Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets

Author summaryThe central nervous system (CNS) is one of the most common extra-respiratory tract sites of infection for influenza A viruses. In ferrets-an animal model used to study the pathogenesis of influenza-highly pathogenic avian influenza H5N1 virus can enter the CNS via the olfactory nerve, resulting in the development of a severe meningo-encephalitis. In the present work, we evaluated the evolutionary dynamics of the virus populations entering and spreading throughout the CNS. We show that once inside the CNS, H5N1 viruses can acquire mutations that increase the polymerase activity in vitro. In vivo, the virus bearing these mutations retained its capacity to infect the CNS but showed reduced spread to other anatomical sites. Analysis of virus populations revealed that infection from the nasal turbinate to the olfactory bulb did not present a genetic bottleneck, suggesting a diffusive passage of viruses from the nasal cavity to the CNS. Inside the CNS, specifically in the brainstem, we found signs of positive selection. These findings support the idea that H5N1 viruses can invade the CNS efficiently via the olfactory nerve, and have the potential to adapt to the CNS. Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.