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doi: 10.1371/journal.pone.0023247. Epub 2011 Aug 3.

Stability of yellow fever virus under recombinatory pressure as compared with chikungunya virus

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Stability of yellow fever virus under recombinatory pressure as compared with chikungunya virus

Charles E McGee et al. PLoS One. 2011.

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 x 106 in BHK-21 (vertebrate) cells and ∼1.05 x 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.

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Conflict of interest statement

Competing Interests: Authors Bruno Guy and Jean Lang are currently employed by Sanofi Pasteur. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Schematic representation of flavivirus and alphavirus recombinant crosses.
A) Yellow fever virus 17D deletion mutant replicon recombinant cross, B) chikungunya virus deletion mutant replicon/defective helper recombinant cross.
Figure 2
Figure 2. Northern blot analysis of chikungunya virus (CHIKV) intra-genic recombinant isolates.
Blot was hybridized with CHIKV-5′UTR BioProbe. Lane 1: CHIKV RNA ladder (size markers indicate CHIKV RNAs of full length genomic, replicon, full length structural gene helper, and capsid only structural helper respectively), Lane 2: CHIKV-LR-3′Δ-Replicon only electroporation, Lane 3: CHIKV-LR-5′Δ-Helper only electroporation, Lanes 4 and 8: CHIKV-LR-3′Δ-Replicon/ CHIKV-LR-5′Δ-Helper co-electroporations, and Lanes 5, 6, 7, 9, and 10: clonal recombinant isolates.
Figure 3
Figure 3. Schematic representation of parental replicon/defective helper recombinant cross (CHIK-LR-3′Δ-Replicon and CHIK-LR-5Δ′-Helper) along with sequence topology analysis of the cross-over region and resulting recombinant topology of clonally isolated recombinant genomes.
Genes not necessarily shown to scale. aa-amino acid, E-envelope glycoprotein, ns-nonstructural, nt-nucleotide, LR-LaReunion, and UTR-untranslated region, @LR location of sequence deletion.
Figure 4
Figure 4. Comparison of post-electroporation growth kinetics of yellow fever viruses (YFV) and deletion mutants in BHK-21 cells.
A) YFV 17D (▪ solid line), YFV 17D GFP (▪ dashed line), and YFV 17D Cherry (○しろまる solid line); B) YFV 17D 5′ ΔE (▴), YFV 17D 3′ ΔE (•), YFV 17D 5′ΔE Cherry (Δ), and YFV 17D 3′ ΔE GFP (○しろまる). Titers expressed as plaque forming units/mL.
Figure 5
Figure 5. Superinfection of YFV Asibi GFP infected Vero and C710 cells with either YFV 17D Cherry or CHIKV 5′ Cherry.
A) YFV 17D-Cherry growth kinetics on naïve (□しろいしかく) and YFV Asibi GFP infected (▪) Vero monolayers. B) YFV 17D Cherry growth kinetics on naïve (○しろまる) and YFV Asibi GFP infected (•) C710 monolayers. C) CHIKV 5′ Cherry growth kinetics on naïve (Δ) and YFV Asibi GFP infected (▴) Vero monolayers. D) CHIKV 5′ Cherry growth kinetics on naïve (◊) and YFV Asibi GFP infected (♦) C710 monolayers. Titers expressed as log10TCID50/mL.

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