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. 2019 Feb 5;93(4):e02057-18.
doi: 10.1128/JVI.02057-18. Print 2019 Feb 15.

Predicting Intraserotypic Recombination in Enterovirus 71

Andrew Woodman  1 , Kuo-Ming Lee  2 , Richard Janissen  3 , Yu-Nong Gong  2 , Nynke H Dekker  3 , Shin-Ru Shih  2   4   5   6 , Craig E Cameron  1
Affiliations

Predicting Intraserotypic Recombination in Enterovirus 71

Andrew Woodman et al. J Virol. .

Abstract

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.

Keywords: EV-A71; conservation; predictive; replicative recombination.

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Figures

FIG 1
FIG 1
Enterovirus 71 (EV-A71) recombination in RD cells is primarily replicative. (A) Cell-based EV-A71 recombination assay. C2 strain firefly luciferase-encoding subgenomic replicon (donor) and full-length EV-A71 C2-MP4 strain genome (acceptor) carrying a lethal deletion of the 3Dpol region were cotransfected in an equimolar ratio into RD cells. A fully functional virus genome can be produced via an RdRp template switch from donor to acceptor (indicated by dashed black arrow). (B) Only upon cotransfection can replication-competent virus be generated (PFU/ml ± standard deviations [SD]; n = 3) (C) Example sequences of plaque-purified recombinant virus from C2/C2 (left panel). Dashed arrows indicate predicted paths of viral RdRp upon template switching. Numbering refers to the position upon the acceptor templates. Lowercase, boldface nucleotides indicate the 5ʹ and 3ʹ boundaries of recombination. The underlined sequences indicate region of homology. (D) Nonreplicative recombination assay. IRES deletion of the C2 donor template inhibits translation. The acceptor template remains the same as in panel A. Viable virus will only be produced via a cell-mediated event. (E) Yield of recombinant virus (PFU/ml ± SD; n = 3) originating from transfection in equimolar ratio of replicative and nonreplicative partners. Statistical analyses were performed using an unpaired, two-tailed t test (***, P = 0.0001).
FIG 2
FIG 2
Mutation to the donor RdRp inhibits recombination and increases susceptibility to ribavirin. (A) L421A mutation does not impact virus yield. Yields of virus are shown for wild-type EV-A71 C2-MP4 and the L421A variant after transfection of RNA (PFU/ml ± SD; n = 3). (B) L421A mutation does not impact donor template replication. Cells were transfected with 250 ng of wild-type EV-A71 replicon and the L421A variant with or without 4 mM guanidine hydrochloride. The luciferase activity is reported in relative light units (RLU) per microgram of total protein in the extract at 8 h posttransfection. (C) L421A inhibits EV-A71 replicative recombination. Yields of recombinant virus following transfection of either wild-type or L421A variant donor template with acceptor RNA in RD cells were determined (PFU/ml ± SD; n = 3). Statistical analyses were performed using an unpaired, two-tailed t test (***, P = 0.0003). (D) EV-A71 L421A population is highly susceptible to ribavirin. RD cells were infected at an MOI 0.1 with wild-type or L421A variant EV-A71 C2-MP4 virus in the presence of various concentrations of ribavirin. After achieving a CPE, virus supernatant was clarified and used for a plaque assay. Results show titer of virus normalized to an untreated control (PFU/ml ± SD; n = 3). Statistical analyses were performed using an unpaired, two-tailed t test (***, P = 0.0004; **, P < 0.005).
FIG 3
FIG 3
Phylogenetic analysis of EV-A71 genotypes B and C. Neighbor-joining phylogenetic analysis of EV-A71 genotypes B and C was based on their P2-P3 genome region, rooted by the coxsackievirus (CV) A16 prototype strain G10 (isolated in 1951). The subtrees show mixed clusters of evolutionary intra- and intertypic recombination events of analyzed EV-A71 sequences (n = 182). EV-A71 subgenotypes and genotypes are depicted in different colors. The subgenotype B5 (orange arrow) is located within the genotype B cluster, showing a ladder-like evolutionary scale. In contrast to other subgenotypes of genotype C, the subgenotype C4 (labeled in green) forms an outgroup of genotype C, close to other recombinogenic EV-A71 strains (e.g., B3 and C2-like) and the prototype CV-A16 sequence. The probabilities of replicate trees in which associated taxa clustered together in the bootstrapped data (1,000 replicates) are shown next to the branches. The phylogenetic tree is drawn to scale, with branch lengths representing the numbers of base substitutions per site.
FIG 4
FIG 4
Alternate donor templates significantly impact viable recombinant frequency in a predictable manner. (A) Single-step growth curve at an MOI 10 of EV-A71 C4 and B5 strains shows no significant difference in replication. (B) C2, C4, and B5 subgenomic replicon firefly luciferase time courses. Cells were transfected with 250 ng of each respective EV-A71 replicon, and the luciferase activity is reported in RLU per microgram of total protein in the extract. (C) EV-A71 recombination assay. RD cells were transfected with the various EV-A71 subgenomic replicon donors and EV71Δ3D RNA. The results show the yield of recombinant virus (PFU/ml ± SD; n = 3). Statistical analyses were performed using an unpaired, two-tailed t test (**, P < 0.01; *, P < 0.05). (D and E) Representative recombinant sequences of plaque-purified recombinant virus from C4/C2 (E) and B5/C2 (F). Dashed arrows indicate predicted paths of viral RdRp upon template switching. Numbering refers to the position on the acceptor templates. Lowercase, boldface nucleotides indicate the 5ʹ and 3ʹ boundaries of recombination. Underlined sequences indicate regions of homology.
FIG 5
FIG 5
Intraserotypic recombination between EV-A71 C2 and C4 subgenotypes requires homology at the recombination junction, but the triggers for template-switching are sequence independent. (A) Positions of template-switching events observed during intratypic recombination between the EV-A71 C2 and C4 subgenotypes. The individual positions of observed strand switching across the P2 genome region are marked in red, including their corresponding nucleotide sequence numbers with respect to the C2 acceptor strand. (B) Sequences of bona fide C2/C4 recombinant viruses. The red border highlights the matching homologous sequences at the recombination sites with lengths between 5 and 11 nucleotides (x̄ = 7 ± 2 nucleotides). The sequences were subject to M-COFFEE, a multiple sequence alignment algorithm, to identify possible gapped sequence motifs. Scores of <50 are considered to exhibit poor sequence consistency (scores range from 0 to 100). The sequence homology for the matching homologous sequences was found to be 58% ± 2%. A consensus sequence with 70% probability is shown below using IUPAC nomenclature. (C) Logo of ungapped de novo sequence motif search using the MEME algorithm that represents a sequence-aligned, position-dependent nucleotide probability matrix. The resulting motif sequence with an E value of 0.06 and a bit value of <1 show no statistical significance and thus failed to find a sequence motif as a recombination trigger. (D) Probability of position-dependent nucleotides at the homologous recombination sequences without sequence alignment. (E to G) The G-C nucleotide density (E), numbers of successive G-C/A-U bases (F), and CpG contents (G) of the homologous recombination sequences and the entire C2 genome (10-nucleotide sequence window) exhibit no significant differences. Statistical analysis was performed using a one-way, two-tailed analysis of variance with comparative Tukey post hoc test (n.s., not significant).
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References

    1. Hyypia T, Hovi T, Knowles NJ, Stanway G. 1997. Classification of enteroviruses based on molecular and biological properties. J Gen Virol 78:1–11. doi:10.1099/0022-1317-78-1-1. - DOI - PubMed
    1. Adams MJ, Lefkowitz EJ, King AMQ, Bamford DH, Breitbart M, Davison AJ, Ghabrial SA, Gorbalenya AE, Knowles NJ, Krell P, Lavigne R, Prangishvili D, Sanfacon H, Siddell SG, Simmonds P, Carstens EB. 2015. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses. Arch Virol 160:1837–1850. doi:10.1007/s00705-015-2425-z. - DOI - PubMed
    1. Cameron CE, Oh HS, Moustafa IM. 2010. Expanding knowledge of P3 proteins in the poliovirus lifecycle. Future Microbiol 5:867–881. doi:10.2217/fmb.10.40. - DOI - PMC - PubMed
    1. Domingo E, Baranowski E, Escarmis C, Sobrino F, Holland JJ. 2002. Error frequencies of picornavirus RNA polymerases: evolutionary implications for virus populations, p 285–298. In Semler B, Wimmer E (ed), Molecular Biology of Picornavirus. ASM Press, Washington, DC. doi:10.1128/9781555817916.ch23. - DOI
    1. Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. 2006. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439:344–348. doi:10.1038/nature04388. - DOI - PMC - PubMed

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