FCV viral RNA was detected at least several times on all items tested. cleaning, but no disinfection took place, FCV viral RNA was detectable using RT-qPCR on all tested items and surfaces, including cat hair. However, only very limited evidence was found of replicating virus using virus isolation. Viral RNA remained demonstrable for at least 28 days after shedding had 20-Hydroxyecdysone ceased in all cats. Disinfection with 5% sodium bicarbonate (and IncidinTM Plus) and barrier measures were effective in that no viral RNA was detectable outside the cat rooms. Our 20-Hydroxyecdysone findings are important for any multicat environment to optimize hygienic measures against FCV infection. and was tested [3,17,18,20]. Feline gammaherpesvirus (FcaGHV)  was tested by PCR with modifications as described in Novacco et al., 2019 . In addition, the 20-Hydroxyecdysone serum samples were tested for antibodies against feline calicivirus, feline herpesvirus, feline parvovirus and feline coronavirus by immunofluorescence assays as previously described [23,24], and for feline immunodeficiency virus by western blot . All cats tested negative for all of these infections prior to the start of the experiment. 2.2. Rabbit polyclonal to Bcl6 Challenge Viruses The isolate for the first challenge, FCV 273, originated from a privately-owned Swiss domestic cat. The cat presented with chronic stomatitis/gingivitis, caudal stomatitis and lingual ulcerations. The isolate for the second challenge, FCV 27, originated from a privately-owned Swiss Norwegian Forest cat that was clinically healthy apart from oral ulceration on the hard palate. The sample had been collected for a previous study . Both FCV isolates were obtained from samples collected by Swiss veterinarians using oropharyngeal cytobrushes that were processed using the method described previously . The veterinarians obtained informed consent from the cat owners . All samples were collected as part of a diagnostic workup; no ethical approval was necessary, in compliance with Swiss regulations . The virus was propagated on Crandell-Rees feline kidney (CRFK) cells and passaged three times. After the third passage, cell culture supernatant was harvested when a cytopathic effect (CPE) was clearly visible, but not fully complete and centrifuged at 800 for 3 min. Aliquots of 1 1 mL were immediately stored at ?80 C. The cell culture supernatants were tested using RT-qPCR and qPCR to confirm the absence of co-infections (FHV-1, FcaGHV, FCoV, FIV, FeLV, FPV, and value 0.05. 3. Results 3.1. Characterization of the Challenge Viruses FCV 273 and FCV 27 The nucleotide sequences of the capsid genes of the two field isolates, FCV 27 and FCV 273, were compared with each other and with the FCV vaccine strain FCV F9. The sequence identity for FCV 27 and FCV 273 was 77.4%. Compared to FCV F9, FCV 27 and FCV 273 had a sequence identity of 75.8% and 74.7% respectively. 3.2. All Cats Shed FCV after the 1st Experimental Infection with FCV 273 Oropharyngeal shedding of FCV was measured using cytobrush material from all ten challenged cats that was tested on CRFK cultures and then performing RT-qPCR on cell culture supernatants. Prior to the FCV challenge, none of the cats was shedding FCV. On days 3 and 9 after the first FCV infection, all samples displayed CPE in cell culture, and the supernatants tested positive for FCV by RT-qPCR (Table 1). Subsequently, CPE was found only in cells incubated with the samples of some cats, but all or most cell culture supernatants still tested positive by FCV RT-qPCR (Table 1). From day 29 onwards, oropharyngeal cytobrushes from six or fewer cats tested FCV positive by RT-qPCR and from day 36 until day 71 only one cat (JJH3) was shedding virus that induced CPE in cell culture. None of the oropharyngeal cytobrush samples tested positive after day 71 following FCV 273.