Lethal H5N1 Virus Invades Medullary Respiratory‐related Neurons Responsible for Respiratory Chemoreflexes and Rhythm/Pattern Generation

Abstract

Highly pathogenic H5N1 avian influenza virus is neurotropic and its infection is lethal mainly due to the respiratory failure. Studies have shown that lethal H5N1 HK483 (A/HK/483/97) virus invades the central nervous system, blunts ventilatory responses to hypoxia and hypercapnia, and induces apneas, leading to death at 6–8 days postinfection (dpi) in mice (Gao et al, Journal of virology 1999, Zhuang et al., ATS 2013, EB 2015). However, the relevant mechanisms remain unclear. We asked if HK483 H5N1 virus: 1) invaded neurons and/or astrocytes in the medullary respiratory‐related nuclei; 2) infected those respiratory neurons expressing neurokinin 1 receptor (NK1R), tyrosine hydroxylase (TH, a marker of catecholaminergic neuron), or tryptophan hydroxylase (TPH, a marker of serotonergic neuron); and 3) altered the these neural expressions of NK1R, TH, or TPH. Female Balb/c mice were intranasally inoculated with vehicle (Ctrl) or HK483 virus (100 PFU) and euthanized at 6–7 dpi. After fixation, the medullary sections were immunohistochemically processed to detect the nucleoprotein of H5N1 influenza A (NP) in neurons and/or astrocytes marked by glial fibrillary acidic protein (GFAP) and its co‐labeling with immunoreactivity (IR) of NK1R, TH, or TPH. We found that: 1) HK483 virus mainly invaded the respiratory‐related nuclei including the nucleus of solitary tract (NTS), ambiguus nucleus (Amb), pre‐Botzinger complex (PBC), Botzinger complex (BC), retrotrapzoid nucleus (RTN) and their neighboring areas with the raphe less infected and the invasion in astrocytes was limited; 2) approximately 20–50% of NK1R neurons (the NTS, PBC, and RTN), 10–30% of TH‐IR neurons (C1/2, A1 areas, the NTS, and locus ceruleus), and < 10% TPH (the raphe) were co‐labeled with NP; and 3) significantly reduced the density of NK1R‐IR in the corresponding nuclei with less effect on reduction of the raphe neural population expressing TPH and no effect on TH‐IR. We conclude that HK483 is capable of invading the medullary respiratory nuclei, particularly those neurons expressing NK1R or TPH to depress NK1R or TPH expression, which likely contributes to depression of the ventilatory responses to hypoxia and hypercapnia and introduction of apneas/respiratory failure, leading to the death.Support or Funding InformationSupported by HL119683 and HL107462