title: Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes.
abstract: BACKGROUND
  Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.
  
  
  RESULTS
  B2 protein expression from SINV (TE/3'2J) inhibited the accumulation of non-specific small RNAs in Aedes aegypti mosquito cell culture and virus-specific small RNAs both in infected cell culture and Ae. aegypti mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2) compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in Ae. aegypti.
  
  
  CONCLUSIONS
  We show that RNAi is active in Ae. aegypti cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. ccirimot@jhsph.edu
authors.forename: Chris M
authors.initials: CM
authors.lastname: Cirimotich
authors.forename: Jaclyn C
authors.initials: JC
authors.lastname: Scott
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Phillips
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Geiss
authors.forename: Ken E
authors.initials: KE
authors.lastname: Olson
mesh_terms: D000330:Aedes / Q000821:virology*; D000524:Alphavirus / Q000472:pathogenicity* / Q000502:physiology; D018354:Alphavirus Infections / Q000821:virology*; D000818:Animals; D002522:Chlorocebus aethiops; D006224:Cricetinae; D015967:Gene Expression Regulation, Viral; D034622:RNA Interference; D012367:RNA, Viral / Q000378:metabolism; D016865:Reassortant Viruses / Q000235:genetics / Q000472:pathogenicity; D012845:Sindbis Virus / Q000235:genetics / Q000472:pathogenicity; D014709:Vero Cells; D014779:Virus Replication / Q000235:genetics*
chemical_list: D012367:RNA, Viral