Michael Coston
Editor, Senior Moderator
CDC Preprint: A Newly Emergent N1 Neuraminidase Associated with Clade 2.3.4.4b HPAI A(H5) Viruses in North America
#19,082
In the fall of 2024 - barely six months after the discovery of the `Bovine' B3.13 genotype of H5N1 circulating in U.S. dairy cattle - another genotype of H5N1 appeared in Canada and the Pacific Northwest - dubbed D1.1 - which very quickly became dominant in wild birds and rapidly swept eastward across the United States and Canada.
Although both B3.13 and D1.1 have spilled over into dozens of (primarily) agriculturally exposed humans, the bovine strain has produced mostly mild symptoms, while a few D1.1 patients saw severe (and even fatal) illness (see here, here, and here).
The exact cause of B3.13's mild presentation remains unclear, but many researchers have posited that - since its N1 Neuraminidase gene is similar to that found in seasonal H1N1 - many people may have some preexisting (albeit, limited) immunity to that genotype.
A few examples include:
Preprint: Cross-Reactive Human Antibody Responses to H5N1 Influenza Virus Neuraminidase are Shaped by Immune History
Preprint: Neuraminidase Imprinting and the Age-related Risk of Zoonotic Influenza
Two EID Journal Articles On Prior Immunity From A(H1N1)pdm09 Infection Against H5N1 (in Ferrets)
The D1.1 genotype, however, carries a different (North American lineage) N1 Neuraminidase, which may account for its ability to cause more severe illness.
And it its opening months, it displayed brief, but worrisome, signs of antiviral resistance.
Our first inkling of all of this came just over a year ago in Emerg. Microbes & Inf: Oseltamivir Resistant H5N1 (Genotype D1.1) found On 8 Canadian Poultry Farms, where Canada's PHAC and CFIA wrote:
Abstract
We report the detection of a clade 2.3.4.4b A(H5N1) reassortant virus with a neuraminidase surface protein derived from a North American lineage low-pathogenic avian influenza virus. This virus caused a widespread and ongoing outbreak across 45 poultry farms in British Columbia, Canada.
Isolates from 8 farms reveal a mutation in the neuraminidase protein (H275Y) that is exceptionally rare among clade 2.3.4.4b viruses (present in 0.045% of publicly available clade 2.3.4.4b isolates). NA-H275Y is a well-known marker of resistance to the neuraminidase inhibitor oseltamivir. We demonstrate that this substitution maintains its resistance phenotype on the genetic background of H5N1 clade 2.3.4.4b viruses.
Since then we've seen multiple spillovers of D1.1 into dairy cattle (Nevada, Arizona & Wisconsin), and some studies (see J.I.D.: Avian influenza virus A(H5N1) genotype D1.1 is better adapted to human nasal and airway organoids than genotype B3.13) have suggested D1.1 may be better suited to human biology than the `bovine' strain.
At this point it's worth noting that there are scores of other HPAI H5 genotypes in circulation - both in North America and around the globe - many of which have yet to be fully (or even partially) characterized.
But, of the H5 genotypes currently on our radar, D1.1 ranks pretty high on our watch list. All of which brings us to a preprint from researchers at the CDC, which looks at what we currently know - and don't know - about this emergent genotype.
I've reproduced the abstract, and an excerpt from the discussion. Follow the link to read the preprint in its entirety. I'll have a postscript after the break.
A newly emergent N1 neuraminidase associated with clade 2.3.4.4b highly pathogenic avian influenza A(H5) viruses in North America
Matthew J Wersebe, Nicole M Paterson, Norman Hassell, Xiao-yu Zheng, Benjamin J Rambo-Martin, Julia C Frederick, Kristine K Lacek, Amanda H Sullivan, Marie Kirby, Rebecca Kondor, Yunho Jang, Sabrina Schatzman, Han Di, C. Todd Davis
doi: https://doi.org/10.64898/2026.03.09.26347929
This article is a preprint and has not been certified by peer review
Preview PDF
Abstract
We investigated the evolutionary history of the newly emergent neuraminidase (am4N1) associated with the D1.1 and D1.2 genotypes of highly pathogenic avian influenza A(H5N1) viruses in North America.
Phylogenetic inference places am4N1 in a sister clade to Eurasian avian, swine, and human A(H1N1)pdm09 viruses and distinct from 1918, pre-2009 human seasonal, and classical swine A(H1N1) lineages.
Am4N1 descends from diverse avian N1 genes endemic to the Americas. Phylodynamic analysis indicates a monophyletic am4N1 lineage with numerous introductions of viruses carrying the am4N1 gene likely originating from western Canada into the United States during emergence of the D1.1 and D1.2 genotypes.
The lineage has diversified and accumulated deletions in the stalk domain. Despite amino acid divergence, structural modeling shows conserved neuraminidase architecture in the globular head. Given its distinct ancestry and amino acid sequence, further studies are needed to assess cross-reactivity of antibodies from prior human A(H1N1)pdm09 infections.
(Continue . . . .)
We are now 18 months since the first detection of D1.1, yet there is still much we don't know about its impact, prevalence, or pandemic potential. Surveillance is spotty at best, and the public release of WGS (Whole Gene Sequencing) and antigenic characterization data continues to be limited.
Remarkably, the exact number of human infections with the D1.1 genotype is unknown, since only a subset (of the roughly 6 dozen) North American human cases have been fully characterized.
A year ago, in Nature: Lengthy Delays in H5N1 Genome Submissions to GISAID. we saw the average delay in submitting sequences to GISAID was 7 months (228 days), with some countries taking nearly 2 years.
And genetic sequences - when they are submitted to GISAID - are often devoid of crucial metadata (i.e. collection date, exact location, host-specific information, etc.), limiting their value to the scientific community.
While I'm sure there are legitimate logistical challenges involved, it is hard to believe this is the best we can do.
https://afludiary.blogspot.com/2026/03/cdc-preprint-newly-emergent-n1.html
#19,082
In the fall of 2024 - barely six months after the discovery of the `Bovine' B3.13 genotype of H5N1 circulating in U.S. dairy cattle - another genotype of H5N1 appeared in Canada and the Pacific Northwest - dubbed D1.1 - which very quickly became dominant in wild birds and rapidly swept eastward across the United States and Canada.
Although both B3.13 and D1.1 have spilled over into dozens of (primarily) agriculturally exposed humans, the bovine strain has produced mostly mild symptoms, while a few D1.1 patients saw severe (and even fatal) illness (see here, here, and here).
The exact cause of B3.13's mild presentation remains unclear, but many researchers have posited that - since its N1 Neuraminidase gene is similar to that found in seasonal H1N1 - many people may have some preexisting (albeit, limited) immunity to that genotype.
A few examples include:
Preprint: Cross-Reactive Human Antibody Responses to H5N1 Influenza Virus Neuraminidase are Shaped by Immune History
Preprint: Neuraminidase Imprinting and the Age-related Risk of Zoonotic Influenza
Two EID Journal Articles On Prior Immunity From A(H1N1)pdm09 Infection Against H5N1 (in Ferrets)
The D1.1 genotype, however, carries a different (North American lineage) N1 Neuraminidase, which may account for its ability to cause more severe illness.
And it its opening months, it displayed brief, but worrisome, signs of antiviral resistance.
Our first inkling of all of this came just over a year ago in Emerg. Microbes & Inf: Oseltamivir Resistant H5N1 (Genotype D1.1) found On 8 Canadian Poultry Farms, where Canada's PHAC and CFIA wrote:
Abstract
We report the detection of a clade 2.3.4.4b A(H5N1) reassortant virus with a neuraminidase surface protein derived from a North American lineage low-pathogenic avian influenza virus. This virus caused a widespread and ongoing outbreak across 45 poultry farms in British Columbia, Canada.
Isolates from 8 farms reveal a mutation in the neuraminidase protein (H275Y) that is exceptionally rare among clade 2.3.4.4b viruses (present in 0.045% of publicly available clade 2.3.4.4b isolates). NA-H275Y is a well-known marker of resistance to the neuraminidase inhibitor oseltamivir. We demonstrate that this substitution maintains its resistance phenotype on the genetic background of H5N1 clade 2.3.4.4b viruses.
Since then we've seen multiple spillovers of D1.1 into dairy cattle (Nevada, Arizona & Wisconsin), and some studies (see J.I.D.: Avian influenza virus A(H5N1) genotype D1.1 is better adapted to human nasal and airway organoids than genotype B3.13) have suggested D1.1 may be better suited to human biology than the `bovine' strain.
At this point it's worth noting that there are scores of other HPAI H5 genotypes in circulation - both in North America and around the globe - many of which have yet to be fully (or even partially) characterized.
But, of the H5 genotypes currently on our radar, D1.1 ranks pretty high on our watch list. All of which brings us to a preprint from researchers at the CDC, which looks at what we currently know - and don't know - about this emergent genotype.
I've reproduced the abstract, and an excerpt from the discussion. Follow the link to read the preprint in its entirety. I'll have a postscript after the break.
A newly emergent N1 neuraminidase associated with clade 2.3.4.4b highly pathogenic avian influenza A(H5) viruses in North America
Matthew J Wersebe, Nicole M Paterson, Norman Hassell, Xiao-yu Zheng, Benjamin J Rambo-Martin, Julia C Frederick, Kristine K Lacek, Amanda H Sullivan, Marie Kirby, Rebecca Kondor, Yunho Jang, Sabrina Schatzman, Han Di, C. Todd Davis
doi: https://doi.org/10.64898/2026.03.09.26347929
This article is a preprint and has not been certified by peer review
Preview PDF
Abstract
We investigated the evolutionary history of the newly emergent neuraminidase (am4N1) associated with the D1.1 and D1.2 genotypes of highly pathogenic avian influenza A(H5N1) viruses in North America.
Phylogenetic inference places am4N1 in a sister clade to Eurasian avian, swine, and human A(H1N1)pdm09 viruses and distinct from 1918, pre-2009 human seasonal, and classical swine A(H1N1) lineages.
Am4N1 descends from diverse avian N1 genes endemic to the Americas. Phylodynamic analysis indicates a monophyletic am4N1 lineage with numerous introductions of viruses carrying the am4N1 gene likely originating from western Canada into the United States during emergence of the D1.1 and D1.2 genotypes.
The lineage has diversified and accumulated deletions in the stalk domain. Despite amino acid divergence, structural modeling shows conserved neuraminidase architecture in the globular head. Given its distinct ancestry and amino acid sequence, further studies are needed to assess cross-reactivity of antibodies from prior human A(H1N1)pdm09 infections.
(SNIP)
Pandemic preparedness countermeasures such as vaccines typically rely on comparing host antibody recognition via Hemagglutinin inhibition assays (HAI) as the HA mediates viral attachment and host cell entry and is the primary surface antigen. However, the NA plays a key role in influenza viral replication as well - allowing the release of progeny virions from infected cells [27, 36].
Neuraminidase activity is targeted by numerous pharmaceutical countermeasures including the widely prescribed oseltamivir which inhibits its enzymatic activity [37]. Currently, Neuraminidase inhibiting pharmaceuticals are stockpiled for the event of an influenza pandemic.
Neuraminidase activity is targeted by numerous pharmaceutical countermeasures including the widely prescribed oseltamivir which inhibits its enzymatic activity [37]. Currently, Neuraminidase inhibiting pharmaceuticals are stockpiled for the event of an influenza pandemic.
Signore et al. [8] showed that am4N1 NAs isolated from Canadian chickens harbored an amino acid substitution at NA:H275Y, a known marker for resistance to oseltamivir and showed that the isolate with NA:H275Y had decreased susceptibility to oseltamivir.
This clade has not shown onward transmission in the samples analyzed here but continued surveillance for NA:H275Y is needed. Most am4N1 NAs do not carry this amino acid substitution and human isolates tested to date are inhibited well by NAI pharmaceuticals [38].
Another critical aspect of pandemic preparedness is fully understanding the genetic makeup and evolutionary history of pathogen threats. Here we provide a description of the phylogenetics and evolution of am4N1 NAs which have contributed to the ongoing epizootic in wild birds, infected numerous mammals, and caused two human fatalities to date.
In addition, we use bioinformatic analysis to predict the structure of diverse NAs and determine how sequence identity changes may have functional implications for am4N1 NAs. Am4N1-like NAs are highly divergent from those previously associated with HPAI A(H5N1) viruses from clade 2.3.4.4b and are phylogenetically distant from NA genes that humans may have immunity to via vaccination and prior infection.
Detailed studies describing the human antibody recognition of viruses with the am4N1 protein following seasonal influenza vaccination or infection are needed to fully understand the public health risk posed by D1.1 viruses and its derivate genotypes sharing this NA lineage.
This clade has not shown onward transmission in the samples analyzed here but continued surveillance for NA:H275Y is needed. Most am4N1 NAs do not carry this amino acid substitution and human isolates tested to date are inhibited well by NAI pharmaceuticals [38].
Another critical aspect of pandemic preparedness is fully understanding the genetic makeup and evolutionary history of pathogen threats. Here we provide a description of the phylogenetics and evolution of am4N1 NAs which have contributed to the ongoing epizootic in wild birds, infected numerous mammals, and caused two human fatalities to date.
In addition, we use bioinformatic analysis to predict the structure of diverse NAs and determine how sequence identity changes may have functional implications for am4N1 NAs. Am4N1-like NAs are highly divergent from those previously associated with HPAI A(H5N1) viruses from clade 2.3.4.4b and are phylogenetically distant from NA genes that humans may have immunity to via vaccination and prior infection.
Detailed studies describing the human antibody recognition of viruses with the am4N1 protein following seasonal influenza vaccination or infection are needed to fully understand the public health risk posed by D1.1 viruses and its derivate genotypes sharing this NA lineage.
(Continue . . . .)
We are now 18 months since the first detection of D1.1, yet there is still much we don't know about its impact, prevalence, or pandemic potential. Surveillance is spotty at best, and the public release of WGS (Whole Gene Sequencing) and antigenic characterization data continues to be limited.
Remarkably, the exact number of human infections with the D1.1 genotype is unknown, since only a subset (of the roughly 6 dozen) North American human cases have been fully characterized.
A year ago, in Nature: Lengthy Delays in H5N1 Genome Submissions to GISAID. we saw the average delay in submitting sequences to GISAID was 7 months (228 days), with some countries taking nearly 2 years.
And genetic sequences - when they are submitted to GISAID - are often devoid of crucial metadata (i.e. collection date, exact location, host-specific information, etc.), limiting their value to the scientific community.
While I'm sure there are legitimate logistical challenges involved, it is hard to believe this is the best we can do.
https://afludiary.blogspot.com/2026/03/cdc-preprint-newly-emergent-n1.html