Primary bovine embryonic fibroblasts support seasonal influenza A virus infection and demonstrate variable fitness of HPAI H5N1

Abstract

The emergence of highly pathogenic avian influenza (HPAI) H5N1 (clade 2.3.4.4b, genotype B3.13) in dairy cattle presents substantial challenges to the agricultural sector and public health. Mechanistic studies of infection and transmission in cattle have proven difficult due to animal handling restrictions as well as limited availability of established cell culture models. Primary Bovine Embryonic Fibroblasts (BeEFs) were collected from a Montana cow and are investigated here as a model to study influenza A virus (IAV) infection dynamics. We compared sialylation profiles, infectious virus production, viral replication, and plaque morphology in both BeEFs and chicken DF-1 cells following infection with the bovine HPAI H5N1 and an earlier 2.3.4.4b genotype (B1.1) isolated in 2022. The data presented here show increased viral fitness of the bovine origin HPAI H5N1 strains across multiple species and bovine susceptibility to human seasonal IAV. This study highlights the ability of BeEFs to serve as a model for studying IAV infections in bovine hosts.

Figure 1:. Characterization of HPAI H5N1 viral growth in BeEFs and DF-1 cells.

(A) A schematic of the experimental design: Infection of BeEF and DF-1 cells with H5N1 isolated before (BEFL) and during (BOOH) the bovine HPAI outbreak. Cells were analyzed for presence of different sialic acid receptors, infectious virus production, and viral replication (B) Sialylation profiling displaying lectin-binding (white) of α−2,3 and α−2,6 specific lectins to BeEF and DF-1 cells. Representative images of each lectin are shown at the same magnification. Scale bar = 100 μm. Growth curves (MOI 0.1) of BOOH and BEFL on BeEFS (C) and DF-1s (D) show increased fitness of BOOH in both cell types. Data represent a minimum of two independent replicates analyzed by two-way ANOVA. Viral replication as measured by RT-qPCR for the viral M gene in total RNA isolated from BeEFs (E) and DF-1s (F). Data represent a minimum of two independent replicates analyzed by two-way ANOVA. (G) Representative images of plaques formed by BOOH and BEFL infection of BeEFs. (H) Comparison of plaque areas reveal significantly reduced plaque sizes for BEFL in both BeEFs and DF-1 compared to BOOH. A minimum of 50 plaques were analyzed for each virus and analyzed by one-way ANOVA. (I) Growth curve of Cal09 H1N1 and UVM-0478 H1N1 on BeEFs. Data represent a minimum of three independent replicates analyzed by two-way ANOVA. * p < 0.05, ** p < 0.01, ***p < 0.001 ****p < 0.0001.