Genetic Characterization of Betacoronavirus Lineage C Viruses in Bats Reveals Marked Sequence Divergence in the Spike Protein of Pipistrellus Bat Coronavirus HKU5 in Japanese Pipistrelle: Implications for the Origin of the Novel Middle East Respiratory Syndrome Coronavirus

While the novel Middle East respiratory syndrome coronavirus (MERS-CoV) is closely related to Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat CoV HKU5 (Pi-BatCoV HKU5) in bats from Hong Kong, and other potential lineage C betacoronaviruses in bats from Africa, Europe, and America, its animal origin remains obscure. To better understand the role of bats in its origin, we examined the molecular epidemiology and evolution of lineage C betacoronaviruses among bats. Ty-BatCoV HKU4 and Pi-BatCoV HKU5 were detected in 29% and 25% of alimentary samples from lesser bamboo bat (Tylonycteris pachypus) and Japanese pipistrelle (Pipistrellus abramus), respectively. Sequencing of their RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes revealed that MERS-CoV is more closely related to Pi-BatCoV HKU5 in RdRp (92.1% to 92.3% amino acid [aa] identity) but is more closely related to Ty-BatCoV HKU4 in S (66.8% to 67.4% aa identity) and N (71.9% to 72.3% aa identity). Although both viruses were under purifying selection, the S of Pi-BatCoV HKU5 displayed marked sequence polymorphisms and more positively selected sites than that of Ty-BatCoV HKU4, suggesting that Pi-BatCoV HKU5 may generate variants to occupy new ecological niches along with its host in diverse habitats. Molecular clock analysis showed that they diverged from a common ancestor with MERS-CoV at least several centuries ago. Although MERS-CoV may have diverged from potential lineage C betacoronaviruses in European bats more recently, these bat viruses were unlikely to be the direct ancestor of MERS-CoV. Intensive surveillance for lineage C betaCoVs in Pipistrellus and related bats with diverse habitats and other animals in the Middle East may fill the evolutionary gap.     

Chimeric Exchange of Coronavirus nsp5 Proteases (3CLpro) Identifies Common and Divergent Regulatory Determinants of Protease Activity

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp''s 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp''s at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.     

CD26/DPP4 Cell-Surface Expression in Bat Cells Correlates with Bat Cell Susceptibility to Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Infection and Evolution of Persistent Infection

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors.     

Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy

Background

Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii.

Methods

Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB).

Results

Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs.

Conclusions

This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity.

     

Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses,Paramyxoviruses, Filoviruses,and Influenza Viruses

Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.     

Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain NL63

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.     

Two Mutations Were Critical for Bat-to-Human Transmission of Middle East Respiratory Syndrome Coronavirus

To understand how Middle East respiratory syndrome coronavirus (MERS-CoV) transmitted from bats to humans, we compared the virus surface spikes of MERS-CoV and a related bat coronavirus, HKU4. Although HKU4 spike cannot mediate viral entry into human cells, two mutations enabled it to do so by allowing it to be activated by human proteases. These mutations are present in MERS-CoV spike, explaining why MERS-CoV infects human cells. These mutations therefore played critical roles in the bat-to-human transmission of MERS-CoV, either directly or through intermediate hosts.     

A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has generated enormous interest in the biodiversity, genomics and cross-species transmission potential of coronaviruses, especially those from bats, the second most speciose order of mammals. Herein, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples of Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3’-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus. Subgenomic mRNA and cellular-level observations demonstrated that the p10 gene is functional and induces the formation of cell syncytia. Therefore, here we report a putative heterologous inter-family recombination event between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, providing insights into the fundamental mechanisms of viral evolution.     

Evidence for ACE2-utilizing coronaviruses (CoVs) related to severe acute respiratory syndrome CoV in bats

In 2002, severe acute respiratory syndrome (SARS)-coronavirus (CoV) appeared as a novel human virus with high similarity to bat coronaviruses. However, while SARS-CoV uses the human angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry, no coronavirus isolated from bats appears to use ACE2. Here we show that signatures of recurrent positive selection in the bat ACE2 gene map almost perfectly to known SARS-CoV interaction surfaces. Our data indicate that ACE2 utilization preceded the emergence of SARS-CoV-like viruses from bats.     

Some Like It Hot: Evolution and Ecology of Novel Endosymbionts in Bat Flies of Cave-Roosting Bats (Hippoboscoidea,Nycterophiliinae)

We investigated previously unknown associations between bacterial endosymbionts and bat flies of the subfamily Nycterophiliinae (Diptera, Streblidae). Molecular analyses revealed a novel clade of Gammaproteobacteria in Nycterophilia bat flies. This clade was not closely related to Arsenophonus-like microbes found in its sister genus Phalconomus and other bat flies. High population infection rates in Nycterophilia across a wide geographic area, the presence of the symbionts in pupae, the general codivergence between hosts and symbionts, and high AT composition bias in symbiont genes together suggest that this host-symbiont association is obligate in nature and ancient in origin. Some Nycterophilia samples (14.8%) also contained Wolbachia supergroup F (Alphaproteobacteria), suggesting a facultative symbiosis. Likelihood-based ancestral character mapping revealed that, initially, obligate symbionts exhibited association with host-specific Nycterophilia bat flies that use a broad temperature range of cave environments for pupal development. As this mutualism evolved, the temperature range of bat flies narrowed to an exclusive use of hot caves, which was followed by a secondary broadening of the bat flies'' host associations. These results suggest that the symbiosis has influenced the environmental tolerance of parasite life history stages. Furthermore, the contingent change to an expanded host range of Nycterophilia bat flies upon narrowing the ecological niche of their developmental stages suggests that altered environmental tolerance across life history stages may be a crucial factor in shaping parasite-host relationships.     

Evolution of genomes, host shifts and the geographic spread of SARS-CoV and related coronaviruses

Severe acute respiratory syndrome (SARS) is a novel human illness caused by a previously unrecognized coronavirus (CoV) termed SARS‐CoV. There are conflicting reports on the animal reservoir of SARS‐CoV. Many of the groups that argue carnivores are the original reservoir of SARS‐CoV use a phylogeny to support their argument. However, the phylogenies in these studies often lack outgroup and rooting criteria necessary to determine the origins of SARS‐CoV. Recently, SARS‐CoV has been isolated from various species of Chiroptera from China (e.g., Rhinolophus sinicus) thus leading to reconsideration of the original reservoir of SARS‐CoV. We evaluated the hypothesis that SARS‐CoV isolated from Chiroptera are the original zoonotic source for SARS‐CoV by sampling SARS‐CoV and non‐SARS‐CoV from diverse hosts including Chiroptera, as well as carnivores, artiodactyls, rodents, birds and humans. Regardless of alignment parameters, optimality criteria, or isolate sampling, the resulting phylogenies clearly show that the SARS‐CoV was transmitted to small carnivores well after the epidemic of SARS in humans that began in late 2002. The SARS‐CoV isolates from small carnivores in Shenzhen markets form a terminal clade that emerged recently from within the radiation of human SARS‐CoV. There is evidence of subsequent exchange of SARS‐CoV between humans and carnivores. In addition SARS‐CoV was transmitted independently from humans to farmed pigs (Sus scrofa). The position of SARS‐CoV isolates from Chiroptera are basal to the SARS‐CoV clade isolated from humans and carnivores. Although sequence data indicate that Chiroptera are a good candidate for the original reservoir of SARS‐CoV, the structural biology of the spike protein of SARS‐CoV isolated from Chiroptera suggests that these viruses are not able to interact with the human variant of the receptor of SARS‐CoV, angiotensin‐converting enzyme 2 (ACE2). In SARS‐CoV we study, both visually and statistically, labile genomic fragments and, putative key mutations of the spike protein that may be associated with host shifts. We display host shifts and candidate mutations on trees projected in virtual globes depicting the spread of SARS‐CoV. These results suggest that more sampling of coronaviruses from diverse hosts, especially Chiroptera, carnivores and primates, will be required to understand the genomic and biochemical evolution of coronaviruses, including SARS‐CoV. © The Willi Hennig Society 2008.     

Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.     

Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation

Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH₄Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.     

Middle East respiratory syndrome coronavirus (MERS-CoV): Impact on Saudi Arabia, 2015

Middle East respiratory syndrome is the acute respiratory syndrome caused by betacoronavirus MERS-CoV. The first case of this disease was reported from Saudi Arabia in 2012. This virus is lethal and is a close relative of a severe acute respiratory syndrome (SARS), which is responsible for more than 3000 deaths in 2002–2003. According to Ministry of Health, Saudi Arabia. The number of new cases is 457 in 2015. Riyadh has the highest number of reports in comparison to the other cities. According to this report, males are more susceptible than female, especially after the age of 40. Because of the awareness and early diagnosis the incidence is falling gradually. The pre-existence of another disease like cancer or diabetic etc. boosts the infection. MERS is a zoonotic disease and human to human transmission is low. The MERS-CoV is a RNA virus with protein envelope. On the outer surface, virus has spike like glycoprotein which is responsible for the attachment and entrance inside host cells. There is no specific treatment for the MERS-CoV till now, but drugs are in pipeline which bind with the spike glycoprotein and inhibit its entrance host cells. MERS-CoV and SAR-CoV are from the same genus, so it was thought that the drugs which inhibit the growth of SARS-CoV can also inhibit the growth of MERS-CoV but those drugs are not completely inhibiting virus activity. Until we don’t have proper structure and the treatment of MERS-CoV, We should take precautions, especially the health care workers, Camel owners and Pilgrims during Hajj and Umrah, because they are at a higher risk of getting infected.     

Comparative review of respiratory diseases caused by coronaviruses and influenza A viruses during epidemic season

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to sweep the world, causing infection of millions and death of hundreds of thousands. The respiratory disease that it caused, COVID-19 (stands for coronavirus disease in 2019), has similar clinical symptoms with other two CoV diseases, severe acute respiratory syndrome and Middle East respiratory syndrome (SARS and MERS), of which causative viruses are SARS-CoV and MERS-CoV, respectively. These three CoVs resulting diseases also share many clinical symptoms with other respiratory diseases caused by influenza A viruses (IAVs). Since both CoVs and IAVs are general pathogens responsible for seasonal cold, in the next few months, during the changing of seasons, clinicians and public heath may have to distinguish COVID-19 pneumonia from other kinds of viral pneumonia. This is a discussion and comparison of the virus structures, transmission characteristics, clinical symptoms, diagnosis, pathological changes, treatment and prevention of the two kinds of viruses, CoVs and IAVs. It hopes to provide information for practitioners in the medical field during the epidemic season.     

Laboratory Investigation and Phylogenetic Analysis of an Imported Middle East Respiratory Syndrome Coronavirus Case in Greece

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1^st and 2^nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient’s history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.     

Alphacoronaviruses in New World bats: prevalence, persistence, phylogeny, and potential for interaction with humans

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.