ArticleThe open virology journal2017
Deep Sequencing Details the Cross-over Map of Chimeric Genes in Two Porcine Reproductive and Respiratory Syndrome Virus Infectious Clones.
Article in The open virology journal, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Authors and funding
3 authors at 1 institution in 2 countries.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundRecombination 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.
objectiveWe utilized 454 sequencing to infer recombination events in this system.
methodTwo 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.
resultsDeep sequencing showed that the nucleotide identities changed from ~37% (in the variable region from 21nt to 165nt) to 20% (T
conclusionThe reverse genetics system incorporating the use of high throughput sequencing creates a genetic platform to study the generation of viable recombinant viruses.
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