Fig. 2. In vitro and in vivo results of cluster 3 TMUV infection in chickens. (a) Growth curves of TMUV SD on HD11, LMH and DF-1 chicken cell lines infected at a MOI of 0.0001. Supernatant samples were collected at designated time points and titrated on BHK cells to determine viral replication efficiency. (b) Viremia in infected chickens: Chickens were intramuscularly inoculated with 10^3.5 TCID₅₀ of the SD strain. The virus litres in blood collected at 2, 3 and 4 dpi were determined by titration on BHK cells. Results indicate viremia development at these time points. (c) Viral shedding detection: Virus litres were assessed in throat and cloacal swabs collected from infected chickens at 2, 3 and 4 dpi, and no virus was detected in all swab samples. (d) Detection of TMUV-specific antibodies in serum samples of inoculated chickens at 14 dpi, contact chickens and non-infected chickens using ELISA. Seroconversion was defined by a PI value of ≥18.4 %, indicating a positive antibody response to the virus.