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.

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.

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.

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.

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.

To further extend these findings, in vivo studies with antibody 227.14 were initiated in HFH4-ACE2–expressing mice. One day before infection, HFH4-ACE2–expressing mice were injected with 200 μg of antibody 227.14 or PBS control as previously described (17); mice were subsequently challenged with either SARS-CoV Urbani or WIV1-CoV and monitored for 7 d. The results indicate that antibody 227.14 protected mice from both lethal SARS-CoV Urbani and WIV-CoV challenge (Fig. 4E); in addition, lung titers revealed no detectable virus in either SARS-CoV or WIV1-CoV–infected HFH4-ACE2–expressing mouse lungs following antibody treatment (Fig. 4F). Together, the in vitro and in vivo data indicate that a mixture of broadly neutralizing antibodies against SARS-CoV would likely provide significant protection if WIV1-CoV–like viruses successfully transmitted to humans.

Previously, whole virion SARS-CoV inactivated by both formalin and UV irradiation (double inactivated virus, DIV) was demonstrated as a potential vaccination candidate based on robust neutralization and protection following homologous SARS-CoV challenge in young mice (18). However, both aged animal and heterologous challenge studies revealed incomplete protection, increased immune pathology, and eosinophilia, indicating the possibility of adverse affects following DIV vaccination (19). To determine if heterologous challenge with WIV1-CoV spike produced a similar affect, 1-y-old BALB/c mice were vaccinated and boosted with DIV or PBS mock control. Mice were then challenged 6 wk postinitial vaccination with WIV1-MA15 and examined over a 4-d time course. Similar to previous experiments, mice infected with WIV1-MA15 had only marginal weight loss and showed no clinical signs of disease with either vaccination group (Fig. 5A). However, viral replication at day 4 was not significantly reduced in DIV-vaccinated groups compared with control (Fig. 5B). In addition, plaque reduction neutralization titers from the serum of aged DIV-vaccinated mice indicated no neutralization of WIV1-MA15, suggesting inadequate protection (Fig. 5C). Importantly, examination of histopathology revealed increased eosinophilia in DIV-vaccinated mice compared with PBS controls, indicating the potential for immune induced pathology due to vaccination. Together, the data indicate that DIV vaccination would not provide significant protection and may cause adverse effects in the context of WIV1-CoV spike-mediated outbreak.

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