PCRPCR-based tests

Tests based on PCR have been used to help diagnose FIP for almost two decades (Li and Scott, 1994). Nested PCR is a method that greatly increases the ability of the test to detect very small amounts of feline coronavirus RNA as cDNA (Gamble et al., 1997). Nested PCR involves amplifying a larger fragment of the viral cDNA in the first step, purifying this PCR product and then amplifying a smaller piece from within the larger amplified DNA in a second reaction. A nested PCR was reportedly >90% sensitive and specific in detecting FIPV in ascites from cats with effusive FIP (Gamble et al., 1997). Although very sensitive, nested PCRs are plagued by DNA contamination with PCR products, which causes false positive reactions. The problem of laboratory contamination with PCR products can be avoided by using real time RT-PCR. It is generally conceded that real time RT-PCR is quite sensitive and specific in detecting and semi-quantitating fecal coronavirus (FECV) shedding in both experimental and naturally infected cats (Pedersen et al., 2008, 2009, 2012; Kipar et al., 2010; Vogel et al., 2010; An et al., 2011; Addie et al., 2012; Amer et al., 2012; Wang et al., 2013).

In one study testing the accuracy, PCR-based testing was found to be only 80– 90% accurate in confirming the presence of FIPV in diseased tissues (Sharif et al., 2010). In another study, only 377/854 (44%) of peritoneal effusion specimens from cats suspected but not confirmed of having FIP tested positive by RT-PCR (Soma et al., 2013), positivity was 78- 92% in purebreds vs. 35% in mixed breed cats. In one study, FECV was identified in blood monocyte/macrophages in 40% of experimentally FECV infected cats by day 14 and 14% remained viremic at day 48 post-infection, which demonstrates that viremia accompanied intestinal infection (Kipar et al., 2010; Vogel et al., 2010). FECV was detected in several internal organs after fecal shedding ceased (Kipar et al., 2010).

The problem of FECV co-infections can be overcome by designing tests that identify FIPV-unique mutations. Mutations in the ORF 3c and at the S1/S2 gene cleavage site are unique to each FIPV and therefore are not good candidates (Licitra et al., 2013; Pedersen et al., 2009). The most FIPV-specific mutations are two single nucleotide changes within the fusion protein region of the spike or surface (S) protein (Chang et al., 2010). Either one of these mutations occurs in >98% of FIPVs detected in diseased tissues, but no results have been reported regarding whether these mutations are present in blood at detectable levels. It is also possible that these mutations could be found in healthy cats with abortive or subclinical infections (Porter et al., 2014). Even if highly sensitive and specific FIPV RNA detection tests could be developed, it appears that many cats with naturally occurring FIP do not have detectable levels of viral RNA in their blood, either within plasma or concentrated in the white cell fraction.

Another study evaluated a discriminating real-time RT-PCR using effusion and/or serum/plasma in the diagnosis of FIP. The results indicate that the detection of the FIPV pathotype with substitution M1058L is very specific for the FIP phenotype and can be a useful tool in the diagnosis of FIP, however, substitution M1058L was also detected in one control cat without FIP. As none of the FIPV-positive effusion samples contained substitution S1060A, it is considered a weak discriminatory factor for the diagnosis of FIP. The fact that in two other control cats FCoV was detected, even though the pathotype could not be determined, shows that FCoV can cause viremia and therefore, traditional non-discriminating RT-PCR is not sufficient to definitively diagnose FIP. Discriminative RT-PCR should be performed in order to minimize the risk of euthanasia of cats suffering from different diseases. The use of serum/plasma is not recommended owing to the low viral load in blood infections (Felten et al., 2017)

Another study performed reverse-transcriptase quantitative-PCR (RT- qPCR) on 102 samples using FCoV-specific primers, followed by sequencing of a section of the S gene on RT-qPCR positive samples. Tissue, fluid, and faecal samples from cats with FIP were more likely to be FCoV RT-qPCR- positive (90.4, 78.4 and 64.6% respectively) than those from cats without FIP (7.8, 2.1 and 20% respectively). Identification of S gene mutated FCoVs as an additional step to the detection of FCoV alone, only moderately increased specificity for tissue samples (from 92.6 to 94.6%) but specificity was unchanged for fluid samples (97.9%) for FIP diagnosis; however, sensitivity was markedly decreased for tissue (from 89.8 to 80.9%) and fluid samples (from 78.4 to 60%) for FIP diagnosis. These findings demonstrate that S gene mutation analysis in FCoVs does not substantially improve the ability to diagnose FIP as compared to detection of FCoV alone (Barker et al., 2017).

Real time reverse transcriptase-PCR for feline enteric coronavirus shedding in faeces

Although cats with FIP will often shed FECV, it is inconsistent, and the virus is shed at lower levels than in healthy cats. Feline coronavirus in these cases is usually of the enteric and not the FIP biotype (Pedersen et al., 2009, 2012; Chang et al., 2010, 2012). The probability that a significant proportion of cats in the cattery will be shedding FECV at any given time is high, while the odds of an adult FECV shedder developing FIP are very low. The higher the proportion of cats in a cattery that shed coronavirus at a given time, and the higher the level of shedding, the more likely FIP will occur in the population (Foley et al., 1997). Large amounts of virus are shed in the faeces for many weeks, and even months, after initial infection, but with time and lack of re-exposure most cats will stop shedding (Pedersen et al., 2008). However, some cats can shed at high levels for prolonged periods of time and some cats that have stopped shedding become susceptible to reinfection (Pedersen et al., 2008).

Hope for the Future!

We hope new studies will give hope for FIP in cats