An Immunocompetent Mouse Model of Zika Virus Infection

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Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B

The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.     

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

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Targeting the NTPase site of Zika virus NS3 helicase for inhibitor discovery

Abstract

The major threats linked to Zika virus (ZIKV) are microcephaly, Guillain-Barre syndrome, and the ability to transfer through sexual transmission. Despite these threats, Zika specific FDA approved drugs or vaccines are not available as of yet. Additionally, the involvement of pregnant women makes the drug screening process lengthy and complicated in terms of safety and minimum toxicity of the molecules. Since NS3 helicase of ZIKV performs the critical function of unwinding double-stranded RNA during replication, it is considered as a promising drug target to block ZIKV replication. In the present study, we have exploited the NTPase site of ZIKV NS3 helicase for screening potential inhibitor compounds by molecular docking, and molecular dynamics (MD) simulation approaches. NS3 helicase hydrolyzes the ATP to use its energy for unwinding RNA. We have chosen twenty natural compounds from ZINC library with known antiviral properties and a helicase focused library (HFL) of small molecules from Life Chemicals compounds. After going through docking, the top hit molecules from ZINC and HFL library were further analysed by MD simulations to find out stable binding poses. Finally, we have reported the molecules with potential of binding at NTPase pocket of ZIKV NS3 helicase, which could be further tested on virus through in vitro experiments to check their efficacy.

Communicated by Ramaswamy H. Sarma     

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

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Dynamic Comparison of Kaplan–Meier Proportions: Monitoring a Randomized Clinical Trial with a Long-Term Binary Endpoint

Summary .   The approach to early termination for efficacy in a trial where events occur over time but the primary question of interest relates to a long-term binary endpoint is not straightforward. This article considers comparison of treatment groups with Kaplan–Meier (KM) proportions evaluated at increasing times from randomization, at increasing calendar testing times. This strategy is employed to improve the ability to detect important treatment effects and provide critical treatments to patients in a timely manner. This dynamic Kaplan–Meier (DKM) approach is shown to be robust; that is, it produces high power and early termination time across a wide range of circumstances. In contrast, a fixed time KM comparison and the log-rank test are both shown to be more variable in performance. Practical considerations of implementing the DKM method are discussed.     

Before, During and After: The Need for Long-term Monitoring in Invasive Plant Species Management

The invasion of non-indigenous plants is considered one of the primary threats to rare and endangered species as well as to the integrity and function of North American ecosystems. However, many of the suspected negative ecosystem impacts are based on anecdotal evidence. For example, there is almost unanimous agreement among natural resource managers of the detrimental ecological impacts of species such as Lythrum salicaria (purple loosestrife), Phragmites australis (common reed) and Alliaria petiolata (garlic mustard) but convincing documentation is scarce. Experimental and theoretical ecology predicts large ecosystem impacts of the most widespread invasive species. However, it is difficult to prioritize control of species that occur at intermediate densities. Long-term monitoring before and during the invasion as well as before, during and after any control attempts can provide valuable ecological information. In particular, it is important to understand how changes in the abundance of species influence ecosystem properties and processes which, in turn, will help guide management decisions. Ideally, this monitoring has to go beyond 'simple impacts on plant communities, involve cross-disciplinary teams of scientists and should incorporate many different taxa and their interactions. Monitoring design and data collection should be sophisticated enough to allow statistically sound data analysis. The available information will be paramount in (1) developing new political and scientific guidelines in invasive species management, (2) helping resolve potential conflicts of interest and (3) helping change public attitudes regarding growth, sale, and control of non-indigenous species.     

The Spectrum of Mutations Causing HPRT Deficiency: An Update

Mutations in the gene encoding hypoxanthine‐guanine phosphoribosyltransferase (HPRT) cause Lesch–Nyhan disease, which is characterized by hyperuricemia, severe motor disability, and self‐injurious behavior. Mutations in the same gene also cause less severe clinical phenotypes with only some portions of the full syndrome. A large database of 271 mutations associated with both full and partial clinical phenotypes was recently compiled. Since the original database was assembled, 31 additional mutations have been identified, bringing the new total to 302. The results demonstrate a very heterogeneous collection of mutations for both LND and its partial syndromes. The differences between LND and the partial phenotypes cannot be explained by differences in the locations of mutations, but the partial phenotypes are more likely to have mutations predicted to allow some residual enzyme function. The reasons for some apparent exceptions to this proposal are addressed.     

The bright and the dark side of human antibody responses to flaviviruses: lessons for vaccine design

Zika and dengue viruses belong to the Flavivirus genus, a close group of antigenically related viruses that cause significant arthropod‐transmitted diseases throughout the globe. Although infection by a given flavivirus is thought to confer lifelong protection, some of the patient's antibodies cross‐react with other flaviviruses without cross‐neutralizing. The original antigenic sin phenomenon may amplify such antibodies upon subsequent heterologous flavivirus infection, potentially aggravating disease by antibody‐dependent enhancement (ADE). The most striking example is provided by the four different dengue viruses, where infection by one serotype appears to predispose to more severe disease upon infection by a second one. A similar effect was postulated for sequential infections with Zika and dengue viruses. In this review, we analyze the molecular determinants of the dual antibody response to flavivirus infection or vaccination in humans. We highlight the role of conserved partially cryptic epitopes giving rise to cross‐reacting and poorly neutralizing, ADE‐prone antibodies. We end by proposing a strategy for developing an epitope‐focused vaccine approach to avoid eliciting undesirable antibodies while focusing the immune system on producing protective antibodies only.     

Exposure to cigarette smoke, a major risk factor for sudden infant death syndrome: effects of cigarette smoke on inflammatory responses to viral infection and bacterial toxins

Exposure to cigarette smoke is a major risk factor for sudden infant death syndrome and also for respiratory infections in children. It has been suggested that toxigenic bacteria colonizing the respiratory tract might play a role in some cases of sudden infant death syndrome and nicotine has been demonstrated to enhance the lethality of bacterial toxins in a model system. Pyrogenic toxins of Staphylococcus aureus have been identified in tissues of infants who died of sudden infant death syndrome. It has been suggested that some of these deaths were due to induction of inflammatory mediators by infectious agents during a period when infants are less able to control these responses. The aim of this study was to assess the effects of a water-soluble cigarette smoke extract on the production of tumor necrosis factor alpha and nitric oxide from human monocytes in response to staphylococcal toxic shock syndrome toxin 1 or infection of the monocytes with respiratory syncytial virus. Cell culture supernatants were examined by a bioassay using mouse fibroblasts (L-929 cell line) for tumor necrosis factor alpha activity and by a spectrophotometric method for nitrite. Compared with monocytes incubated with medium only, monocytes incubated with any of the factors or their combinations tested in the study released higher levels of tumor necrosis factor alpha and lower levels of nitric oxide. Incubation with cigarette smoke extract increased tumor necrosis factor alpha from respiratory syncytial virus-infected cells while it decreased tumor necrosis factor alpha from cells incubated with toxic shock syndrome toxin. Incubation with cigarette smoke extract decreased the nitric oxide production from respiratory syncytial virus-infected cells while it increased the nitric oxide production from cells incubated with toxic shock syndrome toxin. Monocytes from a minority of individuals demonstrated extreme tumor necrosis factor alpha responses and/or very high or very low nitric oxide. The proportion of samples in which extreme responses with a very high tumor necrosis factor alpha and very low nitric oxide were detected was increased in the presence of the three agents to 20% compared with 0% observed with toxic shock syndrome toxin 1 or 4% observed with cigarette smoke extract or respiratory syncytial virus.     

The Dugong Action Plan for the South Pacific: An Evaluation Based on the Need for International and Regional Conservation of Sirenians

Abstract

The purpose of this article is to set out the essential requirements for a successful regional agreement for Sirenians in the South Pacific. To achieve this, the current Dugong Action Plan, which is being formed under the auspice of the South Pacific Regional Environmental Program, will be juxtaposed against the “best practice” in this area, as evinced by current development in international environmental law and policy relating to Sirenians.     

Beyond malaria: The inhibition of viruses by artemisinin-type compounds

Natural products represent valuable chemical scaffolds for drug development. A recent success story in this context was artemisinin, which is not only active against malaria but also to other diseases. This raised the interest of artemisinin's potential for drug repurposing. On the present review, we give an overview on artemisinin's antiviral activity. There is good in vitro and in vivo evidence for the activity of artemisinin and its derivatives against DNA viruses of the Herpesviridae and Hepadnaviridae families such as cytomegaloviruses, human herpesvirus 6, herpes simplex viruses 1 and 2, Epstein-Barr virus and Hepatitis B virus. The evidence is weaker for Polyomaviruses and papilloma viruses. Weaker or no inhibitory activity in vitro has been reported for RNA viruses such as human immunodeficiency viruses 1 and 2, hepatitis C virus, influenza virus and others. Interestingly, the artemisinin derivative artesunate did not exert cross-resistance to ganciclovir-resistant HCMV and exerted synergistic inhibition in combination with several clinically established antiviral standard drugs. The antiviral activity of first generation artemisinin derivatives (e.g. artesunate, artemether, etc.) was enhanced by novel derivatives, including dimer and trimer molecules. First results on patients indicating activity in a subset of HCMV patients. Novel developments in the field of nanotechnology and synthetic biology to bioengineer microorganisms for artemisinin production may pave the way for novel drugs to fight viral infections with artemisinin-based drugs.     

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.     

Traversing the Center: The Politics of Language Use in a Catholic Religious Education Program for Immigrant Mexican Children

This article examines the implementation of educational policy in a religious education program at a Los Angeles Catholic parish. It charts the elimination of Spanish-based classes (doctrina) for Mexican immigrant children in favor of "English-only" instruction. The article offers insights into the politics of language use in everyday practice and examines the resulting educational policy decisions.     

The challenge of using experimental infectivity data in risk assessment for Ebola virus: why ecology may be important

Analysis of published data shows that experimental passaging of Zaire ebolavirus (EBOV) in guinea pigs changes the risk of infection per plaque‐forming unit (PFU), increasing infectivity to some species while decreasing infectivity to others. Thus, a PFU of monkey‐adapted EBOV is 10⁷‐fold more lethal to mice than a PFU adapted to guinea pigs. The first conclusion is that the infectivity of EBOV to humans may depend on the identity of the donor species itself and, on the basis of limited epidemiological data, the question is raised as to whether bat‐adapted EBOV is less infectious to humans than nonhuman primate (NHP)‐adapted EBOV. Wildlife species such as bats, duikers and NHPs are naturally infected by EBOV through different species giving rise to EBOV with different wildlife species‐passage histories (heritages). Based on the ecology of these wildlife species, three broad ‘types’ of EBOV‐infected bushmeat are postulated reflecting differences in the number of passages within a given species, and hence the degree of adaptation of the EBOV present. The second conclusion is that the prior species‐transmission chain may affect the infectivity to humans per PFU for EBOV from individuals of the same species. This is supported by the finding that the related Marburg marburgvirus requires ten passages in mice to fully adapt. It is even possible that the evolutionary trajectory of EBOV could vary in individuals of the same species giving rise to variants which are more or less virulent to humans and that the probability of a given trajectory is related to the heritage. Overall the ecology of the donor species (e.g. dog or bushmeat species) at the level of the individual animal itself may determine the risk of infection per PFU to humans reflecting the heritage of the virus and may contribute to the sporadic nature of EBOV outbreaks.     

Use of Phytoplankton Assemblages for Monitoring Ecological Status of Lakes within the Water Framework Directive: The Assemblage Index

The ingress of an urban stream carrying high contaminant loads into a large coastal river originates a “dispersion plume” subject to the hydrological conditions of a river affected by tidal influences. In the present study 21 sites within the “contaminant plume” of the Riachuelo River in the Rio de la Plata were analysed on the same date in order to evaluate the biological status of the area which receives this strong environmental impact, and to examine its effect on the zoobenthic communities. Diversity, taxonomic richness, abundance, physico-chemical parameters and a biological index (IMRP) were used to assess the responses of the macroinvertebrates. The correlation between exposure and effect was calculated by means of the exposure index (IEX). The relationship between the macroinvertebrate communities and environmental variables was examined using CCA analysis. Conductivity, Cr, BOD and COD, were most strongly correlated with Axis 1, suggesting the existence of a gradient of environmental degradation. The most severely contaminated sites (IMRP= 1.1–2.5; IEX = 100–78%) were all characterized by a reduced community dominated by Nematoda and Oligochaeta. A moderate response was observed between 1400 and 1600 m from the coast (IMRP = 2.6–3.9; IEX = 36%) largely owing to the physico-chemical characteristics of the recipient river which contributed to moderating the effect of the anthopogenic perturbation. For statistical validation, this area was compared with historical physico-chemical and biological data, where OD and COD showed the same tendency throughout the 10-year period.     

The Need for Strategic Environmental Assessment of Fishery Products Regulations in the Taiwan Strait: Taking Health Perspectives of Organochlorine Pesticides in Seafood as an Example

This article proposes an integrated Health Impact Assessment/Strategic Environmental Assessment (HIA/SEA) framework that can be applied to fishery products regulations in Taiwan Strait. In recent years, many studies with regards to organochlorine pesticides (OCPs), especially DDT and its derivatives, in Taiwan's environment and aquatic biota indicated that DDT, DDD, and DDE in seafood, especially oysters, in the Kinmen, Manchu area near China's Fu-Tzien Province, had relatively high concentrations. It was discussed that this may be caused by distribution of OCPs in China's vicinity. In this study, the concentration of DDT, DDD, and DDE reported by many researchers in the last two decades were compared and analyzed. The concentrations of these three chemicals were found to be as high as hundreds of ng/g-dw in seafood produced in Kimmen and Machu near China, whereas those for seafood caught in Taiwan's coastal areas were not significant. The need for trade agreements or regulations on fishery was then discussed. According to the viewpoints of health and environmental sustainability, HIA methodologies were incorporated in the SEA to help identify and analyze the potential impacts on human health caused by OCPs in seafood consumed in Taiwan, which may be caused by the lack of trade regulation mechanisms between Taiwan and China. The integrated HIA-SEA framework for fishery trade agreements or regulations in Taiwan was thus proposed and the procedures were discussed. It is anticipated that the health of Taiwan's residents can be protected through implementing this and the human health perspective can be emphasized in the HIA-SEA procedures.     

The hen's egg test for micronucleus induction (HET-MN): Novel analyses with a series of well-characterized substances support the further evaluation of the test system

The hen's egg test for micronucleus induction (HET-MN) combines the use of the commonly accepted genetic endpoint “formation of micronuclei” with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics—including those that are mutagens or promutagens. This assay procedure is in line with demands for animal protection. In three previous publications we presented the scientific rationale and methodological aspects for this assay as well as results for some well-characterized mutagens and promutagens. Here we present the results of new experiments involving further genotoxic and non-genotoxic model substances. Making a comparison with published data we have to date not found any false negatives or false positives in the experiments presented here and in trials published before, thus demonstrating a promising predictivity of genotoxic effects with this assay.We could confirm relevant genotoxicity for the following substances in the HET-MN: acetylamino-fluorene (2-AAF), acrylamide (ACM), cytarabine (AraC), methotrexate (MTX), cadmium chloride (CD), dipotassium monochromate (DPC), and epirubicine (EPI). Negative results were obtained for azorubin (E122), orange G (OG) and starch (STRC).The micronucleus frequencies (MNE II) of the concurrent negative controls were in agreement with the values of the historical negative control (0.87‰ ± 0.87; average ± s.d.). This value is based upon the scoring of 556,500 erythrocytes from 445 eggs. In historical positive controls the administration of 0.05 mg cyclophosphamide/egg at d8 resulted in an MNE II-frequency of 12.4‰ ± 6.8 (average ± s.d.) at d 10.5. This value is based upon the scoring of 249,250 erythrocytes from 223 eggs.