Orally efficacious broad-spectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses

Article abstract of DOI:10.1128/AAC.00766-18

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N⁴-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5=-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a Guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.

Full text of DOI:10.1128/AAC.00766-18

AAC Accepted Manuscript Posted Online 11 June 2018
Antimicrob. Agents Chemother. doi:10.1128/AAC.00766-18
Copyright © 2018 American Society for Microbiology. All Rights Reserved.
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Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and
Respiratory Syncytial Viruses
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Jeong-Joong Yoon1#, Mart Toots1#, Sujin Lee2,3, Myung-Eun Lee1, Barbara Ludeke4, Jasmina M
Luczo5, Ketaki Ganti6, Robert M Cox1, Zachary M. Sticher7, Vindya Edpuganti7, Deborah. G. Mitchell7,
Mark A. Lockwood7, Alexander A Kolykhalov7, Alexander L Greninger8, Martin L Moore2,3, George R
Painter7, Anice C Lowen6, Stephen M Tompkins5, Rachel Fearns4, Michael G Natchus7,
and Richard K Plemper1,*
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1Institute for Biomedical Sciences, Georgia State University, Atlanta, GA
2Department of Pediatrics, Emory University School of Medicine, Atlanta, GA
3Children’s Healthcare of Atlanta, Atlanta, GA
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4Department of Microbiology, Boston University School of Medicine, Boston, MA
5Center for Vaccines and Immunology, University of Georgia, Athens, GA
6Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA
7Emory Institute for Drug Development, Emory University, Atlanta, GA
8Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA
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Running Title: Inhibitor of influenza-like diseases
#These authors contributed equally to the study
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*Address correspondence to Richard K. Plemper, rplemper@gsu.edu
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Abstract
Morbidity and mortality resulting from influenza-like disease are a threat especially for older adults.
To improve case management, next-generation broad-spectrum antiviral therapeutics are urgently
needed that are efficacious against major drivers of influenza-like disease including influenza viruses
and respiratory syncytial virus (RSV). Using a dual-pathogen high throughput screening protocol for
influenza A virus (IAV) and RSV inhibitors, we have identified N4-hydroxycytidine (NHC) as a potent
inhibitor of RSV, influenza B viruses and IAVs of human, avian, and swine origin. Biochemical in vitro
polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated
into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral
passaging in cell culture in the presence of inhibitor did not induce robust resistance. Pharmacokinetic
profiling demonstrated dose-dependent oral bioavailability of 36-56%, sustained levels of the active 5’-
triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after
extended dosing at 800 mg/kg/day. The compound was orally efficacious against RSV and both
seasonal and highly pathogenic avian IAV in mouse models, reducing lung virus loads and alleviating
disease biomarkers. Oral dosing reduced IAV burden in a guinea pig transmission model and
suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-
spectrum efficacy and pharmacokinetic properties, NHC a promising candidate for future clinical
development as a treatment option for influenza-like diseases.
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Introduction
The clinical burden of respiratory viruses associated with influenza-like diseases is highest for the
elderly, the immunocompromised, and the very young. Patients above 65 years of age, for instance,
are most heavily affected by seasonal influenza, followed by infants (1). Although an inverse patient
age group distribution is seen for respiratory syncytial virus (RSV), a member of the Pneumoviridae
family, the substantial threat caused by severe RSV disease to the elderly is increasingly appreciated
(2) and case fatalities associated with both influenza virus and RSV infections disproportionally affect
older adults (3). Amplifying the need for next-generation antiviral therapeutics for improved
management of respiratory virus infections, the effectiveness of the current tri- or quadrivalent influenza
vaccine is limited to approximately 60% in adults and only 40% in the elderly even under the best
circumstances (4). However, substantially lower vaccine efficacy is seen for instance in the particularly
severe 2017/2018 influenza season (5). No active vaccination is currently available to protect against
RSV infection. Passive monoclonal antibody immunoprophylaxis can be administered to high-risk
patients, but an estimated $300,000 cost to prevent a single RSV hospitalization (6-11) is prohibitive to
broad application.
Since seasonal influenza and RSV outbreaks overlap in temperate regions, clinical symptoms are
often non-specific, and laboratory typing is often not routine outside of clinical centers and/or hampered
by cost constrains (1), an umbrella diagnosis of influenza-like disease with unclear nature of the
etiologic agent remains common. This ambiguous diagnosis compromises the efficacy of antiviral
agents with anticipated narrow therapeutic windows (12-14). We therefore propose that next-generation
therapeutics with a broad antiviral indication spectrum including, as a minimum, influenza viruses and
RSV will be required to improve the management of influenza-like disease.
Generating truly broad-spectrum inhibitors has been a long-coveted goal of antiviral drug
development. Discovery efforts have concentrated mainly on two areas: i) host-targeted antivirals that
are immunomodulatory or interfere with cellular factors required for successful virus replication; and ii)
direct-acting inhibitors targeting a druggable site or activity conserved across different viral families.
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Therapeutic targeting of host factors recruited for virus replication has attracted renewed interest in
the past decade due to the combined promise of expanding the antiviral indication range and reducing
the frequency of viral escape from inhibition (15-18). Although host-directed candidates have largely
met these expectations in experimental settings (15, 16), the approach has yet to deliver approved
therapeutics with safety profiles acceptable for human use against viral diseases such as seasonal
influenza and RSV disease (19). Direct-targeted antivirals typically display more promising initial toxicity
profiles, but the quest for broad-spectrum inhibitory activity has fueled the rediscovery of many
promiscuous, often covalently reactive scaffolds that are associated with unclear mechanisms of
activity (20-23). Based on their demonstrated history of ultimate failure in development, many of these
scaffolds are considered undesirable and were classified as frequently hitting pan-assay interfering
(PAIN) substances (21, 24). As a notable exception, different ribonucleoside analogs have been
identified that combine good clinical promise with a broadened indication spectrum, often showing
remarkable preference for a range of viral over host cell polymerases.
While no nucleoside analog inhibitor is currently in clinical use against influenza viruses in the
United States, the allosteric endonuclease blocker baloxavir marboxil (xofluza) was recently approved
for sale in Japan. Furthermore, ribavirin is licensed for the treatment of RSV infection and T-705
(favipiravir) is conditionally approved for stockpiling in Japan in a situation when a pandemic is caused
by oseltamivir-resistant influenza virus. However, toxicity liabilities and limited efficacy undermine the
clinical use of ribavirin (25, 26) and the potential for teratogenicity (27) may compromise the use of T-
705 for the treatment of influenza. The ribonucleoside analog ALS-8176 was found to be efficacious in
a human RSV challenge study (28), providing important proof-of-concept for the treatment of influenza-
like diseases with competitive polymerase inhibitors, but ALS-8176 did not inhibit influenza virus (29).
Towards the ultimate goal of identifying developable broad-spectrum medications against
influenza-like disease, we have established and validated a replication-competent dual RSV and
influenza A virus (IAV) reporter virus-based high throughput screening (HTS) assay that allows the
simultaneous identification of IAV-specific, RSV-specific, and dually active, potentially broad-spectrum,
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hit candidates (30). The first implementation of this protocol against a large open discovery library of
small-molecule compounds has yielded promising target-specific inhibitors of RSV (31) and influenza
virus (32), but broad-spectrum hits remained limited to undesirable scaffolds and clinically
undevelopable compounds interfering with pyrimidine de novo synthesis (32-34). In this study, we
applied the assay to a collection of ribonucleoside analogs. Having identified a cytidine analog with
potent activity against both target viruses, we initiated mechanistic characterization of RSV and IAV
RNA-dependent RNA polymerase (RdRp) inhibition, evaluated potency against a panel of laboratory
adapted and clinical strains representing RSV, IAVs and influenza B viruses (IBVs) in established cell
lines and primary human bronchial tracheal epithelial cells (HBTECs), determined oral pharmacokinetic
profiles in the murine respiratory tract, assessed potency against RSV and both seasonal and highly
pathogenic IAV subtypes in mouse models, and examined the effect of treatment on influenza virus
spread in the guinea pig IAV transmission model. Collectively, these assays identify the compound as
an orally efficacious broad-spectrum inhibitor of influenza-like disease caused by RSV or influenza virus
infections.
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Results
Having assembled an in-house library of 102 ribonucleoside analog candidates, we sampled the
set in three replicates using our validated dual-pathogen HTS protocol (30). This campaign yielded
three hit candidates that consistently blocked both viral targets in the primary assay, and in addition two
RSV-specific and one influenza virus-specific candidate (Fig. 1A).
Indication spectrum of a dually-active HTS hit candidate
Focusing on the dual-active compounds only, two candidates were excluded based on excessive
toxicity in dose-response potency and toxicity counterscreens (Fig. S1), while the remaining candidate
(dual #1, EIDD-1931 or N4-hydroxycytidine (NHC) Fig. 1B) showed active concentrations in the
nanomolar to low-micromolar range and selectivity indices (SI = CC50/EC50) of 89 against a broad
panel of RSV, IAV, and IBV laboratory strains and isolates (Table 1). This group included clinical RSV
isolates cultured from nasal washes (Fig. 1C), IAVs of human, avian, and swine origin representing
both group 1 and 2 hemagglutinins (HAs), Fig. 1D and Table 1), highly pathogenic H5N1 and emerging
H7N9 avian IAVs (AIVs) (Fig. 1E), and IBVs representing both circulating lineages (35), Victoria and
Yamagata (Fig. 1F). A subset of avian influenza viruses was tested in ovo (Table 1), demonstrating
NHC potency in primary cells. Consistent with high initial SI values in cultured cells, embryo
development in the treated chicken eggs was visually unaffected by the compound (Fig. S2). Whereas
T-705 was inactive in primary HBTECs, antiviral activity of NHC against both RSV and influenza virus
was unchanged in these disease-relevant human primary cells compared to immortalized cell lines (Fig.
1G). Drug combination testing of NHC and the current standard of care (SOC) against IAV infection,
oseltamivir, in cultured cells identified an extended plateau area of medium-level antiviral synergy (HAS
model), while no significant increase in cytotoxicity was noted in the presence of drug combination (Fig.
S3). These data demonstrate activity of NHC against a panel of respiratory viruses associated with
influenza-like diseases. Combined with previously reported antiviral activity of NHC in cell culture
against some Flaviviridae, Coronaviridae, and Togaviridae family members (36-39), they establish
broad-spectrum activity of the compound against different positive- and negative-strand RNA virus
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families and, in the case of IAV infection, spotlight potential for synergistic combination with the current
SOC.
Mechanistic assessment of NHC
Based on reversal of NHC-mediated inhibition of a Chikungunya virus replicon through an excess
of exogenous cytidine and uridine, the compound was thought to behave as a pyrimidine analog in
cellula (37). We first confirmed through time-of-addition variation studies that NHC blocks a post-entry
step in influenza virus and RSV replication cycles (Fig. 2A). Plasmid-based minigenome reporter
assays validated in both cases NHC interference with viral RdRp activity (Fig. 2B). However, an in vitro
activity assay using host cell polymerase  did not reveal inhibition of the cellular polymerase by the
active 5’-triphosphate form of the compound, NHC-TP (Fig. 2C). As previously observed for positive-
strand RNA-virus replicons, inhibition of RSV and influenza virus by NHC can be reversed through
addition of exogenous pyrimidines but not purines (Fig. 2D). To determine whether NHC-TP is
accepted by negative-strand RNA virus RdRps as substitute for CTP or acts as a chain-terminator, we
employed a biochemical assay of RSV polymerase activity that uses purified viral phosphoprotein (P)
and large protein (L) RSV polymerase components, NTP mix, and a synthetic oligonucleotide template,
resulting in a 23-mer product (40). Without CTP in the NTP mix, polymerization is stalled at the first
guanidine residue in the template, releasing 14-mer amplicons. Addition of NHC-TP partially restored
the generation of full-length 23-mer amplicons, demonstrating that the RSV RdRp complex accepts
NHC in place of cytidine and that the compound does not act as an obligatory chain-terminator (Fig.
2E). Rather, we noted a significantly increased frequency of C-to-U, G-to-A, and, in the case of RSV
only, A-to-G transition mutations in viral RNA after single-cycle infection of cells in the presence of 10
μM NHC, followed by subcloning of individual viral RNA-derived cDNA amplicons and Sanger
sequencing (Fig. 2 F). Relative mutation frequencies were increased approximately 7-10-fold (Table 2),
but no specific mutation hot-spots were detected in the fragment analyzed. This observation is
consistent with a postulated ability of NHC to base-pair as either cytosine or uracil (41), which may
drive the replicating viruses into error catastrophe (36, 42).
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Previous attempts to induce robust resistance of two positive strand RNA viruses, Venezuelan
equine encephalitis virus and bovine viral diarrhea virus, from inhibition by NHC were unsuccessful (36,
43). To resistance profile negative-strand RNA virus-derived RdRps, we incubated RSV-A2 and IAV-
WSN in the presence of gradually increasing NHC concentrations for 10 consecutive passages.
However, no robust resistance (> 5-fold increase in EC50 concentrations) emerged, indicating a
universally high barrier against viral escape from NHC, independent of the target virus examined.
Pharmacokinetics
In preparation for in vivo testing, we determined anabolic, pharmacokinetic (PK), and
pharmacodynamic (PD) profiles and lung tissue distributions of the compound in primary HBTECs and
mice. LC/MS/MS analysis after exposure of HBTECs to 20 μM NHC demonstrated effective conversion
to active NHC-TP, represented by an approximately 4 nanomoles/106 cells concentration plateau over
the 24-hour time period examined. By contrast, steady-state levels of free prodrug and NHC-
5’monophosphate (NHC-MP) remained flat at a low ~2 pmoles/106 cells in this period (Fig. 3A).
Subsequent wash-out revealed high metabolic stability of NHC-TP in the HBTECs, resulting in a
calculated half-life exceeding 4 hours (Fig. 3B).
For species consistency with our mouse efficacy models of RSV and IAV infection, we determined
plasma PK of NHC and lung levels of both NHC and the active antiviral agent NHC-TP in mice.
Intraperitoneal (I.P.) and oral (p.o.) administration different dose levels ranging from 10 to 50 mg/kg
(I.P.) and 50 to 500 mg/kg (p.o.) resulted in dose-dependent increases in overall exposure (AUC) to the
prodrug (Fig. 3C) and peak plasma concentrations (Cmax) after oral dosing exceeding 40 μM (Fig. 3D).
Exposure levels corresponded to a dose-dependent oral bioavailability of 36 to 56% (Table 3). Dose-
dependency of overall prodrug exposure extended to respiratory tissue (Fig. 3E), but peak NHC-TP
concentrations in lung saturated above oral dose levels of approximately 150 mg/kg (Fig. 3F). While
this Cmax plateau suggests an anabolism bottleneck of NHC in mouse respiratory tissue at higher
prodrug levels, lung tissue distribution assessment revealed sustained concentrations of active NHC-
TP of approximately 10 nanomoles/g lung tissue for over 8 hours after administration at the 150 and
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500 mg/kg levels. These results highlight the strong potential of NHC for clinical use against influenza-
like diseases.
In vivo efficacy testing against major causes of influenza-like diseases
Based on the PK/PD profiles, we selected as starting point for all experiments oral doses of 100
mg/kg (below NHC-TP lung tissue plateau level) and 400 mg/kg (comfortably within NHC-TP tissue
saturation range), and a twice daily (b.i.d.) dosing regimen for in vivo efficacy testing against RSV and
IAV in mice. For testing against RSV, BALB/cJ mice were infected intranasally (i.n.) with 105 pfu each
of recombinant RSV A2-L19F, which is based on the A2 strain but contains an F protein derived from
the RSV isolate line 19 that increases pathogenicity in the mouse model and better reproduces key
features of human RSV disease such as high lung virus load, extensive mucus production, and
pronounced respiratory distress (44). Treatment was initiated two hours prior to infection and continued
until lung virus titers were determined five days post-infection. At both dose levels, RSV loads were
significantly reduced by more than one order of magnitude compared to vehicle-treated animals (Fig.
4A). However, we did not detect an appreciable difference in progeny titers between the dose groups,
presumably reflecting that even at the lower dose used NHC-TP concentration in lung tissue
approaches saturation levels. Lung histopathology and PAS staining demonstrated complete
suppression of excessive mucin production in the 400 mg/kg group and a partial reduction in animals
dosed with 100 mg/kg (Fig. S4). Exploratory dosing at 30 and 50 mg/kg failed to significantly lower lung
virus load (Fig. S5), but oral doses as low as 30 mg/kg were sufficient to completely ameliorate the
severe respiratory distress experienced by vehicle-treated animals (Fig. 4B). These results
demonstrate effective inhibition of RSV replication by NHC in vivo at higher dose concentrations. The
data furthermore suggest that even minimal pharmaceutical interference with RSV replication can
translate into major changes in RSV disease markers in the mouse model.
To test NHC efficacy against seasonal influenza viruses, we infected BALB/cJ mice i.n. with 103
pfu of mouse-adapted A/Puerto Rico/8/34 (H1N1) (PR8) and assessed lung viral load six days post-
infection as the primary efficacy milestone. In addition, virus-induced hypothermia and selected pro-
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inflammatory cytokines were monitored. Based on the experience with anti-RSV efficacy, we focused
on the 100 and 400 mg/kg dose levels only for these experiments. After prophylactic dosing as before,
we again observed significant reductions in lung virus load in NHC versus vehicle-treated animals (Fig.
4C). All infected animals experienced virus-induced hypothermia, but symptoms were significantly
alleviated in mice treated with high-dose NHC (Fig. S6). To put the antiviral impact of NHC in
perspective to SOC, we orally administered NHC (400 mg/kg dose level only in this experiment) or
oseltamivir to PR8 infected mice and determined lung virus load profiles for each treatment group (Fig.
4D). Consistent with previous observations (45), oseltamivir delayed viral replication in the first 2 days
after infection, but peak lung titers in the oseltamivir group were not significantly different from those in
vehicle-treated animals. In contrast, treatment with NHC caused a significant and sustained reduction in
virus load. RT-qPCR-based quantitation of virus-triggered induction of selected pro-inflammatory
cytokines, IFN-γ and IL-6, revealed a statistically significant decrease in mean induction levels in the
400 mg/kg group (Fig. 4E).
Since PR8 reaches plateau lung titers within 24 hours after infection (Fig. 4D), the time window
provided by the mouse model to assess post-exposure efficacy of NHC is narrow. To gain first insight
into the effect of delayed dosing onset, we initiated treatment at six hours post-infection, again
administering the compounds at the 400 mg/kg dose level only and continuing with a b.i.d. regimen.
Lung virus loads were determined three and six days post-infection, and at both time points showed a
significant reduction compared to vehicle-treated control animals (Fig. 4F). In keeping with the results
obtained after prophylactic dosing, virus-induced hypothermia was significantly reduced in the post-
exposure NHC-treated animals also (Fig. S6), establishing comparable benefit of NHC on management
of influenza virus infection independent of whether treatment followed a prophylactic or post-exposure
regimen.
Efficacy against highly-pathogenic avian influenza virus and effect on IAV transmission
Having obtained proof-of-concept for oral NHC efficacy against PR8 in mice, we employed an
HPAIV/mouse model to examine the potential of the compound to strengthen preparedness against
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highly pathogenic IAVs that constitute a high pandemic threat. Mice were infected i.n. with 6 pfu of
A/Vietnam/1203/2004 (H5N1) (A/Vietnam), treatment initiated prophylactically at the 400 mg/kg dose
level only, and continued as before. Due to pronounced neuropathogenicity of A/Vietnam in the model
(46), both lung and CNS viral loads were determined at day six post-infection in comparison with
vehicle and SOC-treated animals (Fig. 4G). Lung virus titers were again significantly reduced in drug-
treated animals as compared to the vehicle-only group, and virus was undetectable in the CNS in four
out of five animals that had received NHC. This inhibitory effect on A/Vietnam lung virus load was
equivalent to that observed in oseltamivir-treated animals, but NHC more efficiently prevented HPAIV
dissemination to the CNS since three of the five animals in the oseltamivir treatment group presented
with detectable virus in brain tissue.
Since influenza virus does not transmit efficiently in mice (47), we employed the well-established
guinea pig IAV transmission model (48) to evaluate the effect of NHC on virus spread. Similar to
uncomplicated human influenza, virus replication in guinea pigs is limited predominantly to the upper
respiratory tract. Source animals were infected i.n. with 104 pfu of A/Netherlands/602/2009 pH1N1
(NL/09) virus and co-housed with uninfected contact guinea pigs starting 24 hours post-infection.
Treatment of the source group was initiated prophylactically at the 100 and 400 mg/kg dose levels and
continued b.i.d. as before. Source animals showed a significant, dose-dependent reduction in shedding
titers determined from nasal lavages that exceeded two orders of magnitude (Fig. 4H). Vehicle-treated
animals furthermore efficiently passed the virus to contact guinea pigs. Transmitted virus was first
detected at day four post-infection and titers peaked at approximately 106 pfu/ml of nasal wash on day
six after initiation of the study. By contrast, low-dose NHC treatment of source guinea pigs delayed
transmission by approximately one day, and titers remained below 104 pfu/ml of nasal wash throughout
the study (Fig. 4I). We noted an even more pronounced inhibitory effect on virus spread when source
animals were treated at the 400 mg/kg dose level. Weak transmission was detected in this group only
at day eight, and remained limited to two of the four contacts. The other contact animals remained
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virus-free for the duration of the study. These studies demonstrate oral efficacy of NHC against major
pathogens associated with influenza-like illnesses in different animal models.
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Discussion
Influenza-like illnesses show disproportionally high case fatalities in older adults (1-3). To
effectively address this problem, a next-generation therapeutic must be developed for this patient
population frequently suffering from seasonal influenza virus or RSV infection and presenting with
influenza-like symptoms. In addition to a reduction in viral load that is sufficient to prevent disease
progression to severe small airway infection and alleviate acute respiratory distress, a drug candidate
suitable for this patient group should best be orally available to ensure reasonable patient compliance.
Through a dual-pathogen (31, 32) HTS campaign that affords the simultaneous identification of
RSV and IAV inhibitors, we identified NHC, a pyrimidine ribonucleoside analog, as a hit candidate that
integrates promising potency with a broadened indication spectrum. NHC was previously associated
with antiviral activity against positive-strand RNA viruses (36-39), but PK/PD profiles have not been
determined and in vivo efficacy is untested.
The broad overall indication spectrum of NHC is reminiscent of that described for T-705 (favipiravir)
and ribavirin, two compounds that act after phosphoribosylation (T-705) and phosphorylation as purine
analogs, respectively, and are believed to interfere with RNA virus replication through pairing with either
cytidine or uridine (49, 50), resulting in high mutation frequencies and ultimately error catastrophe (42).
While T-705 is conditionally approved in Japan and considered for licensing in the United States, we
found the drug to be compromised by poor antiviral activity in primary human respiratory cells. In
contrast, NHC returned a consistent efficacy profile in immortalized cell lines and in disease-relevant
primary HBTECs, consistent with efficient conversion to active NHC-TP and high metabolic stability of
the 5’-triphosphate also in primary human airway cells.
Early studies suggested that NHC-TP can substitute for uridine or cytidine in RNA polymerase
reactions (51, 52). Three lines of experimental evidence support our hypothesis that the anti-influenza
virus and anti-RSV activity of NHC is predominantly the result of viral error catastrophe: i) an excess of
exogenous cytidine or uridine but not purines reverses compound-mediated inhibition, indicating that
NHC is recognized as a pyrimidine analog by the influenza virus and RSV RdRp complexes; ii) NHC-
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TP can functionally substitute for CTP in a biochemical RSV polymerase assay using purified RdRp
complexes; and iii) growth of IAV-WSN and RSV-A2-L19F in the presence of sub-sterilizing NHC
concentrations resulted in an increased frequency of C-to-U, G-to-A, and A-to-G transition mutations.
The cell-based competition assays indicate that substrate-recognition of NHC-TP by RSV and IAV
polymerase is comparable to that of CTP, since at least equimolar concentrations of exogenous
cytidine were required to reverse NHC-mediated inhibition despite the micromolar levels of endogenous
ribonucleosides naturally present in the cells (53). Previous mutagenesis studies with both positive and
negative sense RNA viruses have shown that viral tolerance for an increase in mutation frequency is
limited (50, 54-56). For instance, an average of three random mutations per viral genome is sufficient
for an 80%-reduction in poliovirus specific infectivity (42). In our biochemical assays, however, NHC-TP
only partially restored RSV polymerase activity in the absence of CTP. This finding suggests that
incorporation of the compound may also reduce polymerase processivity and/or increase the likelihood
of chain termination. Of note, T-705 was also suggested to directly block influenza virus RdRp (57), and
a number of alternative antiviral effects were suggested for ribavirin including lowering of cellular GTP
levels, immunomodulation, blockage of RNA capping, and direct viral polymerase inhibition (58).
Conceivably, antiviral activity of NHC may arise from a combination of lethal mutagenesis and
kinetically impaired or abortive polymerization.
Previous work predicted a mutagenic effect of NHC based on restored growth of cytidine auxotroph
S. typhimurium JL1045 after exposure to the compound (59). However, this conclusion is flawed, since
NHC can be converted directly to cytidine by the mitochondrial amidoxime-reducing component
(mARC) (60), thus the drug itself and not compound-induced reversion mutants serves as pyrimidine
source for JL 1045. Consistent with this view, DNA repair mechanisms are not activated by NHC (61).
The anticipated treatment time of influenza and RSV disease is furthermore short – the recommended
course of oseltamivir, for instance, is 5-7 days (14) – and prolonged exposure to NHC was well
tolerated by both mice and guinea pigs.
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Consistent across both positive- and negative-strand viral targets pursued, viral escape from
inhibition by NHC remains inefficient and extended passaging under conditions that we have
successfully optimized in previous studies for induction of resistance to allosteric antivirals (32, 62, 63)
failed to yield appreciably higher tolerance to NHC. A recent study aimed at inducing escape of
Venezuelan equine encephalitis virus (VEEV), a Togaviridae family member, from NHC inhibition found
that a combination of three distinct mutations in VEEV RdRp was required for partial escape (36).
However, subsequent viral passaging in the absence of compound resulted in rapid loss of resistance.
The appearance of preexisting resistance to available antivirals in circulating influenza viruses is a
major concern, having compromised the M2 ion channel blocker class (64) and affecting, increasingly,
the neuraminidase inhibitors (65-69). Although we have not yet determined in vivo resistance profiles of
the compound, the available data in aggregate suggest that a fitness penalty may prevent the rapid
accumulation of preexisting anti-NHC resistance mutations.
Based on PK profiles in mice indicating sustained high lung tissue concentrations of the antivirally
active NHC-TP anabolite, we chose the mouse models of RSV and IAV infection for small-animal
efficacy testing. While supporting productive infections, tissue tropism differs from human disease in
both models since virus replication occurs predominately in the small airways rather than the upper
respiratory tract (70-73). In the case of the IAV model, mice furthermore develop hypothermia rather
than fever (74) and do not cough or sneeze (47). In keeping with our overall therapeutic premise that
reducing progeny viral load will be paramount in preventing viral spread to the small airways and
severe lower respiratory tract infection in humans, we consider lung viral burden to represent the most
relevant readout to assess treatment efficacy in these models, although additional disease biomarkers
were monitored in parallel. The observed significant lung virus load reductions were consistent with
efficient NHC uptake and anabolism in primary cells, the antiviral activity of the compound in native cell
cultures and in ovo, and the sustained lung tissue concentrations of NHC-TP in mice. Virus titer
reductions were equivalent to, or exceeded those, reported for SOCs ribavirin (75) and oseltamivir (45)
in the mouse model.
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Clinical studies and animal models have implicated a number of pro-inflammatory mediators in
playing a significant role in coordinating the innate immune response to influenza virus infection (76-
78). IL-6 in particular was identified as a promising biomarker for disease severity in the PR8 BALB/cJ
model based on correlation of IL-6 levels with viral titers (45, 79). Consistent with viral load reductions
in NHC treated animals, we found that relative IL-6 expression levels were significantly reduced in the
high-dose NHC treatment group, underscoring therapeutic benefit of the compound against influenza.
No robust small-animal RSV transmission model is available, but guinea pigs support efficient IAV
transmission. Although lacking overt signs of disease, guinea pigs are highly susceptible to infection by
human influenza viruses. Virus replication to high titers in the guinea pig upper respiratory tract,
resembling a hallmark of uncomplicated influenza in humans, furthermore generates a basis for
efficient direct and aerosol transmission (48). In addition to substantially lower viral loads in treated
source animals that corroborated the results of the mouse efficacy studies, reduced IAV transmission
success under NHC treatment generates high promise that human therapy with NHC may accelerate
silencing of virus outbreaks in addition to improving management of influenza-like disease.
NHC emerges from this first in vivo efficacy assessment study as an orally active ribonucleoside
analog with potent activity against influenza viruses and RSV. The compound was highly bioavailable,
efficiently converted to the active NHC-TP form in disease-relevant respiratory tissues, well tolerated,
and did not induce rapid viral escape from inhibition. NHC was effective against seasonal and highly
pathogenic avian IAV strains, IBV strains, and RSV isolates in cell culture. Treatment alleviated clinical
markers of RSV and influenza virus disease in the mouse model and effectively reduced influenza virus
host-to-host spread in a guinea pig transmission model. We consider NHC or a prodrug analog thereof
to be worthwhile of further consideration as a promising candidate for the treatment of influenza-like
diseases.
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Materials and Methods
Cell lines and transfections
Human embryotic kidney cells (293T; ATCC CRL-3216), Madin-Darby canine kidney cells (MDCK;
ATCC CCL-34), HEp-2 cells (ATCC CCL-23) and baby hamster kidney cells (BHK-21; ATCC CCL-10)
stably expressing T7 polymerase (BSR T7/5) were maintained at 37°C and 5% CO2 in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 7.5% fetal bovine serum (FBS). HEp-2 cells are
listed in ICLAC database Version 8.0 of commonly misidentified cell lines, but their use is justified as
these cells are accepted and widely used for studies involving respiratory syncytial virus (RSV).
GeneJuice transfection reagent (Invitrogen) was used for all transfection reactions. Normal primary
Human Bronchial Tracheal Epithelial Cells (HBTECs purchased from LifeLine Cell Technology (Cat.
No. LM-0050), passage number 1-3) were grown in BronchiaLife Cell Culture Medium (LifeLine Cell
Technology). Immortalized cell lines used in this study are routinely checked for microbial
contamination (in approximately 6-month interval). HBTECs were tested for microbial contamination on
July 25, 2017 by LifeLine Cell Technology. These cells were grown for 2 weeks for this study after that
the testing.
Viruses
Influenza viruses A/WSN/33(WSN) (H1N1), WSN-nanoLuc, A/California/7/2009 (H1N1),
A/Georgia/M5081/2012 (H1N1), A/Netherlands/602/2009 (H1N1), A/Panama/2007/99 (H3N2),
A/Wisconsin/67/2005 (H3N2), influenza A/Aichi/2/68 (H3N2), A/swine/Ohio/sw10-132/2010 (H3N2),
B/Yamagata/16/88, and B/Brisbane/60/08 were propagated in MDCK cells for 2 days at 37°C. Influenza
viruses A/duck/Alberta/35/76 (H1N1), A/swine/Spain/53207/2004, and A/Chicken/Potsdam/178-4/83
(H2N2) were propagated in 10-day-old embryonated chicken eggs for 2 days at 37°C. Influenza viruses
A/Vietnam/1203/2004 (H5N1) and A/Anhui/1/2013 (H7N9) were propagated in 9-day-old embryonated
chicken eggs at 37°C for 24 hours. All experiments using live, highly pathogenic H7N9 avian influenza
viruses were reviewed and approved by the institutional biosafety program at the University of Georgia,
were conducted in biosafety level 3 enhanced containment, and followed guidelines for use of Select
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Agents approved by the CDC. Viruses were titrated by standard plaque assays, hemagglutination
assays (80), TCID50 assays, or TCID50-hemagglutination (TCID50-HA) assays in MDCK cells. For
TCID50-HA assays, 10-fold serial dilutions of virus samples in eight replicates each were propagated for
48 hours on MDCK cells in a 96-well plate format, followed by transfer of culture supernatants to
suspensions of chicken red blood cells and scoring of individual wells based on hemagglutination
activity. Clinical RSV isolates were collected from patient nasal washes in 2010, cultured on primary
rhesus monkey kidney (RhMK) cells, and amplified once on HEp-2 cells prior to use in this study.
Recombinant RSV and RSV isolates were grown in HEp-2 cells and titrated by plaque or
immunoplaque assay in HEp-2 cells.
Purification of recombinant reporter virus stocks
Progeny virions were collected from cell culture supernatants (IAV) or released from infected cells
through one freeze-thaw cycle (RSV) and subjected to a clearance centrifugation (4,000 rpm for 20 min
at 4°C). Virions were diluted in DMEM, purified through a 20%-60% one-step sucrose gradient in TNE
buffer (1 mM Tris [pH 7.2], 100 mM NaCl, 10 mM EDTA; 30,000 rpm for 120 min at 4°C), and harvested
from the gradient intersection. Purified virus stocks were stored in aliquots at −80°C.
Automated drug screening
MDCK cells were injected into barcoded white-walled/clear-bottomed 384-well plates using a
MultiFlo automated dispenser (BioTek) equipped with dual 10-μl peristaltic pump manifolds and
incubated for 5 hours at 37°C and 5% CO2. Compounds were added to a final concentration of 5 μM
(20 nl/well) by using a high-density pin tool (V&P Scientific), followed by coinfection with recRSVA2-
L19F-fireSMASh (multiplicity of infection [MOI] = 0.1) and recIAV-nanoLuc (MOI = 0.02) at 10 μl/well by
use of a MultiFlo dispenser unit, and incubation for 48 h at 37°C and 5% CO2. The final vehicle (DMSO)
concentration was 0.05%. The reporter gene activity was recorded 48 hours post-infection with H1
synergy multimode plate reader (BioTek), and compounds showing 80% inhibition of both RSV and
IAV pursued as hit candidates. The MScreen software package was used for library management and
campaign analysis.
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Dose-response antiviral activity and cytotoxicity testing in cultured cells
For automated dose-response testing, three-fold serial dilutions were prepared in 96-well plates in
three replicates each using a Nimbus liquid handler (Hamilton). Target cells as specified were seeded
in white-wall clear-bottom 96-well plates (8×103 cells/well) and the serial dilutions transferred using the
liquid handler, followed by infection with IAV-WSN-nanoLuc (MOI 0.02) or RSV-A2-L19F-fireSMASh
(MOI 0.1). Reporter signals were recorded with the H1 synergy plate reader specified above. To
determine cell viability, PrestoBlue substrate (5 μl/well (Life Technologies)) was added after 48 h
incubation of compound-exposed uninfected cells at 37°C and top-read fluorescence (excitation at 560
nm, emission at 590 nm, instrument gain of 85) recorded after incubation for 45 min at 37°C using the
H1 synergy plate reader. Raw data of all automated dose-response assays were analyzed according to
the formula % inhibition = (XSample-XMin)/(XMax-XMin)×100 with XMin representing the average of four
positive (1 mg/ml cycloheximide) and XMax the average of four negative (DMSO) control wells included
on each plate. Four-parameter variable slope regression modeling was applied to determine 50% active
(EC50) and toxic (CC50) concentrations, using the non-linear regression function in the Prism
(GraphPad) software package. For manual dose-testing of non-reporter viruses, target cells were
seeded in 12-well plates (1.5×105 cells/well) and at approximately 90% confluency infected with the test
virus in the presence of serial compound dilutions. Progeny virus titers were determined 36 to 48 hours
post-infection depending on the virus strain analyzed, and viral titers determined as described above.
Human DNA polymerase inhibition assay
Inhibition of human DNA polymerase  was assayed in a 96-well format containing reaction buffer
(50 mM Tris-HCl (pH 8.7), 10 mM MgCl2, 0.4 mg/ml BSA, 1 mM DTT, 15% glycerol, 0.05 mM dCTP,
0.05 mM dTTP, 0.05 mM dATP, 10 Ci [32P]--dGTP (800 Ci/mmol)), 20 g activated calf thymus DNA,
and NHC-TP in a range of different concentrations. Alphidicolin served as polymerase inhibitor
reference. Reactions were carried out for 30 minutes at 37°C, followed by transfer to filter plates,
precipitation with 10% trichloroacetic acid and repeated washing with 5% trichloroacetic acid and 95%
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ethanol. Incorporation of [-32P] GTP was measured after filter drying using a Microbeta scintillation
counter.
Efficacy against IAV in ovo
Serum pathogen-free (SPF) freshly fertilized chicken eggs were purchased from Hy-Line and
incubated at 37°C, 55-60% humidity for 10 to 11 days. Eggs were candled, disinfected with 70%
ethanol, and NHC in sterile PBS was administered to a final concentration of approximately 10 µM
directly into the allantoic fluid 2 hours prior to infection using a 22-gauge needle. The average volume
of the allantoic fluid was considered to be 50 ml. Eggs were sealed and incubated for 2 hours, followed
by infection with 10 HA units of A/Swine/Spain/53207/2004 (H1N1). After 48-hours incubation, eggs
were cooled to 4°C, virus harvested from the allantoic fluid and titrated using standard hemagglutination
(HA) assays and turkey red blood cells.
Replicon reporter assays
Reporter activities were determined in the presence of three-fold serial dilutions of NHC starting
from 20 M for RSV and 100 M for IAV; treatment was initiated immediately post-transfection.
Luciferase activities in cell lysates were measured in a Synergy H1 microplate reader (BioTek) in top-
count mode using Dual-Glo Luciferase Assay System (Promega). Inhibitory concentrations were
calculated through four-parameter variable slope regression modeling.
Time-of-addition variation studies
HEp-2 cells were infected with 0.1 MOI RSV-A2-fireSMASh and MDCK cells were infected with
0.05 MOI of IAV WSN-nanoLuc. At the specified time points relative to infection, NHC, GRP-71271, AL-
8176, or CL-309623 were added to the culture media to a final concentration of 10 µM. T-705 was
added to a final concentration of 50 µM and volume equivalents of DMSO served as vehicle controls.
Reporter gene expression was measured 24 (IAV WSN-nanoLuc) or 48 (RSV-A2-fireSMASh) hours
post-infection, and the obtained values expressed relative to the vehicle-treated samples.
Nucleotide competition experiments
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HEp-2 cells were infected with 0.1 MOI RSV-A2-fireSMASh and MDCK cells were infected with
0.05 MOI of IAV WSN-nanoLuc. At the time of infection, NHC was added to a final concentration of 10
µM NHC alone, or in combination with 0.1 to 300 µM exogenous nucleosides (Sigma-Aldrich). Volume
equivalents of DMSO served as vehicle control. Reporter gene expression was quantified 24 (IAV-
WSN-nanoLuc) or 48 (RSV-A2-fireSMASh) hours post-infection. Values are expressed relative to the
vehicle-treated samples.
In vitro RSV polymerase assay
RSV large polymerase subunit (L) and phosphoprotein (P) complexes were expressed from a
baculovirus vector and purified by affinity chromatography: L-P complexes were eluted from the Ni-NTA
column with 250 mM imidazole in 50 mM NaH2PO4, pH 7.5, 150 mM NaCl and 0.5% NP-40 followed by
dialysis against 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM DTT and 10% glycerol. L-P hetero-
oligomers were mixed in a Mg2+ buffer with 25-mer RNA oligonucleotide template containing essential
RSV promoter sequences and rNTPs including 0.07 M [-32P]-UTP tracer, but lacking CTP. NHC,
NHC-TP, or CTP were added as specified. Predominant initiation at the +3 position results in up to 23-
mer radiolabeled amplicons, which were subjected to denaturing gel electrophoresis, followed by
autoradiography.
Assessment of mutation frequencies in viral RNA
HEp-2 cells were infected with 0.1 MOI RSV-A2-mKate and MDCK cells were infected with 0.05
MOI of IAV-WSN, followed by growth in the presence of 10 µM NHC for 24 hours; volume equivalents
of DMSO were used as vehicle control. Total RNA was extracted using the ZR Viral RNA Kit (Zymo
Research), cDNA of viral message synthesized with SuperScript III reverse transcriptase (Thermo
Scientific) and oligo dT primers. The PB1 segment of IAV-WSN or an approximately 1500 nucleotide
fragment of the RSV-A2-mKate L ORF were amplified by PCR and subcloned into pUC19 vector. For
each virus and treatment condition, at least 10 distinct subclones (equaling in aggregate at least
approximately 7500 nucleotides each) were Sanger sequenced using universal M13 primers. Data
were analyzed with Sequencer package and mutation frequencies expressed per 5000 nucleotides for
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IAV-WSN and per 10,000 nucleotides for RSV-A2, respectively. Fisher’s exact test was used for
statistical analyses.
Virus adaptation in cell culture
Both dose-escalation and fixed dose adaptation strategies were applied. For dose escalation,
MDCK cells were infected with 0.01 MOI of IAV-WSN and HEp-2 cells were infected with 0.1 MOI of
RSV-A2-mKate. The viruses were passaged in the presence of increasing concentration of NHC,
starting at 250 nM. Dose concentrations were doubled at virus passage up to a final concentration of 10
µM. Six independent passage lines each per target virus were advanced simultaneously for a total of
ten passages, each entailing virus harvest from infected cells, dilution, and reinfection of fresh cell
populations in the presence of compound or vehicle. Virus titers declined significantly towards the end
of the series and no resistant variants emerged. For fixed dose adaptation, MDCK cells were infected
with 0.01 MOI of IAV-WSN or B/Brisbane/60/08, HEp-2 cells infected with 0.1 MOI of RSV-A2-mKate.
The viruses were passaged in the presence of EC99 concentration equivalents of NHC for the
respective target virus, as before for a total of ten passages. Progeny virus titers again declined
towards the end of the cycle and no drug-resistant virus population could be cultivated.
Anabolism and turnover in cultured cells
To screen for cellular uptake and anabolism of NHC to NHC-TP, HBTECs were grown in the
presence of 20 M NHC for 0, 1, 2, 3, 4, 6, 16 and 24 hours. Cells were washed with PBS, lysed with
70% methanol, and clarified samples stored at -20C until analysis. To determine stability of NHC-TP
and other anabolites, the HBTEC cells were grown in the presence of 20 M NHC for 24 hours, media
changed to drug-free BronchiaLife Cell Culture Medium, and cells incubated for an additional 0, 0.5, 1,
2, 3 and 6 hours. Metabolites were extracted with 70% methanol as before and samples kept at -20C
until analysis. NHC anabolites were quantitated using internal standards-based LC-MS/MS on an
Agilent 1200 system (Agilent Technologies) equipped with a SeQuant ZIC-pHILIC column (The Nest
group) column. Mass spectrometry analysis was performed on a QTrap 5500 Mass Spectrometer (AB
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Sciex) using Negative Mode Electrospray Ionization (ESI) in Multiple Reaction Monitoring (MRM) Mode.
Data analysis was performed using Analyst Software (AB Sciex).
PK and PD studies in mice
Female CD-1 mice (6-8 weeks of age) distributed randomly to groups were dosed p.o. with NHC in
240 mM citrate buffer, followed by blood and lung tissue sampling. Plasma was purified from
heparinized blood, tissue samples were snap frozen prior to 70% acetonitrile extraction. Drug
concentrations were determined using 13C5-labeled internal standards for NHC and NHC-TP. Mass
spectrometry was performed as detailed above. For calibration, standard curves were prepared in blank
plasma (concentrations range 25 to 30,000 ng/ml) and blank tissue lysate (concentration range 1.49 to
1,490 ng/ml). Quality-control samples of 30, 500 and 900 ng/ml in blank plasma were analyzed at the
beginning of each sample set. Calibration in each matrix showed linearity with an R2 values >0.99.
Mouse model for RSV infection
Female BALB/cJ mice (5-6 weeks of age) were obtained from the Jack Laboratory or Envigo,
rested for one week, assigned to groups randomly anesthetized by intraperitoneal injection of a
ketamine-xylazine solution and infected i.n. with 1×105 pfu of recRSV A2-L19F. NHC was administered
orally in 240 mM citrate buffer or vehicle volume equivalent 2 hours pre-infection and dosing continued
b.i.d. for up to 8 days. For virus load titration, lungs were extracted, homogenized, homogenates
serially diluted, transferred to HEp-2 cells, and cells overlaid one hour post-infection with minimum
essential medium (MEM) containing 10% FBS, penicillin G, streptomycin sulfate, amphotericin B
solution, and 0.75% methylcellulose. Six days post-infection, cells were fixed with methanol and
plaques visualized by immunodetection. To quantify mucin expression, mice were euthanized eight
days post-infection, heart-lung tissue harvested, and fixed in 10% formalin. Lung tissue sections
embedded in paraffin blocks were stained with periodic acid-Schiff (PAS) stain and slides digitally
scanned using a Zeiss Mirax Midi microscope (Carl Zeiss Microimaging). To determine respiratory
distress noninvasively, a rodent pulse oximeter (MouseOx; Starr Life Sciences Corp., Oakmont, PA)
was applied to the mouse's thigh and arterial O2 saturation, heart rate, pulse rate, pulse distension, and
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breath distension measured every 0.1 seconds for a 1-5 minute overall period. Mean breath distension
for each treatment group was calculated based on mean values of all measurements for each animal in
which all target parameters were present.
Mouse model for IAV infection
Female BALB/cJ mice (6-8 weeks of age) were received from Envigo and housed ABSL-2 (for
infection with IAV-PR8) or ABSL-3 (for infection with HPAIV) facilities in HEPA-filtered microisolator
caging. Mice were rested for one week, weighed, assigned to groups randomly, and infected with 103
pfu of IAV-PR8 or 6 pfu of A/Vietnam/1203/04 (H5N1) as specified in PBS. Treatment was initiated 2
hours pre-infection (prophylactic dosing) or 6 hours post-infection (therapeutic dosing) and continued
for up to 6 days b.i.d. Compounds or vehicle volume equivalents were administered orally in 240 mM
citrate buffer formulation. Animal clinical signs were tracked daily, and animals euthanized at the
indicated time points or when humane endpoints were reached. Lung and, where indicated, brain tissue
were removed, homogenized, clarified by centrifugation, and aliquots frozen at -80°C as outlined above
until virus titration.
Select cytokine mRNA induction in mouse lung tissue
Relative IFN- and IL-6 induction levels present in mouse lung tissue were determined by semi-
quantitative real-time PCR analysis. Total RNA was extracted from lung tissue three days post-infection
of animals with IAV-PR8 or mock-infected (representing day 0). Infected animals were treated orally
with NHC or vehicle (citrate buffer) volume equivalents. RNA was reverse-transcribed with SuperScript
III reverse transcriptase and the resulting cDNAs subjected to real-time PCR using the Fast SYBR
Green Master Mix (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
mRNA served as an internal control, and mRNA induction levels were normalized to the average of the
values obtained determined for mock-infected animals. Each biological repeat was determined in
duplicate and relative changes in transcription levels calculated according to fold change = 2-ΔΔCT
.
Guinea pig IAV infection and transmission model
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Female Hartley strain guinea pigs weighing 250 to 300 g were obtained from Charles River
Laboratories. The animals were assigned to groups randomly, and prior to intranasal inoculation, nasal
lavage, or CO2 euthanasia, the guinea pigs were sedated with a mixture of ketamine and xylazine (30
mg/kg of body weight and 4 mg/kg, respectively). Inoculation and nasal lavage were performed with
phosphate-buffered saline (PBS) as the diluent/collection fluid in each case. Oral treatment of infected
donor animals with NHC or vehicle (240 mM citrate buffer with Ora-Sweet (Paddock Laboratories)) was
initiated two 2 hours pre-infection and continued b.i.d. until the end of day 3 post-infection. Following
inoculation and recovery from sedation, donor guinea pigs were housed in Caron 6040 environmental
chambers (fitted with the optional dryer package) set to 10°C and 20% relative humidity. At 24 hours
post-inoculation of the donor animals, exposed guinea pigs were introduced into the donor animal
cages. Conditions of 10°C and 20% relative humidity were maintained throughout the exposure period,
which ended on day 8 post-inoculation. Virus shedding titers in nasal lavages of source and contact
animals were determined through plaque assays.
Statistical analysis
To assess experimental variation and the statistical significance of differences between sample
means, one-way or two-way analysis of variance (ANOVA) were carried out in combination with
Tukey’s, Dunnett’s, or Sidak’s post hoc test as specified in figure legends, using the Prism (GraphPad)
software package. Fisher’s exact test was used for statistical analyses of mutation frequencies. Results
for individual biological replicates are shown for all in vivo efficacy experiments. When appropriate,
experimental uncertainties are identified by error bars, representing standard deviations (SD).
IACUC approval statement
All animal work was performed in compliance with the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. Mouse work at Georgia State University was approved by
the GSU Institutional Animal Use and Care Committee (IACUC) under protocol A17019, mouse and
guinea pig work at Emory University were approved by the Emory IACUC under protocol numbers
DAR-2003089-ENTRPR-N and DAR-2002738-ELMNTS-A, respectively, and mouse studies with
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HPAIV at the University of Georgia were approved by the UGA IACUC under protocol number A2017
05-009.
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Acknowledgements
We thank A.Mehle and S.Schultz-Cherry for IAV H5N1 and H7N9 minigenome reporter systems,
K.K.Conzelmann for the BSR-T7/5 cell line, C.Jones, S.Johnson, and C.Kyriakis for assistance with the
murine HPAIV studies, G.P.Reddy, J.Marlow, and J.DeBergh for assistance with bioanalytical analysis,
ImQuest BioSciences for assistance with the human DNA polymerase assay, L.Martinez-Sobrido for
IBV isolates, R.T.Jacob for IT support, and A.L.Hammond for critical reading of the manuscript. The
MScreen software package was kindly provided by the Center for Chemical Genomics of the University
of Michigan under a license agreement by the University of Michigan Office of Technology Transfer,
JChem was used for structure database management, search and prediction (JChem 6.2, 2014,
ChemAxon (http://www.chemaxon.com)), and Marvin was employed for drawing, displaying and
characterizing chemical structures, substructures and reactions (Marvin 14.9.22.0, 2014, ChemAxon
(http://www.chemaxon.com)). This work was supported, in part, by contracts HDTRA1-15-C-0075 and
HHSN272201500008C from DTRA and the NIH/NIAID, respectively (to G.R.P.) and by public health
service grants AI119196, AI071002 and HD079327 from the NIH/NIAID and NIH/NICHD, respectively
(to R.K.P.). The funders had no role in study design, data collection and interpretation, or the decision
to submit the work for publication.
Competing interests: The authors declare no competing interests.
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