Construction of an infectious bacterial artificial chromosome clone of a pseudorabies virus variant: Reconstituted virus exhibited wild-type properties in vitro and in vivo

Wang, Tao; Wu, Tong; Ye, Chao; Yu, Zhiqing; Chen, Jing; Gao, Fei; Shan, Tongling; Yu, Hai; Li, Liwei; Li, Guoxin; Tong, Guangzhi; Zheng, Hao

doi:10.1016/j.jviromet.2018.06.004

Cited by 10 publications

(9 citation statements)

References 32 publications

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“…The rapid improvement of biotechnology promoted the research of genetically modified PRV in recent years, such as the application of BAC system in gene deletion (16,(37)(38)(39), and the identification of foreign gene insertion sites in a PRV vector (40) and non-coding regions (UL11-10, UL35-36, UL46-27, or US2-1). Notably, the method that allowed PRV genome manipulation by using the CRISPR/Cas9 system in PK15 cells was developed (23,41) and showed a high positive rate without constructing homology arms, offering a simple and efficient method to manipulate the viral genome in the future, especially in the identification of potential new virulence genes for the highly safe vaccine development to control PR.…”

Section: Discussionmentioning

confidence: 99%

Construction and Immunogenicity of a Recombinant Pseudorabies Virus Variant With TK/gI/gE/11k/28k Deletion

Yan¹,

Huang²,

Bai³

et al. 2022

Front. Vet. Sci.

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In China, the re-emerging pseudorabies virus (PRV) variant has caused large-scale outbreaks of pseudorabies in swine herds with classical PRV vaccine immunization since late 2011. Here, a recombinant PRV with TK/gI/gE/11k/28k deletion was constructed based on variant HN1201 strain isolated in 2012, by the bacterial artificial chromosome infectious clones. Compared with the parental virus, the recombinant PRV rHN1201TK−/gE−/gI−/11k−/28k− showed a similar virus grown curve and exhibited smaller plaques. The vaccination of rHN1201TK−/gE−/gI−/11k−/28k− could elicit an earlier and higher level of gB antibody, and the neutralizing antibodies elicited by rHN1201TK−/gE−/gI−/11k−/28k− were effective against both PRV classical and variant strains. Clinically, the body temperature of the pigs immunized with rHN1201TK−/gE−/gI−/11k−/28k− was significantly lower than that of the classical PRV vaccine immunized pigs, and the recombinant PRV could provide effective protection against the challenge with the PRV variant. These results imply that the rHN1201TK−/gE−/gI−/11k−/28k− could be a promising vaccine candidate for the prevention of the current epidemic of pseudorabies in China.

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Section: Discussionmentioning

confidence: 99%

Construction and Immunogenicity of a Recombinant Pseudorabies Virus Variant With TK/gI/gE/11k/28k Deletion

Yan¹,

Huang²,

Bai³

et al. 2022

Front. Vet. Sci.

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“…Meanwhile, plaque purification of recombination PRVs from parental viruses always takes more than 1 month. Infectious BAC of PRV, another genetic manipulation technology, is efficient for gene insertion and deletion using Red/ET technology in E. coli (Osterrieder et al, 2003;Wang et al, 2018). However, it takes several months to construct BAC clones, but the genetic and phenotypic change occurs in the recombinant virus due to the unstable large genome of PRV in E. coli.…”

Section: Discussionmentioning

confidence: 99%

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

Abid

et al. 2020

Front. Microbiol.

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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.

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“…Human embryonic kidney 293T cells (HEK-293T, ATCC CRL-11268), Rabbit kidney cells (RK13, ATCC CCL-37), Vero cells (ATCC CCL-81), Human embryonic kidney cells (HEK-293A, ATCC CRL-1573), swine testicular cells (ST, ATCC CRL1746), and Porcine kidney cells (PK15, ATCC CCL-33) were maintained in DMEM (Gibco, Waltham, MA, USA) with 10% ( v / v ) fetal bovine serum (ExCell Bio, Canberra, Australia). As previously described, the pseudorabies virus infectious clone was pBac-JS2012 [ 15 ]. Plasmids related to orthogonal translation system, pSD31-pylRS, bjmu-12t-zeo, and pSD31-GFP 39TAG were kindly provided by Professor De-Min Zhou of Peking University [ 14 ].…”

Section: Methodsmentioning

confidence: 99%

“…PRV-PTC construction was performed as previously described [ 15 ]. Briefly, the procedure is as follows.…”

Section: Methodsmentioning

confidence: 99%

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Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology

Wang

Sang

Wang

et al. 2022

Viruses

Self Cite

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Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

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Construction of an infectious bacterial artificial chromosome clone of a pseudorabies virus variant: Reconstituted virus exhibited wild-type properties in vitro and in vivo

Cited by 10 publications

References 32 publications

Construction and Immunogenicity of a Recombinant Pseudorabies Virus Variant With TK/gI/gE/11k/28k Deletion

Construction and Immunogenicity of a Recombinant Pseudorabies Virus Variant With TK/gI/gE/11k/28k Deletion

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

Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology

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