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201 5 Nature America Inc All rights reserved l e t t e r s nature medicine advance online publication The emergence of severe acute respiratory syndrome coronavirus SARS CoV and Middle East respiratory syndrome MERS CoV underscores the threat of cross species transmission events leading to outbreaks in humans Here we examine the disease potential of a SARS like virus SHC014 CoV which is currently circulating in Chinese horseshoe bat populations 1 Using the SARS CoV reverse genetics system 2 we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse adapted SARS CoV backbone The results indicate that group 2b viruses encoding the SHC014 spike in a wild type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II ACE2 replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS CoV Additionally in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis Evaluation of available SARS based immune therapeutic and prophylactic modalities revealed poor efficacy both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein On the basis of these findings we synthetically re derived an infectious full length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo Our work suggests a potential risk of SARS CoV re emergence from viruses currently circulating in bat populations The emergence of SARS CoV heralded a new era in the cross species transmission of severe respiratory illness with globalization leading to rapid spread around the world and massive economic impact 3 4 Since then several strains including influenza A strains H5N1 H1N1 and H7N9 and MERS CoV have emerged from animal populations causing considerable disease mortality and economic hardship for the afflicted regions 5 Although public health measures were able to stop the SARS CoV outbreak 4 recent metagenomics studies have identified sequences of closely related SARS like viruses circulating in Chinese bat populations that may pose a future threat 1 6 However sequence data alone provides minimal insights to identify and prepare for future prepandemic viruses Therefore to examine the emergence potential that is the potential to infect humans of circulating bat CoVs we built a chimeric virus encoding a novel zoonotic CoV spike protein from the RsSHC014 CoV sequence that was isolated from Chinese horseshoe bats 1 in the context of the SARS CoV mouse adapted backbone The hybrid virus allowed us to evaluate the ability of the novel spike protein to cause disease independently of other necessary adaptive mutations in its natural backbone Using this approach we characterized CoV infection mediated by the SHC014 spike protein in primary human airway cells and in vivo and tested the efficacy of available immune therapeutics against SHC014 CoV Together the strategy translates metagenomics data to help predict and prepare for future emergent viruses The sequences of SHC014 and the related RsWIV1 CoV show that these CoVs are the closest relatives to the epidemic SARS CoV strains Fig 1a b however there are important differences in the 14 resi dues that bind human ACE2 the receptor for SARS CoV including the five that are critical for host range Y442 L472 N479 T487 and Y491 ref 7 In WIV1 three of these residues vary from the epidemic SARS CoV Urbani strain but they were not expected to alter binding to ACE2 Supplementary Fig 1a b and Supplementary Table 1 This fact is confirmed by both pseudotyping experiments that meas ured the ability of lentiviruses encoding WIV1 spike proteins to enter cells expressing human ACE2 Supplementary Fig 1 and by in vitro replication assays of WIV1 CoV ref 1 In contrast 7 of 14 ACE2 interaction residues in SHC014 are different from those in SARS CoV including all five residues critical for host range Supplementary Fig 1c and Supplementary Table 1 These changes coupled with A SARS like cluster of circulating bat coronaviruses shows potential for human emergence Vineet D Menachery 1 Boyd L Yount Jr 1 Kari Debbink 1 2 Sudhakar Agnihothram 3 Lisa E Gralinski 1 Jessica A Plante 1 Rachel L Graham 1 Trevor Scobey 1 Xing Yi Ge 4 Eric F Donaldson 1 Scott H Randell 5 6 Antonio Lanzavecchia 7 Wayne A Marasco 8 9 Zhengli Li Shi 4 accepted 8 October published online 9 November 2015 doi 10 1038 nm 3985 201 5 Nature America Inc All rights reserved l e t t e r s advance online publication nature medicine the failure of pseudotyped lentiviruses expressing the SHC014 spike to enter cells Supplementary Fig 1d suggested that the SHC014 spike is unable to bind human ACE2 However similar changes in related SARS CoV strains had been reported to allow ACE2 bind ing 7 8 suggesting that additional functional testing was required for verification Therefore we synthesized the SHC014 spike in the con text of the replication competent mouse adapted SARS CoV back bone we hereafter refer to the chimeric CoV as SHC014 MA15 to maximize the opportunity for pathogenesis and vaccine studies in mice Supplementary Fig 2a Despite predictions from both struc ture based modeling and pseudotyping experiments SHC014 MA15 was viable and replicated to high titers in Vero cells Supplementary Fig 2b Similarly to SARS SHC014 MA15 also required a func tional ACE2 molecule for entry and could use human civet and bat ACE2 orthologs Supplementary Fig 2c d To test the ability of the SHC014 spike to mediate infection of the human airway we examined the sensitivity of the human epithelial airway cell line Calu 3 2B4 ref 9 to infection and found robust SHC014 MA15 replication compa rable to that of SARS CoV Urbani Fig 1c To extend these findings primary human airway epithelial HAE cultures were infected and showed robust replication of both viruses Fig 1d Together the data confirm the ability of viruses with the SHC014 spike to infect human airway cells and underscore the potential threat of cross spe cies transmission of SHC014 CoV To evaluate the role of the SHC014 spike in mediating infection in vivo we infected 10 week old BALB c mice with 10 4 plaque forming units p f u of either SARS MA15 or SHC014 MA15 Fig 1e h Animals infected with SARS MA15 experienced rapid weight loss and lethality by 4 d post infection d p i in contrast SHC014 MA15 infection produced substantial weight loss 10 but no lethality in mice Fig 1e Examination of viral replica tion revealed nearly equivalent viral titers from the lungs of mice infected with SARS MA15 or SHC014 MA15 Fig 1f Whereas lungs from the SARS MA15 infected mice showed robust staining in both the terminal bronchioles and the lung parenchyma 2 d p i Fig 1g those of SHC014 MA15 infected mice showed reduced airway antigen staining Fig 1h in contrast no deficit in antigen staining was observed in the parenchyma or in the overall histology scoring suggesting differential infection of lung tissue for SHC014 MA15 Supplementary Table 2 We next analyzed infection in more susceptible aged 12 month old animals SARS MA15 infected animals rapidly lost weight and succumbed to infection Supplementary Fig 3a b SHC014 MA15 infection induced robust and sustained weight loss but had minimal lethality Trends in the histology and antigen staining patterns that we observed in young mice were conserved in the older animals Supplementary Table 3 We excluded the possibility that SHC014 MA15 was mediating infection through an alternative receptor on the basis of experiments using Ace2 mice which did not show weight loss or antigen stain ing after SHC014 MA15 infection Supplementary Fig 4a b and Supplementary Table 2 Together the data indicate that viruses with the SHC014 spike are capable of inducing weight loss in mice in the context of a virulent CoV backbone Given the preclinical efficacy of Ebola monoclonal antibody therapies such as ZMApp 10 we next sought to determine the efficacy of SARS CoV monoclonal antibodies against infection with TGEV BtCoV HKU9 BtCoV HKU 4 BtCoV HKU 5 2d 2b 2c MERS CoV SA N1 MERS CoV England N1 93 93 2a 100 BCoV HCoV OC43 MHV A59 HCoV HKU1 100 FCoV 100 100 47 88 3 IBV Beaudette 100 97 1 10 0 10 0 100 10 0 100 10 0 1a 1b BtCoV 512 BtCoV 279 BtCoV 273 RsSHC014 WIV 1SARS CoV BtCoV HKU3 BtCoV HKU 8 BtCoV 1BBtCoV 1A PEDV CV7777 HCoV 229E HCoV NL63 a 0 01 Substitution position BtSCoV Rf1 2004 BtCoV 273 2005 BtSCoV Rs 672 2006 BtSCoV HKU3 2 BtSCoV Rp3 BtSCoV Rm1 2004 BtCoV 279 2005 RsSHC014 CoV subgroup 2b SCoV GD01 SCoV CUHK W1 SCoV Urbani SCoV A022 SCoV SZ16 SCoV HC SZ 61 03 WIV 1 b hg 110 100 90 80 70 0 1 2 3 4 5 6 7 Time after infection d Weight of starting weight 9 8 7 6 5 4 3 2 1 0 12 24 Time after infection h Log viral titer 36 48 c e 9 8 7 6 5 4 3 2 Time after infection d Log viral titer 4 7 6 5 4 3 2 1 0 12 24 Time after infection h Log viral titer 36 48 d f Figure 1 SARS like viruses replicate in human airway cells and produce in vivo pathogenesis a The full length genome sequences of representative CoVs were aligned and phylogenetically mapped as described in the Online Methods The scale bar represents nucleotide substitutions with only bootstrap support above 70 being labeled The tree shows CoVs divided into three distinct phylogenetic groups defined as CoVs CoVs and CoVs Classical subgroup clusters are marked as 2a 2b 2c and 2d for the CoVs and as 1a and 1b for the CoVs b Amino acid sequences of the S1 domains of the spikes of representative CoVs of the 2b group including SARS CoV were aligned and phylogenetically mapped The scale bar represents amino acid substitutions c d Viral replication of SARS CoV Urbani black and SHC014 MA15 green after infection of Calu 3 2B4 cells c or well differentiated primary air liquid interface HAE cell cultures d at a multiplicity of infection MOI of 0 01 for both cell types Samples were collected at individual time points with biological replicates n 3 for both Calu 3 and HAE experiments e f Weight loss n 9 for SARS CoV MA15 n 16 for SHC014 MA15 e and viral replication in the lungs n 3 for SARS CoV MA15 n 4 for SHC014 MA15 f of 10 week old BALB c mice infected with 1 10 4 p f u of mouse adapted SARS CoV MA15 black or SHC014 MA15 green via the intranasal i n route g h Representative images of lung sections stained for SARS CoV N antigen from mice infected with SARS CoV MA15 n 3 mice g or SHC014 MA15 n 4 mice h are shown For each graph the center value represents the group mean and the error bars define the s e m Scale bars 1 mm 201 5 Nature America Inc All rights reserved l e t t e r s nature medicine advance online publication SHC014 MA15 Four broadly neutralizing human monoclonal antibodies targeting SARS CoV spike protein had been previously reported and are probable reagents for immu notherapy 11 13 We examined the effect of these antibodies on viral replication expressed as percentage inhibi tion of viral replication and found that whereas wild type SARS CoV Urbani was strongly neutralized by all four antibodies at relatively low antibody concentrations Fig 2a d neutralization varied for SHC014 MA15 Fm6 an antibody generated by phage display and escape mutants 11 12 achieved only background levels of inhibition of SHC014 MA15 replication Fig 2a Similarly antibodies 230 15 and 227 14 which were derived from memory B cells of SARS CoV infected patients 13 also failed to block SHC014 MA15 replication Fig 2b c For all three antibodies differences between the SARS and SHC014 spike amino acid sequences corresponded to direct or adjacent residue changes found in SARS CoV escape mutants fm6 N479R 230 15 L443V 227 14 K390Q E which probably explains the absence of the antibodies neutralizing activity against SHC014 Finally monoclonal antibody 109 8 was able to achieve 50 neutrali zation of SHC014 MA15 but only at high concentrations 10 g ml Fig 2d Together the results demonstrate that broadly neutralizing antibodies against SARS CoV may only have marginal efficacy against emergent SARS like CoV strains such as SHC014 To evaluate the efficacy of existing vaccines against infection with SHC014 MA15 we vaccinated aged mice with double inactivated whole SARS CoV DIV Previous work showed that DIV could neutralize and protect young mice from challenge with a homolo gous virus 14 however the vaccine failed to protect aged animals in which augmented immune pathology was also observed indicating the possibility of the animals being harmed because of the vaccina tion 15 Here we found that DIV did not provide protection from chal lenge with SHC014 MA15 with regards to weight loss or viral titer Supplementary Fig 5a b Consistent with a previous report with other heterologous group 2b CoVs 15 serum from DIV vaccinated aged mice also failed to neutralize SHC014 MA15 Supplementary Fig 5c Notably DIV vaccination resulted in robust immune pathol ogy Supplementary Table 4 and eosinophilia Supplementary Fig 5d f Together these results confirm that the DIV vaccine would not be protective against infection with SHC014 and could possibly augment disease in the aged vaccinated group In contrast to vaccination of mice with DIV the use of SHC014 MA15 as a live attenuated vaccine showed potential cross protec tion against challenge with SARS CoV but the results have important caveats We infected young mice with 10 4 p f u of SHC014 MA15 and observed them for 28 d We then challenged the mice with SARS MA15 at day 29 Supplementary Fig 6a The prior infection of the mice with the high dose of SHC014 MA15 conferred protection against challenge with a lethal dose of SARS MA15 although there was only a minimal SARS CoV neutralization response from the antisera elicited 28 d after SHC014 MA15 infection Supplementary Fig 6b 1 200 In the absence of a secondary antigen boost 28 d p i represents the expected peak of antibody titers and implies that there will be diminished protection against SARS CoV over time 16 17 Similar results showing protection against challenge with a lethal dose of SARS CoV were observed in aged BALB c mice with respect to weight loss and viral replication Supplementary Fig 6c d However the SHC014 MA15 infection dose of 10 4 p f u induced 10 weight loss and lethality in some aged animals Fig 1 and Supplementary Fig 3 We found that vaccination with a lower dose of SHC014 MA15 100 p f u did not induce weight loss but it also failed to protect aged animals from a SARS MA15 lethal dose challenge Supplementary Fig 6e f Together the data suggest that SHC014 MA15 challenge may confer cross protection against SARS CoV through conserved epitopes but the required dose induces pathogenesis and precludes use as an attenuated vaccine Having established that the SHC014 spike has the ability to mediate infection of human cells and cause disease in mice we next synthesized a full length SHC014 CoV infectious clone based on the approach used for SARS CoV Fig 3a 2 Replication in Vero cells revealed no deficit for SHC014 CoV relative to that for SARS CoV Fig 3b however SHC014 CoV was significantly P 0 01 attenu ated in primary HAE cultures at both 24 and 48 h after infection Fig 3c In vivo infection of mice demonstrated no significant weight loss but showed reduced viral replication in lungs of full length SHC014 CoV infection as compared to SARS CoV Urbani Fig 3d e Together the results establish the viability of full length a 120 100 80 60 40 20 0 1 25 2 50 5 00 10 00 20 000 62 Inhibition of plaque formation Antibody concentration g ml b 120 100 80 60 40 20 0 1 25 2 50 5 00 10 000 62 Inhibition of plaque formation Antibody concentration g ml c 120 100 80 60 40 20 0 1 25 2 50 5 00 10 000 62 Inhibition of plaque formation Antibody concentration g ml d 120 100 80 60 40 20 0 1 25 2 50 5 00 10 000 62 Inhibition of plaque formation Antibody concentration g ml Figure 2 SARS CoV monoclonal antibodies have marginal efficacy against SARS like CoVs a d Neutralization assays evaluating efficacy measured as reduction in the number of plaques of a panel of monoclonal antibodies which were all originally generated against epidemic SARS CoV against infection of Vero cells with SARS CoV Urbani black or SHC014 MA15 green The antibodies tested were fm6 n 3 for Urbani n 5 for SHC014 MA15 11 12 a 230 15 n 3 for Urbani n 2 for SHC014 MA15 b 227 15 n 3 for Urbani n 5 for SHC014 MA15 c and 109 8 n 3 for Urbani n 2 for SHC014 MA15 13 d Each data point represents the group mean and error bars define the s e m Note that the error bars in SARS CoV Urbani infected Vero cells in b c are overlapped by the symbols and are not visible 201 5 Nature America Inc All rights reserved l e t t e r s advance online publication nature medicine SHC014 CoV but suggest that further adaptation is required for its replication to be equivalent to that of epidemic SARS CoV in human respiratory cells and in mice During the SARS CoV epidemic links were quickly established between palm civets and the CoV strains that were detected in humans 4 Building on this finding the common emergence paradigm argues that epidemic SARS CoV originated as a bat virus jumped to civets and incorporated changes within the receptor binding domain RBD to improve binding to civet Ace2 ref 18 Subsequent expo sure to people in live animal markets permitted human infection with the civet strain which in turn adapted to become the epidemic strain Fig 4a However phylogenetic analysis suggests that early human SARS strains appear more closely related to bat strains than to civet strains 18 Therefore a second paradigm argues that direct bat human transmission initiated SARS CoV emergence and that palm civets served as a secondary host and reservoir for continued infection Fig 4b 19 For both paradigms spike adaptation in a secondary host is seen as a necessity with most mutations expected to occur within the RBD thereby facilitating improved infection Both theories imply that pools of bat CoVs are limited and that host range mutations are Host range mutation Secondary host reservoir a b c Direct human infection Secondary host reservoir Direct human infection Secondary host reservoir Epidemic strain Epidemic strain Adaptation Human infection Adaptation Adaptation Random Recombination events Generalists receptor orthologs Random Rare Random Rare Figure 4 Emergence paradigms for coronaviruses Coronavirus strains are maintained in quasi species pools circulating in bat populations a b Traditional SARS CoV emergence theories posit that host range mutants red circle represent random and rare occurrences that permit infection of alternative hosts The secondary host paradigm a argues that a nonhuman host is infected by a bat progenitor virus and through adaptation facilitates transmission to humans subsequent replication in humans leads to the epidemic viral strain The direct paradigm b suggests that transmission occurs between bats and humans without the requirement of an intermediate host selection then occurs in the human population with closely related viruses replicating in a secondary host permitting continued viral persistence and adaptation in both c The data from chimeric SARS like viruses argue that the quasi species pools maintain multiple viruses capable of infecting human cells without the need for mutations red circles Although adaptations in secondary or human hosts may be required for epidemic emergence if SHC014 spike containing viruses recombined with virulent CoV backbones circles with green outlines then epidemic disease may be the result in humans Existing data support elements of all three paradigms b d 70 80 90 100 110 0 0 1 2 3 4 Log viral titer 12 24 36 48 Time after infection h Time after infection d Weight of starting weight 1 2 3 4 5 6 7 8 9 e 1 2 3 4 5 6 2 4 Log viral titer Log viral titer 0 12 24 36 Time after infection h Time after infection d 48 0 1 2 3 4 5 6 7 8 ca 0 10 ORF1a SHC014A SHC014B S