SARS-like WIV1-CoV poised for human emergence

Vineet D. Menacherya, Boyd L. Yount Jr.a, Amy C. Simsa, Kari Debbinka,b, Sudhakar S. Agnihothramc, Lisa E. Gralinskia,
Rachel L. Grahama, Trevor Scobeya, Jessica A. Plantea, Scott R. Royala, Jesica Swanstroma, Timothy P. Sheahana,
Raymond J. Picklesc,d, Davide Cortie,f,g, Scott H. Randelld, Antonio Lanzavecchiae,f, Wayne A. Marascoh,
and Ralph S. Barica,c,1
a
Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; bDepartment of Microbiology and Immunology, University
of North Carolina at Chapel Hill, Chapel Hill, NC 27599; cDivision of Microbiology, National Center for Toxicological Research, Food and Drug
Administration, Jefferson, AR 72079; dDepartment of Cell Biology and Physiology and Marsico Lung Institute/Cystic Fibrosis Center, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599; eInstitute for Research in Biomedicine, Bellinzona, Switzerland; fInstitute of Microbiology, Eidgenössische
Technische Hochschule Zurich, Zurich, Switzerland; gHumabs BioMed SA, Bellinzona, Switzerland; and hDepartment of Cancer Immunology and AIDS,
Dana-Farber Cancer Institute–Department of Medicine, Harvard Medical School, Boston MA 02215

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved January 6, 2016 (received for review September 4, 2015)

Outbreaks from zoonotic sources represent a threat to both strategies against SARS were effective against WIV1-CoV spike
human disease as well as the global economy. Despite a wealth of unlike available vaccine approaches. Together, the results highlight
metagenomics studies, methods to leverage these datasets to identify the utility of developing platforms to evaluate circulating zoonotic
future threats are underdeveloped. In this study, we describe an viruses as threats for future emergence and epidemic potential.
approach that combines existing metagenomics data with reverse
genetics to engineer reagents to evaluate emergence and pathogenic Results
potential of circulating zoonotic viruses. Focusing on the severe acute The discovery of SARS-like virus clusters that bridge the gap
respiratory syndrome (SARS)-like viruses, the results indicate that the between the epidemic strains and related precursor CoV strain
WIV1-coronavirus (CoV) cluster has the ability to directly infect and HKU3 virus provided the best evidence for emergence of SARS-
may undergo limited transmission in human populations. However, CoV from Chinese horseshoe bats (5). Comparing the receptor
in vivo attenuation suggests additional adaptation is required for binding domain (RBD), SARS-CoV Urbani and WIV1 share
epidemic disease. Importantly, available SARS monoclonal antibodies homology at 11 of the 14 contact residues with human ACE2
offered success in limiting viral infection absent from available (Fig. 1A); importantly, the three amino acid changes represent
vaccine approaches. Together, the data highlight the utility of a relatively conservative substitution not predicted to ablate
platform to identify and prioritize prepandemic strains harbored in binding (Fig. 1B). Therefore, exploring WIV1 strains allows ex-
animal reservoirs and document the threat posed by WIV1-CoV for
amination of emergence, pathogenesis potential, and adap-
emergence in human populations.
tation requirements. Using the SARS-CoV infectious clone as a
template (7), we designed and synthesized a full-length infectious
SARS | CoV | emergence | Spike | WIV1 clone of WIV1-CoV consisting of six plasmids that could be
enzymatically cut, ligated together, and electroporated into cells
A lthough previously associated with upper respiratory infec-
tions, the emergence of severe acute respiratory coronavirus
(SARS-CoV) in 2002–2003, and more recently, Middle East
to rescue replication competent progeny virions (Fig. S1A). In
addition to the full-length clone, we also produced WIV1-CoV
respiratory syndrome (MERS)-CoV underscores the threat of
Significance
cross-species transmission leading to virulent pandemic viral in-
fections (1, 2). Whereas prevailing research suggests that SARS-
CoV emerged from viruses in the Chinese horseshoe bat, identi- The emergence of severe acute respiratory syndrome coronavirus
fying a progenitor strain that used human angiotensin converting (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV
enzyme 2 (ACE2) had proven elusive (3, 4). However, recent highlights the continued risk of cross-species transmission leading
metagenomics studies isolated several SARS-like virus sequences to epidemic disease. This manuscript describes efforts to extend
that share ≥90% genome-wide homology and represented the surveillance beyond sequence analysis, constructing chimeric and
full-length zoonotic coronaviruses to evaluate emergence poten-
closest sequences to the epidemic strains (5, 6). Importantly, re-
tial. Focusing on SARS-like virus sequences isolated from Chinese
searchers also isolated replication competent virus; WIV1-CoV,
horseshoe bats, the results indicate a significant threat posed by
part of the Rs3306 cluster, could use ACE2 orthologs and medi-
WIV1-CoV. Both full-length and chimeric WIV1-CoV readily repli-
ated low-level replication in human cells (5). Overall, the evidence
cated efficiently in human airway cultures and in vivo, suggesting
indicates that SARS-CoV likely emerged from Chinese horseshoe
capability of direct transmission to humans. In addition, while
bats and that similar viruses are still harbored in these populations.
monoclonal antibody treatments prove effective, the SARS-based
The identification of WIV1-CoV and its capacity to use ACE2
vaccine approach failed to confer protection. Together, the study
orthologs offers a warning for possible reemergence and pro-
indicates an ongoing threat posed by WIV1-related viruses and
vides an opportunity to prepare for a future CoV outbreak. To
the need for continued study and surveillance.
achieve this goal, a new platform is required to translate meta-
genomics findings; the approach must generate critical di- Author contributions: V.D.M., B.L.Y., and R.S.B. designed research; V.D.M., B.L.Y., A.C.S.,
agnostic reagents, define emergence potential of novel strains, S.S.A., L.E.G., T.S., J.A.P., S.R.R., J.S., and T.P.S. performed research; V.D.M., B.L.Y., R.J.P.,
and determine efficacy of current therapeutics. Building on this D.C., S.H.R., A.L., and W.A.M. contributed new reagents/analytic tools; V.D.M., A.C.S.,
K.D., R.L.G., and R.S.B. analyzed data; and V.D.M. and R.S.B. wrote the paper.
premise, we developed a framework to examine circulating CoVs
using reverse genetic systems to construct full-length and chi- The authors declare no conflict of interest.

meric viruses. The results indicate that viruses using WIV1-CoV This article is a PNAS Direct Submission.
spike are poised to emerge in human populations due to efficient Freely available online through the PNAS open access option.
replication in primary human airway epithelial cell cultures. See Commentary on page 2812.
However, additional adaptation, potentially independent of the 1
To whom correspondence should be addressed. Email: [email protected].
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spike protein receptor-binding domain, is required for patho- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
genesis and epidemic disease. Importantly, monoclonal antibody 1073/pnas.1517719113/-/DCSupplemental.

3048–3053 | PNAS | March 15, 2016 | vol. 113 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1517719113

 SEE COMMENTARY
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 epi-
demic 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 in-
fected 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). Simi-
larly, lung antigen staining indicated distinct attenuation of the
WIV1-MA15, with most staining occurring in the airways and
Fig. 1. Full-length and chimeric WIV1 infectious clones produce viruses that absent from large regions of the lungs (Fig. S2 B–D). Together,
replicate in primary human airway epithelial cell cultures. (A) Spike amino these data indicate that WIV1 spike substitution does not pro-
acid residues that interact directly with human ACE2 from SARS-CoV, SARS- gram pathogenesis in the mouse-adapted SARS-CoV backbone.
MA15, and WIV1-CoV spike proteins. Residue changes are highlighted by
Although chimeric studies suggest minimal pathogenesis po-
color. (B) Interaction between S1 domain of SARS-Urbani spike (black) and
WIV1 spike (blue) with human ACE2 (gray). Contact residues highlighted
tential for WIV1 spike, SARS-CoV Urbani spike within the
with consensus amino acids (red) and differences (circled) between SARS and mouse-adapted backbone yielded similar results (8). Therefore,

MICROBIOLOGY
WIV1 spike proteins; human ACE2 contact residues are also highlighted we examined the full-length WIV1-CoV versus the epidemic
(orange). (C ) Viral replication of WIV1-CoV (blue), WIV1-MA15 (blue SARS-CoV Urbani strain in vivo. Ten-week-old BALB/c mice
hatched), and SARS-CoV Urbani (black) following infection of Vero cells at a
multiplicity of infection (MOI) of 0.01. (D) Well-differentiated air–liquid in-
terface primary human airway epithelial cell cultures were infected with
SARS-CoV Urbani (black), SARS-CoV MA15 (black hatched), WIV1-MA15
(blue-white hatched), and WIV-CoV (blue) at (E) MOI of 0.01 in cells from the
same donor at an MOI of 0.01. Samples were collected at individual time
points with biological replicates (n = 3) for all experiments for both C and D.

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 electro-
poration 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 Fig. 2. Viruses using WIV1 spike attenuated relative to SARS spike in vivo.
cleaved spike varied between SARS spikes (Urbani, 1.21; MA15, (A and B) Ten-week-old BALB/c mice were infected with 104 pfu of either
1.44) and WIV1 (full length, 0.61; WIV1-MA15, 0.25) signaling SARS-CoV MA15 (black) or WIV1-MA15 (blue hatched) via the i.n. route and
possible variation in host proteolytic processing (Fig. S1D). examined over a 7-d time course. (A) Weight loss (n = 17 for WIV1-MA15, n =
Overall, the results indicate comparable viral replication, but 9 for SARS-CoV MA15) and (B) lung titer (n = 3 for MA15, n = 4 for WIV1-
possible biochemical differences in processing. 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,
Replication in Primary Human Epithelial Cells. Next, we wanted to
n = 6 for SARS-CoV Urbani) and (D) lung titer (n = 3 for WIV1-CoV, n = 3 for
determine WIV-CoV replication potential in models of the hu- SARS-CoV Urbani) were examined. For each bar graph, center value is rep-
man lung. Previous examination of WIV1-CoV recovered from resentative of group mean and error bars are defined by SEM. P values based
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bat samples demonstrated poor replication in A549 cells (5); on two-tailed Student’s t test of individual time points are marked as in-
however, replication of epidemic SARS-CoV is also poor in dicated: ***P < 0.001.

Menachery et al. PNAS | March 15, 2016 | vol. 113 | no. 11 | 3049
 were infected with 105 pfu of WIV1-CoV or SARS-CoV Urbani
and followed over a 4-d time course. As expected, neither in-
fection 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 in-
fection, contrasting antigen staining throughout the parenchyma
2-d post–SARS-CoV Urbani infection (Fig. S2 E and F). To-
gether, 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 ab-
sence 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 hu-
mans, we generated a mouse that expresses human ACE2 re-
ceptor under control of HFH4, a lung ciliated epithelial cell
Fig. 3. WIV1-Cov still attenuated despite human ACE2 expression in vivo.
promoter (11). However, whereas robust expression was ob- (A) Ten- to twenty-week-old HFH4 ACE2-expressing mice were infected with
served in the lung, other tissues including brain, liver, kidney, 105 pfu of SARS-CoV Urbani (black) or WIV1-CoV (blue) and examined over a
and gastrointestinal tract had varying levels of human ACE2 7-d time course for (A) survival and (B) day-2 lung titer (n = 3 for WIV1-CoV,
expression, indicating greater tissue distribution of HFH4- n = 3 for SARS-CoV Urbani). (C and D) Upon reaching thresholds for humane
mediated expression than initially expected (Fig. S3A). In addi- sacrifice (>20% weight loss) or 7 d postinfection (DPI), endpoint titers were
tion, examination of individual HFH4-ACE2–expressing progeny determined in the (C) lung and (D) brain following infection. P values based
revealed the occasional absence of the human ACE2 gene, on two-tailed Student’s t test of individual time points are marked as in-
dicated: *P < 0.05.
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
to determine if monoclonal antibody therapies could be used to
SARS-CoV Urbani and then followed for a 7-d time course to
lessen disease similar to ZMApp for Ebola (13). We first tested a
determine pathogenesis. The results indicated that WIV1-CoV
SARS-CoV monoclonal derived via phage display and antibody
infection was augmented, but remained attenuated relative to
escape (Fm6) (14) and found both wild-type SARS-CoV Urbani
SARS-CoV Urbani in the presence of human ACE2. Following
and WIV1-MA15 were strongly neutralized at low antibody
SARS-CoV Urbani challenge, HFH4-hACE2–expressing mice
concentrations (Fig. 4A). Similarly, a panel of monoclonal anti-
lost no weight, but then, experienced rapid weight loss and death
bodies derived from B cells from SARS-CoV–infected patients
between days 4 and 5 (Fig. 3A and Fig. S3C). In contrast, WIV1-
CoV produce minimal changes in weight loss until late times also prevented virus infection via WIV1-CoV spike (15, 16).
where animals fell into distinct categories either losing less than Both antibodies 230.15 and 227.14 robustly inhibited WIV1-
or more than 10% of their body weight. Whereas day-2 lung ti- MA15 replication with kinetics similar to or exceeding SARS-
ters were still attenuated relative to SARS-CoV Urbani, titers for CoV Urbani (Fig. 4 B and C). In contrast, antibody 109.8, which
WIV1-CoV were 100-fold higher in the presence of human ACE2 maps outside the receptor binding domain, produced only mar-
compared with wild-type BALB/c, with no similar augmentation ginal neutralization of WIV1-MA15 (Fig. 4D). Whereas the
observed with the epidemic SARS-CoV strain (Fig. 3B). Based on residue associated with prior escape mutants was conserved
pilot studies and previous studies with ACE2 transgenic animals at position 332, the adjacent residue had a significant change
(12), mice experiencing rapid weight loss were predicted to have (K332T) in WIV1-CoV, possibly contributing to reduced efficacy
lethal encephalitis and were humanely killed and harvested for of this antibody.
lung and brain titer if weight loss approached >20% of starting To further extend these findings, in vivo studies with antibody
body weight. All HFH4-ACE2 mice infected with SARS-CoV 227.14 were initiated in HFH4-ACE2–expressing mice. One day
Urbani lost >20% body weight and maintained robust replica- before infection, HFH4-ACE2–expressing mice were injected with
tion in the lung and brain following infection (Fig. 3 C and D). 200 μg of antibody 227.14 or PBS control as previously described
Similarly, mice with >10% weight loss following WIV1-CoV (17); mice were subsequently challenged with either SARS-CoV
infection produced robust viral replication in the brain, but sig- Urbani or WIV1-CoV and monitored for 7 d. The results indicate
nificantly lower titers in the lung. In contrast, mice that main- that antibody 227.14 protected mice from both lethal SARS-CoV
tained minimal weight loss (<10%) following WIV1-CoV Urbani and WIV-CoV challenge (Fig. 4E); in addition, lung titers
infection after 7 d had minimal titers in both the lung and brain, revealed no detectable virus in either SARS-CoV or WIV1-CoV–
suggesting a sufficient adaptive immune response was generated infected HFH4-ACE2–expressing mouse lungs following antibody
to clear virus and survive infection. Together, the data indicate treatment (Fig. 4F). Together, the in vitro and in vivo data in-
that WIV1-CoV maintains attenuation relative to SARS-CoV dicate that a mixture of broadly neutralizing antibodies against
Urbani despite the availability of human ACE2. In addition, SARS-CoV would likely provide significant protection if WIV1-
augmented replication suggests that WIV1-CoV may bind the CoV–like viruses successfully transmitted to humans.
human ACE2 receptor more efficiently that the mouse ACE2,
indicating potential inadequacies in the current mouse models of Vaccine Efficacy Limited Against WIV1 Spike. Previously, whole vi-
SARS pathogenesis. rion SARS-CoV inactivated by both formalin and UV irradiation
(double inactivated virus, DIV) was demonstrated as a po-
Therapeutics Against WIV1 Emergence. Having established a po- tential vaccination candidate based on robust neutralization
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tential threat based on replication in primary human cells and and protection following homologous SARS-CoV challenge in
preference for the human ACE2 receptor in vivo, we next sought young mice (18). However, both aged animal and heterologous

3050 | www.pnas.org/cgi/doi/10.1073/pnas.1517719113 Menachery et al.

 SEE COMMENTARY
Discussion
The recent outbreaks of Ebola, influenza, and MERS-CoV un-
derscore the threat posed by viruses emerging from zoonotic
sources. Coupled with air travel and uneven public health in-
frastructures, it is critical to develop approaches to mitigate these
and future outbreaks. In this paper, we outline a platform that
leverages metagenomics data, synthetic genome design, trans-
genic mouse models, and therapeutic human antibodies to
identify and treat potential prepandemic viruses. Focusing on
SARS-like CoVs, the approach indicates that viruses using the
WIV1-CoV spike protein are capable of infecting HAE cultures
directly without further spike adaptation. Whereas in vivo data
indicate attenuation relative to SARS-CoV, the augmented
replication in the presence of human ACE2 in vivo suggests that
the virus has significant pathogenic potential not captured by
current small animal models. Importantly, therapeutic treatment
with monoclonal antibodies suggests a Zmapp-based approach
would be effective against a WIV1-CoV spike-mediated out-
break. However, failure of SARS DIV vaccine to induce pro-
tection highlights the need for continued development of
additional therapeutics. Overall, the characterization of WIV1-
CoV and its pathogenic potential highlight the utility of this
platform in evaluating currently circulating zoonotic viruses.
Primary human airway epithelial cell cultures derived from
human donors and grown at an air–liquid interface represent the

MICROBIOLOGY
Fig. 4. SARS-CoV monoclonal antibodies have robust neutralization against
WIV1 spike-mediated infection. Neutralization efficacy was evaluated using
percent neutralization assays against SARS-CoV Urbani (black) or WIV1-MA15
(blue) with a panel of monoclonal antibodies: (A) fm6, (B) 230.15, (C) 227.15,
and (D) 109.8, all originally generated against epidemic SARS-CoV. Each data
point is representative of two or more independent neutralization wells. (E and
F) Twenty- to twenty-four-week-old HFH4 ACE2-expressing mice were injected
with 200 μg of anti-SARS human antibody 227.15 (hatched line) or mock (solid
line) 1 d before infection with 1 × 10^5 pfu of SARS-CoV Urbani (black) or WIV1-
CoV (blue) and examined over a 7-d time course for (E) survival (n = 3 for both
antibody-treated groups and mock PBS control WIV1-CoV, n = 2 for mock-
treated SARS-CoV Urbani), (F) day-2 lung titer (n = 3 for all groups). ND signifies
no titers detected. For each bar graph, center value is representative of group
mean and error bars are defined by SEM.

challenge studies revealed incomplete protection, increased im-

mune 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 Fig. 5. Double-inactivated whole SARS-CoV vaccine fails to protect aged
in DIV-vaccinated groups compared with control (Fig. 5B). In animals from chimeric WIV1-CoV infection. Twelve-month-old mice were
addition, plaque reduction neutralization titers from the serum vaccinated and boosted with DIV (dotted line) or PBS (solid line) and infected
of aged DIV-vaccinated mice indicated no neutralization of WIV1- 21 d postboost with 104 pfu of WIV1-MA15 via the i.n. route. (A) Weight loss
MA15, suggesting inadequate protection (Fig. 5C). Importantly, following WIV1-MA15 challenge and (B) viral replication in the lung 4 DPI.
(C) Neutralization of WIV1-MA15 (blue) with serum from aged, DIV-vacci-
examination of histopathology revealed increased eosinophilia
nated mice. (D–H) Histopathology lung sections stained for H&E from DIV-
in DIV-vaccinated mice compared with PBS controls, indicating and mock-vaccinated mice. (D) Eosinophil score (scale 0–4) following DIV or
the potential for immune induced pathology due to vaccination. mock vaccination 4 DPI. (E and F) Representative H&E lung sections for
Together, the data indicate that DIV vaccination would not pro- (E) mock- and (F) DIV-vaccinated mice infected with WIV-MA15. Red arrows
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vide significant protection and may cause adverse effects in the indicate individual eosinophil locations. P values based on two-tailed Stu-
context of WIV1-CoV spike-mediated outbreak. dent’s t test of individual time points are marked as indicated: **P < 0.01.

Menachery et al. PNAS | March 15, 2016 | vol. 113 | no. 11 | 3051
 closest model to the human lung. Therefore, the ability of both based on robust neutralization and protection in young mice
WIV1-CoV and WIV1-MA15 to grow equivalently to the epi- (18). However, studies with DIV in aged animals revealed in-
demic SARS-CoV in these cultures is a major concern for complete protection, significant immune pathology, and eo-
emergence. However, pathogenesis studies in mice suggest that sinophilia (19). Despite these prior results, the efficacy of
further adaptation may be required for epidemic disease. Com- monoclonal antibody treatments made further testing of DIV
pared with SARS equivalents, both full-length and chimeric seemingly worthwhile against WIV1-CoV spike-mediated in-
WIV1 viruses had significant attenuation even with the presence fection. However, the results remained the same, as vaccination
of human ACE2 in the mouse model. Together, the data suggest of aged mice resulted in no protection from WIV1-MA15
that despite using ACE2 and robust replication in primary replication in vivo (Fig. 5). Importantly, increased immune
human airway epithelial cultures, WIV1-CoV likely maintains pathology and observed eosinophilia indicate that broad-based
deficits that impact pathogenesis in mice; therefore, WIV1- vaccination efforts against SARS-CoV–like viruses must con-
mediated infection may have diminished epidemic potential in sider heterologous viruses as well as failure due to senescence in
humans relative to SARS-CoV. the aged host. A number of novel platforms including Venezuelan
A number of factors may contribute to reduced mouse path- Equine Encephalitis Virus Replicon Particle (VRP) and live-
ogenesis observed following WIV1-CoV spike-mediated in- attenuated vaccine approaches show great promise in these
fection. In the context of both the SARS-CoV and MERS-CoV areas, but require further testing and development before de-
outbreaks, focus had been primarily directed to spike binding ployment in an outbreak setting (22, 23).
as the key component of emergence and pandemic potential. Overall, the results from these studies highlight the utility of a
Supported by adaption at Y436H in mouse-adapted SARS spike platform that leverages metagenomics findings and reverse
(10), improved binding to host receptor cannot be discounted as genetics to identify prepandemic threats. For SARS-like WIV1-
a crucial component in emergence. This fact is supported by CoV, the data can inform surveillance programs, improve di-
improved replication of WIV1-CoV in mice expressing human agnostic reagents, and facilitate effective treatments to mitigate
ACE2 compared with control (Fig. 2D versus Fig. 3B). However, future emergence events. However, building new and chimeric
in vivo attenuation of WIV1-CoV relative to SARS-CoV Urbani reagents must be carefully weighed against potential gain-of-
despite efficient infection in primary human airway cultures function (GOF) concerns. Whereas not generally expected to
suggests that additional factors contribute to epidemic emer- increase pathogenicity, studies that build reagents based on
gence. One possibility is that adaptation outside of spike protein viruses from animal sources cannot exclude the possibility of
may lead to emergence via altered host–virus interactions. increased virulence or altered immunogenicity that promote
Whereas WIV1-MA15 was attenuated relative to SARS-MA15 escape from current countermeasures. As such, the potential of a
in vivo, overall titers in the lung were similar to the epidemic threat, real or perceived, may cause similar exploratory studies to
SARS-CoV Urbani in BALB/c mice (Figs. 3 and 4). These data be limited out of an “abundance of caution.” Importantly, the
suggest that CoV backbone changes may account for or com- government pause on GOF studies may have already impacted
pensate for deficits in WIV1-CoV replication compared with the scope and direction of these studies. Whereas previous
SARS-CoV Urbani in vivo. Another possible factor accounting for adaption of the epidemic SARS-CoV strain provided insights
attenuation is changes to spike that are independent of receptor into species-specific changes, bat-derived WIV1 adaptation
binding. Whereas the receptor binding domain had garnered the may identify elements critical for pathogenesis and transition
most interest, changes in the remaining portion of S1 as well as the from reservoir to human host; targets include viral proteins that
S2 portion of spike may also play a critical role in facilitating CoV interact with host machinery or host immunity like NSP1, en-
infection, transmission, and/or pathogenesis (20). Differences in velope, or ORF6 (22, 24, 25). Similarly, WIV1-CoV could be
these regions of spike may yield increased protease targeting, used to drive improved therapeutics, including escape mutants
enhanced spike cleavage, and/or expanded tropism leading to for improved monoclonal antibodies or more broadly neutral-
more robust infection for the epidemic SARS strains. Globally, izing vaccine approaches. However, it remains unclear from the
the in vivo results suggest that any of these areas may have con- current policies and GOF environment if these types of studies
tributed to SARS-CoV emergence. However, even these inter- will be permissible. Although limits and standards for these
pretations must be tempered due to the robust differences types of experiments must be established, erring on the side of
between mouse and human models; further studies in nonhuman caution is not without its own risks and balancing the benefits
primates are required to confirm these results and derive further of these types of studies must also be weighed against the
insight into CoV emergence from zoonotic sources.
potential hazards.
Despite the differences in the backbone genome sequences, Using a novel platform to translate metagenomics findings, the
therapeutics developed against SARS-CoV provide some mea-
WIV1-CoV cluster has been identified as a threat for future
sure of protection in the context of a future outbreak. Testing the
emergence in human populations due to robust replication in
four most broadly neutralizing SARS-CoV antibodies revealed
primary human airway epithelial cell cultures. However, based
effective control of WIV1-MA15 at relatively low concentrations
on in vivo mouse data, additional adaptations will likely be re-
of antibody (14, 15, 21). For two of the four antibodies tested
quired to produce epidemic disease. Notably, whereas current
(Fm6 and 230.15), WIV1-CoV spike-expressing virus was neu-
antibody-based therapies hold great promise in treating WIV1-
tralized equivalently or better than SARS-CoV Urbani. Simi-
CoV spike-mediated infection, failure of SARS-CoV vaccination
larly, only minimal differences at the low end of the neutrali-
approaches presents a major challenge for any efforts to protect
zation curve were noted for antibody 227.14. Whereas antibody
against future emergent viruses. Together, the data illustrate the
109.8 produced only marginal neutralization of WIV1-MA15,
utility of the platform and highlight the need to build and
the overall antibody neutralization data argue that multivalent
monoclonal antibody approaches could limit a WIV1-CoV maintain preparations for future emergence events.
spike-mediated outbreak. As such, a “ZMapp”-based approach Materials and Methods
could have great potential in stemming or preventing a future
Viruses, Cells, and Infection. Wild-type and chimeric CoVs were cultured on
SARS-CoV–like outbreak. Vero E6 cells, grown in DMEM (Gibco) and 5% fetal clone serum (HyClone)
In contrast to the success of monoclonal antibodies, vaccine along with anti/anti (Gibco). Growth curves in Vero and primary human
failure indicated further development and refinement are nec- airway epithelial cells were performed as previously described (26, 27). Hu-
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essary. The development of a DIV SARS-CoV vaccine was man lungs were procured under University of North Carolina at Chapel Hill
buoyed as a possible means to control SARS-CoV outbreaks (UNC) Institutional Review Board-approved protocols.

3052 | www.pnas.org/cgi/doi/10.1073/pnas.1517719113 Menachery et al.

 SEE COMMENTARY
Construction of Chimeric SARS-Like Viruses. Both wild-type and chimeric WIV- Virus Neutralization Assays. Plaque reduction neutralization titer assays were
CoV infectious clones were designed using published sequences and based preformed with previously characterized antibodies against SARS-CoV as
on the SARS-CoV infectious clone (10). Synthetic construction of chimeric previously described (14, 15, 21). Briefly, neutralizing antibodies or serum
mutant and full-length WIV1-CoV were approved by the UNC Institutional were serially diluted twofold and incubated with 100 pfu of the different virus
Biosafety Committee and the Dual Use Research of Concern Committee. 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
Ethics Statement. The study was carried out in accordance with the recom- 37 °C, cells were overlaid with 3 mL of 0.8% agarose in media. Plates were
mendations for care and use of animals by the Office of Laboratory Animal incubated for 2 d at 37 °C and then stained with neutral red for 3 h, and
Welfare (OLAW), National Institutes of Health. The Institutional Animal Care plaques were counted. The percentage of plaque reduction was calculated as
and Use Committee (IACUC) of University of North Carolina (UNC permit no. [1 − (no. of plaques with antibody/no. of plaques without antibody)] × 100.
A-3410-01) approved the animal study protocol (IACUC no. 13–033).
Statistical Analysis. All experiments were conducted contrasting two experi-
Mice and in Vivo Infection. Female 10-wk- and 12-mo-old Balb/cAnNHsD mental groups (either two viruses or vaccinated and unvaccinated cohorts).
mice ordered from the Harlan Labs were infected as previously described Therefore, significant differences in viral titer and histology scoring were de-
(23). For vaccination, young and aged mice were vaccinated and boosted termined by a two-tailed Student’s t test at individual time points. Data were
by footpad injection with a 20-μL volume of either 0.2 μg of double- normally distributed in each group being compared and had similar variance.
inactivated SARS-CoV vaccine (DIV) with alum or mock PBS as previously
described (19). Biosafety and Biosecurity. Reported studies were initiated after the University
of North Carolina Institutional Biosafety Committee approved the experi-
Generation and Infection of ACE2 Tissue-Specific Transgenic Mice. Transgenic mental protocol: project title: Generating infectious clones of Bat SARS-like
mice with airway-targeted overexpression of human ACE2 were generated CoVs; lab safety plan ID: 20145741; schedule G ID: 12279. These studies were
by microinjection of fertilized C3H × C57BL/6 (C3B6) F1 hybrid oocytes with initiated before the US Government Deliberative Process Research Funding
an expression cassette consisting of the HFH4/FOXJ1 lung ciliated epithelial Pause on Selected Gain of Function Research Involving Influenza, MERS, and
cell-specific promoter elements and the coding region of ACE2 cDNA in a SARS Viruses (www.phe.gov/s3/dualuse/Documents/gain-of-function.pdf),
pTG1 vector (11) (UNC Animal Model Core). Founder mice were crossed to and the current paper has been reviewed by the funding agency, the Na-
C3B6, producing human ACE2-transgenic mice that were each previously tional Institutes of Health (NIH). Continuation of these studies has been
tested for transgene expression as described in SI Materials and Methods. requested and approved by the NIH.
ACE2-transgenic mice given i.p. injection of 200 μg of human Ab S227.14 or
PBS control (0.20 mL total volume, five to six mice per group) 1 d before ACKNOWLEDGMENTS. We thank Dr. Zhengli-Li Shi of the Wuhan Institute
infection as previously described (17). 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
Histological Analysis. Lung tissues for histological analysis were fixed in 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.),
buffered formalin phosphate 10% (4–5% wt/wt formaldehyde) (Fisher
and F32AI102561 and K99AG049092 (to V.D.M.). Human airway epithelial
#SF100-20) for at least 7 d, tissues were embedded in paraffin, and 5-μm cell cultures were supported by the National Institute of Diabetes and Di-

MICROBIOLOGY
sections were prepared by the UNC histopathology core facility as previous gestive and Kidney Disease under Award NIH DK065988 (to S.H.R.). Support
described (23). Images were captured using an Olympus BX41 microscope for the generation of the mice expressing human ACE2 was provided by NIH
with an Olympus DP71 camera. Grants AI076159 and AI079521 (to A.C.S.).

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