Detection of feline coronaviruses in cell cultures and in fresh and fixed feline tissues using polymerase chain reaction

Abstract

Feline coronavirus infections in cell cultures and in fresh and fixed feline tissues were detected using a polymerase chain reaction (PCR) test. Cell cultures were inoculated with feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV) or sham inoculum. The tissue samples of liver, kidney and spleen were taken from specific-pathogen-free (SPF) cats that were inoculated intranasally with 10(3) TCID50 of FIPV 79-1146 (n = 10), FIPV UCD1 (n = 3) or sham inoculum (n = 3), from clinical cats (n = 43), and from formalin-fixed archived feline tissues (n = 49), respectively. Additional tissue samples were taken from the FIPV-inoculated cats (n = 6) and were kept at 4 degrees C, room temperatures (20-24 degrees C) and 37 degrees C respectively for 0, 6, 12, 24, 48, 72, and 96 hours before frozen (-70 degrees C) for PCR to evaluate the effects of the ambient temperatures and post-mortem intervals on the test. The samples were also fixed in 10% neutrally buffered formalin, 95% ethanol, and Bouin's solution respectively to evaluate the effects of the fixatives on the test. Positive PCR results were obtained from the cell cultures that were inoculated with FIPV and FECV and from the FIPV-inoculated cats (13/13). Negative PCR results were obtained from the sham-inoculated cell cultures and cats (3/3). Of the 92 clinical cats, 7 of the 8 FIP-suspected cats (87.5%) and 51 of the 84 non-FIP-suspected cats (60.7%) were shown to be virus-positive in at least one of the tissue samples. There was no significant difference in the PCR results between the fresh and the formalin-fixed tissues of the clinical cats (P > 0.05). Of the FIPV inoculated cats, the virus was detectable equally well in fresh and formalin-, Bouin's solution- or ethanol-fixed tissues. However, the amounts of total RNA extracted from the fixed tissues were significantly less than those from fresh tissues (P < 0.01). In tissues that were kept at 4 degrees C, the virus was detectable up to 96 h; at room temperatures, up to 48 h; and at 37 degrees C, up to 24 h, respectively.

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses

“…Furthermore, such steps increase the risk of inaccuracy and contamination. Several RT-PCR assays have been developed to detect FCoV (Li and Scott, 1994;Herrewegh et al, 1995;Gamble et al, 1997). Two of those assays are nested PCRs and in all of those ethidium bromide staining and UV light transillumination of electrophoretically separated PCR products is necessary to detect PCR products.…”

“…Several polymerase chain reaction (PCR) based methods have been suggested in detection of the positive-stranded FCoV RNA, but none of these were designed to be quantitative (Li and Scott, 1994;Herrewegh et al, 1995;Gamble et al, 1997). In addition, conventional PCR methods are time consuming due to several post-amplification steps, contain a certain risk of cross-contamination between the samples due to a separate labor-intensive reverse transcription (RT) step and a second PCR step in nested PCR systems, are limited in sensitivity and allow only relatively few samples to be processed at one time.…”

Detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis

“…In a number of studies, RT-PCR techniques have been used to detect FCoV in samples from clinical FIP cases (Li and Scott, 1994;Egberink et al, 1995;Herrewegh et al, 1995Herrewegh et al, , 1997Fehr et al, 1996;Richter et al, 1996). While some of these studies have involved fresh or formalin-fixed tissue (Li and Scott, 1994), in others plasma, serum, ascitic or pleural fluids have been examined Herrewegh et al, 1995Herrewegh et al, , 1997Fehr et al, 1996;Richter et al, 1996). We found virus in approximately 80% of blood samples from confirmed cases of FIP.…”

Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis

“…The recently developed reverse transcriptase polymerase chain reaction (RT-PCR) assays, using primers targeted to highly conserved regions of the viral genome (3 -UTR (untranslated region) (Herrewegh et al, 1995;Fehr et al, 1996), or S-protein gene (Li and Scott, 1994;Gamble et al, 1997)), which are common to all FCoV strains, became a valuable tool for the detection of FCoV nucleic acid in blood, body cavity effusions, faeces and tissue samples of infected cats.…”