Infectious RHDV and RHDVa particles are shed from infected kittens and virus is transmitted to littermates. Two kittens infected with either RHDV or RHDVa (high dose of inoculum) were placed with four uninfected littermates (bystanders, BS). Each column represents one replicate of the experiment done in triplicates (trial 1: A,D,G; trial 2: B,E,H; trial 3: C,F,I). All kittens were monitored for virus replication via blood and swab samples. Experimentally infected kittens were euthanased at 2 or 4 dpi, and bystanders six days after the trial started. Upper panel: Virus shed over time by kittens infected with RHDV (blue) or RHDVa (red) and bystanders (black); Middle panel: Virus concentration in blood samples of infected and bystander kittens (colour code as above). Lower panel: Virus load in livers of infected kittens and bystanders at the time of autopsy, 2 and 4 dpi. Blue and red bars indicate RHDV and RHDVa genome copy numbers, respectively. SDL (A-F) indicates the genome concentration relative to 20 genome copies detected in the RT-qPCR, which is considered the safe detection limit The average safe detection limit in the liver tissues was 46 genomes/mg (range 30 – 82). Identification and quantification of the virus that infected the bystanders was done by analysing the liver RNA extracts using a RT-qPCR specific for either RHDV or RHDVa (G-I). In case of one bystander (BS 5–3, see G) both PCRs were positive, and a determination of RHDV genome copy numbers with the specific RT-qPCR assay indicated that most (>90 %) of the genomes in its liver were RHDVa genomes (pink bar). Kitten RHDVa 5–6 did not get infected. *indicates kittens that were found dead, both in the morning of days when autopsies were scheduled.