Tuesday, March 14, 2023

PLoS Pathogens: Evolution of Highly Pathogenic H5N1 Influenza A Virus in the Central Nervous System of Ferrets



Although seasonal flu can occasionally cause neurological symptoms (see 2018's Neuroinfluenza: A Review Of Recently Published Studies) it is relatively rare, and often only causes mild, and transient symptoms. 

The exact mechanisms behind these neurological manifestations are largely unknown, as seasonal flu viruses are generally regarded as being non-neurotropic. 

That said, concerns have been raised over the long-term neurological impacts of severe (or repeated) influenza infections (see Nature Comms: Revisiting The Influenza-Parkinson's Link).

But avian H5N1 has a long history of causing severe neurological manifestations both in humans, and in other avian and mammalian hosts.

In 2009, a PNAS study (link below) found that the H5N1 virus was highly neurotropic in lab mice, and in the words of the authors `could initiate CNS disorders of protein aggregation including Parkinson's and Alzheimer's diseases’.

Highly pathogenic H5N1 influenza virus can enter the central nervous system and induce neuroinflammation and neurodegeneration
In 2015, after the death of the first imported H5N1 case in Canada, we saw a study (see CJ ID & MM: Case Study Of A Neurotropic H5N1 Infection - Canada), where the authors wrote: `These reports suggest the H5N1 virus is becoming more neurologically virulent and adapting to mammals'.
In a Scientific Reports study on the genetics of the H5N1 clade virus - Highly Pathogenic Avian Influenza A(H5N1) Virus Struck Migratory Birds in China in 2015 – the authors warned of its neurotropic effects, and that it could pose a ` . . . significant threat to humans if these viruses develop the ability to bind human-type receptors more effectively.'

Over the past 18 months we've seen a steady stream of reports of spillover of avian H5N1 into mammalian hosts, with the vast majority exhibiting severe (often fatal) neurological manifestations. A few of many recent blogs include:

Preprint: Pathology Of HPAI H5N1 clade in Wild Terrestrial Mammals in the United States in 2022

Emerging Microbes & Inf.: Neurotropic HPAI H5N1 Viruses with Mammalian Adaptive Mutations in Free-living Mesocarnivores in Canada

Pathogens: Zoonotic Mutation of Highly Pathogenic Avian Influenza H5N1 Virus Identified in the Brain of Multiple Wild Carnivore Species

Similarly, last September in Clinical Features of the First Critical Case of Acute Encephalitis Caused by Avian Influenza A (H5N6) Virus, we looked at the case of a 6 year-old girl in China who presented with severe neurological symptoms following H5N6 infection. 

All of which brings us to a new study, published in PLoS Pathogens, that finds that when ferrets are experimentally inoculated (via the nasal cavity) with A/Indonesia/5/2005, the virus readily travels to the brain via the olfactory nerve, where it begins to replicate. 

As it replicates, the virus acquired 3 specific mutations (PB1 E177G and A652T and NP I119M) that individually, or collectivelyappear to limit its spread outside of the CNS, while at the same time enhancing polymerase activity locally. 

This may help explain why so many mammals have been found recently with signs of encephalitis or meningoencephalitis, but with little or no signs of pulmonary involvement. 

I've only posted the abstract, and some excerpts, from the study.  Those who want more will want to follow the link to read it in its entirety.  I'll have a brief postscript after the break. 
Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets

Jurre Y. Siegers , Lucas Ferreri , Dirk Eggink, Edwin J. B. Veldhuis Kroeze, Aartjan J. W. te Velthuis, Marco van de Bildt,Lonneke Leijten, Peter van Run, Dennis de Meulder, Theo Bestebroer,Mathilde Richard, Thijs Kuiken, Anice C. Lowen,Sander Herfst, Debby van Riel

Published: March 10, 2023


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.

Author summary

The 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.


In the present study we aimed to understand the basic mechanisms by which highly pathogenic avian influenza H5N1 viruses evolve once they infect the mammalian CNS. Using a ferret model, we found that influenza A/Indonesia/5/2005 (H5N1) virus was not subject to bottlenecks when entering the CNS via olfactory bulb, and that once inside the CNS, positive selection of variants can occur. Although the CNS-associated mutations examined did not appear to be adaptive, the viral dynamics observed suggest that an H5N1 virus can increase in fitness once it infects the CNS.


In summary, here we show that an HPAI H5N1 virus can enter the CNS via the olfactory nerve without a genetic bottleneck, consistent with the high frequency of CNS involvement in mammals infected with these viruses. Efficient dispersal to the CNS via the olfactory nerve was associated with abundant infection and damage in the olfactory mucosa, which may explain reports of CNS infection by H5N1 viruses in the absence of overt respiratory clinical signs [31,32]. Importantly, we found that viral dynamics within the CNS are conducive to positive selection, giving the potential for improvement of viral replicative capacity, which may translate into more severe disease.

          (Continue . . . )

Humans are not ferrets, and there are too many moving genetic parts to know whether a current day human-adapted H5N1 virus would produce the same neurological impacts in people as this parental H5N1 virus does in ferrets.  

Nevertheless, the findings in mammals around the world are concerning, and we ignore them at our own risk. 

Which is why - after examining my dwindling stockpile of N95's - I'm ordering an extra box today, while the availability is high and the prices are low.