Whole Genomic Analysis and Comparison of Two Canine Papillomavirus Type 9 Strains in Malignant and Benign Skin Lesions
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection and Ethical Statement
- Dog 1: Fresh collected and formalin-fixed paraffin-embedded (FFPE) tissue samples from Dog 1 with recurrent SCCs were obtained from the animal hospitals at National Taiwan University in 2018. The sample collections were performed following the guidelines of and under the permissions of the Institutional Animal Care and Use Committees (IACUCs) of National Taiwan University (NTU) with the permission numbers NTU107-EL-00165.
- Dog 2: The FFPE sample from Dog 2 was obtained from the Department of Veterinary Pathology, The University of Tokyo (UT) in 2018 which was deposited for diagnostic purposes and to enable adequate disease prevention strategies. All surgical procedures were performed by educated veterinarians in accordance with Japanese law.
2.2. Case Information
2.3. Histological Descriptions and Immunohistochemistry Staining (IHC)
2.4. DNA Extraction from FFPE and Fresh Tissue
2.5. Polymerase Chain Reaction (PCR) for the Detection and Full Genome Sequencing of CPV
2.6. Sequence Analysis
2.7. RNA Extraction and Genomic DNA Eradication from FFPE Tissue
2.8. Primer-Specific cDNA Synthesis of E6 and E7
2.9. The Detection of mRNA Expression Level of E6 and E7 by PCR and qPCR
3. Results
3.1. Histological Descriptions and Immunohistochemistry Staining
3.2. Complete Nucleotide Sequence of CPV9
3.3. Open Reading Frames (ORFs) and Deduced Amino Acid Comparisons
3.4. Detection of E6 and E7 mRNA by conventional RT-PCR
3.5. Comparison of mRNA Expression Levels of E6 and E7 Using Quantitative PCR (qPCR)
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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ORF | CPV9 Strain | Location | Gene Length | Protein Length |
---|---|---|---|---|
E6 | Dog 1 | 263–718 | 456 bp | 151 a.a. |
Dog 2 | ||||
JF800656.1 | ||||
E7 | Dog 1 | 678–980 | 303 bp | 100 a.a. |
Dog 2 | ||||
JF800656.1 | ||||
E1 | Dog 1 | 970–2862 | 1893 bp | 630 a.a. |
Dog 2 | 970–2862 | 1893 bp | 630 a.a. | |
JF800656.1 | 970–2835 | 1866 bp | 621 a.a. | |
E2 | Dog 1 | 2804–4099 | 1296 bp | 431 a.a. |
Dog 2 | 2804–4267 | 1464 bp | 487 a.a. | |
JF800656.1 | 2777–4240 | 1464 bp | 487 a.a. | |
E8^E2 | Dog 1 | 1352–1389^3409–4099 | 729 bp | 242 a.a. |
Dog 2 | 1352–1389^3409–4267 | 897 bp | 298 a.a. | |
JF800656.1 | 1325–1362^3382–4240 | 897 bp | 298 a.a. | |
E4 | Dog 1 | 3375–4028 | 654 bp | 217 a.a. |
Dog 2 | 3375–4028 | |||
JF800656.1 | 3348–4001 | |||
L2 | Dog 1 | 4180–5697 | 1518 bp | 505 a.a. |
Dog 2 | 4508–6025 | |||
JF800656.1 | 4481–5998 | |||
L1 | Dog 1 | 5750–7255 | 1506 bp | 502 a.a. |
Dog 2 | 6078–7583 | |||
JF800656.1 | 6051–7556 |
Point Mutations | ||||||
---|---|---|---|---|---|---|
Position | Reference Strain (JF800656.1) | Dog 1 (MT265225) | Dog 2 (MT265226) | |||
Nucleotide | Amino Acid | Nucleotide | Amino Acid | Nucleotide | Amino Acid | |
3806 | C | D | T | D | T | D |
4579 | T | I | T | I | C | I |
4927 | C | S | C | S | T | S |
4987 | C | S | C | S | T | S |
5011 | A | V | G | V | A | V |
5080 | T | T | A | T | T | T |
5166 | T | I | T | I | C | T* |
5228 | C | R | A | R | A | R |
5233 | T | R | G | R | T | R |
5281 | C | P | C | P | T | P |
5910 | A | D | G | G* | A | D |
6125 | C | D | C | D | T | D |
6128 | T | S | T | S | A | S |
6722 | A | V | T | V | A | V |
6779 | G | G | A | G | G | G |
7098 | A | T | A | T | T | S* |
7343 | G | P | A | P | G | P |
7542 | C | R | C | R | A | R |
Insertion | ||||||
1113 | Reference Strain | G | ||||
Dog 1 | TAGCTTGGAGGGATCAA ATAGTCAGGGG | |||||
Dog 2 | CAGCTTGGAGGGATCAAATA GTCAGGGG | |||||
Deletion | ||||||
4037–4364 | Dog 1 | 328 bp deletion | ||||
Dog 2 | No deletion |
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Chang, C.-Y.; Yamashita-Kawanishi, N.; Tomizawa, S.; Liu, I.-L.; Chen, W.-T.; Chang, Y.-C.; Huang, W.-H.; Tsai, P.-S.; Shirota, K.; Chambers, J.K.; et al. Whole Genomic Analysis and Comparison of Two Canine Papillomavirus Type 9 Strains in Malignant and Benign Skin Lesions. Viruses 2020, 12, 736. https://doi.org/10.3390/v12070736
Chang C-Y, Yamashita-Kawanishi N, Tomizawa S, Liu I-L, Chen W-T, Chang Y-C, Huang W-H, Tsai P-S, Shirota K, Chambers JK, et al. Whole Genomic Analysis and Comparison of Two Canine Papillomavirus Type 9 Strains in Malignant and Benign Skin Lesions. Viruses. 2020; 12(7):736. https://doi.org/10.3390/v12070736
Chicago/Turabian StyleChang, Chia-Yu, Nanako Yamashita-Kawanishi, Sonoka Tomizawa, I-Li Liu, Wei-Tao Chen, Yen-Chen Chang, Wei-Hsiang Huang, Pei-Shiue Tsai, Kinji Shirota, James K Chambers, and et al. 2020. "Whole Genomic Analysis and Comparison of Two Canine Papillomavirus Type 9 Strains in Malignant and Benign Skin Lesions" Viruses 12, no. 7: 736. https://doi.org/10.3390/v12070736