Product# 17011 rSars Spike(S) Protein (EUK)
|Product# 17101 Murine Anti- SARS Monoclonal Antibody|
The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV highlights the continued risk of cross-species transmission leading to epidemic disease. This manuscript describes efforts to extend surveillance beyond sequence analysis, constructing chimeric and full-length zoonotic coronaviruses to evaluate emergence potential. Focusing on SARS-like virus sequences isolated from Chinese horseshoe bats, the results indicate a significant threat posed by WIV1-CoV. Both full-length and chimeric WIV1-CoV readily replicated efficiently in human airway cultures and in vivo, suggesting capability of direct transmission to humans. In addition, while monoclonal antibody treatments prove effective, the SARS-based vaccine approach failed to confer protection. Together, the study indicates an ongoing threat posed by WIV1-related viruses and the need for continued study and surveillance.
Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.
Although previously associated with upper respiratory infections, the emergence of severe acute respiratory coronavirus (SARS-CoV) in 2002–2003, and more recently, Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission leading to virulent pandemic viral infections (1, 2). Whereas prevailing research suggests that SARS-CoV emerged from viruses in the Chinese horseshoe bat, identifying a progenitor strain that used human angiotensin converting enzyme 2 (ACE2) had proven elusive (3, 4). However, recent metagenomics studies isolated several SARS-like virus sequences that share ≥90% genome-wide homology and represented the closest sequences to the epidemic strains (5, 6). Importantly, researchers also isolated replication competent virus; WIV1-CoV, part of the Rs3306 cluster, could use ACE2 orthologs and mediated low-level replication in human cells (5). Overall, the evidence indicates that SARS-CoV likely emerged from Chinese horseshoe bats and that similar viruses are still harbored in these populations.
The identification of WIV1-CoV and its capacity to use ACE2 orthologs offers a warning for possible reemergence and provides an opportunity to prepare for a future CoV outbreak. To achieve this goal, a new platform is required to translate metagenomics findings; the approach must generate critical diagnostic reagents, define emergence potential of novel strains, and determine efficacy of current therapeutics. Building on this premise, we developed a framework to examine circulating CoVs using reverse genetic systems to construct full-length and chimeric viruses. The results indicate that viruses using WIV1-CoV spike are poised to emerge in human populations due to efficient replication in primary human airway epithelial cell cultures. However, additional adaptation, potentially independent of the spike protein receptor-binding domain, is required for pathogenesis and epidemic disease. Importantly, monoclonal antibody strategies against SARS were effective against WIV1-CoV spike unlike available vaccine approaches. Together, the results highlight the utility of developing platforms to evaluate circulating zoonotic viruses as threats for future emergence and epidemic potential.
The discovery of SARS-like virus clusters that bridge the gap between the epidemic strains and related precursor CoV strain HKU3 virus provided the best evidence for emergence of SARS-CoV from Chinese horseshoe bats (5). Comparing the receptor binding domain (RBD), SARS-CoV Urbani and WIV1 share homology at 11 of the 14 contact residues with human ACE2 (Fig. 1A); importantly, the three amino acid changes represent relatively conservative substitution not predicted to ablate binding (Fig. 1B). Therefore, exploring WIV1 strains allows examination of emergence, pathogenesis potential, and adaptation requirements. Using the SARS-CoV infectious clone as a template (7), we designed and synthesized a full-length infectious clone of WIV1-CoV consisting of six plasmids that could be enzymatically cut, ligated together, and electroporated into cells to rescue replication competent progeny virions (Fig. S1A). In addition to the full-length clone, we also produced WIV1-CoV chimeric virus that replaced the SARS spike with the WIV1 spike within the mouse-adapted backbone (WIV1-MA15, Fig. S1B). WIV1-MA15 incorporates the original binding and entry capabilities of WIV1-CoV, but maintains the backbone changes to mouse-adapted SARS-CoV. Importantly, WIV1-MA15 does not incorporate the Y436H mutation in spike that is required for SARS-MA15 pathogenesis (8). Following electroporation into Vero cells, robust stock titers were recovered from both chimeric WIV1-MA15 and WIV1-CoV. To confirm growth kinetics and replication, Vero cells were infected with SARS-CoV Urbani, WIV1-MA15, and WIV1-CoV (Fig. 1C); the results indicate similar replication kinetics and overall titers between the CoVs. However, Western blot analysis suggests potential differences in spike cleavage/processing of WIV1 and SARS-CoV spike proteins (Fig. S1C); the ratio of full-length to cleaved spike varied between SARS spikes (Urbani, 1.21; MA15, 1.44) and WIV1 (full length, 0.61; WIV1-MA15, 0.25) signaling possible variation in host proteolytic processing (Fig. S1D). Overall, the results indicate comparable viral replication, but possible biochemical differences in processing.
Replication in Primary Human Epithelial Cells.
Next, we wanted to determine WIV-CoV replication potential in models of the human lung. Previous examination of WIV1-CoV recovered from bat samples demonstrated poor replication in A549 cells (5); however, replication of epidemic SARS-CoV is also poor in this cell type, potentially due to ACE2 expression levels (9). Therefore, well-differentiated primary human airway epithelial cell (HAE) air–liquid interface cultures were infected with WIV1-MA15, WIV1-CoV, SARS-CoV Urbani, or SARS-CoV MA15. At 24 and 48 h postinfection, both WIV1-MA15 and WIV1-CoV produce robust infection in HAE cultures equivalent to the epidemic strain and mouse-adapted strains (Fig. 1D). Together, the data demonstrate that the WIV1-CoV spike can mediate infection of human airway cultures with no significant adaptation required.
WIV1 Spike in Vivo.
To extend analysis to pathogenesis, we next evaluated in vivo infection following WIV1-MA15 and WIV1-CoV challenge. Initial studies compared WIV1-MA15 to mouse-adapted SARS-CoV (MA15) to determine spike-dependent pathogenesis. Ten-week-old BALB/c mice were infected with 104 plaque forming units (pfu) of WIV1-MA15 or SARS-CoV MA15 and followed over a 4-d time course. As expected, animals infected with SARS-CoV MA15 experienced rapid weight loss and lethality by day 4 postinfection (Fig. 2A and Fig. S2A) (10). In contrast, WIV1-MA15 induced neither lethality nor notable changes in body weight, indicating limited disease in vivo. Viral titer in the lung also revealed reduced replication following WIV1-MA15 challenge compared with control (Fig. 2B). Similarly, lung antigen staining indicated distinct attenuation of the WIV1-MA15, with most staining occurring in the airways and absent from large regions of the lungs (Fig. S2 B–D). Together, these data indicate that WIV1 spike substitution does not program pathogenesis in the mouse-adapted SARS-CoV backbone.
Viruses using WIV1 spike attenuated relative to SARS spike in vivo. (A and B) Ten-week-old BALB/c mice were infected with 104 pfu of either SARS-CoV MA15 (black) or WIV1-MA15 (blue hatched) via the i.n. route and examined over a 7-d time course. (A) Weight loss (n = 17 for WIV1-MA15, n = 9 for SARS-CoV MA15) and (B) lung titer (n = 3 for MA15, n = 4 for WIV1-MA15. (C and D) Ten-week-old BALB/c mice were infected with 1 × 105 pfu of either SARS-CoV Urbani (black), WIV1-CoV (blue), or SARS-CoV MA15 (gray) and examined over a 4-d time course. (C) Weight loss (n = 6 for WIV1-CoV, n = 6 for SARS-CoV Urbani) and (D) lung titer (n = 3 for WIV1-CoV, n = 3 for SARS-CoV Urbani) were examined. For each bar graph, center value is representative of group mean and error bars are defined by SEM. P values based on two-tailed Student’s t test of individual time points are marked as indicated: ***P < 0.001.
Although chimeric studies suggest minimal pathogenesis potential for WIV1 spike, SARS-CoV Urbani spike within the mouse-adapted backbone yielded similar results (8). Therefore, we examined the full-length WIV1-CoV versus the epidemic SARS-CoV Urbani strain in vivo. Ten-week-old BALB/c mice were infected with 105 pfu of WIV1-CoV or SARS-CoV Urbani and followed over a 4-d time course. As expected, neither infection condition resulted in significant weight loss compared with MA15 (Fig. 2C). However, viral replication was significantly attenuated for WIV1-CoV compared with SARS-CoV Urbani (Fig. 2D); at both days 2 and 4 postinfection, WIV1-CoV titer was reduced nearly 10,000- and 1,000-fold, respectively. Similarly, only minor antigen staining was observed following WIV1-CoV infection, contrasting antigen staining throughout the parenchyma 2-d post–SARS-CoV Urbani infection (Fig. S2 E and F). Together, the data indicate significant attenuation of WIV1-CoV relative to the epidemic SARS-CoV in wild-type mice.
WIV1-CoV in Human ACE2 Expressing Mice.
Whereas studies in wild-type mice provide insight into pathogenesis potential, the absence of clinical disease in the epidemic strains of SARS-CoV suggests that the mouse model may not be adequate to access human disease potential. To test a model more relevant to humans, we generated a mouse that expresses human ACE2 receptor under control of HFH4, a lung ciliated epithelial cell promoter (11). However, whereas robust expression was observed in the lung, other tissues including brain, liver, kidney, and gastrointestinal tract had varying levels of human ACE2 expression, indicating greater tissue distribution of HFH4-mediated expression than initially expected (Fig. S3A). In addition, examination of individual HFH4-ACE2–expressing progeny revealed the occasional absence of the human ACE2 gene, suggesting possible selection against human receptor (Fig. S3B). Therefore, PCR-positive, 10- to 20-wk-old HFH4-ACE2–expressing mice were infected with 105 pfu of WIV1-CoV or SARS-CoV Urbani and then followed for a 7-d time course to determine pathogenesis. The results indicated that WIV1-CoV infection was augmented, but remained attenuated relative to SARS-CoV Urbani in the presence of human ACE2. Following SARS-CoV Urbani challenge, HFH4-hACE2–expressing mice lost no weight, but then, experienced rapid weight loss and death between days 4 and 5 (Fig. 3A and Fig. S3C). In contrast, WIV1-CoV produce minimal changes in weight loss until late times where animals fell into distinct categories either losing less than or more than 10% of their body weight. Whereas day-2 lung titers were still attenuated relative to SARS-CoV Urbani, titers for WIV1-CoV were 100-fold higher in the presence of human ACE2 compared with wild-type BALB/c, with no similar augmentation observed with the epidemic SARS-CoV strain (Fig. 3B). Based on pilot studies and previous studies with ACE2 transgenic animals (12), mice experiencing rapid weight loss were predicted to have lethal encephalitis and were humanely killed and harvested for lung and brain titer if weight loss approached >20% of starting body weight. All HFH4-ACE2 mice infected with SARS-CoV Urbani lost >20% body weight and maintained robust replication in the lung and brain following infection (Fig. 3 C and D). Similarly, mice with >10% weight loss following WIV1-CoV infection produced robust viral replication in the brain, but significantly lower titers in the lung. In contrast, mice that maintained minimal weight loss (<10%) following WIV1-CoV infection after 7 d had minimal titers in both the lung and brain, suggesting a sufficient adaptive immune response was generated to clear virus and survive infection. Together, the data indicate that WIV1-CoV maintains attenuation relative to SARS-CoV Urbani despite the availability of human ACE2. In addition, augmented replication suggests that WIV1-CoV may bind the human ACE2 receptor more efficiently that the mouse ACE2, indicating potential inadequacies in the current mouse models of SARS pathogenesis.
Therapeutics Against WIV1 Emergence.
Having established a potential threat based on replication in primary human cells and preference for the human ACE2 receptor in vivo, we next sought to determine if monoclonal antibody therapies could be used to lessen disease similar to ZMApp for Ebola (13). We first tested a SARS-CoV monoclonal derived via phage display and antibody escape (Fm6) (14) and found both wild-type SARS-CoV Urbani and WIV1-MA15 were strongly neutralized at low antibody concentrations (Fig. 4A). Similarly, a panel of monoclonal antibodies derived from B cells from SARS-CoV–infected patients also prevented virus infection via WIV1-CoV spike (15, 16). Both antibodies 230.15 and 227.14 robustly inhibited WIV1-MA15 replication with kinetics similar to or exceeding SARS-CoV Urbani (Fig. 4 B and C). In contrast, antibody 109.8, which maps outside the receptor binding domain, produced only marginal neutralization of WIV1-MA15 (Fig. 4D). Whereas the residue associated with prior escape mutants was conserved at position 332, the adjacent residue had a significant change (K332T) in WIV1-CoV, possibly contributing to reduced efficacy of this antibody.
Virus Neutralization Assays.
Plaque reduction neutralization titer assays were preformed with previously characterized antibodies against SARS-CoV as previously described (14, 15, 21). Briefly, neutralizing antibodies or serum were serially diluted twofold and incubated with 100 pfu of the different virus strains for 1 h at 37 °C. The virus and antibodies were then added to a six-well plate with 5 ×105 Vero E6 cells per well with n ≥ 2. After a 1-h incubation at 37 °C, cells were overlaid with 3 mL of 0.8% agarose in media. Plates were incubated for 2 d at 37 °C and then stained with neutral red for 3 h, and plaques were counted. The percentage of plaque reduction was calculated as [1 − (no. of plaques with antibody/no. of plaques without antibody)] × 100.
All experiments were conducted contrasting two experimental groups (either two viruses or vaccinated and unvaccinated cohorts). Therefore, significant differences in viral titer and histology scoring were determined by a two-tailed Student’s t test at individual time points. Data were normally distributed in each group being compared and had similar variance.
Biosafety and Biosecurity.
Reported studies were initiated after the University of North Carolina Institutional Biosafety Committee approved the experimental protocol: project title: Generating infectious clones of Bat SARS-like CoVs; lab safety plan ID: 20145741; schedule G ID: 12279. These studies were initiated before the US Government Deliberative Process Research Funding Pause on Selected Gain of Function Research Involving Influenza, MERS, and SARS Viruses (www.phe.gov/s3/dualuse/Documents/gain-of-function.pdf), and the current paper has been reviewed by the funding agency, the National Institutes of Health (NIH). Continuation of these studies has been requested and approved by the NIH.
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We thank Dr. Zhengli-Li Shi of the Wuhan Institute of Virology for access to bat CoV sequences and plasmid of WIV1-CoV spike protein. Research was supported by the National Institute of Allergy and Infectious Disease and the National Institute of Aging of the NIH under Awards U19AI109761 and U19AI107810 (to R.S.B.), AI1085524 (to W.A.M.), and F32AI102561 and K99AG049092 (to V.D.M.). Human airway epithelial cell cultures were supported by the National Institute of Diabetes and Digestive and Kidney Disease under Award NIH DK065988 (to S.H.R.). Support for the generation of the mice expressing human ACE2 was provided by NIH Grants AI076159 and AI079521 (to A.C.S.).
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
See Commentary on page 2812.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1517719113/-/DCSupplemental.
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