RNA Interference Mediated Inhibition of Dengue Virus Multiplication and Entry in HepG2 Cells

Background: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. Methodology/Principal Findings: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8 % for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells

New Marsileaceous Fossils from the Late Cretaceous of South America and a Reevaluation of Marsileaceaephyllum.

Herein we report new macrofossils and associated microfossils representing the aquatic fern family Marsileaceae from the Campanian to Maastrichtian Cañadón del Irupé locality, La Colonia Formation, Chubut Province, Argentina. The macrofossils include compound leaves, detached leaflets, and sporocarps. The venation of the leaflets is consistent with the diagnosis of Marsileaceaephyllum, which we argue is poorly defined. Consequently, the circumscription and diagnosis of Marsileaceaephyllum are emended in order to limit the taxon to include only leaves or rhizomes bearing leaves with four leaflets that most resemble those of Marsilea among extant genera of Marsileaceae. The Cañadón del Irupé leaves and leaflets are assigned to the new genus Mirasolita, erected to encompass compound leaves bearing two petiolulate leaflets, each leaflet having a reniform lamina and reticulate venation. The architecture of Mirasolita leaves and leaflets is distinct from that of leaves of any of the living marsileaceous genera. The stalked sporocarps are assigned to the new genus Lugiomarsiglia. They are attributed to Marsileaceae because each has a sclerenchymatous wall surrounding circular bodies interpreted as sporangia. Associated megaspores are assigned to Molaspora lobata, a geographically widespread, primarily Late Cretaceous dispersed spore taxon. The co-occurrence of Molaspora lobata with an extinct marsileaceous leaf form fits a pattern wherein Cretaceous Regnellidium-like megaspores are associated with or found in situ within marsileaceous macrofossils that display a variety of morphologies.Fil: Hermsen, Elizabeth. Ohio State University; Estados Unidos de América;Fil: Gandolfo, Maria A.. Cornell University; Estados Unidos de América;Fil: Cuneo, Nestor Ruben. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Museo Paleontologico Egidio Feruglio; Argentin