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Quick Start Guide

Overview

The Flu Mutation Explorer project provides influenza A virus phylogenies and associated metadata, plus a database of mammalian adaptation amino acid replacements. Phylogenies are created by clustering Genbank sequences for the 8 genome segments and selecting a representative sequence per cluster, which are then used to construct phylogenetic trees. The Home tab provides a summary table of the numbers of Genbank sequences and representative clusters for each segment.

Tree

The Tree tab provides an interactive phylogenic tree for each segment. Switch between segments by selecting Segment. Within a tree, the tips are named by strain and coloured by various metadata, which may be chosen by selecting Colour by. The tree may be searched for tip names or metadata by entering text into the Search box.

User specified amino acid sequences may be placed into a tree. Paste a sequence into the Query sequence box and click SUBMIT. The closest segment will be determined and then the query sequence will be aligned with a database of represetative sequences, corresponding to the sequences in the tree. A table of hits is then displayed, sorted by closest match. Table rows may be clicked (ctrl/command-click for multiple) and corresponding sequences will be coloured in the tree.

To visualise amino acids using the numbering scheme of a particular reference sequence (for example, to convert between H1 and H3 numbering of the HA prtoein), selected a reference strain in Subtype and enter a position number in Position and click SEARCH. Amino acids aligned to this reference position will be coloured in the tree, using the ClustalX colouring scheme for amino acid chemistry.

Adaptation Mutations

The Adaptation Mutations tab summarises potential mammalian adaptions in an amino acid sequence. Paste an amino acid sequence into the Query sequence box, and click SUBMIT. The closest segment will be determined and a table of any adaption mutations detected displayed. Click on a table row to visualise an interactive chart of the frequencies of amino acids at that position in the cluster representative sequences.

Status

Support

Licence

This work is licensed under the GNU General Public Licence v3.0.
See the GNU GPL v3 for details.

Maximum likelihood phylogeny of the clustered IAV dataset. The curated database for this segment was clustered using MMSeq2 on a 0.95 identity threshold. Representative sequences were aligned using MAFFT. Maximum likelihood trees were obtained with IQ-TREE and midpoint rooted for visualisation.

For Position search, amino acids are numbered based on the selected reference sequence. Amino acid positions for HA mutations are based on mature peptide numbering. Amino acid positions for mutations in all other segments are based on full-length protein numbering.

Sequences with a missing amino acid at the queried position are shown as white nodes in the tree.

Some proteins (e.g., M2 and NEP) share their segment's phylogeny with other proteins. Clustering and the trees are per segment, so this is the tree of the parent segment - the same representatives, in the same clusters - with tips coloured by the amino acid at the position you searched in this reading frame. It is not a phylogeny of the protein.

Amino acid positions for HA mutations are based on mature peptide numbering. Amino acid positions for mutations in all other segments are based on full-length protein numbering. These mammalian adaptation mutations are largely derived from in vitro studies performed in specific viral strain backgrounds. Consequently, their adaptive significance may be context-dependent and may not apply to the submitted query protein sequence.

HA mutation positions use mature peptide numbering for H3 (A/New_York/392/2004) or H5 (A/goose/Guangdong/1/1996), in whichever of the H3 and H5 schemes the sequence matched, while mutations in all other segments use full-length protein numbering based on A/New_York/392/2004. Assessed indicates the number of catalogued mutation positions covered by the submitted sequence. Partial sequences are screened only across the region they span; therefore, none found in a fragment does not indicate that no mutations are present in the complete protein.

These mammalian adaptation mutations are largely derived from in vitro studies performed in specific viral strain backgrounds. Consequently, their adaptive significance may be context-dependent and may not apply to the submitted sequences.

Flu Mutation Explorer

Overview

The inFLUenza Virus Mutation Explorer is a web resource which allows users to search for and visualise sequence variations in the genomes of influenza A viruses (IAVs).

Variation can be assessed using two tools:

Tree

The Tree tool displays an interactive phylogenetic tree, based on nucleotide or protein sequences, for each of the eight segments of the IAV genome. The tree can be coloured to illustrate features including subtype, host taxonomy and amino acid usage at specified positions, and publication-quality images can be downloaded.

Adaptation Mutations

The Adaptation Mutations tool takes a nucleotide or protein protein sequence, and identifies positions previously associated with changes in host species. Segments 7 and 8 each encode two products, and M2 and NEP are listed separately from M1 and NS1 because their catalogued positions are numbered against those proteins. For these positions, the amino acid usage in different host taxa is illustrated, in a graph that can be downloaded as a publication-quality image, and a table provides more detail on sites of adaptation mutations in the sequence, including links to supporting literature.

Batch screening

The Batch screening tab allows for the submission of up to 2000 sequences. The sequences have to be either all nucletoide or all protein sequences. The sequences are scanned against the database of adaptation mutations and for each site with a match to an adaptation mutation, the user can view the barplot frequencies of residues in different hosts or the distribution of residues at that site on the tree.

Methods Summary

Tree visualisation

The IAV phylogenetic trees are visualized using Taxonium, a tool for exploring large phylogenetic trees.

Influenza virus Genome sequence Database

To assemble the sequence database, influenza A virus nucleotide sequences were retrieved from the NCBI Entrez databases using the influenza A virus taxonomic ID, using an in-house Python tool to access the E-utilities API. A BLAST search against a curated IAV reference set was performed to identify the closest reference for each sequence and validate the genome segment number.

Alignments for each genome segment were then obtained as follows:

1. Nextalign was used to obtain sub-alignments of the sequences which shared the same closest reference in the IAV reference set

2. the reference alignment of the IAV reference set was used to guide the insertion of gaps in individual sub-alignments, ensuring consistency with the corresponding reference sequences;

3. sub-alignments were concatenated to obtain the full alignment per segment.

Subtype reference strains

The IAV reference set is made up of the strains below, chosen to give at least one representative of every HA and every NA subtype, with additional strains kept where a subtype is more prevalent or more relevant for research (e.g. H1N1). It is not the full set of subtype combinations reported in GenBank. Strains were taken, in order of priority, from those proposed by Burke & Smith (2014), from the NCBI RefSeq influenza A reference genomes, and from a manual GenBank search where a subtype or a relevant subtype combination was still unrepresented. A dash indicates that no sequence for that segment is included in the reference set.

Strain	Subtype	PB2 (seg 1)	PB1 (seg 2)	PA (seg 3)	HA (seg 4)	NP (seg 5)	NA (seg 6)	M (seg 7)	NS (seg 8)	Source
A/California/07/2009	H1N1	NC_026438	NC_026435	NC_026437	NC_026433	NC_026436	NC_026434	NC_026431	NC_026432	RefSeq
A/New Caledonia/20/1999	H1N1	CY033629	CY033628	CY033627	CY033622	CY033625	CY033624	CY033623	CY033626	Burke & Smith
A/Puerto Rico/8/1934	H1N1	NC_002023	NC_002021	NC_002022	NC_002017	NC_002019	NC_002018	NC_002016	NC_002020	Burke & Smith
A/Korea/426/1968	H2N2	NC_007378	NC_007375	NC_007376	NC_007374	NC_007381	NC_007382	NC_007377	NC_007380	RefSeq
A/New York/392/2004	H3N2	NC_007373	NC_007372	NC_007371	NC_007366	NC_007369	NC_007368	NC_007367	NC_007370	RefSeq
A/Swine/Ontario/01911-1/99	H4N6	AF285892	AF285891	AF285890	AF285885	AF285888	AF285887	AF285886	AF285889	Burke & Smith
A/goose/Guangdong/1/1996	H5N1	NC_007357	NC_007358	NC_007359	NC_007362	NC_007360	NC_007361	NC_007363	NC_007364	RefSeq
A/chicken/Taiwan/0705/99	H6N1	DQ376876	DQ376840	DQ376803	DQ376624	DQ376732	DQ376696	DQ376659	DQ376768	Burke & Smith
A/turkey/Italy/8912/2002	H7N3	CY020612	CY020611	CY020610	CY020605	CY020608	CY020607	CY020606	CY020609	Manual †
A/Netherlands/219/03	H7N7	AY342413	AY340083	AY342418	AY338459	AY342425	AY340079	AY340089	AY342422	Burke & Smith
A/Shanghai/02/2013	H7N9	NC_026422	NC_026423	NC_026424	NC_026425	NC_026426	NC_026429	NC_026427	NC_026428	RefSeq
A/turkey/Ontario/6118/1968	H8N4	CY005831	CY014662	CY005830	CY014659	CY005829	CY014660	CY005828	CY014661	Burke & Smith
A/Hong Kong/1073/99	H9N2	NC_004910	NC_004911	NC_004912	NC_004908	NC_004905	NC_004909	NC_004907	NC_004906	RefSeq
A/Rousettus aegyptiacus/Egypt/381OP/2017	H9N2	PP273148	PP273149	PP273150	PP273151	PP273152	PP273153	PP273154	PP273155	Manual
A/mallard/Bavaria/3/2006	H10N7	–	–	–	FJ183474	DQ792927	FJ183475	FJ743478	–	Burke & Smith
A/duck/England/1/1956	H11N6	GU052209	GU052208	GU052207	GU052203	GU052205	EU429795	GU052204	GU052206	Burke & Smith
A/duck/Alberta/60/1976	H12N5	CY130085	CY130084	CY130083	CY130078	CY130081	CY130080	CY130079	CY130082	Burke & Smith
A/gull/Maryland/704/1977	H13N6	CY014701	CY014700	CY014699	CY014694	CY014697	CY014696	CY014695	CY014698	Burke & Smith
A/mallard/Astrakhan/263/1982	H14N5	CY130101	CY130100	CY130099	CY130094	CY130097	CY130096	CY130095	CY130098	Burke & Smith
A/duck/Australia/341/1983	H15N8	–	–	–	AB295613	–	AB295614	–	–	Burke & Smith
A/wedge-tailed shearwater/Western Australia/2576/1979	H15N9	CY005412	CY005411	CY005410	CY006010	CY005408	CY005407	CY005406	CY005409	Manual
A/black-headed gull/Turkmenistan/13/76	H16N3	–	–	–	EU293864	–	–	–	–	Burke & Smith
A/little yellow-shouldered bat/Guatemala/060/2010	H17N10	CY103889	CY103890	CY103891	CY103892	CY103893	CY103894	CY103895	CY103896	Burke & Smith
A/flat-faced bat/Peru/033/2010	H18N11	CY125942	CY125943	CY125944	CY125945	CY125946	CY125947	CY125948	CY125949	Burke & Smith
A/lesser scaup/CA/1742/2013	H19	–	–	–	OR611723	OR479766	–	OR479890	OR602902	Manual

† Found by manual search and used in place of A/turkey/Italy/220158/02 (Burke & Smith 2014), whose sequences are partial.

Data curation in the pipeline

Several curation steps were applied, including removing non-IAV genomic sequences, eliminating sequences with non-significant BLAST hits, removing sequences shorter than a pre-established length threshold for each segment, and removing sequences that could not be aligned to a reference sequence with Nextalign. Nucleotide alignments were then trimmed to the coding sequences (CDS) for the IAV proteins, which were translated in silico to give protein sequence alignments.

For visualization, the IAV nucleotide sequence database was clustered, and phylogenetic trees were constructed. Clustering was performed on the nucleotide sequences using MMseqs2 with a 95% sequence identity threshold. Representative sequences for each cluster and segment were aligned using MAFFT with default parameters. Maximum likelihood trees were inferred with IQ-TREE using the best-fit model determined by the Bayesian Information Criterion (BIC), and were midpoint-rooted for visualization purposes.

Adaptation mutations

The database of mammalian adaptations is curated and maintained by Daniel Goldhill (Royal Veterinary College).

Media

The virus image on the Home tab is from Naina Nair and Ed Hutchinson, MRC-University of Glasgow Centre for Virus Research (CC-BY 2022).

GitHub

Source code is available on GitHub. Bug reports and feature requests may be submitted via the issue tracker.

Software

Taxonium Sanderson, T. (2022). Taxonium, a web-based tool for exploring large phylogenetic trees. eLife, 11:e82392. DOI: 10.7554/eLife.82392.

E-utilities Kans Jonathan. Entrez Direct: E-utilities on the Unix Command Line. 2013 Apr 23. In: Entrez Programming Utilities Help [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. https://www.ncbi.nlm.nih.gov/books/NBK179288/.

MAFFT Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013 Apr;30(4):772-80. DOI: 10.1093/molbev/mst010. Epub 2013 Jan 16. PMID: 23329690; PMCID: PMC3603318.

MMSEQ2 Steinegger M and Söding J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nature Biotechnology, 35, 1026–1028 (2017). DOI: 10.1038/nbt.3988.

IQ-TREE Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Molecular Biology and Evolution, 32(1), 268–274. DOI: 10.1093/molbev/msu300.

Nextalign Aksamentov, I., Roemer, C., Hodcroft, E. B., & Neher, R. A. (2021). Nextclade: clade assignment, mutation calling and quality control for viral genomes. Journal of Open Source Software, 6(67), 3773. DOI: 10.21105/joss.03773.

Chang W, Cheng J, Allaire J, Sievert C, Schloerke B, Xie Y, Allen J, McPherson J, Dipert A, Borges B (2025). shiny: Web Application Framework for R. R package version 1.11.0, Posit.

Funding

We acknowledge funding from the Medical Research Council (MRC) and Department for Environment, Food and Rural Affairs (Defra, UK) as FluTrailMap-One Health (MR/Y03368X/1); and MRC funding to the MRC-University of Glasgow Centre for Virus Research: MC_UU_00034/5, MC_UU_00034/6, MC_UU_00034/1.