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

It is possible that the calculated relative FCoV copy numbers in this study are an overestimate of viral load. FCoV RT-qPCR assays amplify both cell-associated subgenomic mRNA, as well as cell-associated or virionassociated genomic RNA, with relative abundance determined by the positioning of primers. As viral transcription starts at the 3′ end of the FCoV genome there are more subgenomic mRNAs containing viral 3′ sequence than those containing viral 5′ sequence, hence assays directed at the 5′ end of the genome (e.g. viral replicase complex genes) are less susceptible to viral load overestimation than those directed at the 3′ end of the genome (e.g. 7a/b accessory protein genes). The RT-qPCR assay used in the present study targets the region spanning the membrane-nucleocapsid gene junction (nucleotides positions 26655-26826 of FCoV isolate FIPV 79-1146 DQ010921) [24] . Overestimation is less of an issue in the faecal samples, firstly as faeces has a high level of bacteria, and is thus rich in RNases that would degrade any RNA released from cells shed into the gut lumen, and secondly as the viral RNA is most likely to be present in its virion form, which does not contain subgenomic mRNA and is protected from RNase degradation by the viral envelope. Any cell-associated viral RNA is likely to be degraded due to cell death and the abundant RNases. This study did not determine whether any faecally shed FCoV was infective; this is an important concept as faeces containing mutated FCoV theoretically has increased potential to cause FIP following faecooral transmission, assuming the mutated virus can enter enterocytes and replicate. Previous studies have reported 39-85% of FCoVs detected in tissues from cats with FIP had loss of 3c gene functionality [33] [34] [35] , which has been associated with a loss of ability to replicate in enterocytes and therefore infectivity via the natural route [35, 36] . Enterocyte cell cultures have been used to more accurately assess FCoV infectivity and virus copy number in a recent study [8] . However, use of enterocyte cell cultures was not possible in this study, nor was 3c gene sequencing attempted.