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Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing
Pseudorabies virus (PRV) is a member of Alphaherpesvirinae subfamily, its neurotropism and latency infection attract the attention of many scientists. PRV tagged with a fluorescent reporter gene as a tracker has been used to analyze neuronal circuits, including anterograde and retrograde. In this st...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300229/ https://www.ncbi.nlm.nih.gov/pubmed/32595620 http://dx.doi.org/10.3389/fmicb.2020.01168 |
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author | Qi, Hansong Wu, Hongxia Abid, Muhammad Qiu, Hua-Ji Sun, Yuan |
author_facet | Qi, Hansong Wu, Hongxia Abid, Muhammad Qiu, Hua-Ji Sun, Yuan |
author_sort | Qi, Hansong |
collection | PubMed |
description | Pseudorabies virus (PRV) is a member of Alphaherpesvirinae subfamily, its neurotropism and latency infection attract the attention of many scientists. PRV tagged with a fluorescent reporter gene as a tracker has been used to analyze neuronal circuits, including anterograde and retrograde. In this study, we used fosmid library to construct a rapid and efficient platform to generate recombinant PRV. Firstly, the highly purified PRV ShuangCheng (SC) genomic DNA was sheared randomly into approximately 30–49-kb DNA fragments. After end-blunting and phosphorylation, the DNA fragments were cloned into the fosmid vector and transformed into Escherichia coli. A total of 200 fosmids that cover the complete genome of PRV SC was sequenced. Thirteen fosmid combinations in five groups were transfected into Vero cells, respectively, and each group can successfully rescue PRV strain SC. There was no significant difference between wild type and recombinant in both morphology and growth kinetics. In the next step, an enhanced green fluorescent protein (EGFP) was fused into the amino-terminal of UL36 protein by Red/ET recombination technology, and recombinant rPRV SC-UL36-EGFP was rescued successfully. At last, the single viral particles with green fluorescent were monitored retrograde moving in the axon with an average velocity of 0.71 ± 0.43 μm/s at 0.5–2 h post infection (hpi) and anterograde moving with an average velocity of 0.75 ± 0.49 μm/s at eight hpi. Integration of fosmid library and Red/ET recombination technology in our work was highly efficient and stable for constructing PRV recombinants. This study will accelerate understanding the biology of PRV and the development of novel vaccines. |
format | Online Article Text |
id | pubmed-7300229 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-73002292020-06-26 Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing Qi, Hansong Wu, Hongxia Abid, Muhammad Qiu, Hua-Ji Sun, Yuan Front Microbiol Microbiology Pseudorabies virus (PRV) is a member of Alphaherpesvirinae subfamily, its neurotropism and latency infection attract the attention of many scientists. PRV tagged with a fluorescent reporter gene as a tracker has been used to analyze neuronal circuits, including anterograde and retrograde. In this study, we used fosmid library to construct a rapid and efficient platform to generate recombinant PRV. Firstly, the highly purified PRV ShuangCheng (SC) genomic DNA was sheared randomly into approximately 30–49-kb DNA fragments. After end-blunting and phosphorylation, the DNA fragments were cloned into the fosmid vector and transformed into Escherichia coli. A total of 200 fosmids that cover the complete genome of PRV SC was sequenced. Thirteen fosmid combinations in five groups were transfected into Vero cells, respectively, and each group can successfully rescue PRV strain SC. There was no significant difference between wild type and recombinant in both morphology and growth kinetics. In the next step, an enhanced green fluorescent protein (EGFP) was fused into the amino-terminal of UL36 protein by Red/ET recombination technology, and recombinant rPRV SC-UL36-EGFP was rescued successfully. At last, the single viral particles with green fluorescent were monitored retrograde moving in the axon with an average velocity of 0.71 ± 0.43 μm/s at 0.5–2 h post infection (hpi) and anterograde moving with an average velocity of 0.75 ± 0.49 μm/s at eight hpi. Integration of fosmid library and Red/ET recombination technology in our work was highly efficient and stable for constructing PRV recombinants. This study will accelerate understanding the biology of PRV and the development of novel vaccines. Frontiers Media S.A. 2020-06-11 /pmc/articles/PMC7300229/ /pubmed/32595620 http://dx.doi.org/10.3389/fmicb.2020.01168 Text en Copyright © 2020 Qi, Wu, Abid, Qiu and Sun. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Qi, Hansong Wu, Hongxia Abid, Muhammad Qiu, Hua-Ji Sun, Yuan Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing |
title | Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing |
title_full | Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing |
title_fullStr | Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing |
title_full_unstemmed | Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing |
title_short | Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing |
title_sort | establishment of a fosmid library for pseudorabies virus sc strain and application in viral neuronal tracing |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300229/ https://www.ncbi.nlm.nih.gov/pubmed/32595620 http://dx.doi.org/10.3389/fmicb.2020.01168 |
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