Comparison of Different In Situ Hybridization Techniques for the Detection of Various RNA and DNA Viruses

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.

First report of equine parvovirus‐hepatitis‐associated Theiler’s disease in Europe

“…In this study, liver infection with EqPV‐H was further characterised using a novel ISH technique that has become increasingly useful for associating newly discovered viruses with pathological changes 26 . A modified form of Koch's postulates was developed in 1996 to address the problems of isolation, reinfection and re‐isolation when using sequence‐based approaches that have become increasingly common in the era of high‐throughput next‐generation sequencing 27 .…”

Pestivirus K (Atypical Porcine Pestivirus): Update on the Virus, Viral Infection, and the Association with Congenital Tremor in Newborn Piglets

“…Luxol ®® Fast Blue staining helps to observe the demyelination caused by the APPV in the CNS, primarily located in the cerebellum and spinal cord [16,48,50,52,53]. Histopathology, together with an immunohistochemical technique [46], and in situ hybridization [53,60] enables the detection of the viral agent (protein or nucleic acid) at the lesion site.…”

“…In the United States, APPV was first identified by NGS in pig serum samples collected in 2014 from five different states [ 1 ]. In subsequent years, several studies from different countries reported the circulation of APPV in the US [ 1 , 2 , 51 , 54 , 56 ], Germany [ 53 , 55 , 57 , 58 , 59 , 60 , 61 ], Netherlands [ 10 ], Sweden [ 62 ], Austria [ 50 ], England [ 63 ], China [ 38 , 42 , 44 , 46 , 47 , 61 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ], Spain [ 41 , 74 ], South Korea [ 75 ], Brazil [ 16 , 48 , 49 , 76 ], Great Britain [ 61 ], Italy [ 43 , 61 ], Serbia [ 61 ], Switzerland [ 61 , 77 ], Taiwan [ 61 ], Canada [ 52 ], and Hungary [ 78 ].…”

Recent advances in lab-on-a-chip technologies for viral diagnosis

“…Nevertheless, the co-circulation of closely related flaviviruses such as ZIKV and DENV in number of regions makes the conventional detection of a specific virus using immunoassays a complicated task (Wen and Shresta, 2019). Several techniques can directly detect specific viral DNA or RNA via in-situ hybridization (Pfankuche et al, 2018) (Fig. ), number of copies (c).…”