Covid-19: A Multi-Host Pandemic

SARS-CoV-2, the causal agent of the COVID-19 pandemic, is related to a group of viruses (Sarbecovirus) that circulate in horseshoe bats. Its origin is still uncertain, as there is lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2. Irrespective of its origin, SARS-CoV-2 has been shown to replicate in many mammalian species. So far, over forty species have been found to be susceptible to SARS-CoV-2 infection, and natural infections have been documented in at least 23 species of distant mammalian orders, including Primata, Rodentia, Carnivora, and Arthiodactyla. In two of those species, minks and white tailed deer, continued transmission among conspecifics occurred following introduction of SARS-CoV-2 from humans, at a rate which makes mink farms and deer populations suitable compartments where the virus may be maintained and evolve, and then perhaps spill back to humans or other animals as a new variant, as suggested by molecular evidence. Considering the above, what is truly unique about this pandemic, and adds a major obstacle to attain its control, is its multi-host nature. This is another compelling example of the relevance of the 'One Health' approach. This approach recognizes the inextricable links between people and nature, and visualizes the health and disease phenomenon from an integrative perspective. The COVID-19 pandemic urges us to acknowledge the interconnection between people and the remaining forms of life, and with the environments they share, and demonstrates that the improvement of global health needs a collaborative, multisectoral, and transdisciplinary approach, acting at the local, regional and global levels. This concept becomes paramount when taking into account that most diseases affecting humans in the last decades -not only COVID-19 - have been caused by pathogens originated in animals.Copyright © 2023

Long Term Circulation of Sars-Cov-2 Related Lineages in Bats in Cambodia

Intro: Recent evidence shows the Greater Mekong Subregion to be a hotspot for Sarbecoviruses in bats, especially insectivorous Horseshoe bats (genus Rhinolophus). However, prevalence, maintenance, and evolution of these viruses in Rhinolophids is still poorly understood. Sampling efforts are still limited and generally only cover cross-sectional surveillance at single points in time. Following the detection of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2)-related viruses in Rhinolophus shameli from 2010 in Steung Treng, Cambodia, further active longitudinal surveillance in the same area between 2020-2021 continued the detection of these viruses. Method(s): Live bat capture and sampling has been implemented in several sites located in Stung Treng province. All rectal swabs of bats were tested for the detection of SARS-CoV-2 or Sarbecoviruses by real time RT-PCR. RNA samples from positive RT-PCR bats were then sequenced using a highly multiplexed PCR amplicon approach using new designed primers set guided by the ARTIC Network multiplex PCR primers set (https://artic.network/ncov-2019), on Oxford Nanopore technology. Finding(s): The sarbecoviruses were detected in four Rhinolophus shameli bats, a percentage of similarity ranging at the nucleotide level between 98.8% - 99.1% when compared to two other Cambodian bat sarbecoviruses from 2010 and between 92.4% - 94.5% when compared to human SARS-CoV-2 across the whole genome. Discussion(s): The bat SARS-CoV-2 related virus recently detected in four positive bats in 2020-2021 are genetically homologous with the virus detected in 2010, indicating a geographically/host limited population that is stable over time in the past ten years. Conclusion(s): Overall, our findings indicate further complexity in the diversity and evolution of sarbecoviruses and add intricacy to the search for the origins of the Coronavirus Disease 2019 (COVID-19) pandemic.Copyright © 2023

Isolation of Bat Sarbecoviruses, Japan.

Surveillance of bat betacoronaviruses is crucial for understanding their spillover potential. We isolated bat sarbecoviruses from Rhinolophus cornutus bats in multiple locations in Japan. These viruses grew efficiently in cells expressing R. cornutus angiotensin converting enzyme-2, but not in cells expressing human angiotensin converting enzyme-2, suggesting a narrow host range.

A Review on Catastrophic Evolution of SARS-CoV to SARS-CoV2: A Global Pandemic

The coronaviruses, belonging to the family Coronaviridae, have caused a massive pandemic in December 2019 after their previous outbreaks as SARS-CoV and MERS. The outbreak is believed to have originated from the seafood and live market in the Hubei province of China. The Rhinolophus species are the natural hosts of this virus. This virus caused pneumonia and took away many lives be-fore it was recognized as the novel Coronavirus. Very little information is available about the biology and nature of the novel Coronavirus. This article reviews multiple aspects encompassing its origin, epi-demiology, pathogenesis, symptoms, and the global statistics of spread. Acute respiratory distress syndrome (ARDS) is the key symptom of this condition. Angiotensin-converting enzyme 2 (ACE2) helps in the penetration of the virus into the target cells. Deeper research and understanding are essential for the identification of antibodies that inhibit ACE2 and can prevent viral replication. Drug design and control of disease are crucial. In countries like India, where plant diversity is extensive, it is prudent to focus on plant-based alternative drugs. Many attempts have been made to review and curate the drug discovery attempts using immuno-informatic and bioinformatic tools.Copyright © 2021 Bentham Science Publishers.

COVID-19 Pandemic: Current Scenario, Challenges and Future Perspectives

Background: The new public health emergency of COVID-19 caused by a novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which originated in Wuhan, Hubei province, China in December 2019, evolved into a pandemic in no time and is still in progression. The novel virus mainly targets the lower respiratory system, leading to viral pneumonia, with other associated complications of multi organ failure. Discussion(s): The bats, in particular Rhinolophus affinis, is a natural host of SARS-CoV-2 and the virus is considered to have spread to humans through yet controversial intermediate host pangolins. The incubation period ranges from 2-14 days and mode of person-to-person transmission is primari-ly via the direct contact with the infected person or through the droplets generated by the infected person during coughing or sneezing. The initiation of the infection process by SARS-CoV-2 virus is the invasion of lung type II alveolar cells via a receptor protein called angiotensin-converting enzyme 2 (ACE2) present on the cell membrane with glycosylated spike (S) viral protein that medi-ates host cell invasion. The main diagnostic tools employed are molecular methods based on nucleic acid detection engaging real-time quantitative polymerase chain reaction (RT-qPCR) and a new immunoassays based on antibodies IgM/IgG. Conclusion(s): Due to the lack of specific clinically approved anticovid-19 drugs or vaccines that could be used for its prevention or treatment, the current management approach is essentially sup-portive and symptomatic. The precautionary measures like, social distancing, cleaning hands with soap or sanitizers, using disinfectant solutions to decontaminate the surfaces of things and proper ventilation, wearing masks and other protective gears to curb transmission. The knowledge regard-ing COVID-19 therapies is still evolving and collaborative efforts are being put in to discover definitive therapies on different themes in the form of vaccines, repurposing drugs, RNA interfer-ence, docking studies, etc.Copyright © 2021 Bentham Science Publishers.

Coronavirus Pandemic: Role of Bats And Zoonotic Transmission in Humans

Background: In the past two decades, the human coronavirus (HCoV) outbreaks have gripped the international communities almost six times in different forms [HCoV-OC43 (2001);HCoV-NL63 (2004);SARS-CoV (2003);HCoV HKU1 (2005);MERS-CoV (2012);SARS-CoV--2 (2019)]. These emerging pathogens have been proven very challenging from medical perspec-tives, economic conditions, and psychological impact on human society. Introduction: SARS-CoV-2, a novel coronavirus, has evidenced a historic yet troublesome pandemic across the globe. In humans, its clinical manifestations may range from asymptomatic, severe pneumonia to mortality. Bats are the natural reservoirs of a variety of viruses belonging to the family Coronaviridae. Most of the bats harboring coronaviruses mainly reside in Asian and African regions. Objective(s): The objective was to describe the various characteristic features of all coronaviruses, clinical manifestations, and complications associated with SARS-CoV-2. The major goal was to highlight the involvement of the strong immune system of bats in the cross-species transmission of coronaviruses in intermediate hosts and, finally, zoonotic transmission in humans. Methodology: A systematic literature search was conducted for high quality research and review ar-ticles. We searched the databases for articles published between the year 1972 to 2020 with search terms zoonosis, coronaviruses, zoonotic transmissions, clinical manifestations, and the immune system of bats. Conclusion(s): The domestic and non-domestic animals come in closer contact with humans. Some requisite measures should be taken to decrease the contact with livestock to prevent further threatening viral transmissions. Furthermore, the remarkable immune system of bats is required to in-quire thoroughly to develop novel therapeutics to conquer the evolving coronaviruses in the future.Copyright © 2021 Bentham Science Publishers.

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.

Species-specific molecular barriers to SARS-CoV-2 replication in bat cells

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.

Metagenomic Discovery of a Novel Sarbecovirus in the United Kingdom at the Western Extreme of the Range of Rhinolophidae

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.

Exploring the Natural Origins of SARS-CoV-2 in the Light of Recombination.

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.

SARS-like Coronaviruses in Horseshoe Bats (Rhinolophus spp.) in Russia, 2020.

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.

DEVELOPING AN ORGANOID MODEL TO UNDERSTAND THE BAT GASTROINTESTINAL EPITHELIAL RESPONSE TO THE SEVER ACUTE RESPIRATORY CORONA VIRUS-2 (SARS-COV-2)

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.

Transmission dynamics and susceptibility patterns of SARS-CoV-2 in domestic, farmed and wild animals: Sustainable One Health surveillance for conservation and public health to prevent future epidemics and pandemics.

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.

Cross-species recognition of SARS-CoV-2 to bat ACE2.

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.

The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2.

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.

Detection and Characterization of Bat Sarbecovirus Phylogenetically Related to SARS-CoV-2, Japan.

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.