Distribution of Porcine Cytomegalovirus in Infected Donor Pigs and in Baboon Recipients of Pig Heart Transplantation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals and Transplantations
2.2. Ethics Statement
2.3. Testing for PCMV, HEV and BaCMV, PCR Methods
2.4. Determination of the PCMV Copy Number
2.5. Determination of the BaCMV Copy Number
2.6. PBMC Incubation
2.7. PCMV and HEV Antigens Used for Western Blot Analyses
2.8. Testing for PCMV, HEV and PLHV
2.9. Immunohistochemistry (IHC)
2.10. Sequence Alignment
3. Results
3.1. Time Schedule of the Transplantation Experiments and Testing for PCMV
3.2. Detection of PCMV in Donor Pig and Baboon Recipients
3.3. Distribution of PCMV in Pig Organs
3.4. Development of a New Detection Strategy Using Incubated PBMC
3.5. Distribution of PCMV in Baboon Organs
3.6. Detection of the Baboon Cytomegalovirus (BaCMV) in the Recipient Baboons
3.7. Immunological Cross-Reactivity between PCMV and BaCMV
4. Discussion
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Primers Used for PCR | Sequence 5′-3′ | Nucleotide Position | Accession Number | Reference |
---|---|---|---|---|
PLHV-1,-2 (747) fw PLHV-1,-2 (747) rev | CAYGGTAGTATTTATTCAGACA GATATCCTGGTACATTGGAAAG | 21,146–21,167 21,488–21,467 | AY170317.1 | Ehlers B., 2002 [32] |
PLHV-3 (905) fw PLHV-3 (905) rev | ACAAGAGCCTTAGGGTTCCAAACT GTGTCCAGTGTTGTAATGGATGCC | 13,472–13,495 13,727–13,704 | AY170316.1 | Chmielewicz et al., 2003 [33] |
Primers and probes used for real-time PCR | ||||
pcyclophilin real fw pcyclophilin real rev pcyclophilin probe | TGCTTTCACAGAATAATTCCAGGATTTA GACTTGCCACCAGTGCCATTA Cy5-TGCCAGGGTGGTGACTTCACACGCC-BHQ | 131–158 187–207 161–185 | AY008846 | Duvigneau et al., 2005 [34] |
pGAPDH fw pGAPDH rev pGAPDH probe | ACATGGCCTCCAAGGAGTAAGA GATCGAGTTGGGGCTGTGACT HEX-CCACCAACCCCAGCAAGAG-BHQ1 | 1040–1062 1188–1168 1114–1132 | NM_001206359.1 | Duvigneau et al., 2005 [34] |
JVHEVF JVHEVR JVHEVProbe | GGTGGTTTCTGGGGTGAC TGATTCTCAGCCCTTCGC FAM-TGATTCTCAGCCCTTCGC-BHQ | 5283–5300 5352–5335 5306–5323 | KT633715.1 | Jothikumar et al., 2006 [35] |
PCMV real fw PCMV real rev PCMV probe | ACGAGAAAGATATTCTGACGGTGCA TCTAGACGAAAGGACATTGTTGATA 6FAM-CAGGGCGGCGGTCGAGCTC-TAMRA | 45,962–45,938 45,622–45,646 45,246–45,229 | KF017583.1 | Mueller et al., 2004 [36] |
BaCMV real fw BaCMV real rev BaCMV probe | 5′ GTTTAGGGAACCGCCATTCTG 3′ 5′ GTATCCGCGTTCCAATGCA 3′ 5′ 6FAM-TCCAGCCTCCATAGCCGGGAAGG-BBQ 3′ | 64,375–64,396 64,464–64,483 64,437–64,460 | KR351281 | Mueller et al., 2002 [31] |
BaCCR5 real fw BaCCR5 real rev BaCCR5 probe | 5′ TACCTGCTCAACCTGGCCAT 3′ 5′ TTCCAAAGTCCACTGGGC 3′ HEX-TTTCCTTCTTACTGTCCCCTTCTGGGCTC-BHQ 3′ | Mueller et al., 2002 [31] |
Pig Number | Born | Gender | Material Collected (Date) | PCMV (Real-Time PCR) | HEV (Real-Time RT PCR) | PLHV1/2 (PCR) | PLHV3 (PCR) | PCMV (Western Blot) * | PLHV (Western Blot) ** | HEV (Western Blot) *** |
---|---|---|---|---|---|---|---|---|---|---|
5154 | 1 July 2016 | male | 7 September 2016 | no ct | no ct | no ct | no ct | negative | negative | negative |
5160 | 1 July 2016 | female | 7 September 2016 | no ct | no ct | no ct | no ct | negative | negative | negative |
5160 | 3 February 2017 | no ct | no ct | no ct | no ct | negative | negative | negative | ||
5157 | 1 July 2016 | female | 26 October 2016 | no ct | no ct | no ct | no ct | negative | negative | negative |
Organ | Baboons | ||
---|---|---|---|
57 | 64 | Control 43 | |
Liver | + | + | + * |
Spleen | + | ++ | + * |
Kidney | + | + | + * |
Pig heart | ++ | ++ | + * |
Lung | + | + | + * |
Testis | +++ | + | n.t. |
Lymph node | +++ | +++ | n.t. |
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Fiebig, U.; Abicht, J.-M.; Mayr, T.; Längin, M.; Bähr, A.; Guethoff, S.; Falkenau, A.; Wolf, E.; Reichart, B.; Shibahara, T.; et al. Distribution of Porcine Cytomegalovirus in Infected Donor Pigs and in Baboon Recipients of Pig Heart Transplantation. Viruses 2018, 10, 66. https://doi.org/10.3390/v10020066
Fiebig U, Abicht J-M, Mayr T, Längin M, Bähr A, Guethoff S, Falkenau A, Wolf E, Reichart B, Shibahara T, et al. Distribution of Porcine Cytomegalovirus in Infected Donor Pigs and in Baboon Recipients of Pig Heart Transplantation. Viruses. 2018; 10(2):66. https://doi.org/10.3390/v10020066
Chicago/Turabian StyleFiebig, Uwe, Jan-Michael Abicht, Tanja Mayr, Matthias Längin, Andrea Bähr, Sonja Guethoff, Almuth Falkenau, Eckhard Wolf, Bruno Reichart, Tomoyuki Shibahara, and et al. 2018. "Distribution of Porcine Cytomegalovirus in Infected Donor Pigs and in Baboon Recipients of Pig Heart Transplantation" Viruses 10, no. 2: 66. https://doi.org/10.3390/v10020066