A-Z Index × Submit A-Z Index × Submit A-Z Index Search Dropdown × Submit Facebook Twitter LinkedIn Syndicate Emerging Infectious Disease journal ISSN: 1080-6059 Disclaimer: Early release articles are not considered as final versions. Any changes will be reflected in the online version in the month the article is officially released.
In February 2025, we detected highly pathogenic avian influenza virus A(H5N1) clade 2.3.4.4b virus in a fetal bovine serum lot during routine adventitious agent testing. Sequencing confirmed H5N1 genotype B3.13 virus. We found low viral loads in additional samples from the same lot. Heating at 56°C for 30 minutes completely inactivated the virus.
Highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b genotype B3.13 virus infections were reported in dairy cows in Texas, USA, for the first time in March 2024 ( 1 – 3 ); rapid virus spread followed. As of December 18, 2025, more than 1,084 herds in 19 US states have been affected; most of the herds are in California ( 4 ). We report detection of HPAI H5N1 clade 2.3.4.4b genotype B3.13 virus in a fetal bovine serum (FBS) lot during routine testing.
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.
Figure 1 . Phylogenetic analysis of complete genomes formed by concatenation of all gene segments, confirming highly pathogenic avian influenza A (H5N1) virus genotype B3.13 isolated from fetal bovine serum (FBS), United States,...
We performed viral metagenomic sequencing of the supernatant obtained from Vero cells showing CPE after conducting sequence-independent, single-primer amplification of nucleic acids and library preparation using the ligation sequencing gDNA kit (SQK-LSK109) and Native Barcoding Kit 96 (Oxford Nanopore Technologies, https://www.www.nanoporetech.com ). We conducted sequencing on a MinION flow cell using the GridION platform (both Oxford Nanopore Technologies). We classified a total of 13,599 reads as avian influenza A virus subtype H5N1. Bioinformatic analysis identified the virus as H5N1…