Rapid Screening for Entry Inhibitors of Highly Pathogenic Viruses under Low-Level Biocontainment

Emerging viruses including Nipah, Hendra, Lujo, and Junin viruses have enormous potential to spread rapidly. Nipah virus, after emerging as a zoonosis, has also evolved the capacity for human-to-human transmission. Most of the diseases caused by these pathogens are untreatable and require high biocontainment conditions. Universal methods for rapidly identifying and screening candidate antivirals are urgently needed. We have developed a modular antiviral platform strategy that relies on simple bioinformatic and genetic information about each pathogen. Central to this platform is the use of envelope glycoprotein cDNAs to establish multi-cycle replication systems under BSL2 conditions for viral pathogens that normally require BSL3 and BSL4 facilities. We generated monoclonal antibodies against Nipah G by cDNA immunization in rats, and we showed that these antibodies neutralize both Nipah and Hendra live viruses. We then used these effective Henipavirus inhibitors to validate our screening strategy. Our proposed strategy should contribute to the response capability for emerging infectious diseases, providing a way to initiate antiviral development immediately upon identifying novel viruses

Adenovirus DNA in Guthrie cards from children who develop acute lymphoblastic leukaemia (ALL)

Aims: The aim of this thesis was to increase understanding of how molecular processes influence the development and risk assessment of childhood leukemia. Studies I and II investigates whether a specific virus infection in utero could be involved in a “first hit” in leukemogenesis. Studies III and IV examine whether alterations in protein expression from cell cycle regulating genes may predict a relapse in children with myeloid malignancies undergoing hematopoietic stem cell transplantation (HSCT). Background: Genetic alterations, analyzed at time of diagnosis in children who develop leukemia, have been traced back to neonatal dried blood spots (DBS). This suggests that the majority of chromosome translocations occur in utero during fetal hematopoiesis, generating a “first hit”. A “second hit” is then required to generate a leukemic clone. Today, experiments in vitro, animal models, and clinical observations have revealed that several viruses are oncogenic and capable of initiating a genetic alteration. Smith M postulated the theory that an in utero infection might be the “first hit”, causing genetic aberrations that could later lead to the development of the leukemic clone, which is supported by the early age of onset and space-time clustering data, based on time, place of birth, and diagnosis. Leukemia develops as a result of hematopoietic or lymphoid tissue with uncontrolled cell division. Normally cell division is controlled by the cell cycle, the network of which is complex with numerous regulating proteins both up and down stream, but also containing several feedback loops. The important regulators of this process are tumor suppressor genes, essential for normal cell proliferation and differentiation as well as for controlling DNA integrity. Errors in these genes or their protein expression affect the ability of the cell to check for DNA damage, thus tumors may occur. Proteins from these genes could serve as prognostic markers and predict relapse. Methods: In studies I and II we investigated neonatal DBS by PCR for the presence of adenovirus DNA (243 samples) and the three newly discovered polyomaviruses (50 samples) from children who later developed leukemia but also from controls (486 and 100 samples respectively). In studies III and IV we explored the expression of one (p53) respectively four (p53, p21, p16 and PTEN) cell cycle regulating proteins in bone marrow at diagnosis as well as pre and post HSCT in myeloid malignancies in children. We retrospectively collected clinical data and bone marrow samples from 33 children diagnosed with chronic myeloid malignancies (MDS, JMML and CML), 34 children diagnosed with AML as well as 55 controls. The samples were prepared by tissue micro array (TMA) as well as immunohistochemistry and examined for protein expression in a light microscope. Results: In study I we detected adenovirus DNA in only two patients who later developed leukemia, but in none of the controls. In study II all the samples were negative for KIPyV, WUPyV and MCPyV DNA in both patients and controls. In study III we found an overexpression of p53 protein at diagnosis that significantly predicted relapse after HSCT in children with rare chronic myeloid malignancies. In study IV a significantly higher p53 expression was found in the relapse compared to the non-relapse group at six months post HSCT in children with AML, suggesting that p53 may be used as prognostic markers for predicting a relapse. In addition, the calculated cut off level for p53 at diagnosis (study III) and at six months (study IV) post HSCT was approximately 20%, which indicates that a p53 expression over 20% may predict relapse in children with myeloid malignancies. Conclusion: Although we did not find an association between adenoviruses or the three newly discovered polyomaviruses and the development of childhood leukemia, a virus could still be involved in this process; the virus may have escaped detection, other new viruses could be involved or a virus could precipitate the “second hit”. We suggest that evaluation of p53 protein expression may be used as a supplement to regular prognostic markers both pre and post HSCT. To further evaluate this, a prospective multicenter study has been started

Utilizing associational resistance for biocontrol: impacted by temperature, supported by indirect defence

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Mis-Spliced Transcripts of Nicotinic Acetylcholine Receptor α6 Are Associated with Field Evolved Spinosad Resistance in Plutella xylostella (L.)

The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR) and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxα6), at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxα6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxα6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species

A Critical Assessment of the Effects of Bt Transgenic Plants on Parasitoids

The ecological safety of transgenic insecticidal plants expressing crystal proteins (Cry toxins) from the bacterium Bacillus thuringiensis (Bt) continues to be debated. Much of the debate has focused on nontarget organisms, especially predators and parasitoids that help control populations of pest insects in many crops. Although many studies have been conducted on predators, few reports have examined parasitoids but some of them have reported negative impacts. None of the previous reports were able to clearly characterize the cause of the negative impact. In order to provide a critical assessment, we used a novel paradigm consisting of a strain of the insect pest, Plutella xylostella (herbivore), resistant to Cry1C and allowed it to feed on Bt plants and then become parasitized by Diadegma insulare, an important endoparasitoid of P. xylostella. Our results indicated that the parasitoid was exposed to a biologically active form of the Cy1C protein while in the host but was not harmed by such exposure. Parallel studies conducted with several commonly used insecticides indicated they significantly reduced parasitism rates on strains of P. xylostella resistant to these insecticides. These results provide the first clear evidence of the lack of hazard to a parasitoid by a Bt plant, compared to traditional insecticides, and describe a test to rigorously evaluate the risks Bt plants pose to predators and parasitoids

Different Transcript Patterns in Response to Specialist and Generalist Herbivores in the Wild Arabidopsis Relative Boechera divaricarpa

BACKGROUND: Plants defend themselves against herbivorous insects, utilizing both constitutive and inducible defenses. Induced defenses are controlled by several phytohormone-mediated signaling pathways. Here, we analyze transcriptional changes in the North American Arabidopsis relative Boechera divaricarpa in response to larval herbivory by the crucifer specialist lepidopteran Plutella xylostella (diamondback moth) and by the generalist lepidopteran Trichoplusia ni (cabbage semilooper), and compare them to wounding and exogenous phytohormone application. METHODOLOGY/PRINCIPAL FINDINGS: We use a custom macroarray constructed from B. divaricarpa herbivory-regulated cDNAs identified by suppression subtractive hybridization and from known stress-responsive A. thaliana genes for transcript profiling after insect herbivory, wounding and in response to jasmonate, salicylate and ethylene. In addition, we introduce path analysis as a novel approach to analyze transcript profiles. Path analyses reveal that transcriptional responses to the crucifer specialist P. xylostella are primarily determined by direct effects of the ethylene and salicylate pathways, whereas responses to the generalist T. ni are influenced by the ethylene and jasmonate pathways. Wound-induced transcriptional changes are influenced by all three pathways, with jasmonate having the strongest effect. CONCLUSIONS/SIGNIFICANCE: Our results show that insect herbivory is distinct from simple mechanical plant damage, and that different lepidopteran herbivores elicit different transcriptional responses

Inhibition of Nipah Virus Infection In Vivo: Targeting an Early Stage of Paramyxovirus Fusion Activation during Viral Entry

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses