Feline coronavirus quantitative reverse-transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis. Journal of Feline Medicine and Surgery, 19(2), 240-245. DOI: 10.1177/1098612X15606957

26 Objectives: To determine whether feline coronavirus (FCoV) RNA in effusion samples can be 27 used as a diagnostic marker of feline infectious peritonitis (FIP), and in FCoV RNA positive 28 samples, to examine amino acid codons in the FCoV spike protein at positions 1058 and 1060 29 where leucine and alanine, respectively, have been associated with systemic or virulent (FIP) 30 FCoV infection. 31 Methods: Total RNA was extracted from effusion samples from 20 cats with confirmed FIP and 32 23 cats with other diseases. Feline coronavirus RNA was detected using a reverse transcriptase 33 quantitative polymerase chain reaction assay (qRT-PCR) and positive samples underwent 34 pyrosequencing of position 1058 and Sanger sequencing of position 1060 in the FCoV spike 35 protein. 36 Results: Seventeen (85%) of effusion samples from 20 cats with FIP were positive for FCoV 37 RNA, whereas none of the 23 cats with other diseases were positive. Pyrosequencing of the 17 38 FCoV positive samples showed that 11 (65%) of cats had leucine and 2 (12%) had methionine 39 at position 1058. Of the two samples with methionine, one had alanine at position 1060. 40 Conclusions and relevance: A positive FCoV qRT-PCR result on effusions appears specific 41 for FIP and may be a useful diagnostic marker for FIP in cats with effusions. The majority of 42 FCoVs contained amino acid changes previously associated with systemic spread or virulence 43 (FIP) of the virus. 44 45 Introduction 46 Feline coronavirus (FCoV) infection is common in domestic cat populations worldwide1-3. Most 47 infections are enteric and self-limiting. In a small number of cases, FCoV infection can lead to 48 the development of feline infectious peritonitis (FIP), a significant cause of mortality in young 49 cats. 50 Definitive diagnosis of FIP relies on histopathological examination of affected tissues, ideally 51 with detection of intracellular FCoV antigen by immunostaining1, 4, 5. Obtaining tissue samples is 52 invasive and problematic for ante mortem diagnosis. In many FIP cases, abdominal, pleural 53 and/or pericardial effusions develop2, which can usually be easily obtained for diagnostic 54 testing. Previous studies have reported the use of FCoV antigen staining in effusion samples in 55 the diagnosis of FIP, with sensitivity and specificity of 57-100% and 71.5-100%, respectively6-9. 56 Feline coronavirus RNA can be detected in samples using conventional or quantitative reverse 57 transcriptase polymerase chain reaction assays (qRT-PCR). Studies on tissues using qRT58 PCRs have found that cats with FIP have significantly higher FCoV loads in tissues than healthy 59 or sick (non-FIP) FCoV infected cats5, 10, 11. It is possible that the same is true for effusion 60 samples. Previous studies performing FCoV conventional PCR on effusion samples from cats 61 with FIP have shown promising results, but were limited either by lack of definitive diagnosis of 62 cases12, or lack of control non-FIP cats13. 63 The aim of this study was to perform FCoV qRT-PCR on effusions collected from cats with and 64 without confirmed FIP to investigate whether the presence of FCoV RNA in effusions is helpful 65 in diagnosing FIP. In addition, it has been reported that key amino acid substitutions 66 (methionine to leucine at position 1058 and serine to alanine at position 1060) in the spike 67 protein of FCoV may be associated with FCoV virulence14 or systemic infection11, therefore 68 these substitutions were evaluated in FCoV positive effusions. 69