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

Abstract

Objectives Feline infectious peritonitis (FIP) emerges when feline coronaviruses (FCoVs) mutate within their host to a highly virulent biotype and the immune response is not able to control the infection. FCoV spike (S) gene mutations are considered to contribute to the change in virulence by enabling FCoV infection of and replication in macrophages. This study investigated the presence of FCoV with and without S gene mutations in cats with FIP using two different real-time RT-PCRs on different samples obtained under clinical conditions. Methods Fine-needle aspirates (FNAs) and incisional biopsies (IBs) of popliteal and mesenteric lymph nodes, liver, spleen, omentum and kidneys (each n = 20), EDTA blood (n = 13), buffy coat smears (n = 13), serum (n = 11), effusion (n = 14), cerebrospinal fluid (n = 16), aqueous humour (n = 20) and peritoneal lavage (n = 6) were obtained from 20 cats with FIP diagnosed by immunohistochemistry. Samples were examined by RT-PCR targeting the FCoV 7b gene, detecting all FCoV, and S gene mutation RT-PCR targeting mutations in nucleotides 23531 and 23537. The prevalence of FCoV detected in each sample type was calculated. Results In 20/20 cats, FCoV with S gene mutations was present in at least one sample, but there was variation in which sample was positive. FCoV with mutations in the S gene were most frequently found in effusion (64%, 95% confidence interval [CI] 39-89), followed by spleen, omentum and kidney IBs (50%, 95% CI 28-72), mesenteric lymph node IBs and FNAs (45%, 95% CI 23-67), and FNAs of spleen and liver and liver IBs (40%, 95% CI 19-62). Conclusions and relevanceIn these 20 cats with FIP, FCoVs with S gene mutations were found in every cat in at least one tissue or fluid sample. This highlights the association between mutated S gene and systemic FCoV spread. Examining a combination of different samples increased the probability of finding FCoV with the mutated S gene.

Update on feline infectious peritonitis

“…Therefore, cats without feline infectious peritonitis (FIP) can have detectable virus within blood, effusions and tissues – albeit at a lower frequency and viral copy number. Due to low circulating levels of viraemia, use of RT-PCR of whole blood in cats with suspected FIP is not recommended (Emmler and others 2020).…”

“…Most (18 of 20; 90%) mesenteric lymph node aspirates from cats with non-effusive FIP are RT-PCR positive (Dunbar and others 2018); however, one false-positive result (out of 20 cats) did occur in a cat seropositive for FCoV. Detection of FCoV in cerebrospinal fluid (CSF) by RT-PCR from cats with FIP is variable, ranging from 21% to 86% (Foley and others 1998, Doenges and others 2016, Barker and others 2017, Emmler and others 2020), whereas RT-PCR was negative in all control cats. Detection of FCoV in aqueous humour from cats with FIP was poor (25%), and no control cats were tested (Emmler and others 2020).…”

“…Therefore, cats without feline infectious peritonitis (FIP) can have detectable virus within blood, effusions and tissues – albeit at a lower frequency and viral copy number. Due to low circulating levels of viraemia, use of RT‐PCR of whole blood in cats with suspected FIP is not recommended (Emmler and others 2020). In a recent large study, all but one cat of 57 (98%) with FIP had at least one tissue positive for FCoV by quantitative RT‐PCR, as compared to 12 of 45 cats without FIP (Barker and others 2017); viral copy numbers were also significantly higher in the positive samples from cats with FIP than those without FIP.…”

“…Detection of FCoV in cerebrospinal fluid (CSF) by RT‐PCR from cats with FIP is variable, ranging from 21% to 86% (Foley and others 1998, Doenges and others 2016, Barker and others 2017, Emmler and others 2020), whereas RT‐PCR was negative in all control cats. Detection of FCoV in aqueous humour from cats with FIP was poor (25%), and no control cats were tested (Emmler and others 2020). Additional analysis has been applied to RT‐PCR‐positive samples to determine whether the FCoV present carries genetic mutations that have been said to be associated with FIP.…”

“…Where a highly sensitive method (pyrosequencing) was employed to evaluate the Spike gene, mutations were detected in FCoV‐positive tissue from 15 of 17 (88%) samples from cats without FIP as compared to 202 of 206 (98%) samples from cats with FIP (Barker and others 2017). Other techniques (eg, allelic discrimination) that require a relatively high viral copy number in the sample to generate a result (often not present in cats without FIP), and consider a result where sequencing has failed to be negative, will increase the test specificity by a modest amount by reducing, but not eliminating, the number of false‐positives; however, the detection of true‐positives results in cats with FIP (ie, the test sensitivity) is more markedly reduced (Emmler and others 2020, Felten and others 2017a). In conclusion, although a positive RT‐PCR result on fluid, effusions, aspirates and tissue can provide strong support for a diagnosis of FIP (particularly for CSF), both false positives and false negatives occur such that RT‐PCR should not be solely relied upon to make a diagnosis.…”