The Protein Encoded by the UL3.5 Gene of the Duck Plague Virus Affects Viral Secondary Envelopment, Release, and Cell-to-Cell Spread
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells, Viruses, and Primers
2.2. Antibodies
2.3. Construction of Plasmid and Recombinant BAC Virus Plasmid
2.4. Conventional Polymerase Chain Reaction (PCR)
2.5. Rescue and Identification of Recombinant Virus
2.6. Fluorescent Protein Labeling
2.7. Real-Time Quantitative PCR
2.8. Cell Viability Assessment by CCK-8
2.9. GCV/CHX Drug Inhibition Assay
2.10. Western Blotting Analysis
2.11. Determination of Multi-Step Growth Curve In Vitro
2.12. Virus Adsorption, Invasion, Replication, and Release Assays
2.12.1. Adsorption
2.12.2. Invasion
2.12.3. Replication
2.12.4. Release
2.13. Electron Microscopy
2.14. Analysis of Virus Cell-to-Cell Spread
2.15. Data Analysis
3. Results
3.1. DPV pUL3.5 Is a Multicellular Localization Protein
3.2. DPV UL3.5 Encodes an Early Viral Protein
3.3. Construction and Rescue of DPV-BAC-δUL3.5 and DPV-BAC-RUL3.5 Recombinant Virus Strains
3.4. The Deletion of UL3.5 Leads to the Slow Growth Rate In Vitro
3.5. UL3.5 Protein Participated in the Viral Secondary Envelopment and Virion Release Process
3.6. UL3.5 Exerts a Significant Impact on Cell-to-Cell Spread
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
aa | Amino acid |
AnHV-1 | Anas herpesviruses 1 |
BAC | Bacterial artificial chromosome |
BHV-1 | Bovine herpesvirus 1 |
cDNA | Complementary DNA |
CHX | Cycloheximide |
DEFs | Duck embryo fibroblast |
DEV | Duck enteritis virus |
DMEM | Dulbecco Modified Eagle Medium |
DNA | Deoxyribonucleotide acid |
DP | Duck plague |
DPV | Duck plague virus |
ER | Endoplasmic reticulum |
EGFP | Enhanced green fluorescent protein |
FBS | Fetal bovine serum |
GCV | Ganciclovir |
HSV-1/2 | Herpes simplex virus 1/2 |
ILTV | Infectious laryngotracheitis virus |
MOI | Multiplicity of infection |
NanoLuc | NanoLuc® luciferase |
ORF | Open reading frame |
PBS | Phosphate-buffered saline solution |
PBST | Phosphate-buffered saline with Tween |
PCR | Polymerase chain reaction |
RNA | Ribonucleic Acid |
RT-qPCR | Quantitative real-time PCR |
TBST | Tris-buffered saline with Tween |
Appendix A
Plasmid/Gene | Primer | Primer Sequence (5′ → 3′) |
---|---|---|
pEGFP-N1-UL3.5 | F | AGATCTCGAGCTCAAGCTTCGAATTCGCCACCATGTTCCGGGCGCGGCATT |
R | GACCGGTGGATCCCGGGCCCGCGGTACCGTAATGAGAATGAACCTTATGAT | |
DPV-BAC-NanoLuc-UL3.5 | F | TTTACATCGGCGACAACGTAATAGAGGTCAGTACGCCTCAGTCCTGAAGTGATCGCGAACTATGGATTACAAGGACGACGATGACAAGAACTCCTTCTCC |
R | GCGATTGCTGGTCTATGAGTCGATAACGGCCGGTGTTGATGATTACCTGGTTCAGGCCGCTCGTTTCCGCCGAGTGATCCAGCCAGTGTTACAACCAAT | |
DPV-BAC-δUL3.5 | F | TAGAGGTCAGTACGCCTCAGTCCTGAAGTGATCGCGAACTTAGGGATAACAGGGTAATCGATTT |
R | ATTACCTGGTTCAGGCCGCTCGTTTCCGCCGAGTGATCCAAGTTCGCGATCACTTCAGGACTGAGGCGTACTGACCTCTAGCCAGTGTTACAACCAAT | |
DPV-BAC-RUL3.5 | F | TTACATCGCCGACAACGTAATAGAGGTCAGTACGCCTCAGTCCTGAAGTGATCGCGAACTATGTTCCGGGCGCGGCATTA |
R | GCGATTGCTGGTCTATGAGTCGATAACGGCCGGTGTTGATGATTACCTGGTTCAGGCCGCTCGTTTCCGCCGAGTGATCCAGCCAGTGTTACAACCAAT | |
Identification of UL3.5 | F | GTGGCCACGGCAATAAAAGGC |
R | GTGAGTCGTCATCTAGAACGG | |
ICP27 gene [55] | F | TTGCTCGACCTAAGACAGGA |
R | TCATTAGCAGTAGTTCCGCG | |
UL28 gene [55] | F | TTAAGCTAAGCCGTGCCGAT |
R | TGGAGCCAGTTGTTGAAGCA | |
UL47 gene [55] | F | AACGGAGTTGCTTGGAGAACA |
R | TGGGCGATGAAACAGAGTAGG | |
β-actin [40] | F | GATCACAGCCCTGGCACC |
R | CGGATTCATCATACTCCTGCTT | |
UL30 gene [55] | F | TTTCCTCCTCCTCGCTGAGTG |
R | GTGAGTCGTCATCTAGAACG |
Drug Treatment | Concentration | OD450 Values (mean ± SD) | Cell Viability |
---|---|---|---|
DMSO | 0.2% | 2.91 ± 0.08 | 100.00% |
GCV | 300 μg/mL | 2.89 ± 0.01 | 99.33% |
CHX | 200 μg/mL | 2.89 ± 0.06 | 99.64% |
GCV + CHX | 300 μg/mL + 200 μg/mL | 2.88 ± 0.01 | 99.15% |
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Cao, H.; Tian, B.; Tian, Y.; Cai, D.; Wang, M.; Jia, R.; Chen, S.; Cheng, A. The Protein Encoded by the UL3.5 Gene of the Duck Plague Virus Affects Viral Secondary Envelopment, Release, and Cell-to-Cell Spread. Vet. Sci. 2025, 12, 510. https://doi.org/10.3390/vetsci12060510
Cao H, Tian B, Tian Y, Cai D, Wang M, Jia R, Chen S, Cheng A. The Protein Encoded by the UL3.5 Gene of the Duck Plague Virus Affects Viral Secondary Envelopment, Release, and Cell-to-Cell Spread. Veterinary Sciences. 2025; 12(6):510. https://doi.org/10.3390/vetsci12060510
Chicago/Turabian StyleCao, Huanhuan, Bin Tian, Yanming Tian, Dongjie Cai, Mingshu Wang, Renyong Jia, Shun Chen, and Anchun Cheng. 2025. "The Protein Encoded by the UL3.5 Gene of the Duck Plague Virus Affects Viral Secondary Envelopment, Release, and Cell-to-Cell Spread" Veterinary Sciences 12, no. 6: 510. https://doi.org/10.3390/vetsci12060510
APA StyleCao, H., Tian, B., Tian, Y., Cai, D., Wang, M., Jia, R., Chen, S., & Cheng, A. (2025). The Protein Encoded by the UL3.5 Gene of the Duck Plague Virus Affects Viral Secondary Envelopment, Release, and Cell-to-Cell Spread. Veterinary Sciences, 12(6), 510. https://doi.org/10.3390/vetsci12060510