High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75
Abstract
:1. Introduction
2. Results
2.1. Role of Homology Arm-Length in the Efficiency of Adenoviral Genome Direct Cloning
2.2. Sufficient Amount of Adenovirus Genome Is Required for Successful Cloning
2.3. High-Throughput Cloning of Emerging Adenoviruses Types 70, 73, 74, and 75
2.4. High-Throughput Reporter Insertion
2.5. Screening of Adenovirus Tropisms
2.6. Screening of the Receptor Usage
2.7. New Species D Human Adenoviruses with Lower or Comparable Prevalence of Anti-Adenoviral Antibodies
3. Discussion
4. Materials and Methods
4.1. Generation of Linear Cloning Vector p15A-cm-Ad9HA
4.2. Genomic DNA Isolation for Adenoviral Genome Cloning
4.3. Linear-Linear Homologous Recombination (LL-HR)
4.4. Colony-Polymerase Chain Reaction (PCR) Detection
4.5. Quantitative Real-Time PCR (qPCR) to Detect Virus Genome Copy Numbers (VCN)
4.6. Cell Cultures
4.7. New Species D Human Adenoviruses
4.8. Adenovirus Production
4.9. Evaluation of Adenovirus Transduction Efficiency via Luciferase Assay
4.10. ELISA to Measure Presence of Anti-Adenovirus Antibodies
4.11. Statistics
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
CAR | Coxsackievirus and adenovirus receptor |
CD46 | Cluster of differentiation 46 |
CsCl | Cesium chloride |
CV | Cell/virus lysate |
E3 | Early gene 3 |
gAd | Human adenovirus genome |
GFP | Green fluorescent protein |
GLN | Turbo GFP, nanoLuciferase (Nluc), and neomycin resistance (neo) reporter cassette |
HA | Homology arm |
HAdV | Human adenovirus |
IgG | Immunoglobulin G |
IVIG | Intravenous immunoglobulin |
LLHR | Linear-linear homologous recombination |
ITR | Inverted terminal repeat |
MLP | Major late prompter |
PCR | Polymerase chain reaction |
RLU | Relative light units |
VCN | Virus genome copy number |
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Virus | NCBI Access Number | Penton/Hexon/Fiber | Date/Place | Source (Isolated From) |
---|---|---|---|---|
Ad70 | KP641339 | P70H70F29 | 2014/Leipzig, Germany | Diarrheal feces of a hematopoietic stem cell transplantation recipient [23] |
Ad73 | KY618676 | P67H45F27 | 2015/Leipzig, Germany | Diarrheal feces of a lymphoma patient treated with chemotherapy [24] |
Ad74 | KY618677 | P70H74F51 | 2015/Leipzig, Germany | Diarrheal feces of a hematopoietic stem cell transplantation recipient [24] |
Ad75 | KY618678 | P75H26F29 | 2015/Leipzig, Germany | The feces of an AIDS patient [24] |
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Zhang, W.; Mese, K.; Schellhorn, S.; Bahlmann, N.; Mach, N.; Bunz, O.; Dhingra, A.; Hage, E.; Lafon, M.-E.; Wodrich, H.; et al. High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75. Int. J. Mol. Sci. 2020, 21, 6370. https://doi.org/10.3390/ijms21176370
Zhang W, Mese K, Schellhorn S, Bahlmann N, Mach N, Bunz O, Dhingra A, Hage E, Lafon M-E, Wodrich H, et al. High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75. International Journal of Molecular Sciences. 2020; 21(17):6370. https://doi.org/10.3390/ijms21176370
Chicago/Turabian StyleZhang, Wenli, Kemal Mese, Sebastian Schellhorn, Nora Bahlmann, Nicolas Mach, Oskar Bunz, Akshay Dhingra, Elias Hage, Marie-Edith Lafon, Harald Wodrich, and et al. 2020. "High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75" International Journal of Molecular Sciences 21, no. 17: 6370. https://doi.org/10.3390/ijms21176370