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



Nanhua Chen1, 2, *, Ranjni J. Chand2, Raymond R. R. Rowland2
1 College of Veterinary Medicine, Yangzhou University, Jiangsu 225009, P.R. China.
2 Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, Kansas, United States.


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© 2017 Chen et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, P.R. China, Tel: +86-514-87988323; Fax: +086-514-87972218; E-mail: chnhlh@126.com


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.