Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing

Abstract

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 aminoterminal 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.

Histamine Is Responsible for the Neuropathic Itch Induced by the Pseudorabies Virus Variant in a Mouse Model

“…The PRV SC strain (GenBank accession No. KT809429.1) is a classical virulent PRV strain [ 18 , 19 ]. The PRV TJ strain (GenBank accession No.…”

“…PK-15 cells were seeded into 96-well plates, infected with 10-fold serially diluted PRV suspensions, and cultured at 37 °C, 5% CO 2 , for 72 h. The PRV-infected cells were detected by indirect immunofluorescence assay (IFA), and the number of fluorescent wells was counted, as described previously [ 19 ]. Then, the viral titers were calculated by using the Reed and Muench method, and they were expressed as the median tissue culture infectious dose (TCID 50 ) [ 20 ].…”

Recombinant Pseudorabies Virus Usage in Vaccine Development against Swine Infectious Disease

“…In contrast, fosmid library generation is more efficient. The huge genome of PRV is randomly sheared into various fragments, then each segment is ligated with a fosmid vector to screen out the various combinations covering the whole genome of PRV, and the fosmid combination was co-transfected into virus-susceptible eukaryotic cells to rescue the virus [ 55 , 56 ]. Construction of recombinant virus only needs to modify the corresponding fosmid in E coli.…”

“…In addition, Qi et al constructed a fosmid library for the PRV-SC strain. The EGFP gene was inserted into the N-terminal of the UL 36 gene of PRV-SC via the Red/ET recombination [ 56 ].…”

“…The recombinant virus showed similar biological characteristics to the parental strain, including a stable viral titer and luciferase activity throughout 20 passages. Furthermore, Zhou and Qi constructed the reporter virus based on the PRV variant and classical stain, respectively [ 55 , 56 ]. The EGFP was fused into the amino-terminal of UL36 in these recombinants.…”