Related Experiment Video
Updated: Mar 9, 2026
Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments
Published on: May 17, 2024
Construction of a molecular clone of ovine enzootic nasal tumor virus
Scott R Walsh1,2, María Carla Rosales Gerpe1, Sarah K Wootton3
1Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Background:
Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Transmission experiments performed using virus isolated from cell free nasal tumor homogenates suggest that ENTV-1 is the causative agent of ENA; however, this etiological relationship has not been conclusively proven due to the fact that the virus cannot be propagated in vitro nor is there an infectious molecular clone of the virus.
Methods:
Here we report construction of a molecular clone of ENTV-1 and demonstrate that transfection of this molecular clone into HEK 293T cells produces mature virus particles.
Results:
Analysis of recombinant virus particles derived from the initial molecular clone revealed a defect in the proteolytic processing of Gag; however, this defect could be corrected by co-expression of the Gag-Pro-Pol polyprotein from the highly related Jaagsiekte sheep retrovirus (JSRV) suggesting that the polyprotein cleavage sites in the ENTV-1 molecular clone were functional. Mutagenesis of the molecular clone to correct amino acid variants identified within the pro gene did not restore proteolytic processing; whereas deletion of one proline residue from a polyproline tract located in variable region 1 (VR1) of the matrix resulted in production of CA protein of the mature (cleaved) size strongly suggesting that normal virion morphogenesis and polyprotein cleavage took place. Finally, electron microscopy revealed the presence of spherical virus particles with an eccentric capsid and an average diameter of about 100 nm.
Conclusion:
In summary, we have constructed the first molecular clone of ENTV-1 from which mature virus particles can be produced. Future experiments using virus produced from this molecular clone can now be conducted to fulfill Koch's postulates and demonstrate that ENTV-1 is necessary and sufficient to induce ENA in sheep.
Insights
Researchers created the first molecular clone of Enzootic Nasal Tumor Virus (ENTV-1), an ovine betaretrovirus. This breakthrough allows for the production of mature virus particles, paving the way to prove ENTV-1 causes enzootic nasal adenocarcinoma in sheep.
Area of Science:
- Virology
- Oncology
- Veterinary Medicine
Background:
- Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus implicated in enzootic nasal adenocarcinoma (ENA) in sheep.
- The etiological link between ENTV-1 and ENA remains unproven due to the inability to propagate the virus in vitro or create an infectious molecular clone.
Purpose of the Study:
- To construct the first infectious molecular clone of ENTV-1.
- To produce mature ENTV-1 virus particles from the molecular clone.
- To enable future studies to establish the causal relationship between ENTV-1 and ENA.
Main Methods:
- Construction of a molecular clone of ENTV-1.
- Transfection of the molecular clone into HEK 293T cells.
- Analysis of viral particle production and protein processing using mutagenesis and electron microscopy.
Main Results:
- Transfection of the ENTV-1 molecular clone yielded mature virus particles.
- Defects in Gag polyprotein processing were identified and partially corrected through mutagenesis, particularly by altering a polyproline tract in the matrix protein.
- Electron microscopy confirmed the presence of spherical virus particles approximately 100 nm in diameter.
Conclusions:
- The first molecular clone of ENTV-1 has been successfully constructed, enabling the production of mature virus particles.
- This achievement is a critical step towards fulfilling Koch's postulates to definitively prove ENTV-1 causes ENA in sheep.
More Related Videos
08:49Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein
Published on: October 9, 2011
12:20Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses
Published on: December 29, 2015
