We observed stained cells under an inverted fluorescence microscope (Evos FL; Thermo Fisher Scientific) and calculated the [log.sub.10] 50%
tissue culture infectious dose per milliliter ([TCID.sub.50]/mL) according to the method of Reed and Muench (28).
Tissue Culture Infectious Dose 50-CPE ([TCID.sub.50])-CPE Titre Determinations
We inoculated pigs intranasally and intratracheally with 106
tissue culture infectious dose 50 ([TCID.sub.50]) of MERS-CoV isolate hCoV-EMC/2012 or, for controls (mock-inoculated), with Dulbecco's modified Eagle medium.
Its titer was determined by 50%
tissue culture infectious dose ([TCID.sub.50]) method as described previously.
Virus stocks were grown in Vero E6 cells and titrated by using a 50%
tissue culture infectious dose ([TCID.sub.50]) assay (9).
days viable NHP 1 [log.sub.10] [TCID.sub.50]/mL Drying 27[degrees]C, RH 80% 6.5 5 Drying 21[degrees]C, RH 40% 6.5 1 Liquid 27[degrees]C 7.2 14 Liquid 21[degrees]C 7.2 14 NHP 2 Drying 27[degrees]C, RH 80% 2.8 5 Drying 21[degrees]C, RH 40% 2.8 1 Liquid 27[degrees]C 4.2 11 Liquid 21[degrees]C 4.2 1 NHP 3 Drying 27[degrees]C, RH 80% 7.2 4 Drying 21[degrees]C, RH 40% 7.2 4 Liquid 27[degrees]C 6.5 8 Liquid 21[degrees]C 6.5 14 * [TCID.sub.50], 50%
tissue culture infectious dose; NHP, nonhuman primate; RH, relative humidity.
To evaluate detection limits of the RDTs, we propagated vaccine candidate A(H7N9) virus strain A/Anhui/1/2013 in MDCK cells and determined the mean 50%
tissue culture infectious dose ([TCID.sub.50]) per milliliter on the basis of at least 3 independent assays.
Plates were incubated at 37[degrees]C; CPE was monitored; and 50%
tissue culture infectious dose per milliliter endpoints were calculated.
MERS-CoV strain Jordan-N3/2012 was diluted in unpasteurized milk or nonsupplemented Dulbecco modified Eagle medium (DMEM, GIBCO, Grand Island, NY, USA) to a final median 50%
tissue culture infectious dose of 105.5/mL.
To achieve this goal, we orally inoculated 1 boar (10 years of age), 2 sows (4 and 5 years, respectively), and 1 boar piglet with a 3 x [10.sup.6] 50%
tissue culture infectious dose of the ASFV Caucasus isolate.
To characterize and further assess the pathogenicity profile of the virus, we inoculated 3 groups of 5 ferrets each intranasally with a [10.sup.5.5] 50%
tissue culture infectious dose of A/Cheetah/California/D0912239/2009 or one of the representative strains from humans, A/Tennessee/1-560/09 and A/Ukraine/N6/2009.
Because the median infectious dose of HeV in horses is not known, the virus challenge dose was selected to mimic previous experimental studies (3); virus (2 x 106 50%
tissue culture infectious dose) was administered oronasally to each horse.