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H5N1 genetic sequences - 2024+

sharon sanders

Editor-in-Chief & President
This is a developing thread......


Raj Rajnarayanan
@RajlabN
·
1h
#H5N1 Clade 2.3.4.4b updates | #HPAI PB2 Mutational profile of the 239 WGS sequences uploaded by
@USDA_APHIS

@USDA
[No adaptation signature] Many thanks to
@MichaelWorobey
et al for the consensus sequences from the 239 WGS runs https://public.tableau.com/app/profile/raj.rajnarayanan/viz/H5N1Clade2_3_4_4bPB2Mutations/Dashboard1… 1/n
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Raj Rajnarayanan
@RajlabN
·
1h
Host abbreviations: CAGO - Canada Goose PEFA - Peregrine Falcon
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Raj Rajnarayanan
@RajlabN
·
2h
Timely analysis of the 239 sequences submitted by
@USDA_APHIS

@USDA
H/T
@MichaelWorobey
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KWyLSBcG_mini.jpg

Michael Worobey
@MichaelWorobey
·
2h
Here's a visual of the US H5N1 "Cattle clade", HA plus two internal genes. Strongly suggests to me there was single origin, at least for these sequences. Possibly in late 2023/early 2024.
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Michael Worobey
@MichaelWorobey
OK, I think we're close to decisive evidence that US bovine H5N1 had a single origin from birds, and that when related viruses from birds *have* been found, they are jumps from cattle back into birds. Grackles, blackbirds, chickens all show mammalian adaptation like PB2 M631L.
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5:12 PM · Apr 23, 2024
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Michael Worobey
@MichaelWorobey
·
2h
My understanding is that these bird (and cat) viruses within the "bovine" clade were sampled from farms that had bovine H5N1. So, are the birds on these farms giving this virus to the cattle, or are the cattle giving it to the birds? It is cattle to birds very likely.
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Michael Worobey
@MichaelWorobey
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2h
There is just no good reason to think there's an epizootic of mammalian adapted H5N1 in birds.
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Michael Worobey
@MichaelWorobey
·
2h
Still, the caveat here is that there are still some uncertainties about the (meta)data here.
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Michael Worobey
@MichaelWorobey
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1h
Also, this might merely be the *major* bovine H5N1 lineage. Perhaps we'll find other, minor ones.
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hat tip Pathfinder


Louise Moncla
@LouiseHMoncla
Due to enormous and collaborative effort from the
@nextstrain
team,
@hamesjadfield

@trvrb
and of course
@USDA
, we now have the USDA cattle sequences on Nextstrain: https://nextstrain.org/avian-flu/h5n1/ha/2y?c=host… visualized with new, 2-year builds.
1:54 PM · Apr 25, 2024
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Louise Moncla
@LouiseHMoncla
·
22h
After raw reads were deposited on the SRA, a team of people including
@xrayfoo
rapidly assembles read into consensus genomes and shared them publicly. We used those to update our full genome phylogenies, and built out new builds that allow for 2 year and all-time displays.
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Louise Moncla
@LouiseHMoncla
·
22h
All of the new cattle sequences fall into a single, monophyletic cluster that closely match the original cattle sequences deposited in GISAID. You'll notice stacks of identical sequences across multiple segment trees that fall upstream of other cattle sequences. This pattern ...
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Louise Moncla
@LouiseHMoncla
·
22h
is consistent with a single introduction from birds into cattle, with subsequent spread between farms. Important caveat is that wild birds are chronically undersampled, & further detections in wild birds could disrupt this cluster. However, the genetic similarity among cattle ...
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Louise Moncla
@LouiseHMoncla
·
22h
sequences across segments suggests these genomes are closely related, and likely linked epidemiologically. This matches interpretations and related analyses shared by
@MichaelWorobey
here:
Quote
KWyLSBcG_mini.jpg

Michael Worobey
@MichaelWorobey
·
Apr 23
Here's a visual of the US H5N1 "Cattle clade", HA plus two internal genes. Strongly suggests to me there was single origin, at least for these sequences. Possibly in late 2023/early 2024.
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Louise Moncla
@LouiseHMoncla
·
22h
The human case in Texas still clusters outgrouped to the entire cattle cluster, indicating that they were likely infected prior to the sampled infection from the cattle represented in this tree. This is true across the entire genome.
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Louise Moncla
@LouiseHMoncla
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22h
One big question has been whether these cattle viruses carry known, mammal-adaptive mutations. None of the cattle sequences have PB2 E627K, but all have M631L, an alternative, putative adaptive mutation. https://nextstrain.org/avian-flu/h5n1/pb2/2y?c=gt-PB2_631&gmax=2307&gmin=28&l=scatter&m=div&scatterX =host&scatterY=gt…
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Louise Moncla
@LouiseHMoncla
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22h
Other polymerase mutations of potential interest are PA 497 and 613, both present to some degree in the cattle sequences.
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Louise Moncla
@LouiseHMoncla
·
22h
Moving forward, the
@USDA
decision to test cattle moving between states is an excellent one. Expanded testing in cattle and humans with high risk of exposure, along with more epidemiologic & sequence data on cattle movement, spread, and detection timing and location will all ...
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Louise Moncla
@LouiseHMoncla
·
22h
increase resolution. The information about this outbreak is changing daily, so more interpretation is sure to come. Huge thanks to
@USDA
for depositing these data, to
@xrayfoo
and
@MichaelWorobey
for rapidly sharing assembled genomes, & to
@trvrb
and
@hamesjadfield
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Louise Moncla
@LouiseHMoncla
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22h
@xrayfoo
made the consensus genomes publicly available here: https://github.com/andersen-lab/avian-influenza… and we reformatted them for Nextstrain here: https://github.com/moncla-lab/avian-flu-USDA-cattle/tree/main…. Use however you'd like, but please acknowledge
@xrayfoo
for his awesome work on this.

GitHub - andersen-lab/avian-influenza: Consensus genomes for
From github.com
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JusDayDa
@JusDayDa
·
22h
Where might feline/cats appear on this tree (inside yellow?)
@MichaelWorobey
Also, any complimentary hypothesis to time of shared ancestor? (December 2023 or earlier?) Terrific work and well done!
@statnews

@EricTopol

@PeacockFlu
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Louise Moncla
@LouiseHMoncla
·
19h
The cats are colored yellow here, and are hard to see among so many identical genomes. If you toggle into time view, it is easier to see them!
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Michael Worobey
@MichaelWorobey
·
14h
Great work, Louise! Go Penn-CEIRR/
@CEIRRNetwork
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Michael Worobey
@MichaelWorobey
·
3h
Important update on metadata of H5N1 in cattle (and back to birds): Thanks to the extraordinary detective skills of
@flodebarre
, we are pleased to be able to share this table containing locations and dates for several H5N1 cases in cattle and birds:

avian-influenza/metadata/SraRunTable.csv at master · andersen-lab/avian-influenza
From github.com
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Michael Worobey
@MichaelWorobey
·
12m
Sorry - corrected link here:

avian-influenza/metadata/PRJNA1102327_metadata.csv at master · andersen-lab/avian-influenza
From github.com
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Michael Worobey
@MichaelWorobey
·
1h
We need to talk about that human case of H5N1 in Texas... Here is a bootstrapped (NJ) tree showing how the closest realtive of H5N1 sampled in cattle is a virus the infected an male individual who reportedly worked on a farm with cattle (dairy, I believe).
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Michael Worobey
@MichaelWorobey
·
58m
One last thing - this suggests, but does not prove, that the common ancestor of both the main, and putative minor, bovine H5N1 lineages existed in Texas.​

Michael Worobey
@MichaelWorobey
·
35m
Please check at
@LouiseHMoncla
's analysis on the relevant mutations, on the
@nextstrain
platform!
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NxWZoWyx_mini.jpg

Louise Moncla
@LouiseHMoncla
·
Apr 25
Replying to @LouiseHMoncla
The human case in Texas still clusters outgrouped to the entire cattle cluster, indicating that they were likely infected prior to the sampled infection from the cattle represented in this tree. This is true across the entire genome.
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Michael Worobey
@MichaelWorobey
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1h
If correct, this drives the cattle H5N1 lineage even further back than we had thought, though we may not see the date of the bird to cattle cross-species transmission event change much.
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Michael Worobey
@MichaelWorobey
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1h
It would be good if samples, perhaps environmental samples, could be collected from that farm to directly test these hypotheses.
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Michael Worobey
@MichaelWorobey
My colleagues and I have just posted a "Note about availability of #H5N1 2.3.4.4b consensus sequences from cattle and other species"
@virological_org
. Consensus genomes and metadata.

virological.org
Note about availability of H5N1 2.3.4.4b consensus sequences from cattle and other species:
On Sunday, April 21st, 2024, “in the interest of public transparency and ensuring the scientific community has access to this information as quickly as possible to encourage disease research and...
11:35 AM · Apr 28, 2024
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Michael Worobey
@MichaelWorobey
·
19m
"We hope the H5N1 genome sequences we have assembled from sequence read data generously shared publicly by USDA/APHIS [
@USDA

@APHIS
] will assist in the USDA’s goal of enabling timely outbreak responses and infectious disease research."
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Michael Worobey
@MichaelWorobey
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"We, in turn, encourage researchers globally to use these sequences to support biomedical research, public health, animal health and welfare, and safe and secure food production within the U.S. dairy industry and beyond."
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Michael Worobey
@MichaelWorobey
·
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We are sharing our high quality consensus genomes, as well as date of sampling and location to US state for >100 #H5N1 cases in cattle.
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Michael Worobey
@MichaelWorobey
·
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Karthik Gangavarapu, Jonathan Pekar, Florence Débarre, Kristian Andersen, Gytis Dudas, Daniel Goldhill, Joseph Hughes, Xiang Ji, Jeffrey Joy, Moritz Kraemer, Philippe Lemey, Louise Moncla, Martha Nelson, Stuart Neil,
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Michael Worobey
@MichaelWorobey
·
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Tom Peacock, Oliver Pybus, Andrew Rambaut, Angela Rasmussen, Christopher Ruis, Lorena Mariana Malpica Serrano, Marc Suchard, Joel Wertheim, Michael Worobey​
 
Note about availability of H5N1 2.3.4.4b consensus sequences from cattle and other species:

Influenza virusH5N1-global
Apr 28
1 / 5
Apr 28
37m ago
worobey
2
2h
On Sunday, April 21st, 2024, “in the interest of public transparency and ensuring the scientific community has access to this information as quickly as possible to encourage disease research and development to benefit the U.S. dairy industry”, the National Veterinary Services Laboratories (NVSL) of the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) shared H5N1 sequence reads from 239 Biosamples related to the ongoing H5N1 clade 2.3.4.4b influenza A virus outbreak in U.S. cattle and other species [1]. As they noted, “the sequences were made available with appreciation to the state animal health authorities and National Animal Health Laboratories conducting testing”.

These data were shared as BioProject PRJNA1102327 on the Sequence Read Archive (SRA) of the National Institute of Health’s National Library of Medicine, National Center for Biotechnology Information (NCBI). Sequences posted were from cattle (n=202), cats (9), chickens (18), skunk (1), raccoon (1), grackle (3), blackbird (2), canada goose (1), peregrine falcon (1), and goose (1). APHIS announced that they would continue making additional raw genetic sequences available on a rolling basis at Home - SRA - NCBI (nih.gov ) by searching for “WGS of H5N1” [2].

In order to provide the wider scientific community with consensus sequences required for evolutionary, phylodynamic, molecular, virologic, diagnostic, therapeutic, and other studies, as well as to conduct our own research into the H5N1 outbreak in cattle and other species, we rapidly generated high-quality genome assemblies of each of the eight H5N1 viral genome segments for each of the 239 sets of raw sequencing reads made available by APHIS. We initially shared these consensus genomes in a public GitHub repository [3] on April 22nd, 2024 and have continued to update the repository as new data and metadata become available…

The consensus genome sequences for the eight segments were generated with EPI_ISL_19032063 (GISAID [4]) as a reference, using iVar v1.4.2 [5], a computational package that contains functions broadly useful for viral amplicon-based sequencing. Minimum depth was set at 1, minimum quality at 20, and the minimum frequency to call consensus was set at 50%. Consensus genome sequences, metadata from SRA, and alignments of each genome segment of the 239 viruses are available in this repository

We created an initial table of the metadata associated with the sequencing reads shared in the SRA by APHIS, including host species, which are available in the SRA files [6]. These files do not include information on when (other than year) or where (other than the US) the samples were collected. Dates and locations for a subset of the samples were included in a presentation by Dr. Rosemary Sifford, DVM, Deputy Administrator Veterinary Services, and Chief Veterinary Officer, APHIS, given at a virtual symposium hosted by The Association of State and Territorial Health Officials to facilitate a discussion between public health leaders and scientists driving the U.S. Government’s response to Highly Pathogenic Avian Influenza (HPAI) [7]. We thus updated our table with dates (to day) of sampling and US state of provenance for 152 of the 239 samples [6].

We hope the H5N1 genome sequences we have assembled from sequence read data generously shared publicly by USDA/APHIS will assist in the USDA’s goal of enabling timely outbreak responses and infectious disease research. We, in turn, encourage researchers globally to use these sequences to support biomedical research, public health, animal health and welfare, and safe and secure food production within the U.S. dairy industry and beyond.

Karthik Gangavarapu, Jonathan Pekar, Florence Débarre, Kristian Andersen, Gytis Dudas, Daniel Goldhill, Joseph Hughes, Xiang Ji, Jeffrey Joy, Moritz Kraemer, Philippe Lemey, Louise Moncla, Martha Nelson, Stuart Neil, Tom Peacock, Oliver Pybus, Andrew Rambaut, Angela Rasmussen, Christopher Ruis, Lorena Mariana Malpica Serrano, Marc Suchard, Joel Wertheim, Michael Worobey

References
  1. USDA Publishes H5N1 Influenza A Virus Genetic Sequences on publicly available site. [cited 27 Apr 2024]. Available: https://www.aphis.usda.gov/livestock-poultry-disease/avian/avian-influenza/usda-publishes-h5n1-influenza-virus-genetic 1
  2. Home - SRA - NCBI. [cited 27 Apr 2024]. Available: https://www.ncbi.nlm.nih.gov/sra
  3. GitHub - andersen-lab/avian-influenza: Consensus sequences for U.S. H5N1 clade 2.3.4.4b. In: GitHub [Internet]. [cited 27 Apr 2024]. Available: https://github.com/andersen-lab/avian-influenza
  4. avian-influenza/acknowledgements/gisaid_acknowledge_table_assemby_reference_sequenc es.xls at master · andersen-lab/avian-influenza. In: GitHub [Internet]. [cited 27 Apr 2024]. Available: https://github.com/andersen-lab/avian-influenza/blob/master/acknowledgements/gisaid_acknowledge_table_assemby_reference_sequenc es.xls
  5. GitHub - andersen-lab/ivar: iVar is a computational package that contains functions broadly useful for viral amplicon-based sequencing. In: GitHub [Internet]. [cited 27 Apr 2024]. Available: https://github.com/andersen-lab/ivar
  6. Website. Available: https://github.com/andersen-lab/avi...523b3207e0/metadata/PRJNA1102327_metadata.csv
  7. Highly Pathogenic Avian Influenza Scientific Symposium. 2024. Available: https://www.youtube.com/watch?v=aTizSNagjFI 1
https://virological.org/t/note-abou...s-sequences-from-cattle-and-other-species/967
 
I would like to know how the cats on the dairy farms got infected. It is very important to discover this.

---------------------------------------------------------------------------------------------------------------------------------------

Raj Rajnarayanan
@RajlabN
·

13h
#H5N1 #LessonLearned - Domestic and Farm cats! Common theme among H5N1 clade 2.3.4.4b infected Cats: - Present multiple symptoms almost immediately - Neurologic, Respiratory, rapid decline leading to death (or compassionate euthanasia) - Pick PB2 mammalian adaptations 1/n

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Raj Rajnarayanan
@RajlabN
·
13h
French Cat (2022) Phylogenetic analysis linked the infection closely to virus circulating in the neighboring duck farm, with minor genetic differences. Key mutations: PB2 E627K & NS: E26G Again, rapid selection of mammalian adaptation mutation 2/n
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Raj Rajnarayanan
@RajlabN
Polish Cats (2023) First widespread H5N1 clade 2.3.4.4b infection across a wide geographic area within a country Outbreak affected both indoor/outdoor cats Cat food/ Stork investigated as a likely sources Mammalian adaptations: PB2 E627K & K526R https://twitter.com/RajlabN/status/1675257147238612993… 3/n
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Ngt0V722_mini.jpg

Raj Rajnarayanan
@RajlabN
·
Jul 1, 2023
Key mutations among the #AvianInfluenza sequences isolated from [HASHTAG="t44739"]cats[/HASHTAG] uploaded to #EpiFlu @gisaid from [HASHTAG="c364"]Poland[/HASHTAG] | Analysis using #FluServer tool. Interesting [HASHTAG="c723"]PB2[/HASHTAG] mutations: [HASHTAG="t880"]e627k[/HASHTAG] and #K526R https://cidrap.umn.edu/avian-influe...ficials-probe-h5n1-avian-flu-link-cat-deaths…
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11:01 PM · Apr 27, 2024
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Raj Rajnarayanan
@RajlabN
·
13h
What was once rare..is becoming common Since 2022, H5N1 (Clade 2.3.4.4b) infections in cats were reported from several countries -accumulation of mammalian adaptations in avian and cat isolates -increase in genetic diversity of H5N1 clade 2.3.4.4b owing to reassortment 4/n​

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Raj Rajnarayanan
@RajlabN
·
12h
Adaptations/Reassortments will make it easy to jump across species Not enough testing, lack of genomic sequence data (including appropriate metadata/rapid submission) makes it difficult to track and follow globally 5/n
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Raj Rajnarayanan
@RajlabN
·
12h
For all its worth, the role of #Cats in the current North American Dairy Cattle H5N1 outbreak is still a big puzzle: #FelineFlu (#CatFlu) or #BovineFlu? 6/n
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As a reminder to non-genetic peeps - these genetics analyses are a work-in-progress. It is a developing story. Plus, the genetics of the strains will evolve over time.
 
"All 8 HPAI H5N1 isolates derived from dairy cattle and two cats demonstrated the presence of residues 137A, 158N, and 160A within their HA segments, which may increase binding affinity to the human-type receptor, while none contained residues 192I, 225D, or 228S "

I have not seen this information elsewhere yrt. Maybe i missed it
 
Last edited:
Comment on the paper and HA substitutions from Tom Peacock on X: "the text of that paper is a little misleading (or at least unhelpful) - its completely true the cattle viruses do contain those mutations, but so do all the recent avian 2.3.4.4bs (so its not an adaptation to cattle, its just mutations that are already widespread in the birds)"

So i was a bit too quick in posting the above excerpt
 
Raj Rajnarayanan
@RajlabN
·
3h
#H5N1 #Preliminary Updates Had a quick look at the 87 sequences uploaded (4/29/24) by
@USDA
#NVSL 10 #Cattle, 1 #Pigeon & 2 #Cat sequences have PB2 M631L (+M676A) - likely related to the recent Dairy Cattle Outbreak 6 #Skunk & 6 #Turkey seqs have PB2 E627K (+M676T)


Raj Rajnarayanan
@RajlabN
·
3h
PB2 M676 is mutated to 676T in ~82% of the 87 samples (which also includes E627K adaptation signature in the Skunks/Turkeys); rest of the sequences have 676A (which also includes M631L adaptation signature in Cattle, Pigeon & Cats) Hope we get metadata with dates/location​

 
Roland Baker

@RolandBakerIII
Raj spotted H5N1 M631L & E627K amino acid mutations in the PB2 protein in Kansas dairy cows. These mutations allow for efficient replication in human cells. So far these have not be found in Texas waste water samples in nine cities. Will they show up in Kansas waster water soon?
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Ngt0V722_normal.jpg

Raj Rajnarayanan
@RajlabN
·
May 4
Replying to @RajlabN
One [HASHTAG="c1967"]Kansas[/HASHTAG] #DairyCattle sequence has mammalian adaptations PB2 M631L and E627K The sequence from the specimen collected from the cattle worker has PB2 E627K but lacks M631L This was shown earlier to cluster with the Human isolate (https://gisaid.org/resources/gisaid...fluenza-outbreak-in-the-united-states/#c5130…)
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2:20 PM · May 12, 2024
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Technical Update: Summary Analysis of the Genetic Sequence of a Highly Pathogenic Avian Influenza A(H5N1) Virus Identified in a Human in Michigan


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Updated May 24, 2024

This is a technical summary of an analysis of the genomic sequence of the virus identified in the Michigan case of highly pathogenic avian influenza (HPAI) A(H5N1) virus infection. This analysis supports the conclusion that the overall risk to the general public associated with the ongoing HPAI A(H5N1) outbreak has not changed and remains low at this time. The genome of the virus identified from the patient in Michigan (A/Michigan/90/2024) is publicly posted in GISAID (EPI_ISL_19162802) and has been submitted to GenBank.

May 24, 2024 – CDC has sequenced the influenza virus genome identified in a conjunctival specimen collected from the person in Michigan who was identified to be infected with HPAI A(H5N1) virus and compared each gene segment with HPAI A(H5N1) sequences from cows, wild birds and poultry and the first human case in Texas. The virus HA was identified as clade 2.3.4.4b with each individual gene segment closely related to genotype B3.13 viruses detected in dairy cows available from USDA testing. No amino acid changes were identified in the HA gene sequence from the Michigan patient specimen compared to the HA sequence from the case in Texas and only minor changes were identified when compared to sequences from cows. These data indicate viruses detected in both cows and the two human cases maintain primarily avian genetic characteristics and lack changes that would make them better adapted to infect or transmit between humans. The genome of the human virus from Michigan did not have the PB2 E627K change detected in the virus from the Texas case, but had one notable change (PB2 M631L) compared to the Texas case that is known to be associated with viral adaptation to mammalian hosts, and which has been detected in 99% of dairy cow sequences but only sporadically in birdshttps://www.cdc.gov/flu/avianflu/spotlights/2023-2024/h5n1-technical-update-may-24-2024.html#_edn1. This change has been identified as resulting in enhancement of virus replication and disease severity in mice during studies with avian influenza A(H10N7) viruses[ii]. The remainder of the genome of A/Michigan/90/2024 was closely related to sequences detected in infected dairy cows and strongly suggests direct cow-to-human transmission. Further, there are no markers known to be associated with influenza antiviral resistance found in the virus sequences from the Michigan specimen and the virus is very closely related to two existing HPAI A(H5N1) candidate vaccine viruses that are already available to manufacturers, and which could be used to make vaccine if needed. Overall, the genetic analysis of the HPAI A(H5N1) virus detected in a human in Michigan supports CDC’s conclusion that the human health risk currently remains low. More details of this and other viruses characterized in association with the dairy cow outbreak are available in a previous technical summary.

https://www.cdc.gov/flu/avianflu/sp...n1-technical-update-may-24-2024.html#_ednref1 Thao-Quyen Nguyen, Carl Hutter, Alexey Markin, Megan Thomas, Kristina Lantz, Mary Lea Killian, Garrett M. Janzen, Sriram Vijendran, Sanket Wagle, Blake Inderski, Drew R. Magstadt, Ganwu Li, Diego G. Diel, Elisha Anna Frye, Kiril M. Dimitrov, Amy K. Swinford, Alexis C. Thompson, Kevin R. Snevik, David L. Suarez, Erica Spackman, Steven M. Lakin, Sara C. Ahola, Kammy R. Johnson, Amy L. Baker, Suelee Robbe-Austerman, Mia Kim Torchetti, Tavis K. Anderson Emergence and interstate spread of highly pathogenic avian influenza A(H5N1) in dairy cattle bioRxiv 2024.05.01.591751; doi: https://doi.org/10.1101/2024.05.01.591751

[ii]Zhang X, Xu G, Wang C, Jiang M, Gao W, Wang M, Sun H, Sun Y, Chang KC, Liu J, Pu J. Enhanced pathogenicity and neurotropism of mouse-adapted H10N7 influenza virus are mediated by novel PB2 and NA mutations. J Gen Virol. 2017 Jun;98(6):1185-1195. doi: 10.1099/jgv.0.000770. Epub 2017 Jun 8. PMID: 28597818.

Last Reviewed: May 24, 2024​

https://www.cdc.gov/flu/avianflu/spotlights/2023-2024/h5n1-technical-update-may-24-2024.html
 
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Raj Rajnarayanan
@RajlabN
·
7h


#H5N1 Updates The virus sequence from the patient in Michigan is now publicly available in GISAID (EPI_ISL_19162802) Comparing w/ the sequence from Texas: No changes in HA Michigan sequence has PB2 M631L but no E627K​


Raj Rajnarayanan
@RajlabN
·
7h


#H5N1 #DairyCattleOutbreak Updates PB2 mutations (Sequences from Texas and Michigan human cases)
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Raj Rajnarayanan
@RajlabN
·
7h


Replying to
@RajlabN
Key differences: Presence of PB2 E362G & M631L and the absence of PB2 E627K




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Bloom Lab
@jbloom_lab


In new study led by
@bdadonaite
, we measure how all mutations to H5 influenza HA affect four molecular phenotypes relevant to pandemic risk: https://biorxiv.org/content/10.1101/2024.05.23.595634v1… Results can inform surveillance of ongoing evolution of H5N1.
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9:46 AM · May 25, 2024
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Bloom Lab
@jbloom_lab
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4h


To measure how all HA mutations affect those phenotypes, we created pseudovirus libraries of HA from WHO clade 2.3.4.4b vaccine strain. Pseudoviruses encode no genes other than HA, so can only do a single cycle of infection making them safe for biosafety-level-2.
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Bloom Lab
@jbloom_lab
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First, we measured how all mutations affected HA-mediated cell entry, which is essential for viral fitness See heatmap below, which is easily visualized interactively at https://dms-vep.org/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS/… Some sites constrained (orange); others w many well tolerated mutations (white/blue)
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Bloom Lab
@jbloom_lab
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Here is constraint (average cell entry effect of mutations at each site) on HA structure. These measurements define ideal targets for new approaches (eg https://biorxiv.org/content/10.1101/2024.03.14.585103v1…) to develop antibodies targeting constrained regions of virus.
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Bloom Lab
@jbloom_lab
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Next, we measured how mutations affect HA’s usage of a2-6 vs a2-3 sialic acid receptors (thanks
@PeacockFlu
for idea how to do this) Important because human-transmissible viruses use a2-6. Below are mutations that improve a2-6 usage, and should be monitored for in surveillance
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Bloom Lab
@jbloom_lab
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8 Bloom Lab
@jbloom_lab
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Airborne-transmissible influenza viruses tend to have higher HA stability, so we also measured how all mutations affect stability. As seen below, stability enhancing mutations tend to be located in helices in fusion machinery and interfaces between head & stalk domains.
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Bloom Lab
@jbloom_lab
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Bloom Lab
@jbloom_lab
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In addition to relevance of stability-enhancing mutations for pandemic-risk surveillance, these mutations could be useful to introduce into vaccine immunogens as stabilizing mutations have improved immunogenicity of other viral vaccines.


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Bloom Lab
@jbloom_lab
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We next measured how all HA mutations affected neutralization by sera from vaccinated mice (from
@ScottEHensley
) or ferrets (from Richard Webby). This is important because WHO recommends candidate vaccine strains to H5, which may need to be updated as virus evolves.


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Bloom Lab
@jbloom_lab
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Below are sites in HA where mutations reduce neutralization by mouse or ferret sera (see https://dms-vep.org/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS/for… interactive plots w per-mutation effects) These data make it possible to rapidly identify new viral mutants that are mismatched to current candidate vaccine strains.
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Bloom Lab
@jbloom_lab
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4h


To use these experimental datasets to inform surveillance, Jordan Ort &
@LouiseHMoncla
to integrated them into an interactive Nextstrain tree (https://nextstrain.org/groups/moncla-lab/h5nx/h5-dms/clade-2344b…) that allows you to color nodes by phenotypes (see dropdown at left)


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Bloom Lab
@jbloom_lab
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Such trees make it possible to immediately identify when new viral mutants have potentially relevant HA phenotypic changes; for instance, see below for examples of mutations in dairy cattle cluster that reduce neutralization, or viruses w increased HA stability.
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Bloom Lab
@jbloom_lab
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The datasets are so rich that we recommend viewing interactive plots rather than static figures: - Homepage w all data: https://dms-vep.org/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS/… - Data mapped on HA structure: https://dms-viz.github.io/v0/?data=https%3A%2F%2Fraw.githubusercontent.com%2Fdm s-vep%2FFlu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS%2Fmain%2Fresults%2Fdms-viz%2Fdms-viz.json… - Data mapped on phylogenetic trees: https://nextstrain.org/groups/moncla-lab/h5nx/h5-dms/clade-2344b…


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Bloom Lab
@jbloom_lab
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In addition, we have made a page to help convert between the many confusing numbering schemes used to name HA mutations: https://dms-vep.org/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS/numbering.html… (Throughout we use H3 numbering, but other studies use other schemes)


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Bloom Lab
@jbloom_lab
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Finally, as we discuss in paper, our ability to safely do deep mutational scanning of H5 HA enabled by switch from live-virus to pseudovirus. I thank
@mlipsitch
& others for thoughtful discussion that prompted switch; we welcome continued input on those aspects of our work.
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Bloom Lab
@jbloom_lab
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Thanks to all who contributed to this study:
@bdadonaite
,
@Jenny_Ahn0
, Jordan Ort, Jin Yu, Colleen Furey,
@anniedosey
, Will Hannon, Amy Baker, Richard Webby,
@KingLabIPD
, Yan Liu,
@ScottEHensley
,
@PeacockFlu
,
@LouiseHMoncla
Pre-print is here:

biorxiv.org
Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance
H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple non-human mammalian species. Previous...






 
Preprint: Massive outbreak of Influenza A H5N1 in elephant seals at Peninsula Valdes, Argentina: increased evidence for mammal-to-mammal transmission

Preprint: Massive outbreak of Influenza A H5N1 in elephant seals at Peninsula Valdes, Argentina: increased evidence for mammal-to-mammal transmission






#18,096

Although we are closely watching a distinct genotype (B3.13) of H5N1 clade 2.3.4.4b running rife in American dairy cattle, in South America another genetically distinct H5N1 virus is causing mass mortality in sea lions and seals.

Unlike the American cattle genotype, this H5 virus has spread across multiple countries, killing tens of thousands of its mammalian hosts.​

A few of many past blogs on these events include:
Marine mammals have been long known to be susceptible to influenza A infection (see here, here, here, and here), but previous outbreaks have tended to be smaller, shorter-lived, and geographically limited.

The spread in marine mammals from Peru on the Pacific coast to southern Brazil on the Atlantic side is unprecedented, leading some researchers to suspect mammal-to-mammal transmission.

Today we've a preprint (published June 1st) that presents even more evidence suggestive of mammal-to-mammal transmission, including the identification of a marine-mammal specific clade.

The authors wrote:

Across the genome, we identified more than 64 amino acid changes in the H5N1 HPAI viruses from Península Valdés when compared viruses from birds and mammals from Argentina, other South American countries, Antarctica, North America (genotype B3.2 from 2022–2023) and the original Goose/Guangdong (Gs/Gd) (Supplementary Table 4).
Of the 64 mutations, 18 are potentially associated with increased virulence, transmission or adaptation to mammalian hosts, and fifteen are present in H5N1 viruses from Argentina’s coastal outbreaks in marine mammals and terns but absent in H5N1 (B3.2 genotype) strains from North America and from goose/poultry strains from Argentina (Supplementary Table 4). Of note, eleven of the fifteen common mutations were also present in the human case in Chile (Supplementary Table 5).


This is, as you might imagine, a lengthy (33 Pages) and at times technical report, and while I've posted some excerpts below, is well worth reading in its entirety. I'll have postscript after the break.
Massive outbreak of Influenza A H5N1 in elephant seals at Peninsula Valdes, Argentina: increased evidence for mammal-to-mammal transmission

Marcela M Uhart, Ralph E. T. Vanstreels, Martha I. Nelson, Valeria Olivera, Julieta Campagna, Victoria Zavattieri, Philippe Lemey, Claudio Campagna, Valeria Falabella, Agustina Rimondi
doi: https://doi.org/10.1101/2024.05.31.596774+

PDF
Abstract

H5N1 high pathogenicity avian influenza (HPAI) viruses of the cla(SNIP)de 2.3.4.4b have killed thousands of marine mammals in South America since 2022. In October 2023, following outbreaks in sea lions in Argentina, we recorded unprecedented mass mortality (~17,000 individuals) in southern elephant seals (Mirounga leonina) at Peninsula Valdes. +


Seal pups were disproportionately affected. Adult seals departed early, disrupting social and breeding structure. Frequent interactions with sea lions and scavenging by seagulls were observed. Deaths of terns concurred with seals but peaked weeks later. HPAI H5N1 was confirmed in seals and terns. Moreover, genomic characterization showed viruses from pinnipeds and terns in Argentina form a distinct clade with marine mammal viruses from Peru, Chile and Brazil.

These mammal-clade viruses share an identical set of mammalian adaptation mutations which are notably also found in the terns. Our combined ecological and phylogenetic data support mammal-to-mammal transmission and occasional mammal-to-bird spillover.

To our knowledge, this is the first multinational transmission of H5N1 viruses in mammals ever observed globally. The implication that H5N1 viruses are becoming more evolutionary flexible and adapting to mammals in new ways could have global consequences for wildlife, humans, and/or livestock.

(SNIP)

From a public health perspective, mammal-to-mammal transmission could be a critical stepping-stone in the evolutionary pathway for these viruses to become capable of human-to-human transmission and thus potentially pandemic 67. As mentioned previously, some of the mutations found in the strains of the marine mammal clade are already known to be of concern.

Specifically, the mutation D701N in PB2 has been shown to compensate for the lack of the E627K mutation in PB2 in terms of improved viral growth in mammalian cells and enhanced aerosol transmission of H3N2 and H5N1 viruses68 .

On the other hand, the phenotypic effects of mutations in other gene segments found in the H5 viruses from our study(Supplementary Table 5) are not yet known, and the possibility that some of them may also open evolutionary pathways that enhance the virulence or transmission of these viruses to mammals (including humans) cannot be ruled out.


The fact that the H5N1 HPAI virus detected in a human case in Chile 69 belongs to the marine mammal clade described in this study, highlights the potential risk to public health.

Moreover, given pinniped susceptibility to multiple IAVs (including human-like strains 38–40 ), and their frequent intermingling with other avian and mammalian hosts, co-infections could occur, potentially enabling the emergence of reassorted strains 36,70 .

Hence, while there is no evidence for genomic reassortment occurring in pinnipeds at this time, the broad circulation of H5N1 HPAI viruses in marine mammals is a warning we must not ignore.

In conclusion, as recently demonstrated by the detection of HPAI H5N1 viruses in ruminants 71 , few if any compartments and species are outside the scope of the clade 2.3.4.4b strains. Thus, moving forward, HPAI management requires holistic strategies that recognize the interconnectedness of human, animal, and environmental health and safeguard biodiversity, promote sustainable practices, and enhance resilience globally to emerging infectious diseases.

(Continue . . . )




Although we still don't know if H5N1 is actually capable of sparking a human pandemic (see Are Influenza Pandemic Viruses Members Of An Exclusive Club?), the number of ongoing field experiments around the globe grows with each passing day.

And it is fair to say we are probably only vaguely aware of a fraction of them.​

Even in our own backyard - after > 2 months - we still don't know how widespread genotype B3.13 is in American livestock. After more than a year, there are still no answers to the massive die off of seals in the Caspian sea, and we know very little about what is happening with H5N1 in China, Russia, and most of Africa.

Most of these field experiments will be failures. If it were easy for nature to cobble together a pandemic virus, we'd be hip deep with them all the time.​

But with growing diversity, comes more opportunities for the virus to get `lucky'. And that applies not only to H5N1, but also to a panoply of other subtypes, all embarked on their own evolutionary journeys.

A few recent blogs include:
We live in a threat-rich environment. While the following quote is nearly 20 years old, it sums up the pandemic preparedness dilemma in a nut shell.

Everything you say in advance of a pandemic seems alarmist. Anything you’ve done after it starts is inadequate."- Michael Leavitt, Former Secretary of HHS

https://afludiary.blogspot.com/2024/06/preprint-massive-outbreak-of-influenza.html
 
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