Arbovirus Entry and Characterization of a New Insect Virus

Arboviruses (Arhtropod-borne viruses) include the alphaviruses, flaviviruses and (-) strand RNA bunyaviruses, among them there are over 700 viruses currently known and they are considered a major source of animal and human disease worldwide. Understanding the process of virus infection is fundamental for drug discovery and vaccine development. Different mechanisms have been proposed for Alphavirus and Flavivirus virus entry and genome delivery. Controversial observations led to a general belief that membrane containing viruses such as alphaviruses and flaviviruses can infect cells by either endocytosis or membrane fusion. However for both families alternative entry pathways have been recently proposed. For alphaviruses such as Sindbis the prototype alphavirus, an entry process by direct penetration at the cell surface and neutral pH has been proposed and supported by both electron microscopy and biochemical studies. This mechanism was also proposed for the interaction of flaviviruses such as Dengue and West Nile virus with host cells, and suggests that an alternative pathway may exist for both virus families. Here, the mechanism of Sindbis virus penetration has been revisited using direct observation of the process by electron microscopy under a set of different temperatures non permissive for endocytosis or vesicular transport. Results show that at such temperatures events occur which allow the entry of the virus genome into the cells, allowing the temperature dependence of the process to be observed. Consequently, the delivery of the viral RNA does not require low pH mediated endocytosis or low pH fusion.;An alternative entry process for Flaviviruses has also been investigated by similar ultrastructural techniques. Our preliminary results show that, Dengue virus did not present direct fusion with plasma membrane of host cells and also did not enter by endocytosis. Supporting the hypothesis of entry independent of low pH mediated endocytosis and in agreement with the process proposed for alphaviruses. However due to the high particle/pfu ratio of flaviviruses produced in laboratory, this process was also analyzed by infecting cells in a low volume environment with West Nile virus obtained directly from the mosquito host. Remarkably, the ultrastructural observations support the preliminary results obtained with Dengue virus-cell interactions. These data suggest that medically important flaviviruses infect cells by a mechanism that involves direct penetration of the host cell plasma membrane. In attempting to obtain a more virulent strain of Dengue virus for the studies above, while improving purification methods for Dengue we have observed by electron microscopy a previously unidentified virus. This virus was different from all known flaviviruses and could not be detected by any common procedure. A sequence of the genome has been produced by de novo assembly and was not found to match to any known viral sequence. The composition and three dimensional structure of ESV are presented and its sequence compared to other members of the Birnavirus family. This new virus was named Espirito Santo Virus and was classified as a new Entomobirnavirus which infect insect cells. Interestingly, ESV was found to grow better upon co-infection with a virulent strain of Dengue-2 and to replicate in C6/36 insect cells but not in mammalian Vero cells. Interestingly, during this co-infection, Dengue viral proteins can be detected, but no viral particles are assembled. The uniqueness of this relationship remains to be elucidated.