Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis

Barker, Emily N.; Stranieri, Angelica; Helps, Chris R.; Porter, Emily L.; Davidson, Andrew D.; Day, Michael J.; Knowles, Toby; Kipar, Anja; Tasker, Séverine

Conventional sequencing of the pyrosequencing amplicon was possible in 21 cases in which pyrosequencing failed (n = 48 of 320 samples where pyrosequencing was attempted), significant secondary structure was predicted in these amplicons at the pyrosequencing primer binding site, which could account for the failure to pyrosequence [26] . Mixed non-mutated and mutated FCoV infections may also have been missed by the pyrosequencing assay as the amplification step biases the detected sequence towards the dominant virus type. In addition, both serotype 1 and serotype 2 FCoVs have been associated with the development of FIP; however, only mutations within the S protein fusion peptide of the more prevalent serotype 1 FCoVs have been associated with systemic infection and development of FIP [18, 20] . As the recombination events that result in the formation of serotype 2 FCoVs include the S protein gene, assays that characterise this portion of the genome are not applicable to serotype 2 FCoVs. Therefore, it was predicted that pyrosequencing would fail for those samples containing serotype 2 FCoV; however, both serotype 1 and 2 FCoVs were detected by the RT-qPCR assay used in this study, as this assay targets a section of the genome not affected by the recombination events [24] . In this study only cats that failed pyrosequencing were assessed for the presence of serotype 2 FCoVs, therefore it cannot be excluded that more cats had a dual infection with both serotypes. Spike gene mutation analysis, in combination with FCoV RT-qPCR, results in a modest increase in specificity, but this is offset by a decrease in sensitivity resulting in decreased accuracy. Independent of disease prevalence within a population, there was no significant difference in the proportion of mutated FCoVs detected in tissue and body cavity fluid samples between cats with FIP and cats without FIP that were FCoV RT-qPCR-positive (χ 2 = 2.96, p = 0.086). Overall, the combination of FCoV RT-qPCR and S protein mutation analysis does not enhance the diagnosis of FIP over FCoV RT-qPCR alone, and S gene mutation analysis has significant time and cost implications.