Contemporary highly pathogenic avian influenza (H5N1) viruses retain neurotropism in human cerebral organoids

Background Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 viruses are widely circulating in North America with unprecedented transmission into novel host species. A high incidence of neurologic disease is observed in carnivores infected with clade 2.3.4.4b HPAI H5N1 viruses and historical outbreaks of HPAI H5N1 in humans are also associated with neurologic complications, raising concerns about neurotropism and neurovirulence of clade 2.3.4.4b HPAI H5N1 viruses. Methods We analyzed virus replication kinetics, cellular tropism, and host responses to infection in human cerebral organoids (hCOs) inoculated with clade 2.3.4.4b HPAI H5N1 viruses compared to a historical clade 1 HPAI H5N1 virus and a seasonal influenza A virus. Results HPAI H5N1 viruses replicated to high titers in hCOs, but replication of the seasonal influenza A virus was not detected. Viral antigen and RNA were detected primarily in neuron- and astrocyte-like cells. Interferon responses to infection with HPAI H5N1 viruses were observed in a small population of bystander cells. Higher levels of cell death and proinflammatory cytokines and chemokines were observed in organoids inoculated with the historical HPAI H5N1 isolate. Conclusions Clade 2.3.4.4b HPAI H5N1 viruses exhibit similar neurotropism compared to a historical clade 1 HPAI H5N1 virus. Lower levels of cell death and inflammatory cytokine production induced by clade 2.3.4.4b viruses may indicate reduced neuropathogenic potential of these viruses in humans. Overall design: Human cerebral organoids (hCOs) were inoculated with historical HPAI H5N1 isolate VN2004, contemporary HPAI H5N1 isolates mMT2024 or bOH2024, seasonal influenza A virus isolate BR2007, or mock-inoculated. scRNA-seq aanlysis was conducted at 1 or 3 days post-inoculation. Libraries were prepared using PIPseq T2 3' Single Cell RNA kit v4.0 PLUS (Fluent Biosciences). Samples were indexed using the PIPseq UDI-96 kit (Fluent Biosciences), pooled, and sequenced on an Illumina NextSeq instrument.