Showing papers by "Sina Bavari published in 2017"

Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses

Abstract: The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1′-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3–14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [Nature 2016, 531, 381−385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.

Taxonomy of the order Mononegavirales: update 2017

Abstract: In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys.

Abstract: Ebola virus (EBOV) persistence in asymptomatic humans and Ebola virus disease (EVD) sequelae have emerged as significant public health concerns since the 2013-2016 EVD outbreak in Western Africa. Until now, studying how EBOV disseminates into and persists in immune-privileged sites was impossible due to the absence of a suitable animal model. Here, we detect persistent EBOV replication coinciding with systematic inflammatory responses in otherwise asymptomatic rhesus monkeys that had survived infection in the absence of or after treatment with candidate medical countermeasures. We document progressive EBOV dissemination into the eyes, brain and testes through vascular structures, similar to observations in humans. We identify CD68+ cells (macrophages/monocytes) as the cryptic EBOV reservoir cells in the vitreous humour and its immediately adjacent tissue, in the tubular lumina of the epididymides, and in foci of histiocytic inflammation in the brain, but not in organs typically affected during acute infection. In conclusion, our data suggest that persistent EBOV infection in rhesus monkeys could serve as a model for persistent EBOV infection in humans, and we demonstrate that promising candidate medical countermeasures may not completely clear EBOV infection. A rhesus monkey model may lay the foundation to study EVD sequelae and to develop therapies to abolish EBOV persistence.

Neuropathogenesis of Zika Virus in a Highly Susceptible Immunocompetent Mouse Model after Antibody Blockade of Type I Interferon.

Abstract: Animal models are needed to better understand the pathogenic mechanisms of Zika virus (ZIKV) and to evaluate candidate medical countermeasures. Adult mice infected with ZIKV develop a transient viremia, but do not demonstrate signs of morbidity or mortality. Mice deficient in type I or a combination of type I and type II interferon (IFN) responses are highly susceptible to ZIKV infection; however, the absence of a competent immune system limits their usefulness for studying medical countermeasures. Here we employ a murine model for ZIKV using wild-type C57BL/6 mice treated with an antibody to disrupt type I IFN signaling to study ZIKV pathogenesis. We observed 40% mortality in antibody treated mice exposed to ZIKV subcutaneously whereas mice exposed by intraperitoneal inoculation were highly susceptible incurring 100% mortality. Mice infected by both exposure routes experienced weight loss, high viremia, and severe neuropathologic changes. The most significant histopathological findings occurred in the central nervous system where lesions represent an acute to subacute encephalitis/encephalomyelitis that is characterized by neuronal death, astrogliosis, microgliosis, scattered necrotic cellular debris, and inflammatory cell infiltrates. This model of ZIKV pathogenesis will be valuable for evaluating medical countermeasures and the pathogenic mechanisms of ZIKV because it allows immune responses to be elicited in immunologically competent mice with IFN I blockade only induced at the time of infection.

High Infection Rates for Adult Macaques after Intravaginal or Intrarectal Inoculation with Zika Virus.

Abstract: Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia in 75% of macaques and virus RNA in 100%, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present.

African and asian zika virus isolates display phenotypic differences both in vitro and in vivo

Abstract: Zika virus (ZIKV) is a mosquito-borne member of the Spondweni complex of the genus Flavivirus, family Flaviviridae. The genus Flavivirus comprises many important human pathogens such as Saint Louis encephalitis, yellow fever virus, dengue virus (DENV1–DENV4), West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus.1 The ZIKV genome comprises a single-strand, positive-sense RNA of approximately 11 kb in length. The genome consists of a 5′ untranslated region (UTR) (∼107 nt), an open reading frame (ORF) ∼10.2 kb in length, and a 3′ UTR (∼428 nt). The single ORF encodes three structural proteins: capsid (C), premembrane/membrane (prM), envelope (E), and seven non–structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).1 Similar to other flaviviruses, ZIKV enters cells through receptor-mediated endocytosis, and the low endosomal pH induces fusion to release the genomic RNA into the cytoplasm. The genomic RNA is translated into a single polyprotein that is processed to facilitate replication, virion assembly, and release of mature virions. The mature ZIKV virion structure is similar to that of other flaviviruses, comprising a nucleocapsid core consisting of C protein and genomic RNA and an icosahedral shell consisting of 180 copies of E and M proteins (or prM) embedded in a host-derived lipid bilayer.2 ZIKV was first isolated from the blood of a sentinel rhesus macaque in 1947 in the Zika Forest in Uganda.3 In the following year, ZIKV was isolated from pools of Aedes africanus mosquitoes, and subsequent studies demonstrated virus transmission by Aedes aegypti to both mice and rhesus macaques.4 In the following decades (1950s–1980s), ZIKV was isolated from humans and multiple mosquito species in sub-Saharan Africa and Southeast Asia, and suggesting maintenance in both sylvatic (primarily Aedes spp. mosquitoes and nonhuman primates [NHPs]) and urban cycles (mainly A. aegypti and humans).5–19 ZIKV consists of a single serotype, with isolates comprising two geographic lineages (African and Asian) that have caused sporadic or underreported human outbreaks.20,21 However, starting in 2007, outbreaks were reported in the Yap island in the Federated States of Micronesia and in Gabon, and by 2013 ZIKV was imported from French Polynesia into northeast Brazil resulting in the largest reported epidemic to date and its subsequent expansion to many parts of Americas including mainland USA (Florida and Texas).22–27 Since the start of the 2007 epidemic, ZIKV has spread to or been detected in 84 countries and territories in the Americas, Asia, and Africa, with reported evidence of autochthonous transmission (http://apps.who.int/iris/bitstream/10665/254714/1/zikasitrep10Mar17-eng.pdf?ua=1). Most human ZIKV infections are asymptomatic.28,29 Symptomatic infections typically present as a self-limiting acute febrile illness with symptoms ranging from fever, headache, myalgia, and rash; however, severe clinical manifestations including congenital microcephaly and other fetal defects, Guillain–Barre syndrome (GBS), and death have been reported.30,31 Although ZIKV is transmitted primarily through mosquito/human cycle, the virus can also be transmitted through sexual contact and blood transfusions.32–34 Before its arrival in the Americas, ZIKV was an obscure and understudied pathogen; as a consequence, few virus isolates were accessible for research and vaccine/therapeutic development. In addition, little or no basic in vitro and/or in vivo characterization data were available on accessible isolates. In the present study, we investigated the potential phenotypic differences between low-passage African (ArD 41525) and Asian isolates (CPC-0740 and SV0127-14) in vitro and in vivo.

Discovering drugs for the treatment of Ebola virus

Abstract: Ebola virus, a member of the Filoviridae family, is a causative agent of severe viral hemorrhagic fever in humans. Over the past 40 years, the virus has been linked to several high mortality outbreaks in Africa with the recent West African outbreak resulting in over 11,000 deaths. This review provides a summary of the status of the drug discovery and development process for therapeutics for Ebola virus disease, with a focus on the strategies being used and the challenges facing each stage of the process. Despite the wealth of in vitro efficacy data, preclinical data in animal models, and human clinical data, no therapeutics have been approved for the treatment of Ebola virus disease. However, several promising candidates, such as ZMapp and GS-5734, have advanced into ongoing clinical trials. The gravity of the 2014-2016 outbreak spurred a heightened effort to identify and develop new treatments for Ebola virus disease, including small molecules, immunotherapeutics, host factors, and clinical disease management options. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endoresed by the U.S. Army.

Will There Be a Cure for Ebola

Abstract: Despite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug Administration. However, multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk–pathogen setting. Numerous therapeutics were explored or developed during the outbreak, including repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir, favipiravir, and GS-5734), nucleic acid–based drugs (TKM-Ebola and AVI-7537), and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics progress in the aftermath of the West Africa Ebola virus outbreak and attempt to offer a glimpse of a path forward.

Deubiquitinating enzyme VCIP135 dictates the duration of botulinum neurotoxin type A intoxication

Abstract: Botulism is characterized by flaccid paralysis, which can be caused by intoxication with any of the seven known serotypes of botulinum neurotoxin (BoNT), all of which disrupt synaptic transmission by endoproteolytic cleavage of SNARE proteins. BoNT serotype A (BoNT/A) has the most prolonged or persistent effects, which can last several months, and exerts its effects by specifically cleaving and inactivating SNAP25. A major factor contributing to the persistence of intoxication is the long half-life of the catalytic light chain, which remains enzymatically active months after entry into cells. Here we report that BoNT/A catalytic light chain binds to, and is a substrate for, the ubiquitin ligase HECTD2. However, the light chain evades proteasomal degradation by the dominant effect of a deubiquitinating enzyme, VCIP135/VCPIP1. This deubiquitinating enzyme binds BoNT/A light chain directly, with the two associating in cells through the C-terminal 77 amino acids of the light chain protease. The development of specific DUB inhibitors, together with inhibitors of BoNT/A proteolytic activity, may be useful for reducing the morbidity and public health costs associated with BoNT/A intoxication and could have potential biodefense implications.

Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1

Abstract: A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs.IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires.

Abstract: The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from VDJ recombination and somatic hypermutation (SHM) during B cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable CDR3 region, which plays a critical role in antigen-specificity and binding affinity. Tracking the selection and mutation of the CDR3 region can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present BRILIA (B-cell repertoire inductive lineage and immunosequence annotator), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94% and 70% accuracy, respectively, and it has a 90% SHM positive prediction rate in the CDR3 region of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B cell clusters and yielded inconsistent SHM frequencies. We demonstrate BRILIA’s utility in B cell repertoire studies related to VDJ gene usage, mechanisms for adenosine mutations, and SHM hot spot motifs. Furthermore, we show that the complete gene usage annotation and SHM identification across the entire CDR3 are essential for studying the B cell affinity maturation process through immunosequencing methods.

Implementation of Objective PASC-Derived Taxon Demarcation Criteria for Official Classification of Filoviruses.

Abstract: The mononegaviral family Filoviridae has eight members assigned to three genera and seven species. Until now, genus and species demarcation were based on arbitrarily chosen filovirus genome sequence divergence values (≈50% for genera, ≈30% for species) and arbitrarily chosen phenotypic virus or virion characteristics. Here we report filovirus genome sequence-based taxon demarcation criteria using the publicly accessible PAirwise Sequencing Comparison (PASC) tool of the US National Center for Biotechnology Information (Bethesda, MD, USA). Comparison of all available filovirus genomes in GenBank using PASC revealed optimal genus demarcation at the 55–58% sequence diversity threshold range for genera and at the 23–36% sequence diversity threshold range for species. Because these thresholds do not change the current official filovirus classification, these values are now implemented as filovirus taxon demarcation criteria that may solely be used for filovirus classification in case additional data are absent. A near-complete, coding-complete, or complete filovirus genome sequence will now be required to allow official classification of any novel “filovirus.” Classification of filoviruses into existing taxa or determining the need for novel taxa is now straightforward and could even become automated using a presented algorithm/flowchart rooted in RefSeq (type) sequences.

Sorafenib Impedes Rift Valley Fever Virus Egress by Inhibiting Valosin-Containing Protein Function in the Cellular Secretory Pathway.

Abstract: There is an urgent need for therapeutic development to combat infections caused by Rift Valley fever virus (RVFV), which causes devastating disease in both humans and animals. In an effort to repurpose drugs for RVFV treatment, our previous studies screened a library of FDA-approved drugs. The most promising candidate identified was the hepatocellular and renal cell carcinoma drug sorafenib. Mechanism-of-action studies indicated that sorafenib targeted a late stage in virus infection and caused a buildup of virions within cells. In addition, small interfering RNA (siRNA) knockdown studies suggested that nonclassical targets of sorafenib are important for the propagation of RVFV. Here we extend our previous findings to identify the mechanism by which sorafenib inhibits the release of RVFV virions from the cell. Confocal microscopy imaging revealed that glycoprotein Gn colocalizes and accumulates within the endoplasmic reticulum (ER) and the transport of Gn from the Golgi complex to the host cell membrane is reduced. Transmission electron microscopy demonstrated that sorafenib caused virions to be present inside large vacuoles inside the cells. p97/valosin-containing protein (VCP), which is involved in membrane remodeling in the secretory pathway and a known target of sorafenib, was found to be important for RVFV egress. Knockdown of VCP resulted in decreased RVFV replication, reduced Gn Golgi complex localization, and increased Gn ER accumulation. The intracellular accumulation of RVFV virions was also observed in cells transfected with siRNA targeting VCP. Collectively, these data indicate that sorafenib causes a disruption in viral egress by targeting VCP and the secretory pathway, resulting in a buildup of virions within dilated ER vesicles.IMPORTANCE In humans, symptoms of RVFV infection mainly include a self-limiting febrile illness. However, in some cases, infected individuals can also experience hemorrhagic fever, neurological disorders, liver failure, and blindness, which could collectively be lethal. The ability of RVFV to expand geographically outside sub-Saharan Africa is of concern, particularly to the Americas, where native mosquito species are capable of virus transmission. Currently, there are no FDA-approved therapeutics to treat RVFV infection, and thus, there is an urgent need to understand the mechanisms by which the virus hijacks the host cell machinery to replicate. The significance of our research is in identifying the cellular target of sorafenib that inhibits RVFV propagation, so that this information can be used as a tool for the further development of therapeutics used to treat RVFV infection.

T-cell-dependent mechanisms promote Ebola VLP-induced antibody responses, but are dispensable for vaccine-mediated protection.

Abstract: Humoral responses are essential for the protective efficacy of most Ebola virus (EBOV) candidate vaccines; however, the in vivo development of protective anti-EBOV B-cell responses is poorly defined. Here, by using the virus-like particle (VLP) as a model antigen, we demonstrate that humoral responses are generated through follicular B-cell and T-cell-dependent mechanisms in a mouse model of EBOV infection. In addition, we show that the inclusion of the clinical-grade dsRNA adjuvant known as poly-ICLC in VLP vaccinations both augments and sustains germinal center B-cell reactions, antigen-specific B-cell frequencies and anti-EBOV serum titers. Finally, we used mice that were deficient in either B-cells or T-cell-dependent antibody production to distinguish the contributing roles of EBOV humoral responses. We demonstrate that while anti-EBOV antibody responses promote protection, VLP-vaccinated mice can survive EBOV infection in the absence of detectable anti-EBOV antibodies. Moreover, we found that adjuvant signaling could circumvent the complete requirement for B-cell immunity in protection against EBOV. Collectively, these studies may prove valuable for the characterization and future development of additional EBOV vaccine candidates.

Clinical Laboratory Values as Early Indicators of Ebola Virus Infection in Nonhuman Primates.

Abstract: The Ebola virus (EBOV) outbreak in West Africa during 2013-2016 demonstrated the need to improve Ebola virus disease (EVD) diagnostics and standards of care. This retrospective study compared laboratory values and clinical features of 3 nonhuman primate models of lethal EVD to assess associations with improved survival time. In addition, the study identified laboratory values useful as predictors of survival, surrogates for EBOV viral loads, and triggers for initiation of therapeutic interventions in these nonhuman primate models. Furthermore, the data support that, in nonhuman primates, the Makona strain of EBOV may be less virulent than the Kikwit strain of EBOV. The applicability of these findings as potential diagnostic and management tools for EVD in humans warrants further investigation.

Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses

Abstract: Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 103 to 105 sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) rates of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in 1) properly assessing the statistical significance of repertoire differences, 2) identifying how two repertoires differ, and 3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox’ robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether, but also where, the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated germinal center B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically significant differences between non-immunized, vaccine only, and vaccine-plus-adjuvant groups in terms of Ig properties, including V-family usage, SHM rates, and characteristics of the BCR complementarity-determining region. Most notably, our analyses identified a robust eVLP-specific feature—enhanced IGHV8-family usage in B-cell repertoires. These findings demonstrate the utility of our technique in identifying statistically significant BCR repertoire differences following vaccination. More generally, our approach is potentially applicable to a wide range of studies in infection, vaccination, auto-immunity, and cancer.

Asymptomatic Ebola virus infections-myth or reality?

Abstract: Ebola virus captures the imagination of the public and experts alike. This fascination is in part due to the overall rare occurrence of typically very few outbreaks of Ebola virus disease (EVD) and extremely high case-fatality rates (mean 41·4%). More importantly, the identity of the natural Ebola virus reservoir remains unknown. This lack of knowledge means that novel Ebola virus introductions into human populations cannot be predicted, let alone be prevented, which adds to the enigma of the virus in the public eye. Unsurprisingly, considerable effort has been spent to hone in on the hiding place of the virus. Animals and plants have been screened for Ebola virus infection; ecological niche modelling has been done to predict endemic zones for the Ebola virus; and human sera have been screened for anti-Ebola virus antibodies to identify human populations at risk of infection and consequently to pinpoint geographical areas of endemicity. Unfortunately, the results of these approaches often appear contradictory. Thus far, Ebola virus could not be isolated from any screened wild animal or plant, and next-generation sequencing has not yet yielded at least coding-complete Ebola virus genomes in samples from any wild organism. These results suggest an unusual Ebola virus host. Niche modelling suggests that Ebola virus should be widely distributed over equatorial Africa, including countries from which Ebola virus infections have not been reported. These predictions are puzzling because the high EVD case-fatality rate makes it unlikely that EVD outbreaks have been overlooked repeatedly. The most confusing results, however, stem from a myriad of serosurveys, which revealed anti-Ebola virus antibodies in human beings from all over Africa. Many of these studies were done during the 1980s and 1990s and varied in quality: different assays were used to detect antibodies (eg, immunofluorescence assay, ELISA, western blot); seemingly arbitrary cutoffs were sometimes used to differentiate negative from positive results; different Ebola virus antigens were used for assay development (eg, whole inactivated virions vs individual viral proteins); sample cohort sizes diverged; and proper controls were or could often not be included. However, the results of many of these studies implied high seroprevalence (often >5%) of anti-Ebola virus antibodies throughout Africa among individuals who did not recall having had an EVD-like illness or contact with a suspect EVD case. Plausible explanations for these discrepant serosurvey results are: the serosurveys are artifacts due to crossreaction of Ebola virus antigens with non-antiEbola virus antibodies; the detected antibodies stem from contact with undiscovered, non-pathogenic filoviruses that are endemic in Africa and that are closely related to Ebola virus; or Ebola virus causes widespread subclinical infection in human beings. The last hypothesis has gained popularity, although actual evidence of subclinical Ebola virus infection is sparse and still debated. Recent studies indicate that Ebola virus can persist in some EVD survivors and replicate in immunoprivileged sites in the absence of clinical signs. Notably, though, such replication has never been convincingly demonstrated in people who did not have previous EVD. In The Lancet Infectious Diseases, Judith Glynn and colleagues present a carefully conducted, wellcontrolled serosurvey to take a fresh look at the possibility of subclinical EVD exposure and infection. During the recent large EVD outbreak (over 28000 cases) in western Africa, Sierra Leonean household contacts of people with proven EVD were screened for IgG antiEbola virus antibodies using a newly developed, noninvasive oral fluid capture assay with high specificity and sensitivity. Seroprevalence among household contacts who did not experience clinical signs indicative of EVD was only 2·6%. This value suggests that asymptomatic Ebola virus infections occur rarely, even when individuals have direct contact to individuals infected with Ebola virus. This result is in line with the observation that individuals infected with Ebola virus typically experience grave and frequently lethal disease, and cast further doubt on results of previous serosurveys. Although a single study such as that of Glynn and colleagues does not suffice to come to wide-sweeping conclusions about the possibility and frequency of subclinical individuals infected with Ebola virus infections, their results certainly indicate that such infections are not a typical or widespread phenomenon. The biggest threat to human populations therefore remains another introduction of Ebola virus from its natural host—and not transmission from an apparently healthy person infected with Ebola Kr ist in P al itz a/ DP A/ PA Im ag es

Low potential for mechanical transmission of Ebola virus via house flies (Musca domestica)

Abstract: Ebola virus (EBOV) infection results in high morbidity and mortality and is primarily transmitted in communities by contact with infectious bodily fluids. While clinical and experimental evidence indicates that EBOV is transmitted via mucosal exposure, the ability of non-biting muscid flies to mechanically transmit EBOV following exposure to the face had not been assessed. To investigate this transmission route, house flies (Musca domestica Linnaeus) were used to deliver an EBOV/blood mixture to the ocular/nasal/oral facial mucosa of four cynomolgus macaques (Macaca fascicularis Raffles). Following exposure, macaques were monitored for evidence of infection through the conclusion of the study, days 57 and 58. We found no evidence of systemic infection in any of the exposed macaques. The results of this study indicate that there is a low potential for the mechanical transmission of EBOV via house flies - the conditions in this study were not sufficient to initiate infection.

Broad-spectrum Investigational Agent GS-5734 for the Treatment of Ebola, MERS Coronavirus and Other Pathogenic Viral Infections with High Outbreak Potential

Abstract: Background Recent viral outbreaks with significant mortality such as Ebola virus (EBOV), SARS-coronavirus (CoV), and MERS-CoV reinforced the need for effective antiviral therapeutics to control future epidemics. GS-5734 is a novel nucleotide analog prodrug in the development for treatment of EBOV.

High Infection Rates in Adult Macaques Following Intravaginal or Intrarectal Zika Virus Inoculation

Abstract: Authors: Andrew D. Haddow*, Aysegul Nalca, Franco D. Rossi, Lynn J. Miller, Michael R. Wiley, Unai Perez-Sautu, Samuel C. Washington, Sarah L. Norris, Suzanne E. WollenRoberts, Joshua D. Shamblin, Adrienne E. Kimmel, Holly A. Bloomfield, Stephanie M. Valdez, Thomas R. Sprague, Lucia M. Principe, Stephanie A. Bellanca, Stephanie S. Cinkovich, Luis Lugo-Roman, Lisa H. Cazares, William D. Pratt, Gustavo F. Palacios, Sina Bavari, Margaret L. Pitt, and Farooq Nasar

DDX3 is exploited by Arenaviruses to suppress type I interferons and favor their replication

Abstract: Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication correlated with both a previously un-recognized DDX3 contribution to type I interferon suppression in arenavirus infected cells and a positive effect of DDX3 on viral RNA synthesis. Our results uncover novel mechanisms used by arenavirus to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.

Ebola’s exploits: debilitating host immune responses through interferon inhibiting domains

Abstract: The recent Ebola virus disease (EVD) outbreak in West Africa was unprecedented in human suffering (over 28,000 reported cases, more than 11,000 deaths), timespan [2014–2016], and geographic scope (epidemic spread in Guinea, Liberia, and Sierra Leone with exportation of cases to multiple continents) (1). Ebola virus (EBOV), an etiologic agent of acute hemorrhagic fever, usually results in high case fatality rates. At least part of the virulence of EBOV is attributed to its tropism for cells of the innate immune system: monocytes, macrophages, and immature dendritic cells (2,3). Upon infection, EBOV actively dampens intrinsic antiviral immune defenses, causing systemic immunosuppression, lymphocyte apoptosis, and lymphopenia. In fatal cases, these hallmarks of EVD and the accompanying innate immune disarmament allow unfettered viral replication within the host, resulting in a highly lethal disease with extreme pathogenic characteristics. The 2014 EBOV outbreak renewed interest in characterizing EBOV immunoevasion strategies, primarily occurring through viral antagonism of type I interferons (IFN-I), which may be critical components of vaccine and antiviral therapeutic applications for controlling future EBOV outbreaks.