RNA in-situ hybridization for pathology-based diagnosis of feline infectious peritonitis (FIP): current diagnostics for FIP and comparison to the current gold standard

Abstract

Feline infectious peritonitis (FIP) is a systemic disease of cats caused by a highly pathogenic variant of feline coronavirus, or FCoV. Two distinct genotypes of FCoV exist (also referred to as serotypes): Type 1 viruses constitute the vast majority of FIP cases, while type 2 viruses are responsible for the remaining infections. Immunohistochemistry (IHC) currently serves as the gold standard for diagnosis of FIP; however, IHC is limited by variations in sensitivity. RNA in situ hybridization (RNA ISH) has an established foothold in infectious disease diagnostics and presents a potentially improved method for detection of FIP. This proof-of-concept study evaluated the efficacy of RNA ISH probes targeted to FCoV, as compared to IHC using monoclonal antibody FIP 3-70. Formalin-fixed paraffin-embedded tissues from FIP-positive cats were used for ISH, with the presence of RNA determined chromogenically. ISH tissue slides were then compared to their IHC counterparts, with efficacy determined based on metrics including staining intensity and abundance. Positive ISH staining on tissue was found to be both more intense and abundant than for IHC, suggesting that ISH serves as a highly sensitive method for the detection of FCoV/FIP in comparison to IHC - a finding that awaits further validation.

Molecular detection using hybridization capture and next-generation sequencing reveals cross-species transmission of feline coronavirus type-1 between a domestic cat and a captive wild felid

“…Histological detection like immunohistochemistry (IHC) and in-situ hybridization target the N protein ( i . e ., antibody FIPV3-70) and RdRp (39), respectively. While targeting these genes can be useful for detection purposes ( i .…”

“…For example, commonly used serology tests target the N protein (31), and detection by RT-PCR targets genes like the RNA-dependent RNA polymerase (RdRp, (35) or 7a and 7b (38) (Figure 1). Histological detection like immunohistochemistry (IHC) and in-situ hybridization target the N protein (i.e., antibody FIPV3-70) and RdRp (39), respectively. While targeting these genes can be useful for detection purposes (i.e., they are highly conserved), they are not optimal for differentiating FCoV-1 and -2 (Figure 1).…”

“…For each tissue, an unstained slide of 5 m thick tissue sections were used. A probe directed to the RdRp gene(39) was used to detect FCoV RNA by in-situ hybridization (ISH, RNAscope ® Probe V-FIPV-ORF1a1b, Advanced Cell Diagnostics). The ISH process was carried out at the AHDC in the automated staining platform Ventana Discovery Ultra (Roche Tissue Diagnostics) using the Discovery kits for mRNA Sample Prep, mRNA Red Probe Amplification and mRNA Red Detection and the Ventana Hematoxylin and Ventana Bluing Reagents for counterstaining.Approximately 40 mg of each tissue was used for RNA extraction using the Monarch ® Total RNA Miniprep Kit (New England Biolabs, NEB) according to the manufacturer's instructions and including the suggested DNaseI…”

An Exploratory Bioinformatic Investigation of Cats’ Susceptibility to Coronavirus-Deriving Epitopes

“…To date, these viruses are still considered fatal for young cats, and therapies are mainly based on antiviral nucleotides like remdesivir and its analogs. However, no prophylaxis or vaccine is available [ 2 , 3 ]; in fact, cases of antibody-dependent enhancement (ADE) of FIPV infectivity have been observed following the development of the humoral response, which facilitates the entry of the virus in the macrophages, where the pathogen amplifies its replication [ 4 , 5 , 6 ].…”