Neurotropic influenza A virus infection in the mouse brain : targeting, persistence and functional effects

Abstract: Viral infections of the central nervous system have been associated with sensory, motor and behavioral disturbances both in humans and in animals. An example of this is influenza A virus infection that causes acute respiratory diseases, which occasionally are followed by complications from the central nervous system. Furthermore, influenza A virus infections during the second trimester of pregnancy have in some, but not all, studies been associated with a higher risk for the offspring to develop schizophrenia. The neuroadapted influenza A/WSN/33 strain has been propagated in the mouse brain. This neurotropic strain can target neurons of the substantia nigra and hippocampus following systemic or intracerebral injection and neurons in the anterior olfactory nuclei, and the habenular, thalamic midline and monoamineric brainstem nuclei after injection into olfactory bulbs. The aim of this Ph.D. thesis project was to study how the influenza WSN/33 strain can spread to the mouse brain along olfactory pathways and be restricted by immunological determinants in these areas. Furthermore, the question whether the influenza virus will be eliminated or persist in the brain after both adult and maternal infections is addressed, as well as whether such infections may affect gene expression in the brain and cause behavioral disturbances of the host animal. The present results show that the neurotropic influenza WSN/33 strain can infect neurons in the olfactory epithelia and spread into the olfactory bulb. Viral spread was detected, immunohistochemically, along olfactory projections in the brain to the anterior olfactory nuclei, taenia recta and piriform cortex as well as dorsal endopiriform cortex. Groups of infected neurons also appeared in the hypothalamus and in the upper brainstem at sites corresponding to the dorso-medial raphe nuclei. This indicates that the WSN/33 strain can spread into specific areas of the brain along olfactory pathways after intranasal inoculation. A viral spread along this route is, however, restricted by immunological determinants. IFN-alpha/beta prevented the virus spread from olfactory epithelia, but this restriction appears not to be sufficient since neuroinvasion was detected in both iNOS and CD8+ T cell deficient mice. Viral neuroinvasion into the olfactory bulbs was also prevented by IFN-gamma, which may in part exert its effects through induction of iNOS and the release of NO from macrophages/microglial cells. In the brains of immunodeficient adult mice we detected persistent viral RNA after intranasal installation of the WSN/33 strain. Following injections into the olfactory bulbs of TAP1 -/- mice, viral RNA was present still 17 months later in sections of the brain at the upper brainstem level. Persisting influenza A virus RNA was also detected in brains from offspring to pregnant mice exposed to intranasal infections. Certain genes were found to be differentially expressed in the brains of 90-day-old mice exposed to maternal infection as compared to control animals. Interestingly, these genes were expressed at similar levels in the two groups at the earlier time-points studied. Finally, we observed late-onset behavioral alterations in adult wild-type mice after olfactory bulb inoculation with WSN/33 strain. These mice exhibited changes in measures of anxiety and cognition. Genes encoding synaptic regulatory proteins were differentially expressed in the hypothalamus, the amygdala and the cerebellum. This demonstrates that a transient non-lethal influenza A virus infection in the brain can cause persistent changes in cognitive and emotional behavior of mice associated with differential expression of genes encoding proteins involved in the regulation of synaptic functions in the amygdalahypothalamic areas.