Browsing by Subject "RNA Viruses"
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Item Mechanisms controlling virulence thresholds of mixed viral populations and identification of novel host barriers to poliovirus neuropathogenesis(2012-07-20) Lancaster, Karen; Pfeiffer, Julie K.;Neurotropic viruses comprise some of the worlds most widespread and deadly pathogens, including West Nile virus, rabies virus, and poliovirus. Poliovirus, as a model neurotropic virus, is also an RNA virus. RNA viruses have high mutation rates and a propensity to revert attenuating mutations, contributing to disease and complicating treatment and vaccine development. Despite worldwide epidemics in the early nineteenth century, paralysis from poliovirus is a rare event occurring in less than 1% of poliovirus infections. This suggests the presence of viral and host barriers limiting disease. Here we examined viral barriers by exploring the concept of virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that 1000-fold excess of an attenuated strain of poliovirus was protective against disease induced by the virulent strain. Protection was induced locally, was a poliovirus specific effect, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo, suggesting virulence thresholds may be modulated by competition for viral receptor. Furthermore, we found the attenuated virus became virulent in immune-deficient mice due to enhanced replication and reversion of attenuating mutations. We also identified additional host barriers limiting pathogenesis using a novel hybridization-based viral diversity assay to quantify the efficiency of poliovirus transport from the periphery to the central nervous system. We found viral replication in peripheral axons is limited and the type I interferon response limits viral replication in peripheral tissues, protecting against disease. Significantly, we discovered that retrograde axonal transport of poliovirus in the sciatic nerve was inefficient and only 20% of viral pool members reaching the brain. The efficiency of viral transport increased upon muscle damage, leading to increased viral diversity and pathogenesis. In summary, we identified a viral induced mechanism controlling virulence of mixed viral populations, and characterized three host barriers that restrict poliovirus pathogenesis in the nervous system. The identification of these barriers restricting virulence may help explain the rare incidence of neurological complications following poliovirus infection and aid in our understanding of viral population dynamics and pathogenesis. [Keywords: poliovirus, neurotropic, interferon, retrograde, axonal, transport, virulence, threshold, virus, barrier]Item Viral and Host Genetic Determinants of Hepatitis C Virus Persistence and Interferon Resistance(2006-05-16) Sumpter, Rhea Myers, Jr.; Gale, Michael Jr.Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV), which is an important cause of cirrhosis and hepatocellular carcinoma. HCV replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. The development of the HCV RNA replicon system has allowed the study of persistent HCV RNA replication in tissue culture. We evaluated HCV/IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV genotype 1b subgenomic RNA replicon that differed only at two sites within the NS5A coding region. A replicon with a lysine (K) insertion at HCV codon 2040 (K2040) replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an leucine (L) to serine (S) point mutation at HCV codon 2198 (L2198S) replicated poorly and triggered a cellular response characterized by IFN-! production and low-level interferon-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high passage (HP) variant with 6 additional point mutations throughout the HCV protein-coding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-" treatment and was associated with viral-directed reduction in host cell expression and action of ISG56, an antagonist of HCV RNA translation. We also demonstrated that HCV subgenomic RNA replicons can be used to model the early events of HCV infection. We found that HCV RNA replicons rapidly induce the cellular antiviral response upon their transfection into host Huh7 cells and we determined that intracellular HCV double stranded RNA (dsRNA) is a potent agonist of host dsRNAactivated pathways. A Huh7 derived cell line that is highly permissive for transduction by HCV replicons is specifically defective in the activation of interferon regulatory factor (IRF)-3 by virus infection or HCV dsRNA transfection. We found that a mutation in the caspase recruitment domain (CARD) of the DExH/D-box helicase protein RIG-I, a component of the TLR3-independent intracellular dsRNA-responsive IRF-3 activation pathway, was responsible for this phenotype. Restoration of RIG-I-mediated IRF-3 activation through genetic complementation resulted in decreased permissiveness to HCV RNA replication. These results establish the RIG-I!IRF-3 pathway as a critical determinant of HCV persistence.