Comparison of different in situ hybridization techniques for the detection of various RNA and DNA viruses

Vanessa M. Pfankuche; Kerstin Hahn; Rogier Bodewes; Florian Hansmann; André Habierski; Ann Kathrin Haverkamp; Stephanie Pfaender; Stephanie Walter; Christine Baechlein; Alexander Postel; Eike Steinmann; Paul Becher; Albert Osterhaus; Wolfgang Baumgärtner; Christina Puff

doi:10.3390/v10070384

Comparison of different in situ hybridization techniques for the detection of various RNA and DNA viruses

Vanessa M. Pfankuche, Kerstin Hahn, Rogier Bodewes, Florian Hansmann, André Habierski, Ann Kathrin Haverkamp, Stephanie Pfaender, Stephanie Walter, Christine Baechlein, Alexander Postel, Eike Steinmann, Paul Becher, Albert Osterhaus, Wolfgang Baumgärtner, Christina Puff

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time.

Original language	English
Article number	384
Journal	Viruses
Volume	10
Issue number	7
DOIs	https://doi.org/10.3390/v10070384
Publication status	Published - 20 Jul 2018

Funding

Funding: This study received funding from the European Union’s Horizon 2020 research and innovation program COMPARE (grant agreement no. 643476) and was in part supported by the Niedersachsen-Research Network on Neuroinfectiology (N-RENNT) of the Ministry of Science and Culture of Lower Saxony. This research was performed as part of the Zoonoses Anticipation and Preparedness Initiative (ZAPI project; IMI Grant Agreement no. 115760), with the assistance and financial support of IMI and the European Commission, and in-kind contributions from EFPIA partners. This publication was supported by Deutsche Forschungsgemeinschaft and University of Veterinary Medicine Hannover, Foundation within the funding programme Open Access Publishing.

Keywords

Chromogenic in situ hybridization
Digoxigenin
DNA virus
Fast red
Fluorescent in situ hybridization
RNA virus
Virus discovery

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Pfankuche, V. M., Hahn, K., Bodewes, R., Hansmann, F., Habierski, A., Haverkamp, A. K., Pfaender, S., Walter, S., Baechlein, C., Postel, A., Steinmann, E., Becher, P., Osterhaus, A., Baumgärtner, W., & Puff, C. (2018). Comparison of different in situ hybridization techniques for the detection of various RNA and DNA viruses. Viruses, 10(7), Article 384. https://doi.org/10.3390/v10070384

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abstract = "In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time.",

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Pfankuche, VM, Hahn, K, Bodewes, R, Hansmann, F, Habierski, A, Haverkamp, AK, Pfaender, S, Walter, S, Baechlein, C, Postel, A, Steinmann, E, Becher, P, Osterhaus, A, Baumgärtner, W & Puff, C 2018, 'Comparison of different in situ hybridization techniques for the detection of various RNA and DNA viruses', Viruses, vol. 10, no. 7, 384. https://doi.org/10.3390/v10070384

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AU - Haverkamp, Ann Kathrin

AU - Pfaender, Stephanie

AU - Walter, Stephanie

AU - Baechlein, Christine

AU - Postel, Alexander

AU - Steinmann, Eike

AU - Becher, Paul

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AU - Baumgärtner, Wolfgang

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KW - Digoxigenin

KW - DNA virus

KW - Fast red

KW - Fluorescent in situ hybridization

KW - RNA virus

KW - Virus discovery

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Comparison of different in situ hybridization techniques for the detection of various RNA and DNA viruses

Abstract

Funding

Keywords

UN SDGs

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