Zika, Pregnancy, and the Law

The public health emergency surrounding the spread of the Zika virus has resurrected and brought into sharp relief some of the most vexing questions surrounding the relationship between pregnancy and law: the appropriate circumstances, if any, in which fetal tissue research is permissible; when and how the government may sponsor statements intended to influence reproductive decisions; and how to balance the health and rights of both women and their unborn children when health threats target both

Tissue Remodeling in Health and Disease Caused by Bacteria, Parasites, Fungi, and Viruses

Tissues undergo constant remodeling to maintain architecture during growth, in normal physiology and in response to disease. Interactions of the host with commensals and pathogens may affect immune responses and tissue remodeling, including for example through the generation of neo-epitopes and resulting in damage-associated molecular patterns. Roles for the microbiome, viriome, parasites, and fungi in host-pathogen interactions and in homeostasis is a current topic with considerable interest regarding effects relating to the gut-brain axis, chronic disease, cancer, dysbiosis, host metabolism, and drug metabolism. This E-book contains state-of-the-art primary research studies and review articles from international experts and diverse leading groups in the field to further current understanding of the contributions of commensals and pathogens in tissue remodeling in physiological and pathophysiological processes of the host

Putative Cellular and Molecular Roles of Zika Virus in Fetal and Pediatric Neuropathologies

Although the World Health Organization declared an end to the recent Zika virus (ZIKV) outbreak and its association with adverse fetal and pediatric outcome, on November 18, 2016, the virus still remains a severe public health threat. Laboratory experiments thus far supported the suspicions that ZIKV is a teratogenic agent. Evidence indicated that ZIKV infection cripples the host cells\u27 innate immune responses, allowing productive replication and potential dissemination of the virus. In addition, studies suggest potential transplacental passage of the virus and subsequent selective targeting of neural progenitor cells (NPCs). Depletion of NPCs by ZIKV is associated with restricted brain growth. And while microcephaly can result from infection at any gestational stages, the risk is greater during the first trimester. Although a number of recent studies revealed some of specific molecular and cellular roles of ZIKV proteins of this mosquito-borne flavivirus, the mechanisms by which it produces it suspected pathophysiological effects are not completely understood. Thus, this review highlights the cellular and molecular evidence that implicate ZIKV in fetal and pediatric neuropathologies

Emerging Virus Infections in Adverse Pregnancy Outcomes

In this Special Issue of Viruses, we present original research, reviews and commentaries that contribute to improving our understanding of the viral infection of placenta and fetal cells, or that report on the maternal and fetal outcomes after an emerging viral infection during pregnancy

Prenatal ultrasonography of craniofacial abnormalities

Craniofacial abnormalities are common. It is important to examine the fetal face and skull Epub ahead of print during prenatal ultrasound examinations because abnormalities of these structures may indicate the presence of other, more subtle anomalies, syndromes, chromosomal abnormalities, or even rarer conditions, such as infections or metabolic disorders. The prenatal diagnosis of craniofacial abnormalities remains difficult, especially in the first trimester. A systematic approach to the fetal skull and face can increase the detection rate. When an abnormality is found, it is important to perform a detailed scan to determine its severity and search for additional abnormalities. The use of 3-/4-dimensional ultrasound may be useful in the assessment of cleft palate and craniosynostosis. Fetal magnetic resonance imaging can facilitate the evaluation of the palate, micrognathia, cranial sutures, brain, and other fetal structures. Invasive prenatal diagnostic techniques are indicated to exclude chromosomal abnormalities. Molecular analysis for some syndromes is feasible if the family history is suggestive

Advancing antiviral strategies against emerging RNA viruses by phenotypic drug discovery

Pathogenic RNA viruses can emerge from unexpected sources at unexpected times and cause severe disease in humans, as exemplified by the ongoing coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV) outbreaks from the past decade. Despite the increasing impact of emerging viruses to health and economy worldwide, our preparedness to stand against these diseases is hampered by the lack of approved and effective antiviral therapies. Thus, the development of novel antivirals is of urgent need. To date, antiviral drug discovery has primarily focused on targeting specific viral proteins, but these treatments often suffer from viral resistance and are limited to only one or few viruses. Instead, phenotypic drug discovery enables the identification of drug candidates that are active in the disease-relevant model and not restricted to previously characterized biological processes. As RNA viruses are highly dependent on the host cell pathways due to their relatively small genome, targeting virus vulnerabilities within the host cell has been a promising antiviral strategy for broad spectrum antivirals, but is relatively unexplored so far. In fact, phenotypic approaches can additionally identify host-directed antivirals due to the unbiased nature. The focus of this doctoral thesis was to identify novel antiviral compounds with broad spectrum activity and investigate the compound mechanism of action and target pathways from the host cell and virus perspective. To achieve these goals, multiple cutting-edge phenotype-based methodologies were implemented that additionally advanced the antiviral drug discovery landscape. In Paper I, we developed an image-based phenotypic antiviral assay and screened our in-house chemical library targeting cellular oxidative stress and nucleotide metabolism pathways in Hazara virus (HAZV)-infected cells. Screening hit compounds TH3289 and TH6744 activity was validated by their therapeutic window and both compounds were also active beyond HAZV, especially TH3289 that was tested and displayed activity against EBOV, CCHFV, SARS-CoV-2 and a common cold coronavirus 229E (CoV-229E). We also excluded the intended target 8-oxoguanine DNA glycosylase (OGG1) protein to be responsible for TH6744 antiviral activity and characterized host cell chaperone and co-chaperone network as target pathways of TH6744 by implementing thermal proteome profiling methodology. In Paper II, we transferred our image-based phenotypic assay to ZIKV-infected brain cells in order to screen structural analogs of TH3289 and TH6744 against a pathogenic RNA virus. TH3289 and TH6744 again appeared among the screening hits and presented a promising therapeutic window in various cellular models, further confirming their broad activity. Moreover, TH6744 reduced ZIKV infection and progeny release in cerebral organoid model and impressively rescued ZIKV-induced cytotoxicity in organoids. Additionally, treatment with TH6744 rapidly diminished ZIKV progeny release during late replication cycle stages, elucidating the antiviral mechanism of action. In Paper III, we established an untargeted morphological profiling method to provide in-depth host cell responses during antiviral screening. We combined the Cell Painting protocol with antibody-based virus detection in a single assay followed by automated image analysis pipeline providing segmentation and classification of infected cells and extraction of cell morphological features. We demonstrated how our assay reliably distinguished CoV-229E infected human lung fibroblasts from non-infected controls based on cellular morphological features. Furthermore, our method can be applied in phenotypic drug screening as validated by nine host- and virus-targeting antivirals. Effective antivirals Remdesivir and E-64d treatment reversed the infection-specific signatures in host cells. Thereby, the developed method can be implemented for antiviral phenotypic drug discovery by morphological profiling of drug candidates

Development of electrochemical assays and biosensors for detection of Zika virus

2019 Spring.Includes bibliographical references.Zika virus (ZIKV) emerged as a significant public health concern after the 2015-2016 outbreak in South and Central America. Severe neurological complications and birth defects in adults and children respectively underscore the need for quick and accurate diagnosis so that proper medical observation and intervention can be done. Electrochemical assays and biosensors are attractive as alternative diagnostic tools due to their sensitivity and ease of miniaturization. This dissertation describes three novel electrochemical assays and biosensors to detect ZIKV specific nucleic acid, antibodies, and virus particles. A nuclease protection ELISA (NP-ELISA) was developed for nucleic acid detection by enzymatic readout. The assay was validated using synthetic complementary oligos for absorbance, chemiluminescence, and electrochemical enzymatic readout. Two horseradish peroxidase substrates, 3,3',5,5'-Tetramethylbenzidine (TMB) and hydroquinone, were characterized electrochemically and compared for electrochemical assay use. Electrochemical TMB readout demonstrated better sensitivity compared to all tested detection modalities with a limit of detection of 3.72×103 molecules mL-1, which compares well to the amount of ZIKV RNA in clinical samples and to other approved assays like the CDC's Trioplex assay. For serological analysis, a capacitive microwire biosensor was developed and validated using immunized mouse sera to detect a ZIKV antibody response. Measurements were taken through a wide serial dilution range of 1:1018 to 1:103 and two dilutions (1:1012 and 1:106) were used for analysis for optimal sensitivity. A statistically significant immune response was detected four days after immunization at a 1:1012 dilution and was specific for ZIKV when compared with Chikungunya virus (CHIKV). These results indicate that serological analysis can be performed four days earlier with the wire sensor compared to ELISAs using ultra-dilute samples. The sensor also was used to differentiate between IgG and IgM antibodies and compared well with ELISA results. Lastly, an impedance array sensor was designed and validated for detection of ZIKV particles. The array allows for simultaneous handling of many electrodes, which increases throughput compared to other biosensor designs. The sensor demonstrated good sensitivity with an LOD of 22.4 focus forming units (FFU) which compares well to other reported sensors. In addition, it was optimized for specificity and tested using Sindbis virus (SINV) as a negative control. These novel platforms comprise new advancements in biosensor technology by simplifying existing assays, increasing sensitivity, and providing a new platform for handheld measurements

Investigations into Zika virus-host interactions: A neurological perspective

Zika virus (ZIKV) is a member of the Flavivirus genus of the family Flaviviridae. Members of this genus possess a single-stranded positive-sense RNA genome which are flanked by 5’ and 3’ untranslated regions. Previously, ZIKV infection was thought to cause symptomatic infection in 20% of patients, characterised by a non-purulent rash and a mild fever. However, recent ZIKV outbreaks have seen the emergence of novel neurological sequelae associated with ZIKV infection. These symptoms affected the central and peripheral nervous systems (CNS and PNS, respectively) of neonates and adults (congenital ZIKV syndrome and Guillain-Barré syndrome, respectively). Consequently, prior to commencement of this thesis, the knowledge of ZIKV interactions within the CNS and PNS was limited. Therefore, the primary aim of this thesis was to expand the current knowledgebase of ZIKV infection within cells of the CNS and PNS. The work presented herein assessed available tools to study ZIKV infection in vitro, including testing previously uncharacterised commercial antibodies targeting the ZIKV envelope (ZIKV E) and NS1 proteins. Subsequently, a suitable antibody raised against the ZIKV E was identified and used in downstream analyses. A model system of ZIKV infection of the CNS and PNS using mixed-cell co-cultures derived from mouse spinal cord (CNS) or dorsal root ganglion (PNS) was used to determined cell-type specific susceptibility to ZIKV infection. It was found that cells of the PNS are refractory to ZIKV infection, whereas cells of the CNS are permissive to ZIKV infection. In the CNS, oligodendrocytes and their precursors were the most susceptible cell type to ZIKV infection. Moreover, when infecting CNS co-cultures at a time point which reflects prenatal life through to post-natal life, white matter structures (myelin sheath and axons) are injured. However, neuronal cell bodies remained healthy. Using purified primary neuronal cultures derived from mouse spinal cord, it was determined that neurons were refractory to ZIKV infection in the absence of accompanying glial cells. This suggests axonal damage may not be a result of direct infection of neurons themselves and may be a consequence of oligodendrocyte infection and injury. Transcriptomic analyses of ZIKV-infected CNS co-cultures revealed that ZIKV infection induces the upregulation of genes involved in antiviral responses and inflammatory pathways, including TNF and ROS/NO pathway components; these pathways may be involved in exacerbating injury to white matter structures within ZIKV-infected CNS-co-cultures. In summary, the results described in this thesis show that ZIKV can infect the early post-natal CNS. Furthermore, ZIKV-infection of this murine model demonstrates that infection induces the injury and depletion of myelin and axons. This is likely to be due to a combination of effects such as direct viral infection, cell death of oligodendrocytes, and secreted inflammatory factors. Myelination occurs late in foetal development and carries on into early adulthood. These data, in combination with precedent pathological findings, suggest that ZIKV-infected children born absent of microcephaly may develop other, more subtle, neurological sequelae later in life

Effects of Zika virus on neural precursor cell types and microencephaly in a model of direct embryonic murine brain infection

Prenatal exposure to Zika virus (ZIKV) can result in microencephaly and congenital Zika syndrome but why some brain cells and structures are initially spared by the virus is unknown. Here, a novel murine model of ZIKV infection incorporating in utero electroporation with cell type specific promotors was used to identify the time course of ZIKV infection and to determine which neural precursor cells are initially infected or spared. In vivo time course studies revealed early presence of ZIKV in apical radial glial cells (aRGCs) while infection of basal intermediate progenitor cells climbed after three days of virus exposure. ZIKV-exposed fetal brains exhibited microencephaly as early as 1 day post injection, caused by apoptosis and reduced proliferation, and this change in brain size persisted until birth regardless of developmental age at infection. During infection, 60% of aRGC basal fibers were perturbed while 40% retained normal morphology, indicating that aRGCs are not uniformly vulnerable to ZIKV infection. To evaluate this heterogeneous vulnerability, we generated cell type-specific fate mapping plasmid probes using a previously published single cell RNA-Seq dataset on the E15.5 mouse neocortical wall. The results indicate that one class of aRGC preferentially expresses the putative ZIKV entry receptor AXL, and that these cells are more vulnerable to ZIKV infection than the other aRGC subtypes with low AXL expression. Together, these data highlight important temporal and cellular details of ZIKV fetal brain infection and may be important for prevention strategies and for management of congenital Zika syndrome

Structural and functional analysis of Zika Virus NS5 protein

[eng] Zika virus (ZIKV) belongs to the Flaviviridae family and constitute an important public health concern since ZIKV infection produced devastating effects in new born infants. Flaviviruses present a positive sense single stranded RNA genome flanked by highly structured untranslated regions (UTR) carrying one open reading frame that codifies for three structural proteins (C, prM, E) and five nonstructural proteins (NS1-5). At the most C-terminal end, NS5 protein carries a RNA dependent RNA polymerase (RdRP) and a methyl transferase domain (MTase) for genome copying and 5’ capping activities of the newly synthesized RNA, respectively. Given the crucial role of this enzyme for viral replication, NS5 constitutes an attractive antiviral target to inhibit viral replication. In this study, we determined the structure of the ZIKV NS5 protein using X-Ray crystallography combined with several structural biology approaches to characterize the supramolecular arrangement of the ZIKV NS5 protein. We identified the monomer-monomer and dimer-diner interactions to form fibril-like structures, and evaluated the role of oligomer formation, using in-vitro polymerization assays. We also evaluated the in-vivo effect of NS5-oligomerisation in chicken embryos, stablishing a connection between this protein and microcephaly. One of the most important RNA structures present at the 5’UTR of flavivirus genomes is the 5SLA. This structure was identified previously to bind the NS5 protein, acting as a promoter and being essential for viral replication. We assayed and optimized the NS5-5SLA complex stability using biophysical and biochemical techniques and determined the structure of the complex by single particle cryo-EM. Comparisons between the NS5-5SLA complex and the NS5 crystallographic structure revealed for the first time in flavivirus, important conformational changes in the NS5 RdRP. We identified the residues involved in complex formation and characterized the effect of this binding on NS5 polymerization, shedding new light on the understanding of replication mechanisms in flaviviruses.[spa] El virus Zika (ZIKV) pertenece a la familia Flaviviridae y constituye una amenaza para la salud pública, especialmente debido a las malformaciones provocadas en neonatos. Los flavivirus presentan un genoma RNA de simple cadena con polaridad positiva, flanqueado por regiones no traducidas (UTR) que presentan una elevada estructura secundaria, seguido de una región codificante para una única poliproteína que por proteólisis dará lugar a tres proteínas estructurales (C, prM, E) y cinco proteinas no estructurales (NS1-5). En el extremo C-terminal se encuentra la proteina NS5 que presenta actividad ARN polimerasa dependiente de ARN (RdRP) y un dominio metil-transferasa (MTase) para copiar el genoma y añadir una caperuza al extremo 5’ del nuevo ARN sintetizado, respectivamente. Dado el papel crucial de este enzima en la replicación viral, la proteina NS5 constituye una diana antiviral muy atractiva para inhibir la replicación del virus. En este estudio, determinamos la estructura de la proteína NS5 de ZIKV, usando cristalografía de Rayos-X combinada con diferentes técnicas biofísicas para caracterizar la organización supramolecular de la proteína. Identificamos las interacciones monomero-monomero y dimero-dimero para caracterizar las estructuras fibrilares de la proteína y evaluamos los efectos de la dimerización en la actividad polimerasa in-vitro. También evaluamos los efectos de la oligomerización de NS5 in-vivo en embriones de pollo, estableciendo una conexión entre esta proteína y la aparición de microcefalia en fetos infectados. Una de las estructuras de ARN más importantes presentes en el 5’UTR del genoma de los flavivirus es el 5SLA. Previamente se describió que esta estructura se unía a NS5 y actuaba como un promotor, siendo ademas esencial para la replicación viral. Medimos y optimizamos la estabilidad del complejo NS5-5SLA mediante técnicas biofísicas y bioquímicas y determinamos la estructura del complejo mediante cryo-EM. Las comparaciones entre la estructura cristalográfica y cryo-EM de NS5 revelaron, por primera vez en flavivirus, cambios conformacionales importantes en el dominio RdRP. Identificamos los residuos implicados en la formación del complejo y caracterizamos el efecto de la unión de NS5 a 5SLA sobre su actividad polimerasa. Estos resultados arrojan nueva luz para entender los mecanismos de replicación en los flavivirus