PMID- 28839504
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20200929
IS  - 1874-3579 (Print)
IS  - 1874-3579 (Electronic)
IS  - 1874-3579 (Linking)
VI  - 11
DP  - 2017
TI  - Deep Sequencing Details the Cross-over Map of Chimeric Genes in Two Porcine 
      Reproductive and Respiratory Syndrome Virus Infectious Clones.
PG  - 49-58
LID - 10.2174/1874357901711010049 [doi]
AB  - 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% (T(289)C) 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.
FAU - Chen, Nanhua
AU  - Chen N
AD  - College of Veterinary Medicine, Yangzhou University, Jiangsu 225009, P.R. China.
AD  - Department of Diagnostic Medicine and Pathobiology, College of Veterinary 
      Medicine, Kansas State University, Manhattan, KS 66506, Kansas, United States.
FAU - Chand, Ranjni J
AU  - Chand RJ
AD  - Department of Diagnostic Medicine and Pathobiology, College of Veterinary 
      Medicine, Kansas State University, Manhattan, KS 66506, Kansas, United States.
FAU - Rowland, Raymond R R
AU  - Rowland RRR
AD  - Department of Diagnostic Medicine and Pathobiology, College of Veterinary 
      Medicine, Kansas State University, Manhattan, KS 66506, Kansas, United States.
LA  - eng
PT  - Journal Article
DEP - 20170630
PL  - United Arab Emirates
TA  - Open Virol J
JT  - The open virology journal
JID - 101480213
PMC - PMC5543688
OTO - NOTNLM
OT  - Cross-over map
OT  - Deep sequencing
OT  - Green fluorescent protein
OT  - Infectious clone
OT  - Porcine reproductive and respiratory syndrome virus (PRRSV)
OT  - Viable recombinant
EDAT- 2017/08/26 06:00
MHDA- 2017/08/26 06:01
PMCR- 2017/01/01
CRDT- 2017/08/26 06:00
PHST- 2016/10/06 00:00 [received]
PHST- 2016/11/01 00:00 [revised]
PHST- 2017/02/06 00:00 [accepted]
PHST- 2017/08/26 06:00 [entrez]
PHST- 2017/08/26 06:00 [pubmed]
PHST- 2017/08/26 06:01 [medline]
PHST- 2017/01/01 00:00 [pmc-release]
AID - TOVJ-11-49 [pii]
AID - 10.2174/1874357901711010049 [doi]
PST - epublish
SO  - Open Virol J. 2017 Jun 30;11:49-58. doi: 10.2174/1874357901711010049. eCollection 
      2017.