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Document Type:Latin Dissertation
Language of Document:English
Record Number:52895
Doc. No:TL22849
Call number:‭3275719‬
Main Entry:Rafael Andres Medina Silva
Title & Author:Sin nombre virus-host interactions: In vivo inhibition and viral entry in the deer mouse (Peromyscus maniculatus) modelRafael Andres Medina Silva
College:The University of New Mexico
Date:2007
Degree:Ph.D.
student score:2007
Page No:152
Abstract:Hantaviruses, which are enveloped negative-sense RNA viruses, comprise a genus of the family Bunyaviridae. The virions of hantaviruses contain trisegmented genomes designated the small (S), medium (M) and large segments. The S segment encodes the nucleocapsid protein (N), the M segment encodes the Gc and Gn (also known as G1 and G2) envelope glycoproteins and the L segment encodes the L protein, an RNA dependent RNA polymerase (RdRp). Hantaviruses, which are normally borne by specific rodent carriers, have been associated with two distinct illnesses. Viruses indigenous to Europe and Asia cause hemorrhagic fever with renal syndrome (HFRS), whereas those of the Western hemisphere cause hantavirus cardiopulmonary syndrome (HCPS). Sin Nombre virus (SNV) is the prototypical and most important etiologic agent of HCPS in North America. The natural reservoir for SNV is the deer mouse (Peromyscus maniculatus, Pm), which is found throughout the United States and Canada. Currently there are no disease models for SNV infection and treatments specific to SNV do not exist. In addition, rodent host derived in vitro models have not been developed greatly hampering investigations of the molecular mechanisms involved during hantavirus infection of these cells. Reports have demonstrated that the beta3 subunit of the human alphaVbeta3 (αVβ3) integrin is used by pathogenic hantaviruses to enter human cells. It is currently unknown whether rodent reservoir β3 integrins support hantavirus entry into host cells. However, recent studies have suggested that the murine (Mus musculus) β3 molecule does not confer infectability to non-permissive cells. Using a previously established deer mouse infection model for SNV, we developed novel approaches to evaluate the in vivo inhibitory activities of ribavirin, human convalescent immune plasma (HIP), the anti-beta3 integrin antibody ReoPro and polyclonal antibodies against the SNV-N protein when given concurrently with viral infection. These results demonstrated that ribavirin, HIP and ReoPro were able to reduce the viral loads of SNV infected animals. Titration of ribavirin and HIP, the strongest inhibitors, indicated that this effect was dose dependent. Importantly, the subtle but significant decrease in viral load observed with ReoPro treatment suggested for the first time that the deer mouse β3 might serve as an entry molecule in the rodent host. We therefore, cloned and expressed Pmβ3 in non-permissive Chinese hamster ovary cells (CHO). Our results demonstrate that expression of either human or Pmβ3 was sufficient to increase the permissiveness of CHO cells to SNV infection. To further pursue this question we developed and established an in vitro infection model based on deer mouse cells. We show that primary Pm-astrocytes, which express the β3 integrin subunit, are competent for SNV infection and that replication is supported in those cells. Knock-down of the Pmβ3 mRNA led to a 73% reduction in viral load in Pm-astrocytes. Pre-treatment of these cells with anti-β3 antibodies or with the natural ligand for αVβ3, vitronectin, diminished viral RNA load by 38-46% and activation of integrins by MnCl 2 or dithiothreitol (DTT) decreased RNA load by 42-45%. Altogether, our data indicate that Pmβ3 is used as an entry molecule during SNV infection of host cells in vitro and in vivo, and that our deer mouse infection model serves as a useful and unique tool to assess the antiviral activities of potential therapeutics against SNV.
Subject:Health and environmental sciences; Biological sciences; Beta3 integrin; Deer mouse; Peromyscus maniculatus; Sin nombre virus; Viral entry; Microbiology; Pathology; Virology; 0720:Virology; 0571:Pathology; 0410:Microbiology
Added Entry:B. Hjelle
Added Entry:The University of New Mexico