecovirus from Bulgaria, and (viii) the bat sarbecovirus from Kenya. All these groups can be diagnosed by specific nucleotide compositional features except the one concerned by recombination between SCoVrC and SCoV2rC. In particular, SCoV2rC genomes have less cytosines and more uracils at third codon-positions than other sarbecoviruses, whereas the genomes of pangolin sarbecoviruses show more adenines at third codon-positions. I suggest that taxonomic differences in the imbalanced nucleotide pools available in host cells during viral replication can explain the eight groups of SNC here detected among Sarbecovirus genomes. A related effect due to hibernating bats and their latitudinal distribution is also discussed. I conclude that the two independent host switches from Rhinolophus bats to pangolins resulted in convergent mutational constraints and that SARS-CoV-2 emerged directly from a horseshoe bat sarbecovirus.

A Look inside the Replication Dynamics of SARS-CoV-2 in Blyth's Horseshoe Bat (Rhinolophus lepidus) Kidney Cells.

ABSTRACTBats are considered the natural reservoir of numerous emerging viruses such as severe acute respiratory syndrome coronaviruses (SARS-CoVs). There is a need for immortalized bat cell lines to culture and investigate the pathogenicity, replication kinetics, and evolution of emerging coronaviruses. We illustrate the susceptibility and permissiveness of a spontaneously immortalized kidney cell line (Rhileki) from Blyth’s horseshoe bat (R. lepidus) to SARS-CoV-2 virus, including clinical isolates, suggesting a possible virus-host relationship. We were able to observe limited SARS-CoV-2 replication in Rhileki cells compared with simian VeroE6 cells. Slower viral replication in Rhileki cells was indicated by higher ct values (RT-PCR) at later time points of the viral culture and smaller foci (foci forming assay) compared with those of VeroE6 cells. With this study we demonstrate that SARS-CoV-2 replication is not restricted to R. sinicus and could include more Rhinolophus species. The establishment of a continuous Rhinolophus lepidus kidney cell line allows further characterization of SARS-CoV-2 replication in Rhinolophus bat cells, as well as isolation attempts of other bat-borne viruses.IMPORTANCE The current COVID-19 pandemic demonstrates the significance of bats as reservoirs for severe viral diseases. However, as bats are difficult to establish as animal models, bat cell lines can be an important proxy for the investigation of bat-virus interactions and the isolation of bat-borne viruses. This study demonstrates the susceptibility and permissiveness of a continuous kidney bat cell line to SARS-CoV-2. This does not implicate the bat species Rhinolophus lepidus, where these cells originate from, as a potential reservoir, but emphasizes the usefulness of this cell line for further characterization of SARS-CoV-2. This can lead to a better understanding of emerging viruses that could cause significant disease in humans and domestic animals.

Epidemiology and Genomic Characterization of Two Novel SARS-Related Coronaviruses in Horseshoe Bats from Guangdong, China.

ABSTRACTSevere acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and SARS-CoV-2, the causative agents of SARS, which broke out in 2003, and coronavirus disease 2019 (COVID-2019), which broke out in 2019, probably originated in Rhinolophus sinicus and R. affinis, respectively. Rhinolophus bats are important hosts for coronaviruses. Many SARS-related coronaviruses (SARSr-CoVs) have been detected in bats from different areas of China; however, the diversity of bat SARSr-CoVs is increasing, and their transmission mechanisms have attracted much attention. Here, we report the findings of SARSr-CoVs in R. sinicus and R. affinis from South China from 2008 to 2021. The full-length genome sequences of the two novel SARSr-CoVs obtained from Guangdong shared 83 to 88% and 71 to 72% nucleotide identities with human SARS-CoV and SARS-CoV-2, respectively, while sharing high similarity with human SARS-CoV in hypervariable open reading frame 8 (ORF8). Significant recombination occurred between the two novel SARSr-CoVs. Phylogenetic analysis showed that the two novel bat SARSr-CoVs from Guangdong were more distant than the bat SARSr-CoVs from Yunnan to human SARS-CoV. We found that transmission in bats contributes more to virus diversity than time. Although our results of the sequence analysis of the receptor-binding motif (RBM) and the expression pattern of angiotensin-converting enzyme 2 (ACE2) inferred that these viruses could not directly infect humans, risks still exist after some unpredictable mutations. Thus, this study increased our understanding of the genetic diversity and transmission of SARSr-CoVs carried by bats in the field.

First evidence that an emerging mammalian alphacoronavirus is able to infect an avian species.

A novel swine enteric alphacoronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), related to Rhinolophus bat CoV HKU2 in the subgenus Rhinacovirus emerged in southern China in 2017, causing diarrhoea in newborn piglets, and critical questions remain about the pathogenicity, cross-species transmission and potential animal reservoirs. Our laboratory's previous research has shown that SADS-CoV can replicate in various cell types from different species, including chickens. Here, we systematically explore the susceptibility of chickens to a cell-adapted SADS-CoV strain both in vitro and in vivo. First, evidence of SADS-CoV replication in primary chicken cells, including cytopathic effects, immunofluorescence staining, growth curves and structural protein expression, was proven. Furthermore, we observed that SADS-CoV replicated in chicken embryos without causing gross lesions and that experimental infection of chicks resulted in mild respiratory symptoms. More importantly, SADS-CoV shedding and viral distribution in the lungs, spleens, small intestines and large intestines of infected chickens were confirmed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The genomic sequence of the original SADS-CoV from the pig source sample in 2017 was determined to have nine nucleotide differences compared to the cell-adapted strain used; among these were three nonsynonymous mutations in the spike gene. These results collectively demonstrate that chickens are susceptible to SADS-CoV infection, suggesting that they are a potential animal reservoir. To our knowledge, this study provides the first experimental evidence of cross-species infection in which a mammalian alphacoronavirus is able to infect an avian species.

Molecular phylogeny of coronaviruses and host receptors among domestic and close-contact animals reveals subgenome-level conservation, crossover, and divergence

BackgroundCoronaviruses have the potential to cross species barriers. To learn the molecular intersections among the most common coronaviruses of domestic and close-contact animals, we analyzed representative coronavirus genera infecting mouse, rat, rabbit, dog, cat, cattle, white-tailed deer, swine, ferret, mink, alpaca, Rhinolophus bat, dolphin, whale, chicken, duck and turkey hosts; reference or complete genome sequences were available for most of these coronavirus genera. Protein sequence alignments and phylogenetic trees were built for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The host receptors and enzymes aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2), sialic acid synthase (SAS), transmembrane serine protease 2 (TMPRSS2), dipeptidyl peptidase 4 (DPP4), cathepsin L (and its analogs) and furin were also compared.ResultsOverall, the S, E, M, and N proteins segregated according to their viral genera (α, β, or γ), but the S proteins of alphacoronaviruses lacked conservation of phylogeny. Interestingly, the unique polybasic furin cleavage motif found in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) but not in severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) exists in several β-coronaviruses and a few α- or γ-coronaviruses. Receptors and enzymes retained host species-dependent relationships with one another. Among the hosts, critical ACE2 residues essential for SARS-CoV-2 spike protein binding were most conserved in white-tailed deer and cattle.ConclusionThe polybasic furin cleavage motif found in several β- and other coronaviruses of animals points to the existence of an intermediate host for SARS-CoV-2, and it also offers a counternarrative to the theory of a laboratory-engineered virus. Generally, the S proteins of coronaviruses show crossovers of phylogenies indicative of recombination events. Additionally, the consistency in the segregation of viral proteins of the MERS-like coronavirus (NC_034440.1) from pipistrelle bat supports its classification as a β-coronavirus. Finally, similarities in host enzymes and receptors did not always explain natural cross-infections. More studies are therefore needed to identify factors that determine the cross-species infectivity of coronaviruses.

The COVID-19 pandemic from a One Health perspective

The "One Health" approach is essential to better understand and manage a pandemic of animal origin. Sensitive geopolitical considerations seem to hamper the investigations into the origin of the pandemic, but everything points to the Rhinolophus bat as the starting point of this devastating pandemic. Through a phenomenon of reverse zoonosis, several hundred cases of contamination of animals by SARS-CoV-2 have been identified worldwide, involving about twenty species of mammals. The virus has also passed from animals to humans in the case of infected mink farms in Denmark or through contact with hamsters in Hong Kong. For the development of vaccines and treatments and to help detect COVID-19 in train stations or airports, the animal has confirmed its role as a valuable auxiliary resource for humans in the fight against the pandemic.

Spatial epidemiology and genetic diversity of SARS-CoV-2 and related coronaviruses in domestic and wild animals.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed susceptibility to diverse animal species. We conducted this study to understand the spatial epidemiology, genetic diversity, and statistically significant genetic similarity along with per-gene recombination events of SARS-CoV-2 and related viruses (SC2r-CoVs) in animals globally. We collected a number of different animal species infected with SARS-CoV-2 and its related viruses. Then, we retrieved genome sequences of SARS-CoV-2 and SC2r-CoVs from GISAID and NCBI GenBank for genomic and mutational analysis. Although the evolutionary origin of SARS-CoV-2 remains elusive, the diverse SC2r-CoV have been detected in multiple Rhinolophus bat species and in Malayan pangolin. To date, human-to-animal spillover events have been reported in cat, dog, tiger, lion, gorilla, leopard, ferret, puma, cougar, otter, and mink in 25 countries. Phylogeny and genetic recombination events of SC2r-CoVs showed higher similarity to the bat coronavirus RaTG13 and BANAL-103 for most of the genes and to some Malayan pangolin coronavirus (CoV) strains for the N protein from bats and pangolin showed close resemblance to SARS-CoV-2. The clustering of animal and human strains from the same geographical area has proved human-to-animal transmission of the virus. The Alpha, Delta and Mu-variant of SARS-CoV-2 was detected in dog, gorilla, lion, tiger, otter, and cat in the USA, India, Czech Republic, Belgium, and France with momentous genetic similarity with human SARS-CoV-2 sequences. The mink variant mutation (spike_Y453F) was detected in both humans and domestic cats. Moreover, the dog was affected mostly by clade O (66.7%), whereas cat and American mink were affected by clade GR (31.6 and 49.7%, respectively). The α-variant was detected as 2.6% in cat, 4.8% in dog, 14.3% in tiger, 66.7% in gorilla, and 77.3% in lion. The highest mutations observed in mink where the substitution of D614G in spike (95.2%) and P323L in NSP12 (95.2%) protein. In dog, cat, gorilla, lion, and tiger, Y505H and Y453F were the common mutations followed by Y145del, Y144del, and V70I in S protein. We recommend vaccine provision for pet and zoo animals to reduce the chance of transmission in animals. Besides, continuous epidemiological and genomic surveillance of coronaviruses in animal host is crucial to find out the immediate ancestor of SARS-CoV-2 and to prevent future CoVs threats to humans.

Longitudinal monitoring in Cambodia suggests higher circulation of alpha and betacoronaviruses in juvenile and immature bats of three species

Recent studies suggest that coronaviruses circulate widely in Southeast Asian bat species and that the progenitors of the SARS-Cov-2 virus could have originated in rhinolophid bats in the region. Our objective was to assess the diversity and circulation patterns of coronavirus in several bat species in Southeast Asia. We undertook monthly live-capture sessions and sampling in Cambodia over 17 months to cover all phases of the annual reproduction cycle of bats and test specifically the association between their age and CoV infection status. We additionally examined current information on the reproductive phenology of Rhinolophus and other bat species presently known to occur in mainland southeast China, Vietnam, Laos and Cambodia. Results from our longitudinal monitoring (573 bats belonging to 8 species) showed an overall proportion of positive PCR tests for CoV of 4.2% (24/573) in cave-dwelling bats from Kampot and 4.75% (22/463) in flying-foxes from Kandal. Phylogenetic analysis showed that the PCR amplicon sequences of CoVs (n = 46) obtained clustered in Alphacoronavirus and Betacoronavirus. Interestingly, Hipposideros larvatus sensu lato harbored viruses from both genera. Our results suggest an association between positive detections of coronaviruses and juvenile and immature bats in Cambodia (OR = 3.24 [1.46–7.76], p = 0.005). Since the limited data presently available from literature review indicates that reproduction is largely synchronized among rhinolophid and hipposiderid bats in our study region, particularly in its more seasonal portions (above 16° N), this may lead to seasonal patterns in CoV circulation. Overall, our study suggests that surveillance of CoV in insectivorous bat species in Southeast Asia, including SARS-CoV-related coronaviruses in rhinolophid bats, could be targeted from June to October for species exhibiting high proportions of juveniles and immatures during these months. It also highlights the need to develop long-term longitudinal surveys of bats and improve our understanding of their ecology in the region, for both biodiversity conservation and public health reasons.