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H5N1 genetic sequences - 2024+

Welcome SeniorLearner! Thank you for posting this substack essay. ​

I also agree with this snip:

"I don’t want to drone on. We have a relatively new zoonotic H5N1 2.3.4.4b D1.3 genotype that has killed over 19 million chickens on 84 farms in a small area in less than 2 1/2 months! It put one poultry worker in the hospital. No one has even bothered to publicly announce the new genotype to my knowledge! CDC has likely sequenced it from the hospitalized human patient yet has failed to deposit the sequence or even inform the public of its findings."

https://substack.com/home/post/p-159188160
​

edited to add links to pertinent threads...

Please see:

Ohio - Avian flu in poultry 2024-2025​

Indiana: 2024 & 2025 Avian Flu in poultry​

Ohio reports state's first human case of bird flu - February 12, 2025​
 
SeniorLearner said:
Substack Home - Hogvet51’s Livestock Emerging and Zoonotic Disease Forum​

H5N1 2.3.4.4b D1.3 in Ohio-Indiana Poultry Outbreak with Associated Undisclosed Human Sequence Data

Background phylogenetic work on USDA raw sequence submissions reveals a surprise unpublicized emerging avian genotype (D1.3) as a common source in the large multi-state poultry outbreak

more....
​
Click to expand...

Update by the CDC today addresses the above issue (among others):


CDC A(H5N1) Bird Flu Response Update March 19, 2025
​
 
4/1/25

@RajlabN
​Quick analysis of 2 #AvianFlu sequences sampled from #Bears (Collection date: 2025; Location: unknown) to
@gisaid
from #USA | D.1.1 genotype PB2 mutations: K389R, V478I, E627K, M676T Ref: A/Duck/Guangdong/E1/2012​

​
Screenshot 2025-04-01 at 6.53.12 AM.png
 
4/1/25
@RajlabN

NA mutations: T8I, V16I, V20I, M23V, Y44N, Q45H, P48T, I53V, V67I, F74L, L75I, V80I, T81D, S82P, T84A, N221S, V234I, V241I, K257R, D259E, E268D, P272S, G286S, D287E, I288V, N329S, S336G, M338V, E395A Ref: A/AmericanWigeon/SouthCarolina/22-000345-001/2021​

Click image for larger version Name: Screenshot 2025-04-01 at 6.58.33 AM.png Views: 1 Size: 777.4 KB ID: 1010877
 
Raj Rajnarayanan
@RajlabN
·
8h




#H5N1 #AvianInfluenza Quick analysis of the sequence uploaded to
@gisaid
from Khett Kampong Spoe, #Cambodia (11 yr, male patient) HA mutations: E2K, K3R, S12N, H44Q, T87I, D136N, G179D, R205K, S233P, V285A, E286Q, N325S, I351K, I390V, Q506R Ref: Duck/VietnamNCVD-1584/2012
Quote
Ngt0V722_normal.jpg

Raj Rajnarayanan
@RajlabN
·
22h
[HASHTAG="c2811"]H5N1[/HASHTAG] updates [HASHTAG="c1781"]Cambodia[/HASHTAG] reports 4th fatal human case this year! Will update sequence information as soon as its available x.com/CIDRAP/status/…


1


9


33

1.3K




Raj Rajnarayanan
@RajlabN
·
8h




NA mutations: A46T, L60V, V79I, I129V, I191L, N250D, R293Q, R312T, I376M, N396D Ref: A/Cambodia/NPH230032/2023 2/n


1


4


10

479




Raj Rajnarayanan
@RajlabN




HA mutation 205 is flagged in literature as antigenic drift / escape mutant and virulence
​
https://pubmed.ncbi.nlm.nih.gov/31635227/​
 
from my email: [TABLE="class: yiv2212227425gd_tbl_wrap, align: center, width: 0"]
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[TD="class: yiv2212227425abe-column-block-padding"]DATCPanimals@wisconsin.gov | (608) 224-4872 | datcp.wi.gov [/TD]
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[TD="class: yiv2212227425abe-column-block-padding"]Update on Highly Pathogenic Avian Influenza (HPAI)

Today, the U.S. Department of Agriculture (USDA) announced information on the genetic sequencing of the highly pathogenic avian influenza (HPAI) that was detected in Dodge County, Wisconsin last week. Access USDA's press release here.
On December 17, the National Veterinary Services Laboratories (NVSL) completed whole genome sequencing and confirmed that the virus is H5N1 clade 2.3.4.4b genotype D1.1. Analysis indicates that this detection is a new spillover event from wildlife into dairy cattle, separate from previous events.
Some key points from USDA's news release include:
  • Most detections in U.S. dairy herds have resulted from movements linked to the original spillover event that occurred in the Texas Panhandle in late 2023, involving the B3.13 strain.
  • In early 2025, through the National Milk Testing Strategy, USDA detected two spillover events in Arizona and Nevada dairy herds. Both were identified early, with limited spread to other farms. These events involved the D1.1 strain.
  • The Wisconsin herd, also detected through the National Milk Testing Strategy, represents a new, separate spillover event and involves the D1.1 strain. At this time, no additional dairy herds have been identified as infected in association with this event.
Wisconsin and the National Milk Testing Strategy (NMTS)
With help from industry partners, Wisconsin is sampling milk at the farm level for the mandatory USDA Animal and Plant Health Inspection Service (APHIS) NMTS. When Wisconsin began NMTS testing in May 2025, the goal was to test each farm approximately every month. Wisconsin achieved unaffected Gold Status under the NMTS in September 2025. In October, DATCP reduced its testing frequency to 50%.
Wisconsin will resume NMTS testing at a 100% frequency statewide. DATCP will continue to utilize milk samples that are already collected through existing, routine sampling to streamline the NMTS testing process. The DATCP HPAI in Dairy Cattle webpage will continue to be updated weekly as the testing continues.
Biosecurity and Monitoring
It is important to remain vigilant and utilize enhanced biosecurity measures.
H5N1 Protocol: What to Do if H5N1 is Confirmed on Your Dairy Farm
DATCP created a resource document in case of a detection of H5N1 on a dairy farm. The document is intended to serve as a general reference for industry, but individual needs and response actions may differ for H5N1 detections depending on the operation and specific circumstances. Access the H5N1 Protocol: What to Do if H5N1 is Confirmed on Your Dairy Farm document.
Human Health Precautions
Human cases of H5N1 have been reported following exposure to infected animals. Access the confirmed cases and exposure sources for human infections.
The Wisconsin Department of Health Services (DHS) advises those working with animals to take precautions and wear PPE per CDC guidance. According to the Centers for Disease Control and Prevention (CDC), the public health risk from HPAI detections remains low.
Additional Resources:
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​:datcp@service.govdelivery.com
 
Zoonotic Implications of the Co-Circulation of Clade 2.3.4.4b and 2.3.2.1a H5N1 Avian Influenza Viruses in Nepal in 2023

​ 2025 Nov 6;17(11):1481. doi: 10.3390/v17111481​

Abstract

Samples collected from two avian influenza outbreaks in Bagmati Province in central Nepal between January and March 2023 were positive for H5N1. Full genomes were generated for both viruses, which revealed that one of the viruses was very similar to clade 2.3.4.4b H5N1 identified in Bangladesh in 2021/2022. The second virus was a reassortant H5N1 virus consisting of four genes (HA, NA, NP, and M) originating from a clade 2.3.2.1a H5N1 and the remaining four genes (NS, PB1, PB2, and PA) originating from a 2.3.4.4b H5N1. Notably, this second virus had a high identity with 2.3.2.1a clade viruses identified in humans and cats in India in 2024–2025. These are the first full genome sequences of H5N1 avian influenza viruses from Nepal and given the recent human infections by 2.3.2.1a H5N1 viruses in the region, these data will be of interest to both public health and veterinary authorities.
​
https://pmc.ncbi.nlm.nih.gov/articles/PMC12656878/
 
A Global Review of Highly Pathogenic Avian Influenza (HPAI) and Control Strategies in Nepal

​Submission received: 22 January 2026 / Revised: 1 March 2026 / Accepted: 25 March 2026 /Published: 1 April 2026​

Simple Summary

Highly pathogenic avian influenza (HPAI) remains one of the most significant viral diseases affecting poultry production in Nepal. Since 2009, the country has experienced repeated outbreaks, mainly due to high poultry density, migratory wild birds, and informal cross-border trade. These outbreaks have caused major economic losses and continue to threaten animal and public health. This review summarizes the global outbreak patterns of avian influenza and evaluates current surveillance, diagnostic capacity, and control measures in Nepal. It also identifies key gaps and highlights the need for stronger biosecurity, improved preparedness, and better coordination through a One Health approach to reduce the impact of avian influenza in Nepal.

-snip-
2. Influenza A Viruses

​... Among all avian influenza subtypes, H5 and H7 subtypes are of particular concern due to their ability to mutate into highly pathogenic forms, causing severe losses in poultry production and posing significant zoonotic risks to humans [21]. H9N2 viruses have zoonotic potential and play a critical role as internal gene donors for other reassortant influenza viruses, including H5 and H7 subtypes. Thus, H9N2 represents a persistent endemic and evolutionary reservoir risk rather than an acute high-mortality threat [22].​

... In Nepal, HPAI H5 viruses have been identified from multiple genetic clades, reflecting repeated introductions over time. The principal clades detected include clade 2.2, clade 2.3.2, and clade 2.3.2.1, with subclades 2.3.2.1a and 2.3.2.1c representing the predominant lineages in earlier outbreaks. More recently, viruses belonging to clade 2.3.4.4, particularly subclade 2.3.4.4b, have been reported [23]. Overall, clade 2.3.2.1 has historically dominated HPAI circulation in Nepal, whereas clade 2.3.4.4b represents more recent incursions, occasionally detected alongside reassortant H5N1 viruses [24].​

continued: https://www.mdpi.com/2813-0227/6/2/11
​
 
J Virol . Isolation and characterization of a clade 2.3.4.4b genotype D1.1 H5N1 virus from dairy cattle in Wisconsin

J Virol


. 2026 Jul 10:e0076126.
doi: 10.1128/jvi.00761-26. Online ahead of print.
Isolation and characterization of a clade 2.3.4.4b genotype D1.1 H5N1 virus from dairy cattle in Wisconsin

Daria Mezhenskaia[SUP] 1 [/SUP], Lavanya Babujee[SUP] 1 [/SUP], Ailam Lim[SUP] 2 [/SUP], Lizheng Guan[SUP] 1 [/SUP], Dani Nguyen[SUP] 1 [/SUP], Chunyang Gu[SUP] 1 [/SUP], Gabriele Neumann[SUP] 1 [/SUP], Keith Poulsen[SUP] 2 [/SUP], Amie J Eisfeld[SUP] 1 [/SUP], Yoshihiro Kawaoka[SUP] 1 3 4 5 [/SUP]


Affiliations
Abstract

Highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses of clade 2.3.4.4b have recently been detected in U.S. dairy cattle following multiple spillover events from avian reservoirs. In December 2025, HPAI H5N1 virus was identified in a dairy herd in Wisconsin through the National Milk Testing Strategy. Here, we report the isolation of a clade 2.3.4.4b, genotype D1.1 H5N1 virus, A/dairy cow/Wisconsin/25G05743-001/2025 (WI5743-H5N1), from bulk milk associated with the affected herd, describe its phylogenetic relationships, and assess its pathogenicity in mice. Infectious virus was recovered following blind passage in embryonated chicken eggs. Phylogenetic analysis demonstrated that WI5743-H5N1 is distinct from previously reported D1.1 viruses detected in dairy cattle in Nevada and Arizona, supporting an independent introduction into cattle, and indicating a likely local avian source. Compared with closely related avian viruses, WI5743-H5N1 encoded the mammalian-adapting substitution PB2-E627K and additional amino acid differences in HA, PB1-F2, and NS1. In mice, WI5743-H5N1 replicated efficiently in respiratory tissues and was detectable in the brain but exhibited lower lethality relative to other recent clade 2.3.4.4b, genotype B3.13 viruses. Together, these findings highlight the genetic and phenotypic diversity of HPAI H5N1 viruses infecting dairy cattle and underscore the importance of continued surveillance and functional characterization of emerging strains.IMPORTANCEHighly pathogenic avian influenza A(H5N1) viruses have recently entered U.S. dairy cattle through multiple spillover events from avian reservoirs, creating new opportunities for viral adaptation in mammals. Here, we describe the isolation and characterization of a clade 2.3.4.4b, genotype D1.1 H5N1 virus from bulk milk collected during a spillover event in Wisconsin in December 2025. Phylogenetic analyses demonstrated that this virus represents an independent introduction into dairy cattle distinct from previously reported D1.1 viruses identified in Nevada and Arizona. Although the virus encoded the mammalian-adapting PB2-E627K substitution, it exhibited comparatively low lethality in mice, highlighting the complexity of mammalian adaptation and pathogenicity in H5N1 viruses. These findings expand current understanding of the genetic and phenotypic diversity of H5N1 viruses infecting dairy cattle and emphasize the importance of continued surveillance and functional characterization of emerging strains.

Keywords: H5N1; PB2-E627K; clade 2.3.4.4b; dairy cattle; genotype D1.1; influenza A virus; mammalian adaptation.

​
 
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