Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

A Critical Analysis of the Evidence for the SARS-CoV-2 Origin Hypotheses.

When humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.

Identification and Genetic Characterization of MERS-Related Coronavirus Isolated from Nathusius' Pipistrelle (Pipistrellus nathusii) near Zvenigorod (Moscow Region, Russia).

Being diverse and widely distributed globally, bats are a known reservoir of a series of emerging zoonotic viruses. We studied fecal viromes of twenty-six bats captured in 2015 in the Moscow Region and found 13 of 26 (50%) samples to be coronavirus positive. Of P. nathusii (the Nathusius' pipistrelle), 3 of 6 samples were carriers of a novel MERS-related betacoronavirus. We sequenced and assembled the complete genome of this betacoronavirus and named it MOW-BatCoV strain 15-22. Whole genome phylogenetic analysis suggests that MOW-BatCoV/15-22 falls into a distinct subclade closely related to human and camel MERS-CoV. Unexpectedly, the phylogenetic analysis of the novel MOW-BatCoV/15-22 spike gene showed the closest similarity to CoVs from Erinaceus europaeus (European hedgehog). We suppose MOW-BatCoV could have arisen as a result of recombination between ancestral viruses of bats and hedgehogs. Molecular docking analysis of MOW-BatCoV/15-22 spike glycoprotein binding to DPP4 receptors of different mammals predicted the highest binding ability with DPP4 of the Myotis brandtii bat (docking score -320.15) and the E. europaeus (docking score -294.51). Hedgehogs are widely kept as pets and are commonly found in areas of human habitation. As this novel bat-CoV is likely capable of infecting hedgehogs, we suggest hedgehogs can act as intermediate hosts between bats and humans for other bat-CoVs.

Host range and structural analysis of bat-origin RshSTT182/200 coronavirus binding to human ACE2 and its animal orthologs.

Bat-origin RshSTT182 and RshSTT200 coronaviruses (CoV) from Rhinolophus shameli in Southeast Asia (Cambodia) share 92.6% whole-genome identity with SARS-CoV-2 and show identical receptor-binding domains (RBDs). In this study, we determined the structure of the RshSTT182/200 receptor binding domain (RBD) in complex with human angiotensin-converting enzyme 2 (hACE2) and identified the key residues that influence receptor binding. The binding of the RshSTT182/200 RBD to ACE2 orthologs from 39 animal species, including 18 bat species, was used to evaluate its host range. The RshSTT182/200 RBD broadly recognized 21 of 39 ACE2 orthologs, although its binding affinities for the orthologs were weaker than those of the RBD of SARS-CoV-2. Furthermore, RshSTT182 pseudovirus could utilize human, fox, and Rhinolophus affinis ACE2 receptors for cell entry. Moreover, we found that SARS-CoV-2 induces cross-neutralizing antibodies against RshSTT182 pseudovirus. Taken together, these findings indicate that RshSTT182/200 can potentially infect susceptible animals, but requires further evolution to obtain strong interspecies transmission abilities like SARS-CoV-2.

The Origins of Severe Acute Respiratory Syndrome-Coronavirus-2.

An outbreak of severe pneumonia of unknown cause was identified in Wuhan, China in December 2019: the causative agent was a novel betacoronavirus, severe acute respiratory syndrome-cotonavirus-2 (SARS-CoV-2), a virus that joins a list of coronaviruses causing severe (e.g., SARS and Middle East respiratory syndrome) or milder (e.g., 229E, OC43, NL63, and HKU1) respiratory tract infection. The World Health Organization (WHO) classified the spreading outbreak as a pandemic on March 11, 2020. Many SARS-related coronaviruses (SARSr-CoVs) have been identified in bats, particularly in Rhinolophus horseshoe bats, animals that are common in southern China and Southeast Asia. Many of the features of SARS-CoV-2 that facilitate human infection-the furin cleavage site, the receptor binding domain that binds to the human ACE2 receptor-can be identified in SARSr-CoVs. Related coronaviruses can be detected in pangolins and other animals, and human SARS-CoV-2 itself can infect various animals, some of which can transmit SARS-CoV-2 back to humans. Investigation by the WHO and others pointed to the initial outbreak being centered on the Huanan wet market in Wuhan where wild and farmed animals were sold, and where environmental testing revealed widespread SARS-CoV-2 contamination. This supports the hypothesis that bats, probably via an intermediate animal, are the origin of SARS-CoV-2. Other possible origins have been postulated, such as an accidental or deliberate laboratory leak, or virus present in frozen foods, but evidence for these ideas has not surfaced. Study of the origins of SARS-CoV-2 have been complicated by intense media and political commentary, features that may slow the studies required to understand the viral origins. Such studies are complex and may be slow: international openness and co-operation is vital. Origins explanations are needed to predict or prevent future pandemics and support the "One Health" approach to disease.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors.

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.

Wildlife trade is likely the source of SARS-CoV-2.

Multiple transmissions from wildlife at a market in Wuhan probably led to SARS-CoV-2 emergence.

Lethal Swine Acute Diarrhea Syndrome Coronavirus Infection in Suckling Mice.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a recently emerging bat-borne coronavirus responsible for high mortality rates in piglets. In vitro studies have indicated that SADS-CoV has a wide tissue tropism in different hosts, including humans. However, whether this virus potentially threatens other animals remains unclear. Here, we report the experimental infection of wild-type BALB/c and C57BL/6J suckling mice with SADS-CoV. We found that mice less than 7 days old are susceptible to the virus, which caused notable multitissue infections and damage. The mortality rate was the highest in 2-day-old mice and decreased in older mice. Moreover, a preliminary neuroinflammatory response was observed in 7-day-old SADS-CoV-infected mice. Thus, our results indicate that SADS-CoV has potential pathogenicity in young hosts. IMPORTANCE SADS-CoV, which likely has originated from bat coronaviruses, is highly pathogenic to piglets and poses a threat to the swine industry. Little is known about its potential to disseminate to other animals. No efficient treatment is available, and the quarantine strategy is the only preventive measure. In this study, we demonstrated that SADS-CoV can efficiently replicate in suckling mice younger than 7 days. In contrast to infected piglets, in which intestinal tropism is shown, SADS-CoV caused infection and damage in all murine tissues evaluated in this study. In addition, neuroinflammatory responses were detected in some of the infected mice. Our work provides a preliminary cost-effective model for the screening of antiviral drugs against SADS-CoV infection.

Wildlife susceptibility to infectious diseases at global scales.

Disease transmission prediction across wildlife is crucial for risk assessment of emerging infectious diseases. Susceptibility of host species to pathogens is influenced by the geographic, environmental, and phylogenetic context of the specific system under study. We used machine learning to analyze how such variables influence pathogen incidence for multihost pathogen assemblages, including one of direct transmission (coronaviruses and bats) and two vector-borne systems (West Nile Virus [WNV] and birds, and malaria and birds). Here we show that this methodology is able to provide reliable global spatial susceptibility predictions for the studied host-pathogen systems, even when using a small amount of incidence information (i.e., [Formula: see text] of information in a database). We found that avian malaria was mostly affected by environmental factors and by an interaction between phylogeny and geography, and WNV susceptibility was mostly influenced by phylogeny and by the interaction between geographic and environmental distances, whereas coronavirus susceptibility was mostly affected by geography. This approach will help to direct surveillance and field efforts providing cost-effective decisions on where to invest limited resources.

Distinct evolutionary trajectories of SARS-CoV-2-interacting proteins in bats and primates identify important host determinants of COVID-19.

The coronavirus disease 19 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a coronavirus that spilled over from the bat reservoir. Despite numerous clinical trials and vaccines, the burden remains immense, and the host determinants of SARS-CoV-2 susceptibility and COVID-19 severity remain largely unknown. Signatures of positive selection detected by comparative functional genetic analyses in primate and bat genomes can uncover important and specific adaptations that occurred at virus-host interfaces. We performed high-throughput evolutionary analyses of 334 SARS-CoV-2-interacting proteins to identify SARS-CoV adaptive loci and uncover functional differences between modern humans, primates, and bats. Using DGINN (Detection of Genetic INNovation), we identified 38 bat and 81 primate proteins with marks of positive selection. Seventeen genes, including the ACE2 receptor, present adaptive marks in both mammalian orders, suggesting common virus-host interfaces and past epidemics of coronaviruses shaping their genomes. Yet, 84 genes presented distinct adaptations in bats and primates. Notably, residues involved in ubiquitination and phosphorylation of the inflammatory RIPK1 have rapidly evolved in bats but not primates, suggesting different inflammation regulation versus humans. Furthermore, we discovered residues with typical virus-host arms race marks in primates, such as in the entry factor TMPRSS2 or the autophagy adaptor FYCO1, pointing to host-specific in vivo interfaces that may be drug targets. Finally, we found that FYCO1 sites under adaptation in primates are those associated with severe COVID-19, supporting their importance in pathogenesis and replication. Overall, we identified adaptations involved in SARS-CoV-2 infection in bats and primates, enlightening modern genetic determinants of virus susceptibility and severity.

Species-Specific Molecular Barriers to SARS-CoV-2 Replication in Bat Cells.

Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. 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. 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 the absence of bACE2 binding in these cells. 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. Our data highlight the existence of species-specific and cell-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. IMPORTANCE Bats are host ancestors of several viruses that cause serious disease in humans, as illustrated by the ongoing SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions has been hampered by a limited number of available bat cellular models. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The various permissivities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by overexpression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bat cells and viruses.

Present and future distribution of bat hosts of sarbecoviruses: implications for conservation and public health.

Global changes in response to human encroachment into natural habitats and carbon emissions are driving the biodiversity extinction crisis and increasing disease emergence risk. Host distributions are one critical component to identify areas at risk of viral spillover, and bats act as reservoirs of diverse viruses. We developed a reproducible ecological niche modelling pipeline for bat hosts of SARS-like viruses (subgenus Sarbecovirus), given that several closely related viruses have been discovered and sarbecovirus-host interactions have gained attention since SARS-CoV-2 emergence. We assessed sampling biases and modelled current distributions of bats based on climate and landscape relationships and project future scenarios for host hotspots. The most important predictors of species distributions were temperature seasonality and cave availability. We identified concentrated host hotspots in Myanmar and projected range contractions for most species by 2100. Our projections indicate hotspots will shift east in Southeast Asia in locations greater than 2°C hotter in a fossil-fuelled development future. Hotspot shifts have implications for conservation and public health, as loss of population connectivity can lead to local extinctions, and remaining hotspots may concentrate near human populations.

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

Severe 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. IMPORTANCE Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 probably originated from the SARS-related coronaviruses (SARSr-CoVs) carried by Rhinolophus bats from Yunnan, China. Systematic investigations of the reservoir hosts carrying SARSr-CoVs in Guangdong and the reservoir distribution and transmission are urgently needed to prevent future outbreaks. Here, we detected SARSr-CoV in Rhinolophus bat samples from Guangdong in 2009 and 2021 and found that the transmission of SARSr-CoV from different host populations contributes more to increased virus diversity than time. Bat SARSr-CoVs in Guangdong had genetic diversity, and Guangdong was also the hot spot for SARSr-CoVs. We once again prove that R. sinicus plays an important role in the maintenance of the SARS-CoVs. Besides, the SARSr-CoVs are mainly transmitted through the intestines in bats, and these SARSr-CoVs found in Guangdong could not use human ACE2 (hACE2), but whether they can pass through intermediate hosts or directly infect humans requires further research. Our findings demonstrate the ability of SARSr-CoVs to spread across species.

Bat coronaviruses related to SARS-CoV-2: what about their 3CL proteases (MPro)?

Despite a huge effort by the scientific community to determine the animal reservoir of SARS-CoV-2, which led to the identification of several SARS-CoV-2-related viruses both in bats and in pangolins, the origin of SARS-CoV-2 is still not clear. Recently, Temmam et al. reported the discovery of bat coronaviruses with a high degree of genome similarity with SARS-CoV-2, especially concerning the RBDs of the S protein, which mediates the capability of such viruses to enter and therefore infect human cells through a hACE2-dependent pathway. These viruses, especially the one named BANAL-236, showed a higher affinity for the hACE2 compared to the original strain of SARS-CoV-2. In the present work, we analyse the similarities and differences between the 3CL protease (main protease, Mpro) of these newly reported viruses and SARS-CoV-2, discussing their relevance relative to the efficacy of existing therapeutic approaches against COVID-19, particularly concerning the recently approved orally available Paxlovid, and the development of future ones.

Computational Inference of Selection Underlying the Evolution of the Novel Coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2.

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that recently emerged in China is thought to have a bat origin, as its closest known relative (BatCoV RaTG13) was described previously in horseshoe bats. We analyzed the selective events that accompanied the divergence of SARS-CoV-2 from BatCoV RaTG13. To this end, we applied a population genetics-phylogenetics approach, which leverages within-population variation and divergence from an outgroup. Results indicated that most sites in the viral open reading frames (ORFs) evolved under conditions of strong to moderate purifying selection. The most highly constrained sequences corresponded to some nonstructural proteins (nsps) and to the M protein. Conversely, nsp1 and accessory ORFs, particularly ORF8, had a nonnegligible proportion of codons evolving under conditions of very weak purifying selection or close to selective neutrality. Overall, limited evidence of positive selection was detected. The 6 bona fide positively selected sites were located in the N protein, in ORF8, and in nsp1. A signal of positive selection was also detected in the receptor-binding motif (RBM) of the spike protein but most likely resulted from a recombination event that involved the BatCoV RaTG13 sequence. In line with previous data, we suggest that the common ancestor of SARS-CoV-2 and BatCoV RaTG13 encoded/encodes an RBM similar to that observed in SARS-CoV-2 itself and in some pangolin viruses. It is presently unknown whether the common ancestor still exists and, if so, which animals it infects. Our data, however, indicate that divergence of SARS-CoV-2 from BatCoV RaTG13 was accompanied by limited episodes of positive selection, suggesting that the common ancestor of the two viruses was poised for human infection.IMPORTANCE Coronaviruses are dangerous zoonotic pathogens; in the last 2 decades, three coronaviruses have crossed the species barrier and caused human epidemics. One of these is the recently emerged SARS-CoV-2. We investigated how, since its divergence from a closely related bat virus, natural selection shaped the genome of SARS-CoV-2. We found that distinct coding regions in the SARS-CoV-2 genome evolved under conditions of different degrees of constraint and are consequently more or less prone to tolerate amino acid substitutions. In practical terms, the level of constraint provides indications about which proteins/protein regions are better suited as possible targets for the development of antivirals or vaccines. We also detected limited signals of positive selection in three viral ORFs. However, we warn that, in the absence of knowledge about the chain of events that determined the human spillover, these signals should not be necessarily interpreted as evidence of an adaptation to our species.

Genomic Bootstrap Barcodes and Their Application to Study the Evolution of Sarbecoviruses.

Recombination creates mosaic genomes containing regions with mixed ancestry, and the accumulation of such events over time can complicate greatly many aspects of evolutionary inference. Here, we developed a sliding window bootstrap (SWB) method to generate genomic bootstrap (GB) barcodes to highlight the regions supporting phylogenetic relationships. The method was applied to an alignment of 56 sarbecoviruses, including SARS-CoV and SARS-CoV-2, responsible for the SARS epidemic and COVID-19 pandemic, respectively. The SWB analyses were also used to construct a consensus tree showing the most reliable relationships and better interpret hidden phylogenetic signals. Our results revealed that most relationships were supported by just a few genomic regions and confirmed that three divergent lineages could be found in bats from Yunnan: SCoVrC, which groups SARS-CoV related coronaviruses from China; SCoV2rC, which includes SARS-CoV-2 related coronaviruses from Southeast Asia and Yunnan; and YunSar, which contains a few highly divergent viruses recently described in Yunnan. The GB barcodes showed evidence for ancient recombination between SCoV2rC and YunSar genomes, as well as more recent recombination events between SCoVrC and SCoV2rC genomes. The recombination and phylogeographic patterns suggest a strong host-dependent selection of the viral RNA-dependent RNA polymerase. In addition, SARS-CoV-2 appears as a mosaic genome composed of regions sharing recent ancestry with three bat SCoV2rCs from Yunnan (RmYN02, RpYN06, and RaTG13) or related to more ancient ancestors in bats from Yunnan and Southeast Asia. Finally, our results suggest that viral circular RNAs may be key molecules for the mechanism of recombination.