Abstract
Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are mosquito-borne pathogens that cause significant human diseases. A novel group of insect-specific flaviviruses (ISFs), which only replicate in mosquitoes, have also been identified. However, little is known about the mechanisms of ISF host restriction. We report the generation of infectious cDNA from two Australian ISFs, Parramatta River virus (PaRV) and Palm Creek virus (PCV). Using circular polymerase extension cloning (CPEC) with a modified OpIE2 insect promoter, infectious cDNA was generated and transfected directly into mosquito cells to produce infectious virus indistinguishable from wild-type virus. When infectious PaRV cDNA under transcriptional control of a mammalian promoter was used to transfect mouse embryo fibroblasts, the virus failed to initiate replication even when cell entry steps were by-passed and the type I interferon response was lacking. We also used CPEC to generate viable chimeric viruses between PCV and WNV. Analysis of these hybrid viruses revealed that ISFs are also restricted from replication in vertebrate cells at the point of entry. The approaches described here to generate infectious ISF DNAs and chimeric viruses provide unique tools to further dissect the mechanisms of their host restriction.
Highlights
Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are mosquito-borne pathogens that cause significant human diseases
We report the use of the circular polymerase extension cloning (CPEC) approach to generate chimeric viruses between insect-specific flaviviruses (ISFs) and vertebrate-infecting flaviviruses (VIFs), further expanding the tools to investigate mechanisms of ISF restriction as well as providing potential platform for generating recombinant viruses between ISFs and VIFs as candidates for safe vaccines and diagnostic antigens for flaviviral diseases
The modified Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) untranslated regions (UTRs)-linkers were assembled by CPEC with cDNA fragments from WNVKUN and directly transfected into C6/36 cells
Summary
Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are mosquito-borne pathogens that cause significant human diseases. The advent of deep sequencing methods, sensitive reverse transcription (RT) PCR assays using flavivirus generic primers and the development of broad-spectrum diagnostic tools, such as monoclonal antibodies (mAbs) to viral dsRNA intermediates, have seen the isolation of many new ISFs from various regions around the world[3, 6,7,8,9,10,11] These interesting viruses provide a unique model to investigate the molecular basis of their restriction to an insect host and efficient mode of vertical transmission. We report the use of the CPEC approach to generate chimeric viruses between ISFs and VIFs, further expanding the tools to investigate mechanisms of ISF restriction as well as providing potential platform for generating recombinant viruses between ISFs and VIFs as candidates for safe vaccines and diagnostic antigens for flaviviral diseases
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