2.9 Viral challenge assay in cattle
At 90 dpv, cattle were challenged by aerosol exposition as described previously (Romera et al., 2014; Quattrocchi et al., 2017) with a total of 5 mL in each nostril of 1x106.81TCID50/mL with BoHV-1 LA strain. At 0, 3, 5, 6, 7, 8, and 12 dpc, calves were clinically examined, and rectal temperature was recorded. Clinical score after viral challenge was established according to: Grade 0 = normal; grade 1 = slight rhinitis with serous mucus with or without mild serous conjunctivitis; grade 2 = moderate/heavy rhinitis with fibrinous serous mucus with or without moderate serous conjunctivitis; grade 3 = fibrinopurulent mucus with moderate or severe conjunctivitis; grade 4 = rhinotracheitis with or without conjunctivitis (Romera et al., 2014).
To measure viral excretion, nasal swabs were obtained at 0, 3, 5, 6, 7, 8, and 12 dpc by inserting tampons into each nostril and dipping them in MEM containing 5,000 IU/mL penicillin, 2,500 µg/mL streptomycin, and 10 µg/mL amphotericin B. For virus titration in nasal swabs, samples were serially diluted and inoculated onto MDBK cell monolayers, which were inspected for cytopathic effect (cpe). Virus titration was performed by the end point dilution method (Reed and Muench, 1938).