Since early 2024, highly pathogenic avian influenza (HPAI) H5N1 viruses, which belong to hemagglutinin (HA) clade 2.3.4.4b genotype B3.13, have been causing outbreaks in dairy cattle in the United States. These outbreaks have resulted in transmission from cattle to other mammalian species, including humans, on affected dairy farms. Notably, cow H5N1 viruses isolated from humans have been shown to transmit among ferrets through respiratory droplets, posing a threat to public health.

Unpasteurized milk from affected cows has been shown to contain high levels of virus. Pathological examination of the mammary glands of naturally or experimentally infected dairy cows has revealed the presence of viral antigens in the alveolar epithelial cells, and higher viral RNA loads have been detected in the mammary glands compared with the lung. These findings suggest that the mammary gland is the primary target of HPAI H5N1 virus replication. Although Halwe et al. showed that an avian HPAI H5N1 virus can replicate in the mammary gland of lactating cows, studies comparing the replication of the bovine H5N1 viruses in the bovine mammary gland and teat ex vivo with that of viruses isolated from other species are lacking. Here, we compared the replicative capacity and cell tropism of cow H5N1, chicken H5N1, and human H1N1 2009 pandemic (H1N1pdm) viruses in ex vivo explant cultures of the mammary gland and teat from lactating cows. We also examined viral receptor expression in these organs.