Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Rapid in vitro activity of telavancin against Bacillus anthracis and in vivo protection against inhalation anthrax infection in the rabbit model.

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 µg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection.

Inhibition of phosphodiesterase 12 results in antiviral activity against several RNA viruses including SARS-CoV-2.

The 2',5'- oligoadenylate synthetase (OAS) - ribonuclease L (RNAseL) - phosphodiesterase 12 (PDE12) pathway is an essential interferon-induced effector mechanism against RNA virus infection. Inhibition of PDE12 leads to selective amplification of RNAseL activity in infected cells. We aimed to investigate PDE12 as a potential pan-RNA virus antiviral drug target and develop PDE12 inhibitors that elicit antiviral activity against a range of viruses. A library of 18 000 small molecules was screened for PDE12 inhibitor activity using a fluorescent probe specific for PDE12. The lead compounds (CO-17 or CO-63) were tested in cell-based antiviral assays using encephalomyocarditis virus (EMCV), hepatitis C virus (HCV), dengue virus (DENV), West Nile virus (WNV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro. Cross reactivity of PDE12 inhibitors with other PDEs and in vivo toxicity were measured. In EMCV assays, CO-17 potentiated the effect of IFNα by 3 log10. The compounds were selective for PDE12 when tested against a panel of other PDEs and non-toxic at up to 42 mg kg-1 in rats in vivo. Thus, we have identified PDE12 inhibitors (CO-17 and CO-63), and established the principle that inhibitors of PDE12 have antiviral properties. Early studies suggest these PDE12 inhibitors are well tolerated at the therapeutic range, and reduce viral load in studies of DENV, HCV, WNV and SARS-CoV-2 in human cells and WNV in a mouse model.

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence.

UNLABELLED: Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, within vivo pathogenesis often not being correlated within vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. IMPORTANCE: The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a significant role for this sequence in determining the pathogenesis of the virus within the mammalian host. Our studies demonstrate that these chimeras are viable and suggest that such recombinant viruses may be useful for investigation of domain-specific antibody responses and the more extensive definition of the contributions of EIII to the tropism and pathogenesis of WNV or other flaviviruses.

Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs.

Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

Thermodynamic mechanism for the evasion of antibody neutralization in flaviviruses.

Mutations in the epitopes of antigenic proteins can confer viral resistance to antibody-mediated neutralization. However, the fundamental properties that characterize epitope residues and how mutations affect antibody binding to alter virus susceptibility to neutralization remain largely unknown. To address these questions, we used an ensemble-based algorithm to characterize the effects of mutations on the thermodynamics of protein conformational fluctuations. We applied this method to the envelope protein domain III (ED3) of two medically important flaviviruses: West Nile and dengue 2. We determined an intimate relationship between the susceptibility of a residue to thermodynamic perturbations and epitope location. This relationship allows the successful identification of the primary epitopes in each ED3, despite their high sequence and structural similarity. Mutations that allow the ED3 to evade detection by the antibody either increase or decrease conformational fluctuations of the epitopes through local effects or long-range interactions. Spatially distant interactions originate in the redistribution of conformations of the ED3 ensembles, not through a mechanically connected array of contiguous amino acids. These results reconcile previous observations of evasion of neutralization by mutations at a distance from the epitopes. Finally, we established a quantitative correlation between subtle changes in the conformational fluctuations of the epitope and large defects in antibody binding affinity. This correlation suggests that mutations that allow viral growth, while reducing neutralization, do not generate significant structural changes and underscores the importance of protein fluctuations and long-range interactions in the mechanism of antibody-mediated neutralization resistance.

Co-circulation of West Nile virus variants, Arizona, USA, 2010.

Molecular analysis of West Nile virus (WNV) isolates obtained during a 2010 outbreak in Maricopa County, Arizona, USA, demonstrated co-circulation of 3 distinct genetic variants, including strains with novel envelope protein mutations. These results highlight the continuing evolution of WNV in North America and the current complexity of WNV dispersal and transmission.

Comparison of Routes of Administration, Frequency, and Duration of Favipiravir Treatment in Mouse and Guinea Pig Models of Ebola Virus Disease.

Favipiravir is a ribonucleoside analogue that has been explored as a therapeutic for the treatment of Ebola Virus Disease (EVD). Promising data from rodent models has informed nonhuman primate trials, as well as evaluation in patients during the 2013-2016 West African EVD outbreak of favipiravir treatment. However, mixed results from these studies hindered regulatory approval of favipiravir for the indication of EVD. This study examined the influence of route of administration, duration of treatment, and treatment schedule of favipiravir in immune competent mouse and guinea pig models using rodent-adapted Zaire ebolavirus (EBOV). A dose of 300 mg/kg/day of favipiravir with an 8-day treatment was found to be fully effective at preventing lethal EVD-like disease in BALB/c mice regardless of route of administration (oral, intraperitoneal, or subcutaneous) or whether it was provided as a once-daily dose or a twice-daily split dose. Preclinical data generated in guinea pigs demonstrates that an 8-day treatment of 300 mg/kg/day of favipiravir reduces mortality following EBOV challenge regardless of route of treatment or duration of treatments for 8, 11, or 15 days. This work supports the future translational development of favipiravir as an EVD therapeutic.

Vaccine value profile for Chikungunya.

Chikungunya virus (CHIKV) a mosquito-borne alphavirus is the causative agent of Chikungunya (CHIK), a disease with low mortality but high acute and chronic morbidity resulting in a high overall burden of disease. After the acute disease phase, chronic disease including persistent arthralgia is very common, and can cause fatigue and pain that is severe enough to limit normal activities. On average, around 40% of people infected with CHIKV will develop chronic arthritis, which may last for months or years. Recommendations for protection from CHIKV focus on infection control through preventing mosquito proliferation. There is currently no licensed antiviral drug or vaccine against CHIKV. Therefore, one of the most important public health impacts of vaccination would be to decrease burden of disease and economic losses in areas impacted by the virus, and prevent or reduce chronic morbidity associated with CHIK. This benefit would particularly be seen in Low and Middle Income Countries (LMIC) and socio-economically deprived areas, as they are more likely to have more infections and more severe outcomes. This 'Vaccine Value Profile' (VVP) for CHIK is intended to provide a high-level, holistic assessment of the information and data that are currently available to inform the potential public health, economic and societal value of vaccines in the development pipeline and vaccine-like products.This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations. All contributors have extensive expertise on various elements of the CHIK VVP and collectively aimed to identify current research and knowledge gaps.The VVP was developed using only existing and publicly available information.

Molecular evolution of lineage 2 West Nile virus.

Since the 1990s West Nile virus (WNV) has become an increasingly important public health problem and the cause of outbreaks of neurological disease. Genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in New York in 1999 and its neuroinvasive phenotype. Until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. However, recent epidemics of lineage 2 in Europe (Greece and Italy) and Russia have shown the increasing importance of this lineage. There are very few genetic studies examining isolates belonging to lineage 2. We have sequenced the full-length genomes of four older lineage 2 WNV isolates, compared them to 12 previously published genomic sequences and examined the evolution of this lineage. Our studies show that this lineage has evolved over the past 300-400 years and appears to correlate with a change from mouse attenuated to virulent phenotype based on previous studies by our group. This evolution mirrors that which is seen in lineage 1 isolates, which have also evolved to a virulent phenotype over the same period of time.

Complete genome sequence of Koutango virus strain DakAnD5443 isolated from Tatera kempi in 1968.

Koutango virus (KOUV), a close relative of West Nile virus, is highly neuroinvasive in animal models and has been associated with human disease. The complete genome of the KOUV prototype strain DakAnD5443 is reported here and may facilitate development of infectious clones for further characterization of this novel flavivirus.

Baseline mapping of Oropouche virology, epidemiology, therapeutics, and vaccine research and development.

Oropouche virus (OROV) is an arthropod-borne orthobunyavirus found in South America and causes Oropouche fever, a febrile infection similar to dengue. It is the second most prevalent arthropod-borne viral disease in South America after dengue. Over 500,000 cases have been diagnosed since the virus was first discovered in 1955; however, this is likely a significant underestimate given the limited availability of diagnostics. No fatalities have been reported to date, however, up to 60% of cases have a recurrent phase of disease within one month of recovery from the primary disease course. The main arthropod vector is the biting midge Culicoides paraensis, which has a geographic range as far north as the United States and demonstrates the potential for OROV to geographically expand. The transmission cycle is incompletely understood and vertebrate hosts include both non-human primates and birds further supporting the potential ability of the virus to spread. A number of candidate antivirals have been evaluated against OROV in vitro but none showed antiviral activity. Surprisingly, there is only one report in the literature on candidate vaccines. We suggest that OROV is an undervalued pathogen much like chikungunya, Schmallenberg, and Zika viruses were before they emerged. Overall, OROV is an important emerging disease that has been under-investigated and has the potential to cause large epidemics in the future. Further research, in particular candidate vaccines, is needed for this important pathogen.

Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate.

The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.

Persistent infection with West Nile virus years after initial infection.

West Nile virus (WNV) RNA was demonstrated in 5 (20%) of 25 urine samples collected from convalescent patients 573-2452 days (1.6-6.7 years) after WNV infection. Four of the 5 amplicons sequenced showed >99% homology to the WNV NY99 strain. These findings show that individuals with chronic symptoms after WNV infection may have persistent renal infection over several years.

In Vivo Activity of Repurposed Amodiaquine as a Host-Targeting Therapy for the Treatment of Anthrax.

Anthrax is caused by Bacillus anthracis and can result in nearly 100% mortality due in part to anthrax toxin. Antimalarial amodiaquine (AQ) acts as a host-oriented inhibitor of anthrax toxin endocytosis. Here, we determined the pharmacokinetics and safety of AQ in mice, rabbits, and humans as well as the efficacy in the fly, mouse, and rabbit models of anthrax infection. In the therapeutic-intervention studies, AQ nearly doubled the survival of mice infected subcutaneously with a B. anthracis dose lethal to 60% of the animals (LD60). In rabbits challenged with 200 LD50 of aerosolized B. anthracis, AQ as a monotherapy delayed death, doubled the survival rate of infected animals that received a suboptimal amount of antibacterial levofloxacin, and reduced bacteremia and toxemia in tissues. Surprisingly, the anthrax efficacy of AQ relies on an additional host macrophage-directed antibacterial mechanism, which was validated in the toxin-independent Drosophila model of Bacillus infection. Lastly, a systematic literature review of the safety and pharmacokinetics of AQ in humans from over 2 000 published articles revealed that AQ is likely safe when taken as prescribed, and its pharmacokinetics predicts anthrax efficacy in humans. Our results support the future examination of AQ as adjunctive therapy for the prophylactic anthrax treatment.

Erratum: Publisher Correction: New international guidance on quality, safety and efficacy of DNA vaccines.

[This corrects the article DOI: 10.1038/s41541-020-0199-0.].

Long-term, West Nile virus-induced neurological changes: A comparison of patients and rodent models.

West Nile virus (WNV) is a mosquito-borne virus that can cause severe neurological disease in those infected. Those surviving infection often present with long-lasting neurological changes that can severely impede their lives. The most common reported symptoms are depression, memory loss, and motor dysfunction. These sequelae can persist for the rest of the patients' lives. The pathogenesis behind these changes is still being determined. Here, we summarize current findings in human cases and rodent models, and discuss how these findings indicate that WNV induces a state in the brain similar neurodegenerative diseases. Rodent models have shown that infection leads to persistent virus and inflammation. Initial infection in the hippocampus leads to neuronal dysfunction, synapse elimination, and astrocytosis, all of which contribute to memory loss, mimicking findings in neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). WNV infection acts on pathways, such as ubiquitin-signaled protein degradation, and induces the production of molecules, including IL-1ß, IFN-γ, and α-synuclein, that are associated with neurodegenerative diseases. These findings indicate that WNV induces neurological damage through similar mechanisms as neurodegenerative diseases, and that pursuing research into the similarities will help advance our understanding of the pathogenesis of WNV-induced neurological sequelae.

Baseline mapping of severe fever with thrombocytopenia syndrome virology, epidemiology and vaccine research and development.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emergent tick-borne bunyavirus first discovered in 2009 in China. SFTSV is a growing public health problem that may become more prominent owing to multiple competent tick-vectors and the expansion of human populations in areas where the vectors are found. Although tick-vectors of SFTSV are found in a wide geographic area, SFTS cases have only been reported from China, South Korea, Vietnam, and Japan. Patients with SFTS often present with high fever, leukopenia, and thrombocytopenia, and in some cases, symptoms can progress to severe outcomes, including hemorrhagic disease. Reported SFTSV case fatality rates range from ~5 to >30% depending on the region surveyed, with more severe disease reported in older individuals. Currently, treatment options for this viral infection remain mostly supportive as there are no licensed vaccines available and research is in the discovery stage. Animal models for SFTSV appear to recapitulate many facets of human disease, although none of the models mirror all clinical manifestations. There are insufficient data available on basic immunologic responses, the immune correlate(s) of protection, and the determinants of severe disease by SFTSV and related viruses. Many aspects of SFTSV virology and epidemiology are not fully understood, including a detailed understanding of the annual numbers of cases and the vertebrate host of the virus, so additional research on this disease is essential towards the development of vaccines and therapeutics.

Teixobactin Provides Protection against Inhalation Anthrax in the Rabbit Model.

The use of antibiotics is a vital means of treating infections caused by the bacteria Bacillus (B.) anthracis. Importantly, with the potential future use of multidrug-resistant strains of B. anthracis as bioweapons, new antibiotics are needed as alternative therapeutics. In this blinded study, we assessed the protective efficacy of teixobactin, a recently discovered antibiotic, against inhalation anthrax infection in the adult rabbit model. New Zealand White rabbits were infected with a lethal dose of B. anthracis Ames spores via the inhalation route, and blood samples were collected at various times to assess antigenemia, bacteremia, tissue bacterial load, and antibody production. Treatments were administered upon detection of B. anthracis protective antigen in the animals' sera. For comparison, a fully protective dose of levofloxacin was used as a positive control. Rabbits treated with teixobactin showed 100% survival following infection, and the bacteremia was completely resolved by 24-48 h post-treatment. In addition, the bacterial/spore loads in tissues of the animals treated with teixobactin were either zero or dramatically less relative to that of the negative control animals. Moreover, microscopic evaluation of the tissues revealed decreased pathology following treatment with teixobactin. Overall, these results show that teixobactin was protective against inhalation anthrax infection in the rabbit model, and they indicate the potential of teixobactin as a therapeutic for the disease.