High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Naturally acquired feline bocavirus type 1 and 3 infections in cats with neurologic deficits.

Feline bocaviruses (FBoVs) have been recognized as novel feline pathogens associated with gastrointestinal diseases. Although bocavirus infections in humans and animals present a broad range of clinical symptoms including neurologic diseases, the neuropathology caused by FBoV infection in cats is unknown. This study aims to investigate the presence of bocavirus in the brain samples of 78 cats showing neurologic deficits and 41 healthy cats using polymerase chain reaction (PCR) and to present the pathological findings of FBoV infection in brain tissues. Only five (6.41%, five out of 78) cats with neurological deficit were FBoV positive on PCR screening and were characterized as FBoV-1 (four out of five) and FBoV-3 (one out of five) by sequencing. Among FBoV-positive cases, viral DNA were detected by PCR in the cerebrum and brain stem of all FBoV-positive cases and rarely detected in the cerebellum of some cases. Histologically, all FBoV-positive cases revealed a variety of inflammatory responses. Among these, 80% (four out of five cases) showed multifocal neuronal vacuolation, mainly found in the cerebrum and brain stem. Eosinophilic inclusion-like materials were found within the nuclei of glial cells in the FBoV-3-positive case. In situ hybridization revealed FBoV DNA in oligodendroglia and vacuolated neurons detected using dual labelling with Olig-2 and NeuN immunohistochemistry, respectively. Transmission electron microscopy confirmed the presence of FBoV-3 virions in the nuclei of glial cells. Apart from localization in brain tissues, the FBoV DNA were also detected in multiple lymph nodes (five out of five) and some intestines (two out of five) of such positive cases, suggesting both parenteral and enteral infections. Complete genome sequence analysis revealed genetic diversity of detected FBoV-1, which were closely related to the strains found in China and Hong Kong, while the detected FBoV-3 presented distant monophyletic clade to previously detected FBoV-3 sequences. The FBoVs, together, should be considered a neurotropic virus and a possible cause for neuronal vacuolation in cats with neurologic deficits.