In Vitro Antiviral Screening for Henipaviruses at BSL4.

In vitro screening for antivirals is an essential step in the development of effective treatments against new and emerging pathogens. Here, we describe a simple, cell-based screening assay for evaluating antiviral effectiveness against Hendra and Nipah live virus infection under BSL4 conditions.

A systematic review of invasive pneumococcal disease vaccine failures and breakthrough with higher-valency pneumococcal conjugate vaccines in children.

INTRODUCTION: The pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV/PCV10) and 13-valent pneumococcal conjugate vaccine (PCV13) protect against vaccine-serotype invasive pneumococcal disease (VT IPD). However, VT IPD can still occur in fully or partially vaccinated children (vaccine failure or breakthrough). We performed a systematic review of vaccine failures and breakthrough IPD with PCV10 and PCV13 in ≤5-year-olds. AREAS COVERED: We searched Scopus/Medline/EMBASE to retrieve articles/abstracts published between 1/2008-7/2019. We excluded reports only including data from ≥6-year-olds, exclusively assessing PCV7-vaccinated children or children with comorbidities. Twenty-six reports (20 PCV13, 1 PCV10, 5 both), covering studies with various designs in six continents, using different schedules, were included. Collectively, they reported 469 VT IPD cases classified as vaccine failures and 403 as breakthrough. Vaccine failure and breakthrough rates were low: 8.4% and 9.3%, respectively, of all IPD in vaccinated children, consistent with the vaccines' high effectiveness. The main serotypes associated with vaccine failure/breakthrough were 19A, 3 and 19F for PCV13 and 14, 6B and vaccine-related 19A and 6A for PCV10. EXPERT OPINION: As we move to vaccines with more serotypes, it is not only important to consider which serotypes are added, but also monitor and address incomplete protection against specific serotypes.

PLAIN LANGUAGE SUMMARYWhat is the context?Pneumococcal conjugate vaccines have been given to children for over 20 years to prevent infections caused by the bacterium Streptococcus pneumoniae (such as pneumonia, meningitis and sepsis).At least 100 different types of S. pneumoniae, so called serotypes, exist, but a relatively small number causes most disease.Two current vaccines (Synflorix, GSK and Prevnar 13, Pfizer) protect against 10 to 13 serotypes and have significantly reduced pneumococcal disease caused by these serotypes.A rise in serotypes not targeted by these vaccines has lessened the vaccines' expected impact.As no vaccine is 100% protective, some serotypes targeted by the current vaccines continue to circulate.What is new?We performed a systematic literature review to evaluate which serotypes are most often associated with invasive disease occurring after receives all planned pneumococcal vaccine doses (vaccine failure) or after a child receives part of the planned vaccine doses (breakthrough).We found that vaccine failures and breakthrough disease were uncommon with both vaccines, irrespective of the administered schedule.A small number of serotypes were responsible for most vaccine failures and breakthrough disease with both vaccines.What is the impact?The low rate of vaccine failures and breakthrough disease observed with the current vaccines confirms their high effectiveness in preventing pneumococcal disease.The primary consideration in developing pneumococcal conjugate vaccines that include more than 13 serotypes will be how additional protection they can provide.Reduced protection against individual serotypes remains a risk.The evaluation of current vaccines demonstrates that incomplete protection against specific serotypes should also be addressed.

A systematic review of the burden of pertussis in South Korea.

This review summarizes the published data on epidemiology and burden of pertussis in South Korea as these may be under-categorized. A systematic literature review of PubMed, SCOPUS, EMBASE and KMBASE was performed to identify published literature in South Korea since 2000. Pertussis detection rates among 19 eligible studies range from 0.7% to 100% across different age groups, detection methods and study settings. Highest rates are observed in infants, while adolescents and adults with pertussis infection may suffer from persistent coughing. Vaccination uptake of pertussis booster dose among adolescents and adults remains low while seropositivity (detection of anti-pertussis immunoglobulin G), is high among adults. This review reveals a high burden of vaccine-preventable pertussis in South Korea. Besides primary childhood vaccination, strategies like maternal immunization and decennial revaccination of adults should be considered. Active testing, reporting and better utilization of vaccine registries may provide insights for decision-makers nationwide.

A Cost-Effectiveness Analysis of the 10-Valent Pneumococcal Non-Typeable Haemophilus influenzae Protein D Conjugate Vaccine (PHiD-CV) Compared to the 13-Valent Pneumococcal Conjugate Vaccine (PCV13) for Universal Mass Vaccination Implementation in New Zealand.

OBJECTIVES: Invasive pneumococcal disease (IPD), pneumonia and acute otitis media (AOM) still represent a significant medical burden in children < 5 years of age in New Zealand (NZ), with marked disparities across socio-economic and ethnic groups. This cost-effectiveness evaluation aims to compare the potential impact of two childhood universal immunisation strategies: vaccination with a 3 + 1 schedule of the 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV, Synflorix, GSK) and the 13-valent pneumococcal conjugate vaccine (PCV13, Prevenar 13, Pfizer). METHODS: A static Markov-process cohort model was used to simulate the epidemiological and economic burden of pneumococcal diseases on a single-birth cohort over its lifetime. Costs and outcomes were discounted annually at 3.5%. Epidemiological and cost inputs were extracted from the most recently available NZ data, or derived from the most relevant reference countries' sources. The most updated evidence on the efficacies of the corresponding vaccines were used, particularly the significant effectiveness for PHiD-CV against IPD caused by serotype 19A. RESULTS: The model estimated that both vaccines have a broadly comparable impact on IPD-related diseases and pneumonia. Due to the additional benefits possible through broader impact on AOM, PHiD-CV is estimated to potentially provide additional discounted cost offsets of approximately NZD 0.8 million over the lifetime of the birth cohort. CONCLUSIONS: To ensure health equity in children, given the substantial burden of pneumonia and AOM, decision-makers should also take into account the impact of PCVs on these diseases for decisions relating to routine infant immunization. GSK STUDY IDENTIFIER: HO-15-16775.

Literature review of the epidemiology of influenza B disease in 15 countries in the Asia-Pacific region.

Influenza control strategies focus on the use of trivalent influenza vaccines containing two influenza A virus subtypes and one of the two circulating influenza type B lineages (Yamagata or Victoria). Mismatches between the vaccine B lineage and the circulating lineage have been regularly documented in many countries, including those in the Asia-Pacific region. We conducted a literature review with the aim of understanding the relative circulation of influenza B viruses in Asia-Pacific countries. PubMed and Western Pacific Region Index Medicus were searched for relevant articles on influenza type B published since 1990 in English language for 15 Asia-Pacific countries. Gray literature was also accessed. From 4834 articles identified, 121 full-text articles were analyzed. Influenza was reported as an important cause of morbidity in the Asia-Pacific region, affecting all age groups. In all 15 countries, influenza B was identified and associated with between 0% and 92% of laboratory-confirmed influenza cases in any one season/year. Influenza type B appeared to cause more illness in children aged between 1 and 10 years than in other age groups. Epidemiological data for the two circulating influenza type B lineages remain limited in several countries in the Asia-Pacific, although the co-circulation of both lineages was seen in countries where strain surveillance data were available. Mismatches between circulating B lineages and vaccine strains were observed in all countries with available data. The data suggest that a shift from trivalent to quadrivalent seasonal influenza vaccines could provide additional benefits by providing broader protection.

Anti-hepatitis B activity of isoquinoline alkaloids of plant origin.

Hepatitis B virus (HBV) is the causative agent of B-type hepatitis in humans, a vaccine-preventable disease. Despite the availability of effective vaccines, globally, 2 billion people show evidence of past or current HBV infection, of which 350 million people are persistently infected, with an estimated annual increase of 1 million. There is no cure for chronic HBV infections, which are associated with cirrhotic liver failure and with an increased risk of developing hepatocellular carcinoma. Hepatitis antiviral research has focused primarily on the development of inhibitors of viral polymerase through the use of nucleoside analogues. Therefore, there is an urgent need for the development of non-nucleoside compounds to be used as an alternative or to complement the current therapy. To address this need, 18 isoquinoline alkaloids were evaluated for their potential antiviral activity against HBV in vitro.

Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors.

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Off Label Antiviral Therapeutics for Henipaviruses: New Light Through Old Windows.

Hendra and Nipah viruses are recently emerged zoonotic paramyxoviruses for which there is no vaccine or protective therapy available. While a number of experimental therapeutics and vaccines have recently been reported, all of these will require lengthy approval processes, limiting their usefulness in the short term. To address the urgent need for henipavirus therapeutics, a number of currently licensed pharmaceuticals have been evaluated for off label efficacy against henipavirus replication in vitro. Initially it was observed that compounds which released intracellular calcium stores induced a potent inhibition of henipaviruses replication, prompting the evaluation of known drugs with a similar effect on calcium mobilisation. Of the eight compounds randomly selected based on existing literature, seven inhibited virus replication in the micromolar range while the remaining compound also inhibited virus replication but only at millimolar concentrations. Pretreatment experiments with various calcium chelators, channel antagonists or endoplasmic reticulum release inhibitors supported a calcium mediated mechanism of action for five of these compounds. The mechanism of antiviral action for the remaining three compounds is currently unknown. Additionally, a number of other modulators of calcium flux, including calcium channel and calmodulin antagonists also exhibited potent antiviral activity in vitro providing a broad range of potential therapeutic options for the treatment of henipavirus infections. Importantly, as many of these compounds are currently licensed drugs, regulatory approval should be a much more streamlined process, with the caveat that appropriate in vivo efficacy can be demonstrated in animal models.

A novel L-ficolin/mannose-binding lectin chimeric molecule with enhanced activity against Ebola virus.

Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.

Characterization of the antiviral and inflammatory responses against Nipah virus in endothelial cells and neurons.

Nipah virus (NiV) is a highly pathogenic paramyxovirus which causes fatal encephalitis in up to 75% of infected humans. Endothelial cells and neurons are important cellular targets in the pathogenesis of this disease. In this study, viral replication and the innate immune responses to NiV in these cell types were measured. NiV infected endothelial cells generated a functionally robust IFN-beta response, which correlated with localization of the NiV W protein to the cytoplasm. There was no antiviral response detected in infected neuronal cells. NiV infection of endothelial cells induced a significant increase in secreted inflammatory chemokines, which corresponded with the increased ability of infected cell supernatants to induce monocyte and T-lymphocyte chemotaxis. These results suggest that pro-inflammatory chemokines produced by NiV infected primary endothelial cells in vitro is consistent with the prominent vasculitis observed in infections, and provide initial molecular insights into the pathogenesis of NiV in physiologically relevant cells types.

Antiviral activity of gliotoxin, gentian violet and brilliant green against Nipah and Hendra virus in vitro.

BACKGROUND: Using a recently described monolayer assay amenable to high throughput screening format for the identification of potential Nipah virus and Hendra virus antivirals, we have partially screened a low molecular weight compound library (>8,000 compounds) directly against live virus infection and identified twenty eight promising lead molecules. Initial single blind screens were conducted with 10 microM compound in triplicate with a minimum efficacy of 90% required for lead selection. Lead compounds were then further characterised to determine the median efficacy (IC50), cytotoxicity (CC50) and the in vitro therapeutic index in live virus and pseudotype assay formats. RESULTS: While a number of leads were identified, the current work describes three commercially available compounds: brilliant green, gentian violet and gliotoxin, identified as having potent antiviral activity against Nipah and Hendra virus. Similar efficacy was observed against pseudotyped Nipah and Hendra virus, vesicular stomatitis virus and human parainfluenza virus type 3 while only gliotoxin inhibited an influenza A virus suggesting a non-specific, broad spectrum activity for this compound. CONCLUSION: All three of these compounds have been used previously for various aspects of anti-bacterial and anti-fungal therapy and the current results suggest that while unsuitable for internal administration, they may be amenable to topical antiviral applications, or as disinfectants and provide excellent positive controls for future studies.

Characteristics of Nipah virus and Hendra virus replication in different cell lines and their suitability for antiviral screening.

We have recently described the development and validation of a high throughput screening assay suitable for henipavirus antiviral identification. While we are confident this assay is robust and effective, we wished to investigate assay performance in a range of alternative cell lines to determine if assay sensitivity and specificity could be improved. We evaluated ten different cell lines for their susceptibility to Hendra and Nipah virus infection and their sensitivity of detection of the effects of the broad spectrum antiviral, ribavirin and nine novel antivirals identified using our initial screening approach. Cell lines were grouped into three categories with respect to viral replication. Virus replicated best in Vero and BSR cells, followed by Hep-2, HeLa, BHK-21 and M17 cells. The lowest levels of RNA replication and viral protein expression were observed in BAEC, MMEC, A549 and ECV304 cells. Eight cell lines appeared to be similarly effective at discriminating the antiviral effects of ribavirin (<2.7-fold difference). The two cells lines most sensitive to the effect of ribavirin (ECV304 and BAEC) also displayed the lowest levels of viral replication while Vero cells were the least sensitive suggesting excess viral replication may limit drug efficacy and cell lines which limit viral replication may result in enhanced antiviral efficacy. However, there was no consistent trend observed with the other nine antivirals tested. While improvements in antiviral sensitivity in other cell lines may indicate an important role in future HTS assays, the slightly lower sensitivity to antiviral detection in Vero cells has inherent advantages in reducing the number of partially effective lead molecules identified during initial screens. Comparison of a panel of 54 novel antiviral compounds identified during routine screening of an in-house compound library in Vero, BHK-21 and BSR cells suggests no clear advantage of screening in either cell type.

Human parainfluenza virus infection of the airway epithelium: viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity.

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN's receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN's F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN's F-triggering activity may have promise in vivo.

Kinetic dependence of paramyxovirus entry inhibition.

Peptides derived from conserved heptad repeat (HR) regions of paramyxovirus fusion (F) proteins inhibit viral fusion by interfering with the formation of the fusogenic six-helix bundle structure. Peptide efficacy is affected by the strength of the peptide association with the target virus's complementary HR region. Here, we show that a second basis for peptide efficacy lies in the kinetics of F activation by the homotypic attachment protein: efficient F activation by the attachment protein shortens the period during which antiviral molecules targeting intermediate states of F may act, thereby modulating the effectiveness of inhibitory peptides. These results highlight new issues to be considered in developing strategies for fusion inhibitors.

Simulating henipavirus multicycle replication in a screening assay leads to identification of a promising candidate for therapy.

Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic paramyxoviruses that cause encephalitis in humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a screen for antivirals for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. The screening of a small compound library identified several effective molecules, including the well-known compound chloroquine, as highly active inhibitors of pseudotyped virus infection. Chloroquine inhibited infection with live HeV and NiV at a concentration of 1 microM in vitro (50% inhibitory concentration, 2 microM), which is less than the plasma concentrations present in humans receiving chloroquine treatment for malaria. The mechanism for chloroquine's antiviral action likely is the inhibition of cathepsin L, a cellular enzyme that is essential for the processing of the viral fusion glycoprotein and the maturation of newly budding virions. Without this processing step, virions are not infectious. The identification of a compound that inhibits a known cellular target that is important for viral maturation but that had not previously been shown to have antiviral activity for henipaviruses highlights the validity of this new screening assay. Given the established safety profile and broad experience with chloroquine in humans, the results described here provide an option for treating individuals infected by these deadly viruses.

Determination of the henipavirus phosphoprotein gene mRNA editing frequencies and detection of the C, V and W proteins of Nipah virus in virus-infected cells.

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic zoonotic paramyxoviruses. Like many other paramyxoviruses, henipaviruses employ a process of co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternate reading frame. Sequence analysis of multiple, cloned mRNAs showed that the mRNA editing frequencies of the P genes of the henipaviruses are higher than those reported for other paramyxoviruses. Antisera to synthetic peptides from the P, V, W and C proteins of NiV were generated to study their expression in infected cells. All proteins were detected in both infected cells and purified virions. In infected cells, the W protein was detected in the nucleus while P, V and C were found in the cytoplasm.

Inhibition of Henipavirus infection by RNA interference.

Nipah virus (NiV) and Hendra virus (HeV) are recently emerged zoonotic paramyxoviruses exclusively grouped within a new genus, Henipavirus. These viruses cause fatal disease in a wide range of species, including humans. Both NiV and HeV have continued to re-emerge sporadically in Bangladesh and Australia, respectively. There are currently no therapeutics or vaccines available to treat Henipavirus infection and both are classified as BSL4 pathogens. RNA interference (RNAi) is a process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells. Small interfering RNAs (siRNAs) mediate RNAi by inhibiting gene expression of homologous mRNA and our preliminary studies suggest RNAi may be a useful approach to developing novel therapies for these highly lethal pathogens. Eight NiV siRNA molecules (four L and four N gene specific), two HeV N gene specific, and two non-specific control siRNA molecules were designed and tested for their ability to inhibit a henipavirus minigenome replication system (which does not require the use of live virus) in addition to live virus infections in vitro. In the minigenome assay three out of the four siRNAs that targeted the L gene of NiV effectively inhibited replication. In contrast, only NiV N gene siRNAs were effective in reducing live NiV replication, suggesting inhibition of early, abundantly expressed gene transcripts may be more effective than later, less abundant transcripts. Additionally, some of the siRNAs effective against NiV infection were only partially effective inhibitors of HeV infection. An inverse correlation between the number of nucleotide mismatches and the efficacy of siRNA inhibition was observed. The demonstration that RNAi effectively inhibits henipavirus replication in vitro, is a novel approach and may provide an effective therapy for these highly lethal, zoonotic pathogens.

Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus.

There are currently no antiviral drugs approved for the highly lethal Biosafety Level 4 pathogens Nipah and Hendra virus. A number of researchers are developing surrogate assays amenable to Biosafety Level 2 biocontainment but ultimately, the development of a high throughput screening method for directly quantifying these viruses in a Biosafety Level 4 environment will be critical for final evaluation of antiviral drugs identified in surrogate assays, in addition to reducing the time required for effective antiviral drug development. By adapting an existing immunoplaque assay and using enzyme linked immunodetection in a microtitre plate format, the current experiments describe a simple two step assay protocol involving an overnight virus inoculation of Vero cell monolayers (with or without antiviral drug treatment) at Biosafety Level 4, followed by cell fixation and virus inactivation enabling removal of plates from the Biosafety Level 4 laboratory and a subsequent immunodetection assay using a chemiluminescent horse radish peroxidase substrate to be performed at Biosafety Level 2. The analytical sensitivity (limit of detection) of this assay is 100 tissue culture infectious dose50/ml of either Nipah or Hendra virus. In addition this assay enables linear quantitation of virus over three orders of magnitude and is unaffected by dimethyl sulfoxide concentrations of 1% or less. Intra-assay coefficients of variation are acceptable (less than 20%) when detecting a minimum of 1000 tissue culture infectious dose50/ml of either virus although inter-assay variation is considerably greater. By an assessment of efficacies of the broad spectrum antiviral Ribavirin and an experimental fusion inhibitory peptide, this assay reveals a good correlation with previously published fluorescent immunodetection assays. The current experiments describe for the first time, a high throughput screening method amenable for direct assessment of live henipavirus antiviral drug activity.