Quantitative real time polymerase chain reaction (qRT-PCR) and RNAscope in situ hybridization (RNA-ISH) as effective tools to diagnose feline herpesvirus-1-associated dermatitis

Background: Felid herpesvirus type 1 (FHV-1)-associated dermatitis is characterized by facial and nasal involvement; clinical and histopathological manifestations may overlap with other dermatitides. Objective: To evaluate the realibility of qRT-PCR-2−ΔΔCq and RNAscope in situ hybridization (RNA-ISH) methods to diagnose FHV-1-associated dermatitis, in formalin-fixed paraffin-embedded (FFPE) tissues. Animals: Sixteen FFPE samples from cats with facial dermatitis and four controls were studied. Methods and materials: Based on histopathological features, cases were separated into: Group 1, samples with herpetic dermatitis (four); Group 2, samples with nonherpetic facial dermatitis (six); Group 3, samples with facial dermatitis of ambiguous nature (allergic or viral) (six); and Group 4, samples from healthy cats (four). A relative quantification using the 2−ΔΔCq method was used to estimate the “upregulation” of each FHV-1 target viral gene copies (glycoprotein-B and thymidine-kinase) relative to reference gene. Detection of FHV-1 mRNA was performed using the RNAscope 2.5 detection kit. Results: By 2−ΔΔCq analysis, upregulation of both FHV-1 genes was observed in all samples from Group 1 and two of six from Group 3. No upregulation was identified in samples from groups 2 and 4. Positive mRNA hybridization signal was observed in all cases from Group 1 and two cases of Group 3. No positivity was observed in samples from groups 2 and 4. Conclusions and clinical importance: QRT-PCR 2−ΔΔCq analysis and RNA-ISH can identify the FHV-1 genome as causative agent of the associated dermatitis, even where inclusion bodies are not detectable. Both techniques are functional in retrospective studies, have greater specificity than conventional PCR, and may be proposed for research and diagnostic purposes.