1,721,069 research outputs found
Towards in vitro and in vivo model systems for Hepatitis B and Delta Viruses
No full textHepatitis B virus (HBV) is one of the oldest viruses known to afflict humankind, having been discovered in the remains of ancient Egyptian and Korean mummies. Today, HBV remains a global scourge, infecting approximately 257 million individuals worldwide, of whom about 887,000 die of complications each year. Besides fundamental interest into the basic biology of this virus, a need to develop curative therapeutics motivates biomedical research. For the past several decades, study of this virus and development of therapies has been hampered by a severe lack of model systems, both in vitro and in vivo. While in vitro systems cannot support robust infection for more than a week, there exist few in vivo systems for the virus at all, save for the chimpanzee, research on which a recent NIH moratorium halted, and the transgenic mouse, which while certainly helpful is genetically dissimilar to humans. In this thesis, we make progress on both the in vitro and in vivo fronts. For an in vitro model, we employ the self-assembling primary human hepatocyte co-culture (SACC-PHH) model system, which takes advantage of non-parenchymal fibroblasts to allow primary human hepatocytes to support HBV infection for up to four weeks, far longer than current systems and without suppression of antiviral immunity. We utilize this model for several applications, including co-infection with the satellite hepatitis Delta virus, testing of antiviral therapeutics, RNA sequencing, and mathematical modeling of infection. For an in vivo model, we pursue a viral adaptation project. Here, we mutagenize residues of the HBV surface protein, preS1, to discover variants capable of utilizing a cynomolgus macaque hepatocyte receptor for entry. Altogether, we anticipate that our results will empower other researchers to pursue some of the most pressing questions in HBV biology today, whether they concern the host immune response to infection, transcriptomic changes undergone by infected hepatocytes, or the development of curative therapeutics.
Identification and characterization of positive regulators governing hepatitis C virus host tropism
No full textHepatitis C virus (HCV) continues to affect millions of lives worldwide. Although the advent of highly effective direct-acting antivirals (DAAs) has revolutionized clinical management of chronic hepatitis C as the great majority of affective individuals can be cured of their infection, DAAs are very expensive and HCV transmission remains high in most countries, including the United States. Therefore, the development of a vaccine is essential to contain HCV transmission more efficiently. However, systematic testing of vaccine candidates is significantly hindered because HCV can only infect humans and chimpanzees. Understanding HCV’s limited host tropism is critical towards the development of an animal model for HCV infection. This study hypothesizes that human-specific host factors exist which are not expressed in murine cells or whose murine orthologues are not functional, that can boost HCV permissiveness in murine cells. Prior studies have implicated Tripartite Motif Containing 26 (TRIM26), and cyclophilin A (CypA) to modulate HCV replication efficiency across different species. However, our results do not confirm that their expression augments HCV infection in murine cells. By utilizing the human ORFeome library, a gain of function screen was conducted to identify additional human factors that augment HCV infection in murine cells. In parallel, transgenic mice were generated to express human factors and infected with HCV to assess the effect of these human factors on HCV infection in vivo. Identifying and characterizing positive regulators that govern HCV host tropism will lead to the construction of an HCV-susceptible mouse that can be used for further research which will pave the way for the development of a vaccine for HCV
Probing the dynamics of Usutu virus infection, replication, and spread using an in vitro cell culture model
No full textAnalysis of the Impact of Site-specific Demethylation of N6-methyladenosine on Interferon Beta
No full textEpitranscriptomics is a growing field of study that refers to the factors outside of genes that affect expression via direct regulation of RNA molecules, by chemical modification. The most abundant and well-studied RNA modification is the methylation of the N6 position on adenosine, or N6-methyladenosine (m6A). The placement of this modification is mediated by the methyltransferase complex, METTL3 and METTL14. Our lab has amassed data demonstrated that the targeted disruption of METTL14 in mouse hepatocytes in vivo causes liver damage, including fibrosis and cirrhosis. It is presently unknown which specific transcript(s) – being subject to epitranscriptomic regulation – are responsible for the observed phenotype. To enable demethylation of specific transcripts, we constructed a CRISPR dCas13-FTO construct that can be directed to specific sites using a guide-RNA. As a proof-of-concept, we target interferon- (IFNB), which has been well studied in its relationship with METTL14 depletion in the liver, to demethylate three m6A modified sites on its RNA transcript. Ultimately, vector-mediated delivery of CRISPR dCas13-FTO can be used to specifically remove m6A modifications from other RNA transcripts in the liver that are up- or downregulated in response to METTL14 depletion
Characterization of the Role of Type I and III Interferon Receptor Signaling in Controlling Flavivirus Infection and the Development of Tools for Tracking Virally Infected Cells In Vivo
No full textYellow fever virus (YFV) is an arbovirus that contributes significantly to human
morbidity and mortality. The live-attenuated YFV vaccine strain, YFV-17D, is derived from
the virulent YFV Asibi clinical isolate. One of the most efficient vaccines ever developed,
YFV-17D represents a prototypical flavivirus convenient for the study of host control of
infection. Although the role of type I and II IFNs in controlling YFV-17D infection has been
previously described in vivo, the role of type III IFN is incompletely understood. We
characterized the clinical phenotype of mice deficient for type I, type III, and type I and III
IFN receptors upon challenge with YFV-17D. We observed that type III IFN contribution to
the control of YFV-17D infection was additive to type I as only type I-III IFN knock-out
(KO) mice exhibited a significant mortality (>50%). Although both type I and type I-III KO
mice displayed high viral load in serum and evidence of adaptive immune response activation,
type I-III KO mice showed significant viral neuroinvasion and partial evasion of the adaptive
immune response. In this work, we propose a model where type III IFN signaling is critical
in preventing blood brain barrier permeability during failure of the type I IFN-mediated innate
immune response against YFV-17D. In tandem, we recognized the general dearth of molecular
tools for the study of host immune control of YFV-17D infection, and made progress in
developing two tools for this purpose. A YFV-17D recombinant reporter strain and a platform
for the detection of intracellular YFV-17D RNA in single cells, while unique in their use and
applicability to in vitro and in vivo experimentation, will nonetheless prove valuable tools for the
investigation of host and flavivirus biology
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Identifying the role of DNA ligase I in hepatitis B virus cccDNA formation in human hepatoma cells
No full textOver 257 million people are chronically infected with hepatitis B virus (HBV) worldwide, and these patients are at a 100-fold higher risk for developing cirrhosis and hepatocellular carcinoma compared to the general population. Current treatment can only suppress the virus but rarely lead to a cure. Limitations to current treatment options are largely due to the persistence of the viral covalently closed circular DNA (cccDNA)—the critical viral replication intermediate. While previous studies have identified cccDNA elimination as the key to curing HBV infection, the mechanism for cccDNA formation and maintenance remains to be fully elucidated. Recent findings identified a minimal set of host factors necessary for cccDNA formation by biochemically reconstituting this reaction. To validate these findings in a cellular environment that more closely resembles the native biological context of an HBV infection, we first established a system to rapidly and conditionally deplete DNA ligase I, one of the five identified host factors, in HBV-susceptible human hepatoma cells by dually introducing CRISPR knockout and the degron system. Following HBV infection in this tissue culture model, we observed that neither LIG1 overexpression nor partial knockout demonstrated any effects on cccDNA levels, and the challenges of establishing a complete knockout precluded us from drawing definitive conclusions on the role LIG1 plays in cccDNA formation. The findings in this study extend the applicability of the degron system to studying the role of essential host factors in viral-host interactions and contribute to current work in decoding the mechanisms of cccDNA formation. By establishing an experimental framework to interrogate the effects of essential host factors in cccDNA formation, this study lays the foundation for identifying promising therapeutic targets for chronic HBV patients
Development of platforms to dissect interactions between the transcriptional networks of chronic viral hepatitis B and Plasmodium parasite co-infections in the liver
No full textespite significant progress in the last decade, malaria remains an important and
difficult global health challenge, with over 438,000 deaths in the last year alone. Five species
of the Plasmodium parasite, transmitted by the female Anopheles mosquito vector, cause
malaria in humans. Most regions affected by malaria are also highly co-endemic with
hepatitis B virus (HBV) infections, which affect over 240 million people worldwide. Malaria
parasites and HBV both infect the liver, specifically at the hepatocyte level which serves as
the obligate site for all human Plasmodium liver stage development and the unique host
reservoir for HBV. Despite their high co-endemicity, little is known how these hepatotropic
pathogens interact at the transcriptomic level or collectively influence host response and
disease severity. Research efforts have been thwarted by a lack of reliable models that
physiologically recapitulate and support hepatotropic infection in vivo.
To this end, the Ploss Lab has developed the novel human liver chimeric FAH-/-
NOD-Rag1-/-/IL-2Rγnull (FNRG) mouse model to study co-infections of the different human
Plasmodium species and HBV. Given the tremendous logistical challenges with obtaining
malaria parasites, we first sought to develop the scientific infrastructure for infecting FNRG
mice with Plasmodium species. Here, we demonstrate established pipelines and procedures for
dissecting mosquito salivary glands to obtain Plasmodium falciparum sporozoites, for infecting
mice via live feeding of Plasmodium vivax-infected mosquitoes in Iquitos, Peru, and for
quantifying relative parasite abundance of Plasmodium malariae and Plasmodium ovale in coinfected
patients in Blantyre, Malawi. Furthermore, we demonstrate that the FNRG model is
capable of supporting high levels of persistent HBV infection, which is a requisite for
subsequent analysis of hepatotropic co-infections. In addition, to successfully visualize
infection, we have developed indirect immunofluorescence assays for Plasmodium and HBV
antigens and applied the fluorescent lipophilic dye DiR for tracing infection in vivo. Lastly, we
have optimized a laser capture microdissection protocol to extract small numbers of cells for
subsequent RNA quantification. These platforms, encompassing the diagnosis of malaria
patients, generation of mosquitoes, development of sporozoites, infection of mice,
quantification and visualization of viremia and parasitemia, and isolation of infected cells,
demonstrate the vast potential of this framework toward the ultimate aim of characterizing
the transcriptomic networks of malaria and HBV co-infections in the human liver
Of Mice and Men: Characterizing Dengue Virus Infection in Tissue Specific STAT1 Knockout Mice
No full textDengue Virus (DENV) is a positive sense RNA virus with four serotypes and transmitted via Aedes aegypti and Aedes albopictus mosquitos. Over half of the world¿s population is at risk for contracting DENV, which has symptom severity manifesting anywhere from flu-like symptoms to hemorrhagic fever and death. Secondary, heterologous infection is particularly concerning because no cross-protection exists between serotypes and disease symptoms during secondary infections are often more severe. There are no specific treatments for Dengue Virus infection, though promising steps have been made towards developing a tetravalent vaccine. Study of the complex biology of Dengue disease as well as the development of more effective therapies has been hampered by the lack of a suitable small animal model that faithfully recapitulates the human disease phenotype. In this paper we describe a series of assays and use them to characterize a novel model for DENV infection and pathology¿mice that are STAT1 deficient within all hematopoietic cells. These mice are both susceptible and permissive to DENV infection and replication but show immunity when re-challenged with a homologous serotype. Serum isolated from VAV-Cre mice also shows elements of antibody-mediated memory protection. While we have started to characterize this model, more work must be done. Our long-term objectives are to define the barriers of Dengue species tropism and to develop a model system where Dengue can replicate in the presence of a fully competent immune system
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