The Junction Between nsp1β and nsp2 in the Porcine Reproductive and Respiratory Syndrome Virus Genome Is a New Site for the Insertion and Expression of Foreign Genes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell, Virus, and Serum Samples
2.2. Construction of a Full-Length Infectious cDNA Clone of the HP-PRRSV SH01 Strain
2.3. Construction of Full-Length Infectious cDNA Clones of Recombinant PRRSV Expressing Foreign Genes Inserted Between nsp1β and nsp2
2.4. Rescue of Recombinant PRRSVs
2.5. Plaque Assays
2.6. Immunofluorescence Assay, Western Blotting, and RT–PCR
2.7. Replication Kinetics of PRRSV and Viral Titration
2.8. Fluorescence Focus Unit Reduction-Based Assay for Detection of Neutralizing Antibodies
2.9. Statistical Analysis
3. Results
3.1. Construction and Recovery of the Recombinant Virus rPRRSV-SH01
3.2. Construction and Recovery of the Recombinant Virus rPRRSV-SH01-eGFP Expressing eGFP Inserted Between nsp1β and nsp2
3.3. Analysis of the Genetic Stability of rPRRSV-SH01-eGFP
3.4. Application of rPRRSV-SH01-eGFP as a Reporter Virus for Detection of Immune Response Against PRRSV in Vitro
3.5. Construction and Recovery of the Recombinant Virus rPRRSV Expressing iLOV3 or TEVp Inserted Between nsp1β and nsp2
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Name | Sequence (5′ to 3′) | Usage |
---|---|---|
PRRSV Sac I | ggtctatataagcagagctcgtttagtgaaccgtatgacgtataggtgttgg | Construction of the intermediate plasmid pCI-F1 |
PRRSV Pst I | ctgctcgaagcggccgccggcggagctttgtttaaacccaactgcagttacataaacac | |
PRRSV Pst I nsp3 | atcgtcagtattgactgcagtggggt | Construction of the intermediate plasmid pClone007-F2 |
Infnsp8 | gtgtctgaggctcgcgctagcagtttaaacac | |
1b Pme I | gtttaaactgctagccg | Construction of the intermediate plasmid pClone007-F3 |
1b Asc I | cgccggcggaattcgatatcgaattcaggcgcgcccgaaac | |
PRRSV Asc I | tgattacgccaagctggcgcgccag | Construction of the intermediate plasmid pUC19-F4-42A |
PRRSV3UTR | aattacggccgcatg | |
42AF | catgcggccgtaattaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagg | |
InPRRSV SVR | gacggccagtgaattcgccggcgttaacttgtttattgcagc | |
seqNar I nsp2stu | cccaaacctgaggac ccttactctttcaggaaggg | Construction of the intermediate plasmid pCI-F1-target gene |
1br 1bGFP P2Ar P2Ansp2 | gcctgcaccgtacc ggtacggtgcaggcgtgagcaagggc aggtcctgggttttcctccacgtctc gtggaggaaaacccaggacctgccggaaagagag | Construction of the intermediate plasmid pCI-F1-eGFP |
P2Af | ggcagcggagcc | Construction of the intermediate plasmids pCI-F1-iLOV3 and pCI-F1-TEVp |
1biLOV3 | ggtacggtgcaggcgagaagtcctttgtg | Construction of the intermediate plasmid pCI-F1-iLOV3 |
iLOV3P2A | gtggctccgctgccgacatgatcggagccg | |
1bTEVp | ggtacggtgcaggcgagtctctgtttaagg | Construction of the intermediate plasmid pCI-F1-TEVp |
TEVpP2A | gtggctccgctgccgttcatcagctgggtg | |
nsp1bPLAG | acgtcaccactggctgg | Detection of inserted target genes |
nsp2ATKH | gcttcgtggcctgcc |
Percentage of Reduction in Fluorescent Focus Units (%) | ||||||
---|---|---|---|---|---|---|
Serum Sample | 1:4 | 1:8 | 1:16 | 1:32 | 1:64 | |
Vaccinated pigs | 23# | 99.27 ± 0.46 | 99.18 ± 0.16 | 98.95 ± 0.05 | 98.90 ± 0.08 | 74.54 ± 5.49 |
24# | 99.73 ± 0.22 | 98.76 ± 0.48 | 97.92 ± 0.56 | 72.44 ± 0.32 | 17.25 ± 3.22 | |
25# | 99.58 ± 0.25 | 99.46 ± 0.21 | 68.66 ± 4.70 | 15.68 ± 3.88 | 13.74 ± 6.21 | |
26# | 99.61 ± 0.17 | 99.71 ± 0.29 | 92.63 ± 0.61 | 55.20 ± 4.03 | 16.64 ± 2.34 | |
Naïve pigs | 33# | 21.55 ± 2.14 | 18.81 ± 2.34 | 16.51 ± 2.35 | 15.61 ± 1.80 | 12.29 ± 1.68 |
34# | 19.89 ± 3.08 | 21.16 ± 3.25 | 23.00 ± 0.90 | 19.02 ± 3.29 | 15.34 ± 1.82 | |
35# | 19.43 ± 4.15 | 19.67 ± 9.76 | 15.74 ± 5.20 | 4.02 ± 13.21 | 6.66 ± 9.32 | |
36# | 20.82 ± 3.56 | 5.63 ± 25.72 | 16.79 ± 3.86 | 22.17 ± 1.77 | 13.12 ± 8.28 |
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Xiao, C.; Lin, Y.; Zhang, H.; Li, Z.; Liu, K.; Li, B.; Shao, D.; Qiu, Y.; Ma, Z.; Wei, J. The Junction Between nsp1β and nsp2 in the Porcine Reproductive and Respiratory Syndrome Virus Genome Is a New Site for the Insertion and Expression of Foreign Genes. Viruses 2025, 17, 656. https://doi.org/10.3390/v17050656
Xiao C, Lin Y, Zhang H, Li Z, Liu K, Li B, Shao D, Qiu Y, Ma Z, Wei J. The Junction Between nsp1β and nsp2 in the Porcine Reproductive and Respiratory Syndrome Virus Genome Is a New Site for the Insertion and Expression of Foreign Genes. Viruses. 2025; 17(5):656. https://doi.org/10.3390/v17050656
Chicago/Turabian StyleXiao, Changguang, Yafang Lin, Hailong Zhang, Zongjie Li, Ke Liu, Beibei Li, Donghua Shao, Yafeng Qiu, Zhiyong Ma, and Jianchao Wei. 2025. "The Junction Between nsp1β and nsp2 in the Porcine Reproductive and Respiratory Syndrome Virus Genome Is a New Site for the Insertion and Expression of Foreign Genes" Viruses 17, no. 5: 656. https://doi.org/10.3390/v17050656
APA StyleXiao, C., Lin, Y., Zhang, H., Li, Z., Liu, K., Li, B., Shao, D., Qiu, Y., Ma, Z., & Wei, J. (2025). The Junction Between nsp1β and nsp2 in the Porcine Reproductive and Respiratory Syndrome Virus Genome Is a New Site for the Insertion and Expression of Foreign Genes. Viruses, 17(5), 656. https://doi.org/10.3390/v17050656