This is the first multiyear surveillance project of CoVs in wild bats in North America. CoV RNA was detected in approximately 7% of all bats sampled (likely an underestimate of prevalence, Text S1
), comparable to the prevalence of CoV RNA detected in various species of bats reported in other parts of the world (ranging from 2–55%) 
. In our study no CoV RNA was detected in bats in 13 of the 17 species we sampled (also likely biased negatively). Failure to detect CoVs in bats of these species could be related to the smaller numbers sampled. However, a relatively high prevalence of CoV RNA was detected in bats of 2 species collected at several different sites: 12% for big brown bats and 8% for long-legged bats, and at lower prevalence, 3% in little brown bats and 2% in western long-eared bats.
In marked contrast to the enormous diversity of CoV genomes found in Old World bats 
, in this and several other CoV surveillance studies of New World bats 
, all CoVs detected were alphacoronaviruses. Our data indicate that nucleotide sequences of alphacoronaviruses harbored by Colorado bats are distinct from those found in Old World bats. Two recent studies of the bat guano virome using next generation sequence technology also only detected alphacoronaviruses in the New World bats of the species tested, as well as a diverse array of other types of viruses 
. Thus, so far there appears to be much more limited CoV diversity in New World bats of the species tested than in Old World bats.
Betacoronaviruses have only been detected in Old World bat species belonging to the families Pteropodidae (Rousettus
spp) and Rhinolophidae
spp.) which belong to the chiropteran suborder Yinpterochiroptera. Based on available evidence, betacoronaviruses could be restricted to hosts in the suborder Yinpterochiroptera (families Pteropodidae, Rhinolophidae, Megadermatidae, Craseonycteridae, Rhinopomatidae). No bat families of the suborder Yinpterochiroptera occur in the New World. 
. The finding of only alphacoronaviruses in our study may be because bats of these species are resistant to other CoVs and/or bats from different parts of the New World have yet to be tested for CoV infection, as we sampled bats from only a subset of the hundreds of species that reside in the New World.
These observations also support the hypothesis that coronaviruses may have co-evolved with their bat hosts, as no species of bat is found both in the New World and Old World 
. To date, however, only a small subset of New World species of bats has been tested for coronavirus infection. As 75% of living genera of all bats worldwide are found in the New Worlds tropics alone, further CoV surveillance in bats of additional species from different regions in the Western hemisphere may reveal hitherto undetected varieties of coronaviruses.
The seasonal epidemiology and persistence of New World CoV infections in individual bats and within bat populations has not been elucidated. The most comprehensive epidemiological investigation of CoVs to date in Old World bat populations showed that the prevalence of SARS-Rh-BatCoVs in rhinolophid bats over a four-year period at collection sites in Hong Kong SAR and China peaked in the spring and varied from year to year. We found similar results in New World bats. At site #4 long-legged bats had an alphacoronavirus RNA prevalence of 31% in 2007, 19% in 2009, but only 6% in 2008. In all five of the urban maternity roosts sampled, CoVs persisted in bat roosts throughout the course of the non-hibernating part of the year (spring/summer) and persisted from year to year. We also found that the prevalence of CoV infection in these bat roosts tended to peak in late spring/early summer. The prevalence of infection with human CoVs also shows significant annual variations 
, possibly depending on environmental conditions and/or fluctuating CoV antibody levels in the population. Possible seasonal variation in CoV infection rates may explain why in our initial 2006 study we found a high prevalence (50%) of alphacoronavirus RNA in occult bats 
, but in 2007 we did not detect any positive individuals (22 tested in the same region).
The majority of the bats sampled in our study were adult females because they were primarily captured from maternity roosts. The highest prevalence of infection was noted in juvenile bats. In Germany, CoV infection was also found to be associated with young age and was more common in female bats from maternity roosts compared to female bats found at foraging or swarming sites 
. These findings support the hypothesis that younger bats may be more susceptible to CoV infection and may serve to propagate and maintain these viruses within bat colonies.
No overt clinical manifestations of disease were observed in any of the captured bats, including those that were infected with CoVs. In the small subset of bats that were tagged and recaptured, no individual bat remained persistently positive for CoV RNA after 6 weeks. Similar findings were made in rhinolophid bats in Asia that harbor SARs-like-bat-CoVs 
and in fruit bats experimentally infected with bat CoVs which showed no signs of illness 
. These data suggest that although CoVs persist within bat populations, individual bats may experience only self-limited infections with CoVs without apparent illness.
Phylogenetic studies of CoV genomes in Old World bats in Asia and Europe have suggested that some bat CoVs may infect bats of only one species or several closely related species. In Asia and Germany, different species of bats roosting in the same cave were found to host different CoVs, whereas bats of the same species in different locations harbored similar CoVs 
. In Europe, strict associations were found between bat CoV deduced amino acid sequences in an 816 bp fragment of the RdRp gene and their specific bat hosts 
. In Africa, CoVs found in one species of bat were not detected in bats of different species co-roosting in the same cave 
. Similarly, our study showed that New World bats of the same species in geographically distinct locations and over the course of several years harbor similar CoVs. In contrast to these findings, in Kenya some CoVs appear to be able to infect Old World bats of several different species 
. Our preliminary nucleotide sequence data also suggests that we found very closely related CoV nucleotide sequences in New World bats from three different species of Myotis
, M. evotis
, and M. occultus
). Furthermore, in site #4, we found similar nucleotide sequences in the spike and replicase genes in CoV RNAs from both a big-brown bat and a long-legged bat, suggesting that at least some New World bat CoVs may be able to infect bats of different genera. These findings are notable, as recent phylogenetic studies of rabies viruses in bats suggest that host species barriers play a key role in cross species transmission of viruses 
To assess the potential for zoonotic transmission of bat CoVs, we focused part of this present work on North American bats that have the closest contact with humans and sampled roosts where big brown bats had histories of contact or potential for contact with people 
. Big brown bats are common inhabitants of buildings in cities and towns in Colorado and across the United States, and are the primary species encountered by humans in terms of potential exposure to disease agents 
These bats had a high prevalence of CoV infection, ranging from 0–67% (overall 10%) depending on the site and time of year. Big brown bats submitted to the CDPHE for rabies testing because of known direct contact with humans also had a very high prevalence (19%) of CoV infection. Because bats which have known or potential contact with humans have such a high prevalence of CoV infection, opportunities exist for potential transmission of these viruses to humans.
Following the SARS epidemic, intensive surveillance detected a great diversity of CoVs throughout the animal kingdom. CoVs can undergo a high frequency of RNA recombination, both in vitro
and in vivo
, which may play an important role in their evolution and virulence 
. Old World bat CoVs of several different genotypes were found to co-exist in a single bat 
. Thus recombination between different bat CoVs could potentially occur in vivo
, giving rise to new CoV genomes. Two strains of HCoV- HKU1 have recombined to yield a novel HCoV-HKU1 genotype 
, and recombination between different strains of SARS-CoV-like viruses in bats may have given rise to civet SARS-CoV 
. The great diversity of CoVs, their high frequency of RNA recombination, their ability to persist in bat populations, and the finding that some CoVs can apparently infect bats of divergent genera, suggest that ongoing evolution of CoVs in bats may pose a continuing threat for emergence of novel CoVs into new hosts.