Influenza A virus H5N1 genotypes B3.13 and D1.1 show temperature-dependent restriction of replication in primary human respiratory epithelial cell cultures derived from the upper and lower respiratory tract

Abstract

H5N1 clade 2.3.4.4b avian influenza A viruses pose a significant threat to wild animal populations, domesticated animals, and potentially, the human population. For H5N1s to infect and transmit among mammalian species, mutations for improved utilization of mammalian receptors and enhanced replication at the lower temperatures of the upper respiratory tract need to be acquired. A human H1N1pdm09-like virus was compared to H5N1 genotypes B3.13 and D1.1 for replication at 33°C, 37°C, and 39°C – temperatures consistent with the upper and lower respiratory tract in humans, and dairy cow udder tissue. All H5N1 viruses had increased plaque sizes on MDCK cells at 37°C and 39°C compared to H1N1pdm09. In primary, differentiated human nasal and bronchial epithelial cultures, all H5N1 viruses show restricted infectious virus production compared to H1N1 at 33°C. While H5N1 D1.1 also showed restricted replication at 37°C and 39°C, the H5N1 B3.13 replicated to nearly equivalent titers as H1N1pdm09. All H5N1 viruses demonstrated similar cell tropism in cells from the upper and lower respiratory tract, infecting more ciliated than non-ciliated cells relative to H1N1pdm09. H1N1, H5N1 B3.13 D1.1 infection induced similar innate immune factors, with nasal epithelial cells producing higher levels compared to bronchial epithelial cells. These data suggest that genotype B3.13 and D1.1 H5N1 viruses show different temperature dependent replication patterns compared to H1N1pdm09. IMPORTANCE The ongoing H5N1 outbreaks have increased the number of mammalian infections with H5N1, thus increasing the chances of the virus adapting to mammals. Two H5N1 genotypes that have recently infected dairy cows, commercial poultry flocks, and a limited number of humans in the United States showed decreased replication at 33°C but not 37°C or 39°C compared to human H1N1 virus. H5N1 virus replication was detected at all temperatures, suggesting temperature and not host species may be the primary barrier preventing H5N1 infection in humans.