Abstract
Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4×106 in BHK-21 (vertebrate) cells and ∼1.05×105 in C710 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely.
Highlights
The family Flaviviridae contains three genera of viruses of significant human and veterinary importance: Hepacivirus (Human Hepatitis C virus), Pestivirus (bovine viral diarrhea virus (BVDV) and classical swine fever virus), and Flavivirus
Analysis of chikungunya virus (CHIKV) intra-genic recombination To validate that our strategy of using viral genomes containing intra-open reading frame (ORF) deletions was capable of detecting intra-genic recombination of positive sense single stranded RNA viruses, the relative efficiency of CHIKV intra-genic recombination was evaluated using a replicon/defective helper system that contains complementary 59 and 39 deletions in the structural ORF (Fig. 1B)
CHIKV intra-genic recombinant isolates were competent for replication in both Vero and C6/36 cells and reached average titers of 2.966107 pfu/mL by 48 h post-infection in C6/36 cells, which are comparable to titers observed post-infection with infectious clone derived wt CHIKV
Summary
The family Flaviviridae contains three genera of viruses of significant human and veterinary importance: Hepacivirus (Human Hepatitis C virus), Pestivirus (bovine viral diarrhea virus (BVDV) and classical swine fever virus), and Flavivirus. The flavivirus genome is ,11–12 Kb, which is comprised of a positive sense single stranded RNA molecule that contains a 59methylguanosine cap but no poly-adenosine tail. The flavivirus virion is ,50 nm in diameter and is comprised of an electron dense nucleocapsid core surrounded by an endoplasmic reticulum derived lipid bilayer [6,7,8]. Within this lipid bilayer are embedded the mature membrane and E proteins. The flavivirus virion contains 180 E proteins associated as dimers that are oriented parallel to the lipid bilayer in an icosaheldral symmetry [12]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
