Monday, January 30, 2023

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

Flu Virus binding to Receptor Cells – Credit CDC


Unlike in mammals, where influenza viruses generally produce a respiratory infection, influenza in birds is predominantly a gastrointestinal malady. The virus attaches to - and replicates in – the avian gut, and is spread mostly via infected droppings.
Avian flu viruses are well adapted to attack the kind of cells commonly found within the avian intestinal tract; α2,3-linked sialic acid avian receptor cells and to replicate efficiently at the higher temperatures found in the avian gut. 

In order to infect and transmit among mammals, avian viruses need to be able to attach to the α2,6-linked receptor cells commonly found in their respiratory tract, and to replicate at the lower temperatures found there. 

Many avian viruses have an affinity for both avian and mammalian receptor cells, which is why they are occasionally able to jump species. Once in a mammalian host, however, further `host adaptations' are needed for the virus to flourish.  

One of the mutations that we know to look for is PB2-E627K; the swapping out of Glutamic acid (E) for Lysine (K) at position 627 in the PB2 protein, which allows the virus to replicate at a lower temperature. 

Additional adaptations are needed to make an avian virus a genuine pandemic threat (some we know about, while others we may not), but PB2-E627K is believed to be an important stepping stone. 

Last week, in ASM J.: HPAI H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations, we looked at a report from the Netherlands on 3 red foxes with severe neurological manifestations, who were found to be infected with HPAI H5N1. 

The report stated virus was ` . . . mainly present in the brain, with limited or no detection in the respiratory tract or other organs' and they reported finding a mixture of the avian (PB2-627E) and the mammalian (PB2-627K) variants in each host. 

Today many of the same authors are back with another report, published this time in the journal Pathogens, which describes additional findings in a large array of small mammals (fox, polecat, otter and badger) in the Netherlands. 

Once again, these 11 infected animals displayed severe neurological symptoms, and testing showed the virus was primarily detected in their brain tissue.  As before, the PB2-E627K mutation was identified in most of the samples.

Since these all appear to be unrelated events, the finding of the same mutation across a wide selection of non-avian hosts suggests the virus quickly adapts to mammals. 

That, along with the frequently reported unusual neurological involvement (see also here, here, and here) - and the recently reported mink-to-mink transmission of the virus in Spain - has helped elevate concerns over the potential public health threat from H5N1. 

I've only posted the Abstract and some excerpts from a much longer report. Follow the link to read it in its entirety. 

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

Sandra Vreman 1,*,†,Marja Kik 2,3,†,Evelien Germeraad 1,Rene Heutink 1,Frank Harders 1,Marcel Spierenburg 4,Marc Engelsma 1,Jolianne Rijks 2,Judith van den Brand 2,3,‡ and Nancy Beerens 1,*,‡

The authors contributed equally to the manuscript.
Pathogens 2023, 12(2), 168;
Received: 23 December 2022 / Revised: 11 January 2023 / Accepted: 13 January 2023 / Published: 20 January 2023


Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021–2022 outbreak in the Netherlands included red fox (Vulpes vulpes), polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals were submitted for testing because they showed neurological signs. 

In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal. 

Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1 virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness.


Genetic analysis of the carnivore viruses in this study identified the zoonotic mutation PB2-E627K in 8 out of 11 cases. The fact that this mutation was not detected in any of the wild bird sequences during the 2021–2022 epizootic in the Netherlands suggests this mutation quickly arises upon infection of mammals. In one fox (nr. 2), the avian E627-variant was still present in the viral genome as a minority population, further supporting the emergence of the mutation within this mammal. A previous analysis of two fox viruses also supported that the mutation arose after infection of the mammals, as minority variants were detected at position PB2-627 [8].
Mutation E627K is likely an adaptation to the lower body temperature in the mammalian upper respiratory tract compared to that of avian species. We previously showed that this mutation increases the replication of the HPAI H5N1 virus in mammalian cell lines at lower temperatures [8]. The fact that the mammalian adaptation marker E627K was found in many of the carnivore viruses suggests that this virus can rapidly adapt to replication in mammals.
However, previous research has indicated that a combination of viral adaptations is required for efficient air-borne transmission of HPAI viruses between mammals [13]. Although the chance that such mutations will arise in an infected animal is very small, the impact of the emergence of a zoonotic virus with potentially pandemic characteristics may be large.

In this observational wildlife study, carnivore carcasses were sampled for different surveillance purposes and therefore carcasses showed variation in degree of autolysis and also different sampling strategies were applied. In the future, a more universal wildlife sampling approach within different institutes will enable structural analysis with more reliable results. Another important caveat of sampling found dead or euthanized wild animals compared to an experimental infection is the unknown time point of infection, which hampers the evaluation of the HPAI H5N1 pathogenesis. Finally, many of the wild carnivores were infested with lung parasites [33]. Especially in red foxes, the presence of A. vasorum larvae in the lungs was a common finding [34] and may have overshadowed changes due to HPAI lung pathology. Furthermore, the lung pathology and the clinical relevance of these parasites is variable in wildlife and it is unclear if these co-infections influenced susceptibility to HPAI. Besides these limitations, our results can be used to improve HPAI wildlife surveillance.

          (Continue . . . )

While H5N1 has been knocking at our door for the past 20 years - and was the reason I started this blog 17 years ago - it hasn't managed to find the right combination of mutations to turn it into a viable pandemic threat, at least, not yet.

And perhaps, as some have suggested, there is some sort `species barrier' that protect us. 

But H5N1 has never been as well entrenched around the world (in birds) as it is today, it is reassorting and churning out new genotypes at a record pace, and it appears to be expanding its host range (including to humans), and has shown the ability to produce severe (even fatal) illness in mammals. 

All reasons why we can't afford to ignore the insistent tapping at our door.