Emergence of an Antigenically Drifted and Reassorted Influenza B Virus at the end of the 2024-25 Influenza Season

Elgin Akin, David A Villafuerte, Anne P. Werner, Matthew Pinsley, Amary Fall, Omar Abdullah, Julie M Norton, Richard Eric Rothman, Katherine Fenstermacher, Yu-Nong Gong, Eili Klein, Heba H Mostafa, Andrew Pekosz

BioRxiv Preprint doi: https://doi.org/10.1101/2025.07.24.666632

Organization of this repository

This repository houses all source code for the 8 segment and 1 genome Nextstrain Build for Assessing C.3 Reassortment Rates along with all reassortment tanglegrams for the 2024-25 Influenza B season.

Important

Repository Code of conduct for ALL collaborators using this repo:

1. DO NOT push commits to main. Please open a pull request.
2. Please document any problems or issues you encounter by opening a github issue

Warning

This repository does NOT house the necessary data to construct the 9 IBV C.3-enriched nextstrain builds. These data are availible on a private OneDrive. Contact Elgin Akin (eakin1@jh.edu) for access.

This repository houses the nextstrain segment build and genome build snakemake pipeline. It functionally represents a typical Pekosz Lab seasonal Influenza B nextstrain build but replaces the automated data ingest pipeline with a manual data ingest pipeline. This pipeline can be found in the in the 01_ingest.qmd notebook in order to accomodate sequences from GISAID. In the future, efforts will be made to automate the cleaning of publically sourced data.

Tanglegram Quick Links

Instructions: Click on each link to visualize a HA:{segment} tanglegram in auspice.

Warning

You must have access to the Pekosz Lab private nextstrain account. Contact Dr. Andy Pekosz apekosz1@ jh.edu and cc Elgin Akin (eakin1@jh.edu) for access

4 Probable C.5.1 HA reassortments with a C.3 HA in NA, NP, NS and PA

• HA:NA
• HA:NP
• HA:NS
• HA:PA

3 Non C.3 Reassortments

• HA:PB2
• HA:PB1
• HA:MP

How to execute this build

1. Archive the current version of the repository.
   • This is important because the nextstrain pipeline will overwrite files in the data/ and source/intermediate/ directories.
   • Make a new directory in the snapshots folder naming it according to the date of the new build YYYMMDD (e.g. 20250615)
   • Copy the entire contents of your project into this new directory
     • a script has been written to accomplish this: execute python scripts/snapshot.py -s . -o snapshots/{YYYYMMDD} changing the date to the current date.
   • Once you have manually confirmed that the new directory is a complete copy of the project, you can safely delete the contents of the data/, results, and source/intermediate/ directories using our safety-enhanced script:
     • Run python scripts/snapshot.py.
2. Download updated Influenza B Genomic data from GISAID
   • Dates should be filtered to 2020-10-05 to the Present Date.
   • Rename the sequences.fasta file to gisaid_vic_sequences.fasta
   • Rename the metadata.tsv file to gisaid_vic_metadata.tsv
   • Place these files in the source/ directory of your project directory (this repository)
3. Manually curate the sequences and metadata files using the 01_ingest.qmd notebook.
4. Execute the snakemake pipeline to build the 8 segment and 1 genome builds.
   • The snakemake pipeline is located in the workflow/ directory of this repository.
   • The snakemake pipeline can be executed by running the following command in your terminal:

snakemake --cores 8

How are these data organized?

The 01_ingest.qmd explains how sequences were down-selected and curated to result in a high quality genomes for 8 segment-specific builds in augur.

The most recently curated consensus dataset can be found in the data/ directory of the most recent data snapshots HERE: Link to OneDrive

This folder represents various snapshots of data downsampling and will be periodically updated with circulating Influenza B strains deposited on GISAID. However, all current and historical builds will be deposited into this folder.

Critically, the data/ directory houses 8 sequence.fasta and 8 metadata.tsv which contain equivalent numbers of genomes across all 8 segment files.

Instructions to view non-HA:{segment} reassortments in Auspice/Nextstrain

1. In your browswer, click the URL window and delete its contents.
2. The formula for building a tanglegram in your browser is as follows:
   • https://nextstrain.org/groups/PekoszLab/akine/ibvc3/vic/**segment**:groups/PekoszLab/akine/ibvc3/vic/**segment**
   • The segment must be in all lowercase. Options: pb2, pb1, pa, ha, np, na, mp, ns.

For example: a PB2:PA tanglegram can be build and visualized by entering the following URL into your browswer: (https://nextstrain.org/groups/PekoszLab/akine/ibvc3/vic/pb2:groups/PekoszLab/akine/ibvc3/vic/pa)

Build Specific Enhancements and TODOs

Steps required needed for Public Release

• refactor this repository's version fludb to accomodate authorship into the nextstrain build
• Add a numerical conversion of glycosylation for HA and NA.
  • We really should consider wrapping this up in a package to centralize version control across projects...

Enter Pekosz Lab Nextstrain Pipeline Enhancements into the seasonal builds

• Custom ingest process for piping gisaid and jhh hospital sequence and metadata
• update fludb upload scrupt upload_jhh.py: JHH location from "JHH" to GISAID-formatted Region / Country / Division / Location: "North America/United States/Maryland/Baltimore"
• upload_gisaid.py enhancements:
  • strain name spaces are removed. Former logic was to replace " " with "_".
  • Change the default location entry from "JHH" to "North America / United States / Maryland / Baltimore" to match GISAID location formatting.
• Augur export - 'locations' renamed to 'area' - need to update lat_long.tsv config file

To preview builds locally:

auspice view \
    --datasetDir auspice/vic

Find and kill local server

lsof -i tcp:4000

kill -9 <PID> 

Upload to private nextstrain

python scripts/c.3_nextstrain_upload_private_genomes.py

quick export edit

snakemake --cores 8 --forcerun export