Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia

Viruses. 2020 Sep 11;12(9):1017. doi: 10.3390/v12091017.


To monitor the arthropod-borne virus transmission in mosquitoes, we have attempted both to detect and isolate viruses from 3304 wild-caught female mosquitoes in the Livingstone (Southern Province) and Mongu (Western Province) regions in Zambia in 2017. A pan-flavivirus RT-PCR assay was performed to identify flavivirus genomes in total RNA extracted from mosquito lysates, followed by virus isolation and full genome sequence analysis using next-generation sequencing and rapid amplification of cDNA ends. We isolated a newly identified Barkedji virus (BJV Zambia) (10,899 nt) and a novel flavivirus, tentatively termed Barkedji-like virus (BJLV) (10,885 nt) from Culex spp. mosquitoes which shared 96% and 75% nucleotide identity with BJV which has been isolated in Israel, respectively. These viruses could replicate in C6/36 cells but not in mammalian and avian cell lines. In parallel, a comparative genomics screening was conducted to study evolutionary traits of the 5'- and 3'-untranslated regions (UTRs) of isolated viruses. Bioinformatic analyses of the secondary structures in the UTRs of both viruses revealed that the 5'-UTRs exhibit canonical stem-loop structures, while the 3'-UTRs contain structural homologs to exoribonuclease-resistant RNAs (xrRNAs), SL-III, dumbbell, and terminal stem-loop (3'SL) structures. The function of predicted xrRNA structures to stop RNA degradation by Xrn1 exoribonuclease was further proved by the in vitro Xrn1 resistance assay.

Keywords: 3′-UTR; 5′-UTR; Barkedji virus; Barkedji-like virus; RNA secondary structure; dISFVs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Cell Line
  • Culex / virology
  • Culicidae / virology
  • Exoribonucleases / chemistry
  • Exoribonucleases / classification
  • Exoribonucleases / genetics*
  • Female
  • Flavivirus / enzymology*
  • Flavivirus / genetics*
  • Flavivirus / isolation & purification
  • Genome, Viral
  • Insect Proteins / genetics
  • Insecta / virology*
  • Israel
  • Phylogeny
  • Zambia


  • 3' Untranslated Regions
  • Insect Proteins
  • Exoribonucleases