Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture
Abstract
:1. Introduction
2. Materials and Methods
2.1. Computer Analyses of SAV3 E1 and E2 Sequences
2.2. Cell Cultures
2.3. Plasmid Constructs
2.4. Transfection
2.5. Indirect Fluorescent Antibody Technique
2.6. Recovery and Passage of Mutated Clones
2.7. Replication of Viral RNA
2.8. Analysis of Virus Titers
2.9. RNA Isolation and RT-qPCR
3. Results
3.1. Prediction of N-glycosylation Sites in SAV3 E1 and E2
3.2. Recovery of Recombinant Viruses
3.3. Presence of Viral RNA in Culture Medium after Passage in CHH-1 Cells
3.4. Cytopathic Effects
3.5. Quantification of Intracellular Viral RNA from Adherent CHH-1 Cells
3.6. Titer of Infectious Virus Produced by CHH-1 Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Plasmid Constructs | Mutation(s) |
---|---|
prSAV3 | - |
prSAV3 E135Q | E135N→Q |
prSAV3 E135A | E135N→A |
prSAV E2319Q | E2319N→Q |
prSAV E2319A | E2319N→A |
prSAV3 E135Q E2319Q | E135N→Q/E2319N→Q |
prSAV3 E135A E2319A | E135N→A/E2319N→A |
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Aksnes, I.; Markussen, T.; Braaen, S.; Rimstad, E. Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture. Viruses 2020, 12, 1071. https://doi.org/10.3390/v12101071
Aksnes I, Markussen T, Braaen S, Rimstad E. Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture. Viruses. 2020; 12(10):1071. https://doi.org/10.3390/v12101071
Chicago/Turabian StyleAksnes, Ida, Turhan Markussen, Stine Braaen, and Espen Rimstad. 2020. "Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture" Viruses 12, no. 10: 1071. https://doi.org/10.3390/v12101071