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. 2017 Jun 30:11:49-58.
doi: 10.2174/1874357901711010049. eCollection 2017.

Deep Sequencing Details the Cross-over Map of Chimeric Genes in Two Porcine Reproductive and Respiratory Syndrome Virus Infectious Clones

Nanhua Chen  1   2 , Ranjni J Chand  2 , Raymond R R Rowland  2
Affiliations

Deep Sequencing Details the Cross-over Map of Chimeric Genes in Two Porcine Reproductive and Respiratory Syndrome Virus Infectious Clones

Nanhua Chen et al. Open Virol J. .

Abstract

Background: Recombination is an important contributor to the genetic diversity of most viruses. A reverse genetics system using green fluorescence protein (GFP)- and enhanced GFP (EGFP)-expressing infectious clones was developed to study the requirements for recombination. However, it is still unclear what types of cross-over events occurred to produce the viable offspring.

Objective: We utilized 454 sequencing to infer recombination events in this system.

Method: Two porcine reproductive and respiratory syndrome virus (PRRSV) infectious clones, P129-EGFP-97C and P129-GFPm-d (2-6), were co-transfected into HEK-293T cells. P129-EGFP-97C is a fully functional virus that contains a non-fluorescent EGFP. P129-GFPm-d (2-6) is a defective virus but contains a fluorescent GFPm. Successful recombination was evident by the appearance of fully functional progeny virus that expresses fluorescence. Total RNA was extracted from infected cells expressing fluorescence, and the entire fluorescent gene was amplified to prepare an amplicon library for 454 sequencing.

Results: Deep sequencing showed that the nucleotide identities changed from ~37% (in the variable region from 21nt to 165nt) to 20% (T289C) to ~38% (456-651nt) then to 100% (672-696nt) when compared to EGFP. The results indicated that cross-over events occurred in three conserved regions (166-288nt, 290-455nt, 652-671nt), which were also supported by sequence alignments. Remarkably, the short conserved region (652-671nt) showed to be a cross-over hotspot. In addition, four cross-over patterns (two single and two double cross-over) might be used to produce viable recombinants.

Conclusion: The reverse genetics system incorporating the use of high throughput sequencing creates a genetic platform to study the generation of viable recombinant viruses.

Keywords: Cross-over map; Deep sequencing; Green fluorescent protein; Infectious clone; Porcine reproductive and respiratory syndrome virus (PRRSV); Viable recombinant.

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Figures

Fig. (1)
Fig. (1)
Recombination between two PRRSV infectious clones P129-EGFP-97C and P129-GFPm-d (2-6). P129-EGFP-97C is a fully functional non-fluorescent virus and P129-GFPm-d (2-6) is a fluorescent defective virus lacking ORF2-6. Successful recombination between the two parental viruses is evidenced by producing viable fluorescent progeny viruses.
Fig. (2)
Fig. (2)
Evidence for the occurrence of cross-over events. The percentages of nucleotide identity to EGFP gene decreased from 37% (21bp-165bp variable region) to 20% (C289T substitution), then increased to 38% (456bp-651bp variable region) and to 100% (672bp-696bp variable region). The changes indicated that cross-over events occurred in three conserved regions: 166bp-288bp, 290bp-455bp, and 652bp-671bp.
Fig. (3)
Fig. (3)
Four potential cross-over patterns used for producing recombinants. Two single cross-over Figs. (3A and B) and two double cross-over Figs. (3C and D) could be used to generate the viable fluorescent recombined viruses in this in vitro reverse genetics system.
Fig. (4)
Fig. (4)
The alignment analysis identified two cross-over events. The cross-over events in the 123bp conserved region (4A) and 20bp conserved region (4B) were identified. The recombinants are identical to GFPm in region-1 but identical to EGFP in region-3, which were highlighted in red dotted line box. The conserved and cross-over regions are in region-2.
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References

    1. Perez-Losada M, Arenas M, Galan JC, et al. Recombination in viruses: Mechanisms, methods of study, and evolutionary consequences. Infection, genetics and evolution. journal of molecular epidemiology and evolutionary genetics in infectious diseases 30C. 2015:296–307. - PMC - PubMed
    1. Simon-Loriere E., Holmes E.C. Why do RNA viruses recombine? Nat. Rev. Microbiol. 2011;9:617–626. - PMC - PubMed
    1. Lunney J.K., Fang Y., Ladinig A., Chen N., Li Y., Rowland R.R., Renukaradhya G.J. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV): Pathogenesis and Interaction with the Immune System. Annu. Rev. Anim. Biosci. 2016;4:129–154. - PubMed
    1. Pasternak A.O., Spaan W.J., Snijder E.J. Regulation of relative abundance of arterivirus subgenomic mRNAs. J. Virol. 2004;78:8102–8113. - PMC - PubMed
    1. Galetto R., Giacomoni V., Veron M., Negroni M. Dissection of a circumscribed recombination hot spot in HIV-1 after a single infectious cycle. J. Biol. Chem. 2006;281:2711–2720. - PubMed

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