Isolation of Infectious Highly Pathogenic Avian Influenza A(H5N1) Virus from Fetal Bovine Serum, United States, 2025

In February 2025, a lot of FBS was received by the Virology Laboratory at the Cornell Animal Health Diagnostic Center (AHDC; Ithaca, NY, USA) for routine extraneous agents testing, per requirements of Title 9 of the Code of Federal Regulations (9CFR) for animal origin biologic products (§113.53, §113.46, §113.47). The tested FBS consisted of a pool of samples collected in 5 states (Pennsylvania, Kansas, Nebraska, South Carolina, and California). We tested the sample as prescribed in 9CFR, by seeding Vero cells (ATCC CCL-81) and primary fetal bovine kidney (FBK) cells, developed by the AHDC Virology Laboratory, in T75 tissue culture flasks in Eagle minimum essential medium supplemented with 2% penicillin/streptomycin and 15% (final concentration) of the test FBS sample. We maintained cells for 7 days and monitored for cytopathology. FBK cells did not exhibit viral cytopathic effect (CPE) but were positive for noncytopathic bovine viral diarrhea virus through virus-specific immunofluorescence assay (IFA) staining on day 7. We observed CPE in Vero cells on day 7 post-inoculation. Subsequent IFA testing of the Vero cells was negative for the bovine viruses listed in 9CFR (i.e., bovine viral diarrhea virus, bovine parvovirus, bluetongue virus, reovirus, bovine adenovirus, bovine respiratory syncytial virus, and rabies virus). We tested FBK and Vero cells cultured in the presence of 15% control gamma-irradiated FBS in accordance with 9CFR regulations; the cells remained negative for CPE and IFA virus.