key: cord-0791247-labwc87m
authors: Lo, Michael K; Spengler, Jessica R; Krumpe, Lauren R H; Welch, Stephen R; Chattopadhyay, Anasuya; Harmon, Jessica R; Coleman-McCray, Joann D; Scholte, Florine E M; Hotard, Anne L; Fuqua, Joshua L; Rose, John K; Nichol, Stuart T; Palmer, Kenneth E; O’Keefe, Barry R; Spiropoulou, Christina F
title: Griffithsin Inhibits Nipah Virus Entry and Fusion and Can Protect Syrian Golden Hamsters From Lethal Nipah Virus Challenge
date: 2020-02-10
journal: J Infect Dis
DOI: 10.1093/infdis/jiz630
sha: 566a7d2ab68fa23838d89b1fb30e00cf3f451075
doc_id: 791247
cord_uid: labwc87m

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from across 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

Nipah virus (NiV) is a zoonotic henipavirus harbored by pteropid bats, and it is responsible for causing near-annual outbreaks of fatal human encephalitis and respiratory disease in Bangladesh and India [1] . Although several therapeutics protect against lethal NiV challenge in animals [2] [3] [4] , none is currently approved for human use. Inhibitors of NiV entry and spread generally target protein interactions involved in receptor binding or membrane fusion processes [5] [6] [7] [8] [9] [10] , but the glycosylation of NiV glycoproteins presents another viable target for therapeutic development [11] [12] [13] . Griffithsin (GRFT) is a homodimeric high-mannose oligosaccharide binding lectin currently being evaluated in clinical trials as a topical microbicide against human immunodeficiency virus 1 (HIV-1) [14, 15] , and it has also demonstrated in vivo broad-spectrum activity against viruses including severe acute respiratory syndrome coronavirus, hepatitis C virus, and Japanese encephalitis virus [16] [17] [18] [19] . In this study, we characterized the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) [20] against NiV and other viruses from across 4 virus families, and we also tested the in vivo prophylactic potential of 3mG and an oxidation-resistant form of GRFT (Q-GRFT) against lethal NiV challenge in the Syrian golden hamster model. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

All work with infectious virus or infected animals was conducted in a biosafety level 4 (BSL-4) laboratory at the Centers for Disease Control and Prevention (CDC) following established BSL-4 standard operating procedures approved by the Institutional Biosafety Committee.

Griffithsin, monomeric GRFT (mGRFT), mGRFT tandemers, and Q-GRFT were expressed and purified as described previously [20] [21] [22] [23] . Codon-optimized mCherry, Malaysian NiV-F, and NiV-G coding sequences were synthesized and cloned into pCAGGS expression plasmids (GenScript). A pTM1-based plasmid encoding enhanced green fluorescent protein (eGFP) under the control of a T7 polymerase promoter (pT7-eGFP) was a gift from Dr. Mark E. Peeples (The Research Institute at Nationwide Children's Hospital).

HeLa, HEK293T/17, HT-1080, Vero, Huh7, and BSRT7/5 cells [24] were cultivated as previously described [25, 26] . CHO-K1 cells were maintained in F-12K medium supplemented with 10% fetal bovine serum. All cells were incubated at 37°C in 5% CO 2 . Wild-type NiVs (NiV Malaysia [NiV-M], NiV Bangladesh [NiV-B]), Hendra virus (HeV), recombinant reporter NiVs (rNiV-RLuc [formerly named NiV-Luc2AM], rNiV-ZsG [formerly named NiV-GFP2AM]) [27] , measles vaccine virus (rMV EZ EGFP(3)) [28] , mumps virus (rMuV-EGFP, Iowa 2006 strain) [29] , human parainfluenza virus 3 (hPIV3-GFP, JS strain) [30] , human metapneumovirus (hMPV-GFP, CAN97-83 strain) [31] , Lassa virus (rLASV-ZsG) [32] , and Crimean-Congo hemorrhagic fever virus (rCCHFV-ZsG) [33] were propagated in Vero E6, Vero, or Huh7 cells and were quantitated by 50% tissue culture infections dose (TCID 50 ) assay [34] . Unless stated otherwise, all infections were performed at multiplicity of infection of 0.25.

Recombinant reporter virus assays were performed as previously described [27, 35] . In brief, 10 4 cells pretreated with serial dilutions of GRFT for 1 hour were infected with the above-mentioned reporter viruses (without washing) for 24-72 hours and then assayed either for GFP, GFP + cells, or for Renilla luciferase activity. A nanoluciferase (NLuc)-based NiV minigenome assay was conducted as previously described [25] . A total of 10 4 BSRT7/5 cells [24] (per well) were transfected using LT-1 transfection reagent (Mirus Bio) with appropriate amounts of NiV support plasmids (see Supplemental Methods for details). Griffithsin was added directly to the media 4 hours posttransfection, and at 48 hours NLuc activity was measured.

A total of 10 4 cells infected with NiV-M, NiV-B, or rNiV-ZsG for 1 hour were washed and treated with GRFT or 3mG. At 24-48 hours postinfection (hpi), viral titers from supernatants were quantitated by TCID 50 assay. Fluorescent micrographs of rNiV-ZsG-infected cells were captured using the EVOS FL Cell Imaging System (Thermo Fisher Scientific) at ×4 magnification. For immune fluorescence, infected cells were fixed in 10% formalin, stained using GRFT at 1 µg/mL and a monoclonal anti-NiV nucleoprotein antibody [36] , followed by staining with anti-GRFT polyclonal rabbit antibody, and then detected with antirabbit Dylight488 and antimouse Dylight550 antibodies (1:1000 dilution; Bethyl Laboratories) along with 4' ,6-diamidino-2-phenylindole (DAPI) nuclear stain (1:1000 dilution). Fluorescence micrographs were taken using a Nikon Axioscope Ti inverted fluorescence microscope at ×4 magnification.

Nipah Virus (NiV)-F and NiV-G Glycoprotein Expression, Purification, and Detection HEK293T/17 cells (3 × 10 5 ) were transfected with 3 µg of NiV-F AU1, NiV-G His6×, or mCherry plasmid. At 48 hours posttransfection, cells were harvested in 250 µL radioimmunoprecipitation buffer/well (Thermo Fisher Scientific). Nipah virus-F AU1 was purified from cell lysates using an anti-AU1 affinity bead column according to the manufacturer's recommendations (BioLegend). Nipah virus-G His6× was purified using nickel-nitrilotriacetic acid affinity resin. Cell lysates and purified proteins were run on SDS-PAGE gels (Supplemental Figure 1A) , transferred onto polyvinylidene difluoride membranes, and probed with either anti-AU1 (Bethyl) or anti-6His6× (Cell Signaling Technology) rabbit antibodies, or with GRFT along with anti-GRFT polyclonal rabbit sera, and detected using goat antirabbit 680RD secondary antibodies (LI-COR) (Supplemental Figure 1B and C). Protein bands were visualized using LI-COR imaging.

Griffithsin binding enzyme-linked immunosorbent assays (ELISAs) were performed as previously described [15] . In brief, 1 picomole (or 50 ng) of purified NiV-F AU1 or NiV-G His6× was bound to a 96-well plate, which was washed and blocked with bovine serum albumin. Between requisite washing steps, plates were incubated with increasing amounts of GRFT or 3mG, followed by incubation with a 1:1000 dilution of the anti-GRFT rabbit polyclonal antibody preparation. The bound GRFT was determined using goat antirabbit antibodies conjugated to horseradish peroxidase (HRP) and a TMB peroxidase substrate system. Peroxidase activity was quenched using 1 M hydrochloric acid and absorbance was measured at 450 nm. Additional details are found in the Supplemental Methods.

A replication-deficient vesicular stomatitis virus (VSV) expressing eGFP in place of the VSV attachment glycoprotein (VSV-ΔG-eGFP) was pseudotyped with either VSV G (VSVpt) or NiV-F and NiV-G glycoproteins (NiVpt) as previously described [37] . Fifty particles of VSVpt or NiVpt were used to either infect Vero cells in conjunction with GRFT/3mG treatment for 24 hours or to infect Vero cells that were pretreated with GRFT/3mG for 1 hours and then removed before infection. At 24 hpi, numbers of eGFP expressing cells were visualized and counted [37] .

Inhibition of virus-induced cytopathic effect (CPE) was assayed as previously described using CellTiter-Glo 2.0 reagent (Promega) in a BioTek HD1 Synergy plate reader [25] . Luminescence values were normalized to uninfected cell controls according to % viability as follows: % viability = [(specific value-reference value)/(dimethyl sulfoxide control value − reference value)] × 100. Reference values were derived from control wells without cells. Uninfected cell control viability values (after subtraction of reference values) were set at 100% inhibition of CPE.

A total of 10 4 HT-1080 cells were transfected for 4 hours with 0.1 µg each of NiV-F AU1 and NiV-G HA expression plasmids, washed, and replenished with growth media containing GRFT or 3mG. At 24 hours posttransfection, cells were stained using CellMask Green (Thermo Fisher Scientific) and DAPI and viewed using a Nikon Axioscope Ti inverted fluorescence microscope at ×4 magnification. For quantitative fusion assay, 2 × 10 4 CHO-K1 cells were transfected for 4 hours with 0.1 µg each of NiV-F AU1, NiV-G HA, and pT7-eEGFP plasmids. Negative control CHO-K1 cells were transfected with 0.2 µg of NiV-F AU1 and 0.1 µg of pT7-eGFP. Cells then were washed and replenished with growth media containing GRFT or 3mG. BSRT7/5 cells (2 × 10 4 ) were then overlaid onto CHO-K1 cells. At ~16 hours postoverlay, green fluorescent NiV glycoproteininduced syncytia were visualized at ×2.5 magnification on a Cytation5 instrument (BioTek) and counted from photograph montages of each well using Gen5 Software. Fluorescent objects that were both >75 µm in diameter and had a fluorescence reading >5000 were classified as positive syncytia. Additional details are found in the Supplemental Methods.

All animal experiments were approved by the CDC Institutional Animal Care and Use Committee and performed in an AAALAC-International approved facility. Groups of 5-10 female HsdHan:AURA golden Syrian hamsters (6 weeks of age; Envigo no. 8903F) were inoculated intranasally (IN) with Dulbecco's modified Eagle's medium or NiV-B (10 7 TCID 50 ). On 1 and 2 days postinfection (dpi) and/or -1 and -2 dpi, animals were treated IN with vehicle control (phosphate-buffered saline [PBS]), GRFT (10 mg/kg), or 3mG (10 mg/kg). Hamsters were humanely euthanized with isoflurane vapor when they met euthanasia criteria or at the completion of the study (28 dpi). Additional clinical scoring details are found in the Supplementary Methods.

Ribonucleic acid (RNA) was extracted from blood and homogenized tissue samples using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher Scientific) on a 96-well ABI MagMAX extraction platform with a DNaseI treatment step according to manufacturer's instructions. The RNA was quantitated using one-step real-time reverse-transcription polymerase chain reaction (RT-PCR) targeting the N gene sequence [38] and was standardized to 18S ribosomal RNA with a SuperScript III Platinum One-Step qRT-PCR Kit (Thermo Fisher Scientific) according to manufacturer's instructions.

Total NiV-specific immunoglobulin G (IgG) and neutralizing antibodies were measured as previously described [2] . Gammairradiated plasma were serially diluted 1:100, 1:400, 1:1600, and 1:6400 and incubated for 1 hour on plates coated with inactivated NiV lysate or Vero cell control lysate at 37°C. With requisite washing steps, plates were then incubated with antibody detector protein A/G conjugated with HRP (Thermo Fisher Scientific) for 1 hour at 37°C, followed by incubation with ABTS substrate (KPL) for one-half hour at 37°C. Absorbance was read at 410 nm and 490 nm. Well-specific 490 nm absorbance values were subtracted from their corresponding 410 nm values. The resulting values from Vero control lysates were then subtracted from those taken from NiV-infected lysates. Cutoff values were derived from adding the mean of 6 negative control plasma to 3 times the standard deviation of the mean. Samples were considered true IgG positives if their values were above cutoff values for both 1:100 and 1:400 dilutions. Additional details are found in the Supplementary Methods. Two-fold dilutions of plasma were incubated with 200 TCID 50 of NiV-B in duplicate for 1 hour and then transferred onto Vero cells for 6 days at 37°C. Neutralizing titers were recorded as the lowest reciprocal dilution at which all wells lacked CPE.

All statistical analyses were performed using GraphPad Prism 8.1.2. Animal survival was analyzed by log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon test. Quantitative fusion assay and virus yield comparisons between GRFT and 3mG were analyzed using multiple t tests with adjusted P values. Fifty percent effective concentrations and 50% cytotoxic concentrations were calculated using 4-parameter variable slope nonlinear regression fitting of mean values of all antiviral assays.

We initially tested GRFT against a reporter NiV expressing Renilla luciferase (rNiV-RLuc) [27] . Pretreatment of 4 different cell lines with increasing amounts of GRFT 1 hour before rNiV-RLuc infection resulted in dose-dependent decreases in reporter activity assayed at 24 hpi, with 50% effective inhibitory concentration (EC 50 ) values ranging between 0.5 and 1.5 µg/mL (~20-60 nM) ( Figure 1A , Table 1 ). We then measured wild-type recombinant NiV (rNiV) infectious virus yield at 24 hpi from cells treated with GRFT immediately after infection and observed a dosedependent decrease >2 log 10 TCID 50 ( Figure 1B ). To determine whether GRFT interferes with NiV replication directly at the level of RNA transcription and replication, we tested increasing concentrations of GRFT against a reporter NiV minigenome expressing NLuc and observed no inhibition of reporter signal, consistent with no direct inhibition of viral RNA levels ( Figure 1C ).

Because GRFT preferably binds to high mannose N-linked oligosaccharides found commonly on viral envelope glycoproteins [39, 40] , we sought to characterize the interaction of GRFT with purified NiV-M fusion (NiV-F AU1) and attachment (NiV-G His6×) surface glycoproteins (Supplemental Figure  S1A -C). We used GRFT to probe against NiV-F AU1 and NiV-G His6× by Western blot, and we observed detection of both proteins ( Figure 2A , left panel, well no. 6 NiV-F AU1, well no. 10 NiV-G). To further characterize these interactions, we performed ELISA binding assays by titrating increasing levels of GRFT against the purified glycoproteins, and we observed comparatively greater binding to NiV-G than to NiV-F (Figure 2A , right panel). We then conducted time-of-addition experiments to determine how long after infection could GRFT still inhibit virus replication. We either incubated serial dilutions of GRFT with rNiV-RLuc for 2 hours before infection or added GRFT at 0, 2, 6, or 12 hpi. Although a 2-hour preincubation of GRFT with rNiV-RLuc showed negligible differences in inhibition compared with GRFT treatment at 0 hpi, gradually increasing reporter activity correlated with longer delays before GRFT treatment ( Figure  2B , left panel). It is notable, however, that even when GRFT was added 12 hpi, at concentrations of ≥~40 nM GRFT reporter activity was still reduced compared with untreated control infected cells. Dose-response curves for reporter activity indicated an approximately 5-to 6-fold decrease in potency between 0 and 12 hpi treatments, with GRFT treatments at 2 or 6 hpi showing an intermediate 2-to 3-fold comparative decrease to 0 hpi treatment ( Figure 2B, right panel) .

To determine whether GRFT inhibition of NiV could be overcome by more virus, we used increasing amounts of rNiV-RLuc to infect GRFT-pretreated cells. We observed a dose-dependent increase in reporter activity against ~40 nM of GRFT in 2 cell types, but at higher concentrations of GRFT (~120-360 nM), there were only minimal increases in reporter signal ( Figure 2C , left and right panels). This indicated that higher concentrations of GRFT may better inhibit NiV entry and spread indirectly via binding to cellular glycoproteins.

To visualize GRFT-mediated inhibition of NiV spread, we performed immune fluorescence assay of cells infected with rNiV before treatment with GRFT. In untreated infected cells, we observed large holes in the cell monolayer caused by NiVinduced syncytia, with widespread NiV nucleoprotein antigen present ( Figure 2D, top leftmost panel) . With increasing amounts of GRFT, we observed smaller syncytia concomitant with a visible decrease in infected cells ( Figure 2D , top 3 right panels). Cells treated with GRFT were positively stained with an anti-GRFT polyclonal antibody, indicating GRFT binding to cell-surface glycoproteins ( Figure 2D , bottom 3 right panels).

The presence of more than 1 oligosaccharide-binding face in the dimeric form of GRFT is crucial to maintain its potent anti-HIV-1 activity [41, 42] . Several synthetically linked tandemers of mGRFT have shown up to 10-fold greater in vitro potency than GRFT [20] . We compared the antiviral activities of GRFT to mGRFT, to a synthetic dimer (2mG), a synthetic trimer (3mG), and a Q-GRFT containing 1 amino acid change (M78→Q), and we found that 3mG was most potent, Q-GRFT was equivalently potent, 2mG was less potent, and that mGRFT had lost all antiviral activity against rNiV-RLuc ( Figure 3A , left and right panels, Table 1 ). To confirm these findings, we tested GRFT, 2mG, 3mG, and Q-GRFT against wild-type NiV-B, NiV-M, and HeV, and we observed similar trends as seen for rNiV-RLuc, with 3mG having the greatest antiviral potency ( Figure 3B , Table 1 ). Among these compounds, only 3mG showed mild cytotoxicity at higher concentrations. We also observed varied antiviral potencies for GRFT and 3mG against other respiratory and hemorrhagic fever viruses of human public health importance, ranging from no activity to potent activity with sub-nanomolar EC 50 values (Table 1 , Supplementary Figures S2A-D and S3A and B) . To understand the greater in vitro activity of 3mG compared with GRFT, we performed ELISA protein binding assays against purified NiV-M F and G glycoproteins. We observed greater binding by GRFT to both glycoproteins than by 3mG, which was contrary to their virus inhibition profiles ( Figure 3C ). We then compared the abilities of GRFT and 3mG to block virus entry by replication-deficient, eGFP-expressing VSVs pseudotyped with either the VSV G glycoprotein (VSVpt) or with both NiV-M F and G glycoproteins (NiVpt). We did not observe significant differences in antiviral potencies at equivalent compound concentrations under both continuous GRFT/3mG treatment or with only 1 hour of pretreatment before infection ( Figure 3D , left and right panels, respectively).

We tested the abilities of GRFT and 3mG to block NiV-F and NiV-G glycoprotein-induced syncytia formation by treating transfected cells expressing both NiV-F and NiV-G glycoproteins with either compound, and we observed that 3mG visibly inhibited syncytia formation more efficiently than GRFT at equivalent concentrations ( Figure 4A ). To confirm this result, we developed a quantitative image-based syncytia counting assay. Whereas cells transfected only with NiV-F plasmid had minimal background syncytia counts, cells transfected with both NiV glycoproteins had >1000 syncytia counts ( Figure 4B , left panel). Using this assay, we observed a dose-dependent decrease in syncytia counts that correlated with increased levels of GRFT or 3mG, but we noted that 3mG more efficiently reduced syncytia than GRFT at higher concentrations ( Figure  4B , right panel). When Vero cells were treated with GRFT or 3mG after infection by either green fluorescent reporter NiV (rNiV-ZsG) or NiV-B, 3mG reduced virus yield more efficiently than GRFT, similarly reflecting their differing abilities to block syncytia formation ( Figure 4C , left and right panels, respectively). Accordingly, 3mG visibly delayed or reduced the size of syncytia formation more efficiently than GRFT in rNiV-ZsG infected cells ( Figure 4D ).

Finally, we explored the in vivo prophylactic potential of 3mG and of the Q-GRFT [21] against a uniformly lethal intranasal challenge dose of NiV-B (~10 7 TCID 50 ) in Syrian golden hamsters [43] . Groups of 10 animals each received intranasal doses of either Q-GRFT (10 mg/kg), 3mG (10 mg/kg) on days 1 and 2 before challenge (for 2× dose and 4× dose treatment groups), and on days 1 and 2 postchallenge (for 4× dose treatment groups only). Vehicle control animals received an intranasal equivalent dose volume of PBS on the same 4 treatment days as the 4× dose treatment groups. Animals receiving either 2 or 4 doses of Q-GRFT treatment groups showed partial but significant protection from NiV-B challenge (~35% survival rate across Q-GRFT treatment groups) ( Figure 5A , top panel), whereas only 3 animals across 3mG treatment groups survived infection (~15% overall survival rate) ( Figure 5A , bottom panel). More than 70% of Q-GRFT treated survivors had no clinical signs by the end of the study, whereas only one 3mG treated survivor lacked clinical signs (Supplemental Figure S4C) . Nipah virus RNA was detected in most tissues sampled from euthanized or dead animals ( Figure 5B , filled shapes in all 3 panels), but it was only detected in the eyes and brains of Q-GRFT and 3mG treated survivors ( Figure 5B , open triangles and squares in top 2 panels). All survivors from Q-GRFT and 3mG treatment groups developed neutralizing antibody titers against NiV ( Figure 5C , Supplemental Table) .

In this study, we characterized the in vitro antiviral activity of GRFT and its 3mG tandemer against NiV and a series of other important human viral pathogens. Similar to in vitro observations for HIV-1, GRFT and 3mG can block both NiV entry and cellto-cell spread [15, 20] . Although 3mG consistently outperformed GRFT in its ability to block both NiV glycoprotein-induced cell-to-cell fusion and NiV infectious yield, it had comparatively weaker ELISA binding profiles against purified NiV glycoproteins. This observation could be explained by differences in anti-GRFT polyclonal antibody binding to 3mG because it is not an obligate dimer as is wild-type GRFT, against which the polyclonal antibodies were generated. Future biochemical and structural studies should not only identify the N-glycosylation sites on NiV-F and NiV-G that are important for antiviral susceptibility to GRFT/3mG, but also investigate the molecular interactions of GRFT/3mG with NiV-F and NiV-G proteins to determine how these may alter the biophysical properties of a NiV virion. Despite the superior in vitro ability of 3mG to block NiV syncytia formation and virus yield, 3mG protected less than half as many animals as did Q-GRFT from in vivo challenge. This can be attributed to 3mG likely having a shorter in vivo half-life compared with Q-GRFT, which is significantly more resistant to oxidation compared with wild-type GRFT [21] . The 3mG may require re-engineering to achieve better in vivo efficacy, because 3mG contains flexible linkers that are more susceptible to proteolytic cleavage than GRFT [44] . Although all survivors developed significant neutralizing antibodies against NiV, only 1 survivor treated with 3mG tested positive for total anti-NiV IgG (Supplemental Table) , suggesting that the majority of NiV-specific antibodies generated in Q-GRFT/3mG-treated survivors target the surface glycoproteins. The GRFT and other lectins represent another avenue towards developing NiV therapeutics. Because NiV glycoprotein glycosylation modulates syncytia formation and shields against neutralizing antibodies [45, 46] , retaining these glycosylation sites likely maintains viral fitness. A potential treatment regimen combining GRFT with both monoclonal antibody m102.4 and nucleotide prodrug remdesivir would provide an additional constraint against development of NiV escape mutants.

Our findings, taken together with the favorable pharmacokinetic and safety profiles of GRFT [22, [47] [48] [49] , make Q-GRFT an excellent candidate for further preclinical evaluation against NiV infection.

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Nipah virus outbreaks: still small but extremely lethal

GS-5734) protects African green monkeys from Nipah virus challenge

Pathogenic differences between Nipah virus Bangladesh and Malaysia strains in primates: implications for antibody therapy

T-705) protects against Nipah virus infection in the hamster model

Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody

Fusion inhibitory lipopeptides engineered for prophylaxis of Nipah virus in primates

Broad spectrum antiviral activity for paramyxoviruses is modulated by biophysical properties of fusion inhibitory peptides

A smallmolecule inhibitor of Nipah virus envelope proteinmediated membrane fusion

Inhibition of Henipavirus fusion and infection by heptad-derived peptides of the Nipah virus fusion glycoprotein

Heparan sulfatedependent enhancement of henipavirus infection

Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells

Endothelial galectin-1 binds to specific glycans on Nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation

Novel innate immune functions for galectin-1: galectin-1 inhibits cell fusion by Nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines

Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp

Studies in a murine model confirm the safety of griffithsin and advocate its further development as a microbicide targeting HIV-1 and other enveloped viruses

Griffithsin inhibits Japanese encephalitis virus infection in vitro and in vivo

Broadspectrum in vitro activity and in vivo efficacy of the S12 • jid 2019:XX (XX XXXX) • Lo et al antiviral protein Griffithsin against emerging viruses of the family Coronaviridae

Antiviral lectins from red and blue-green algae show potent in vitro and in vivo activity against hepatitis C virus

Griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus

Kramzer LF, inventors; The United States of America, as represented by the Secretary, Department of Health and Human Services

Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component

Improving the large scale purification of the HIV microbicide, griffithsin

Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter

GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses

Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses

Evaluation of luciferase and GFP-expressing Nipah viruses for rapid quantitative antiviral screening

Live-attenuated measles virus vaccine targets dendritic cells and macrophages in muscle of nonhuman primates

Rescue of wild-type mumps virus from a strain associated with recent outbreaks helps to define the role of the SH ORF in the pathogenesis of mumps virus

Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium

Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes

Lassa and Ebola virus inhibitors identified using minigenome and recombinant virus reporter systems

Identification of 2'-deoxy-2'-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

A simple method of estimating fifty percent endpoints

4'-Azidocytidine (R1479) inhibits henipaviruses and other paramyxoviruses with high potency

Use of monoclonal antibodies against Hendra and Nipah viruses in an antigen capture ELISA

Complementing defective viruses that express separate paramyxovirus glycoproteins provide a new vaccine vector approach

Characterization of Nipah virus from outbreaks in Bangladesh

Crystallographic, thermodynamic, and molecular modeling studies of the mode of binding of oligosaccharides to the potent antiviral protein griffithsin

Crystallographic studies of the complexes of antiviral protein griffithsin with glucose and N-acetylglucosamine

Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity

The griffithsin dimer is required for highpotency inhibition of HIV-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism

Foodborne transmission of Nipah virus in Syrian hamsters

Degradation of naturally occurring and engineered antimicrobial peptides by proteases

N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry

N-Glycans on the Nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization

Pharmacokinetics of the antiviral lectin griffithsin administered by different routes indicates multiple potential uses

Activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models

Investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate

We thank Tatyana Klimova for assistance with editing the manuscript. We thank Jennifer Wilson (National Cancer Institute) for antiviral activity testing.Disclaimer. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.