INTRODUCTION
Bats are known to be reservoir hosts for several human viruses, including rabies, Marburg, Nipah, Hendra, and the severe acute respiratory syndrome coronavirus (SARS-CoV) (
5). In addition, virome studies have shown unprecedented numbers of viruses present in the fecal samples of this ancient mammalian species (
11,
24). However, little is known about the genetic architecture of most bat species, the virus variation and gene flow that occur through different species, the potential of different bat species to support human virus replication, the differences between the bat and human immune systems, or the potential of bat viruses to undergo zoonotic transmission to humans and other mammals.
Coronaviruses are the largest known RNA viruses; these viruses contain single-stranded plus sense genomes and are classified in the family
Coronaviridae, which is divided into three genera, including
Alphacoronavirus (α-CoV),
Betacoronavirus (β-CoV), and
Gammacoronavirus (γ-CoV). Five CoVs are known to cause human disease, including the β-CoVs SARS-CoV, human CoV (HCoV)-OC43, and HCoV-HKU1 and the α-CoVs HCoV-229E and HCoV-NL63 (
35). Three of these HCoVs have been shown to or have been predicted to have spilled over from zoonotic reservoirs, including SARS-CoV, which likely emerged from the Chinese horseshoe bat (Rhinolophidae) (
26), HCoV-OC43, which probably emerged from bovine CoV (BCoV) (
50), and HCoV-229E (
36), which was predicted by molecular clock analysis to share a most recent common ancestor (MRCA) just over 200 hundred years ago with a bat CoV found in the leaf-nosed bat (
Hipposideros caffer ruber) bat in Ghana (
36). The close link between bat and human CoVs has led to the speculation that all human, and perhaps mammalian, CoVs may have originated in bats (
19,
36,
49).
HCoV-NL63 was first discovered in 2004 as a new HCoV isolated from a 7-month-old baby suffering from bronchiolitis (
48). A similar virus was isolated around the same time in samples derived from an 8-month-old baby with pneumonia (
16). Since 2004, this HCoV has been detected in 1.0 to 9.3% of respiratory tract samples collected from several different countries (
15), which indicates that HCoV-NL63 is distributed worldwide. There is no known reservoir for this virus, and little is known about its evolutionary history prior to 2004, although phylogenetic evidence suggests that HCoV-NL63 has infected humans for centuries, as it is predicted to have diverged from HCoV-229E approximately 1,000 years ago (
38).
There are more than 1,100 species of bats, with bat populations inhabiting every continent except Antarctica. Bats belong to the order Chiroptera and are further divided into the suborders of Yinpterochiroptera, which include mostly the megabats (formerly known as the megachiroptera) and Yangochiroptera, which includes most of the microbat (formerly known as the microchiroptera) families (
46). In the United States, most bat species are small nocturnal bats that belong to the family Vespertilionidae, and all are insectivores. In Maryland, previous surveys have yielded fecal samples from the big brown bat (
Eptesicus fuscus), the little brown myotis (
Myotis lucifugus), the northern long-eared myotis (
Myotis septentrionalis), the eastern small-footed myotis (
Myotis leibii), the hoary bat (
Lasiurus cinereus), the red bat (
Lasiurus borealis), and the tricolored bat (
Perimyotis subflavus). In our previous work, we have shown that five of these species shed unique α-CoV sequences (
11); however, the reagents necessary to isolate these viruses and determine if North American bat CoVs pose a threat to public health are currently lacking.
In this study, we identified nucleic acid sequences that potentially indicate the presence of a novel α-CoV that is predicted to share an RCA with HCoV-NL63 in the tricolored bat. We developed an immortalized lung cell line for this U.S. bat species and show that human CoVs grow in these bat cells. This observation suggests that human CoVs are capable of infecting multiple mammalian hosts, potentially establishing zoonotic-reverse zoonotic cycles that allow CoVs to maintain viral populations in multiple hosts, evolve novel recombinant viruses with viral genes derived from human and animal CoVs, and traffic back into human populations at a later date. In addition, these results suggest that HCoV-NL63 may have originated in bats and crossed the species barrier to infect humans roughly 563 to 822 years ago. Supporting a growing body of literature, our data support the hypothesis that cross-species transmission events and the emergence and colonization of new species represent common features of the Coronaviridae.
DISCUSSION
Bats are important reservoir hosts for a large number of emerging viruses that cause human disease. However, only a small number of species (∼36 of 1,100) (
5,
11,
24) have been studied and shown to harbor RNA viruses, and many of these viruses share sequence similarity to human strains. The fact that CoVs have been found in multiple Old World and New World bat species suggests that bats may be particularly suited for maintaining CoVs in nature. Including the findings in this study, at least three human CoVs have been linked to bat CoVs (
26,
36), and there is mounting evidence that all mammalian CoVs may have originated from bats (
19,
36,
49), particularly human coronaviruses.
Although the exact number of infections is unknown, HCoV-NL63 has been shown to be distributed worldwide; it infects a significant number of children and elderly persons each year, and HCoV-OC43 and -229E account for nearly one-third of all colds. Moreover, SARS-CoV infected over 8,000 people worldwide, with a mortality rate approaching 10% (
34). These observations indicate that CoVs are important human pathogens that cause significant morbidity and frequent mortality. The fact that over 1,000 bat species have not been assessed for the presence of CoVs or other RNA and DNA viruses may have enormous public health consequences in an outbreak setting. Given that there are more than 1,100 species of bats and nearly one-half of the 36 bat species that have been studied for viruses have shown evidence of CoV sequences, there may be hundreds of novel coronaviruses in bats throughout the world. If this is the case, then the next CoV spillover from bats to humans is just a matter of ecological opportunity.
North American bats have been shown to harbor α-CoV sequences (
10,
11,
24,
31,
33); however, no α-CoVs have been isolated from these bats, and in fact, no full-length CoV sequences have been obtained or reported from North American bats. Of note, in our metagenomics study of North American bats in the Eastern United States, we identified α-CoV sequences in multiple bat species, including the big brown bat and the tricolored bat (
11). In this study, we detected α-CoV sequences in a big brown bat population in New York and from a tricolored bat population sampled in Maryland (
Fig. 2). Interestingly, the two sequences derived from big brown bats (ARCoV.1 and NECoV) were nearly identical in a >2,200-nt fragment of the replicase gene, even though the two populations were from different regions (New York and Maryland) (
Fig. 2B). In contrast, the same sequence fragment obtained from tricolored bats was significantly different (74% identity at the nucleotide level), suggesting that different bat species harbor CoVs that have adapted specifically to that species (
Fig. 2B). Whether or not a compartmentalized CoV from one species is more likely to infect humans, as occurs with the rabies virus (
6,
32,
40), is yet to be determined. However, it is clear that specific bat species harbor bat SARS-like viruses that are more closely related to SARS-CoV (
26).
The fact that some of the Roche 454 sequences derived from the tricolored bat were more closely related to HCoV-NL63 than any other α-CoV suggested that this novel North American α-CoV may be related to HCoVs. Therefore, we conducted molecular clock analysis using BEAST to determine the relatedness of this α-CoV to other CoVs, by adding it to previous data sets used to determine ancestry for other CoVs (
36,
50). Of note, ARCoV.2 and HCoV-NL63 appear to have an MRCA that occurred just over 560 years ago, based on a 650- to 800-nt portion of the highly conserved replicase gene. This analysis was limited to this fragment so that we could use the data obtained from previous studies to calibrate the clock and validate our results. If these predictions are correct, this observation suggests that HCoV-NL63 may have originated from bats between 1190 and 1449 CE. However, it is possible that recombination between bat and human CoVs or other mammalian strains may have occurred in this region, and therefore, more sequence information is necessary to work out the evolutionary history of these CoVs. Comparing the two trees in
Fig. 2 suggests that recombination has likely occurred, as the tree topologies are slightly different even in the nonstructural gene sequences that are known to be highly conserved among CoVs.
Immortalized cell lines are important reagents for investigating the similarities and differences between viruses that grow in bats and other mammals. To date, only a few immortalized bat cell lines have been developed, and the majority of these are for fruit bats, which allows for further studies with the Nipah and Hendra viruses (
4,
7,
22). In this study, we successfully immortalized bat cells (
Fig. 3) from a bat species known to harbor a unique α-CoV that appears to share common ancestry with HCoV-NL63. The facts that SARS-CoV and related strains replicate (
Fig. 7 and
8) and that HCoV-NL63 grows in the PESU-B5L immortalized lung cell line indicate the importance of developing robust reagents to a number of different bat species. To our knowledge, this is the first bat cell line that supports growth of HCoVs. We are currently in the process of characterizing the innate immune response in these cells, and we have primary cells available for three additional New World bat species that will be targeted for immortalization. These cell lines will be used to generate reagents for directly studying CoVs in bats and for assessing the potential of these viruses to infect humans. Our data suggest that the lentivirus-based immortalization system reported herein would also be successful for multiple North American bat species and likely represents a robust tool for rapidly establishing continuous cell lines from bats and other zoonotic species.
Only a small number of immortalized bat cell lines currently exist, and these were derived both from Old World bats, including the Egyptian fruit bat (
Rousettus aegyptiacus) (
22), the straw-colored fruit bat (
Eidolon helvum) (
4), and the black flying fox (
Pteropus alecto) (
7), and from New World bats, such as the Brazilian free-tailed bat (
Tadarida brasiliensis) (ATCC CCL-88). In these cases, the cell lines were immortalized using three different methods, including the simian virus 40 (SV40) large tumor antigen (
4,
7), the adenovirus serotype 5 E1A and E1B genes driven from the promoters for human phosphoglycerate kinase and thymidine kinase of herpes simplex virus (
22), and hTERT (
7). Initially, we attempted to immortalize the various primary bat cells using hTERT, but these attempts did not result in immortalized cells (data not shown). Interestingly, it has been shown that hTERT alone cannot immortalize primary human bronchial epithelial (hBE) cells (
30), possibly due to suboptimal culture conditions. Moreover, while hTERT in combination with viral oncogenes has been used to immortalize mammalian cell lines, these immortalizations have often resulted in immortalized cell lines that were limited in differentiation capacity and that frequently exhibited other morphological and genetic abnormalities (
18). To circumvent these limitations, we used a viral-oncogene-independent approach that was successfully used to immortalize hBE cells for the study of cystic fibrosis (
18). The primary difference in this approach was that it used the expression of the murine Bmi-1 gene (
18), which is a proto-oncogene that normally maintains stem cell populations but has also been shown to recapitulate normal cell structure and function (
18). The PESU-B5L cells immortalized by this approach have shown no morphological modifications over time and appear to be a homogenous population of lung epithelial cells.
The isolation and growth of novel α-CoVs from the North American and other global bat populations continue to be a difficult problem. We have attempted to isolate α-CoVs of several bat species from fecal samples that were positive by PCR for an α-CoV by inoculating freshly filtered fecal supernatants from these samples onto a variety of cell types, including PESU-B5L lung cells. To date, we have been unsuccessful in isolating a CoV using this approach. Therefore, we have been using high-throughput and Sanger sequencing approaches to identify and fill in genomic sequences that can be engineered into infectious clones that will allow us to study these viruses in the laboratory. We previously used this synthetic approach to resurrect Bat CoV HKU3, the SARS-like CoV identified in the Chinese horseshoe bat (
26), and showed that the only block to replication of this bat virus in primate cells was the 180-amino-acid portion of the spike glycoprotein known as the receptor-binding domain (RBD) (
3).
In this paper, we report the first evidence that traditional human CoVs are capable of growth in bat cells. Interestingly, both HCoV-NL63 and SARS-CoV and its related viruses replicated in these cells; however, replication occurred over multiple passages with HCoV-NL63. Recent studies have demonstrated that SARS-CoV and HCoV-NL63 use angiotensin-1 converting enzyme-2 (ACE2) as a receptor for entry into permissive cells (
25,
28,
37,
45). However, crystallography studies have shown that these HCoVs interact with ACE2 using different receptor binding domains in the spike glycoproteins of each virus to interact with different regions of the ACE2 molecule (
23,
27,
52). In fact, we would predict that it is differences in these interactions that impact the ability of SARS-CoV to efficiently replicate upon passage in these cells. Interestingly, while SARS-CoVgfp replicated leader-containing transcripts at 24 h, transcription was not detected at 48 h and no GFP fluorescence was observed in these cultures. However, MA15 and the two chimeric human SARS-CoVs bearing spike genes from the early stage of the SARS epidemic appeared to replicate leader-containing transcripts more efficiently and exhibited more cytopathology, indicating that these viruses grew better in these cells than did SARS-CoVgfp. This observation suggests that the spike protein may be the primary determinant for infectivity in these cells and that spike proteins that are more adapted to human ACE2, such as SARS-CoV, are less capable of establishing infection in these bat cells. However, HCoV-NL63 appeared to replicate more efficiently than any of the SARS-CoV variants, suggesting that the HCoV-NL63 interaction site on the tricolored bat ACE2 may be the primary determinant of infection. The interactions between PESU-B5L cells and various spike proteins are currently being explored in more detail. Interestingly, a recent study has shown that signatures of recurrent positive selection in the bat ACE2 gene map almost perfectly to known SARS-CoV interaction surfaces, suggesting that ACE2 utilization preceded the emergence of SARS-CoV-like viruses from bats (
9).
In addition, the sequence of the orthologous ACE2 receptor of the tricolor bat has not been determined but likely presents the determinants of cross-species transmission, allowing HCoVs to infect bats. Supporting this hypothesis, several bat ACE2 receptors were recently shown to function as receptors for SARS-CoV docking and entry, and notably, HCoV-NL63 receptor usage was not evaluated in these studies (
21). Of note, most α-CoVs use aminopeptidase N (APN) molecules as the cellular receptor, and the exogenous expression of feline APN in cells that are refractory to infection has rendered these cells susceptible to most α-CoVs (
47). The fact the HCoV-NL63, which is an α-CoV, uses ACE2 is an unusual feature of this HCoV, although it is not know which receptors the bat α-CoVs use. We are currently in the process of cloning and sequencing all of the known CoV receptor orthologues for this bat species to determine which receptor(s) is used by SARS-CoV and HCoV-NL63 and to assess the risk of reverse zoonotic transmission from humans to bats. We also plan to evaluate PESU-B5L infectivity and receptor usage by other coronaviruses such as HCoV-229E and HKU3 in future experiments. Attempts to obtain these viruses for this study were unsuccessful.
CoVs have a long history of cross-species transmission (
35). BCoV and HCoV-OC43 are closely related, with an MRCA predicted to have occurred ∼100 years ago (
2,
50,
51). BCoV strains have also spread to alpaca and wild ruminants. These observations suggest that HCoV-OC43 arose from a cross-species transmission of BCoV into humans, although it is equally likely that the transmission happened in reverse (
2,
50,
51). In the α-CoV genus, canine CoV (CCoV), feline CoV (FCoV), and the porcine CoV transmissible gastroenteritis virus (TGEV) carry genetic evidence of recombination with each other, suggesting that these CoVs likely originated from or infected the same host prior to becoming host range restricted. In fact, early strains CCoV-1 and FCoV-1 are thought to have diverged from a common ancestor, and multiple recombination events between these strains and an unknown CoV (in an unknown host) likely resulted in the novel CoVs CCoV-II and FCoV-II (
20a). Sequence analysis looking at the similarities between CCoV-II and TGEV suggests that TGEV emerged from a cross-species transmission of CCoV-II from an infected canine to pigs (
29).
SARS-CoV emerged from the Chinese wet markets, where the virus is thought to have crossed the species barriers from Chinese horseshoe bats to masked palm civets (
Paguma larvata) and raccoon dogs (
Nyctereutes procyonoides) to humans (
20). However, given that the human and civet SARS-CoV genomes were 96% identical and that the primary restriction to host range occurred at a small number of amino acid positions in the RBD, SARS-CoV may have been present in humans first and then been transmitted to civets and other animals in the markets (
20). This is particularly intriguing, as the SARS-CoV takes a more generalist approach to host range, whereby it can infect cells expressing human, civet, and bat ACE2 (
42,
43). In contrast, the civet SARS-CoV is restricted to infecting cells expressing civet ACE2 (
42,
43). This study provides evidence that HCoV-NL63 is also a generalist, as it can infect bat cells as well as primate (
48) and human (
12) cells. Interestingly, HCoV-NL63 does not grow in mice.
These are important observations because they indicate that some human and animal CoVs are generalists that can grow in a variety of different mammals, which is in contrast to many other mammalian CoVs that appear to be restricted to a single host. Recognizing that few mammalian coronaviruses have been tested in bat cell lines, these data suggest that humans and perhaps other select mammals may serve as the gateway species for the evolutionary expansion of mammalian CoVs. In addition, this observation suggests that human and/or other mammalian CoVs could potentially circulate back and forth between bats and humans, establishing a zoonotic-reverse zoonotic cycle that may allow the virus to maintain viral populations in multiple hosts, exchange genetic information to alter pathogenesis or transmission characteristics, and potentially evolve variants that are capable of efficiently infecting humans or other mammals. Further work is necessary to determine the risks of such a cycle, evaluate the impact that this could have on public health, and determine whether or not this represents a general feature of the mammalian Coronaviridae, which are predicted to have originated from bats.