Detection of feline coronavirus mutations in paraffin-embedded tissues in cats with feline infectious peritonitis and controls

Abstract

Objectives The amino acid substitutions M1058L and S1060A in the spike protein of feline coronavirus (FCoV) have been postulated to be responsible for the development of the pathogenic feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP). The aim of the following study was to investigate the presence of mutated virus in tissue samples of cats with and without FIP. Methods The study population consisted of 64 cats, 34 of which were diagnosed with FIP and 30 control cats. All cases underwent autopsy, histopathology and immunohistochemistry (IHC) for FCoV. Furthermore, a genotype-discriminating quantitative reverse transcriptase PCR (RT-qPCR) was performed on shavings of paraffin-embedded tissues to discriminate between cats with FIP and controls, and the sensitivity and specificity of this discriminating RT-qPCR were calculated using 95% confidence intervals (CIs). Results Specificity of genotype-discriminating RT-qPCR was 100.0% (95% CI 88.4-100.0), sensitivity was 70.6% (95% CI 52.5-84.9). In cats with FIP, 24/34 cats tested positive for FIPV. In samples of three control cats and in seven cats with FIP, FCoV was found, but genotyping was not possible owing to low FCoV RNA concentrations. Out of the positive samples, 23 showed the amino acid substitution M1058L in the spike protein and none the substitution S1060A. One sample in a cat with FIP revealed a mixed population of non-mutated FCoV and FIPV (mixed genotype). For one sample genotyping was not possible despite high viral load, and two samples were negative for FCoV. Conclusions and relevance As none of the control animals showed FCoV amino acid substitutions previously demonstrated in cats with FIP, it can be presumed that the substitution M1058L correlates with the presence of FIP. FCoV was detected in low concentration in tissues of control animals, confirming the ability of FCoV to spread systemically. The fact that no negative controls were included in the IHC protocol could potentially lead to an underestimation of the sensitivity of the RT-qPCR.

Feline coronavirus with and without spike gene mutations detected by real-time RT-PCRs in cats with feline infectious peritonitis

“…5 Later studies confirmed these findings and only detected few [25][26][27] or no FCoV at all with S1060A. 33 As such, M1058L is the more common S protein substitution, which is also reflected by the results of the present study.…”

“…This has already been observed in other studies using the same method. 27,33 Another reason could be the absence of the particular S gene mutations examined here and the presence of other mutations involved in FIP pathogenesis inst ead. 6,8,9,14,15,38,39 Some other mutations, such as in the 3c gene, have been discussed as playing a role in FIP pathogenesis, but a clear causal relationship to FIP still has not been identified.…”

“…32 Additional studies have evaluated RT-PCR detection of FCoV mutations in paraffin-embedded tissues and effusion from cats with confirmed FIP. 27,33 Briefly, highly specific hydrolysis probes were used, detecting either the mutation at position 3174 (A → C/T) or 3180 (T → G) on the FCoV genome, corresponding to amino acid positions 1058 and 1060, nucleotide 23531 and 23537, and M1058L and S1060A of reference sequence FJ938051, respectively, or non-mutated sequences by using an allelic discrimination approach (IDEXX Laboratories, unpublished data). Probes for mutated and non-mutated S gene sequences were fluorophore-labelled (6-FAM and VIC, respectively).…”

Feline Infectious Peritonitis Virus Nsp5 Inhibits Type I Interferon Production by Cleaving NEMO at Multiple Sites

“…Spontaneous mutations of the S gene [6], ORF3abc [7,8], and ORF7ab [9,10] in the FCoV genome allow virions to efficiently replicate in macrophages and monocytes, ultimately resulting in FIPV [11]. Detection of FCoV S gene mutations is considered as a tool to diagnose FIPV [12,13]. FIP is typically characterized by a fibrinous and granulomatous serositis, protein-rich serous effusions, and pyogranulomatous lesions in several organs [14,15].…”

Development and evaluation of a SYBR Green I RT-qPCR assay for Feline Infection Peritonitis virus detection

“…At present, TaqMan based real-time PCR and conventional PCR assays for detecting FCoV have been previously reported [12][13][14]. However, conventional PCR methods are not quantitative and can sometimes include non-specific products of the same size.…”