1. Why is it important to predict HPAI H5N1 HA clades?
    Different names have been used in publications to describe emerging lineages of the highly pathogenic avian influenza A (HPAI) H5N1 viruses. This has made discussion and comparison of the various lineages difficult. For that reason, a unified nomenclature system was designed for the HPAI H5N1 viruses so that interpretation of sequence/surveillance data from different labs becomes easier. In addition, the system should help to remove stigmatizing labelling of clades by geographical reference, provide for easy future expansion of the A/goose/Guangdong-like lineage phylogenetic tree, and offer a starting point for a more extensive system to follow based upon antigenic variation and reassortment into multiple genotypes.
2. What is the procedure used by the H5N1 Evolution Working Group for the HA clade designation of H5N1 viral strains?
    Nucleotide sequences of the highly pathogenic H5N1 hemagglutinin (HA) (only nearly complete sequences) were collected from the publicly available NCBI database. A large alignment consisting of 884 HA sequences, each approximately 1,659 nucleotides, was generated using ClustalX. From this alignment a phylogenetic tree was generated by the neighbor-joining method using both MEGA (Version 3.1) and PAUP (Version 4.0) with the Kimura 2-parameter. The large tree was rooted to the highly pathogenic historical Eurasian H5 isolates (A/turkey/England/91 and A/chicken/Scotland/59). 1000 bootstrap replicates were performed to support tree topology. Based on this tree topology and other criteria, clade assignments were made for each group of viruses in the lineage.
3. What are the criteria for clade designation?
  1. Maintain previously designated clade numbers when possible (i.e., Clade 2.2 remains 2.2 and Clade 1 remains 1)
  2. New clade designations based on phylogenetic tree topology derived from the large tree
    • H5N1 progenitors (closest to gs/Guangdong/1/96) designated as Clade 0
    • Subsequent clades numbered starting from Clade 3 (i.e., Clades 3-9)
    • Clades designated by the presence of a distinct common node shared by at least 4 isolates
    • Subclades/sub-subclades designated as a single clade evolves into more than one distinct lineage (based on sharing of a common node)
  3. Average percentage pairwise distances between and within clades (using the Kimura 2-parameter):
    • Distinct clades should have ≥ 1.5% average distances between other clades
    • Distinct clades should have ≤ 1.5% average distances within the clade (may be slightly higher in clades that have highly evolved outliers (i.e., ck/ Shanxi/2/06 in Clade 7)
  4. Bootstrap support for clade defining node (based on 1000 replicates)
    • >60 at clade defining node
  5. Antigenic properties as measured by the hemagglutination inhibition assay should be used as a correlate of clade designation when the data is available
4. How do I use the H5N1 HA web tool for clade prediction?

    To use the H5N1 HA Web tool you first need a file with sequences. These sequences must be in FASTA format. An example of this format can be found Here. After you have this file, go to the Clade Predition Tool web page.

    Before selecting your file, you must first choose some options. Our reconmended setting are choosen by default, and can be left alone if you are unsure about what to change. We reconmend using the BLAST algorithm as it's just as fast as megablast with a small sequence set. After you have chosen the options, enter the number of files you want to process with the H5N1 HA web tool, and then press ok.

    Here you will be presented with a number of text and file upload boxes. From here you can either choose to copy and paste your sequences, or click the 'Browse' button to find the file to upload. After you have completed this step for the number of files you choose to upload, press 'Ok' to start processing your files.

    Depending on how many sequences are being processed, the H5N1 HA webtool can take anywhere from 2 to 20 minutes to complete. After your sequences have been processed, you will see a table with the resulting information.

5. How do I deal with a new sequence that is predicted as an unknown clade?

    When a sequence is predictted as an unknown clade, you will be presented with the option to submit this sequence to FluGenome for annotation and prediction. All submitted sequences will be processed manually by FluGenome to determine what the best clade is, and then manually added to our database. We greatly appreciate all submissions, and thank you in advance for helping make the H5N1 HA web tool the best it can be.

6. What is the relationship between FluGenome and the current H5N1 HA clade prediction tool?

    FluGenome and the H5N1 HA clade prediction tool are both developed and maintained by the same staff. This is a very good thing as both projects deal with the same subject area, and FluGenome already has experience with the FluGenome tools. The current staff of FluGenome are Ruben Donis (Centers for Disease control, Influenza Division; Project Leader), Guoqing Lu (University of Nebraska @ Omaha, Department of Biology; Project Leader), Thaine Rowley (University of Nebraska @ Omaha; Key Personnel; Programming/Design), and Todd Davis (Centers for Disease Control, Influenza Division; Key Personnel; Virology).

7. How often is your H5N1 database updated and where can I get the information of latest update?

    The H5N1 database is updated every night around the times of 10pm to midnight. This update is accomplished via an automatic updating program. Due to human error and missing data, some sequences may be rejected during this update. Therefore the FluGenome staff regularly checks these rejected sequences to fix errors and improve the automatic updater. A page has been dedicated to these updates Here.

8. If I find a bug, how to report it?

    There are two main ways to report bugs. The first method (preferred) is to send an e-mail to admin@flugenome.org. With this method the FluGenome staff may reply to request more information if needed. The second method is to fill out the form Here. This method allow anonymos contact with FluGenome. If you choose this method you can either submit a return e-mail address or not.

    Also, any comments, suggestions, and feedback may be submitted with either of these two methods. The FluGenome staff thanks you in advance for any information you provide to make the H5N1 HA clade prediction tool even better.

9. What is the difference between lineage and clade?
In the context of this database/browser, the viral lineage is classified at the gene segment level and refers to a monophyletic group of virus isolates that share a common ancestor and have evolved independently from other monophyletic groups of related viruses. For example, at the HA gene level and within the H5 subtype there are many different lineages that characterize isolates that share a common ancestor (e.g., the A/goose/Guangdong-like lineage is described as lineage 5J, whereas H5 subtype viruses from Mexico are described as 5B). Within each gene segment lineage, the larger monophyletic group can be further characterized into smaller groups known as clades. Each lineage may be broken up into only a few or several clades that describe distinct groups within a lineage that share a common ancestor (or node on a phylogenetic tree) at a more finite level (usually defined by the nucleotide distance between two or more distinct clades). For example, within the A/goose/Guangdong-like lineage of H5N1 viruses, there are many clades that have been defined for small, evolutionarily distinct groups of isolates (e.g., Clade 2.1 H5N1 viruses make up a distinct clade of isolates from Indonesia). Very few of the gene segment lineages of influenza A have been defined at the clade-level. Therefore, the FluGenome clade prediction tool is useful only for predicting which clade of the A/gs/Guangdong-like lineage a virus belongs.
10. How to cite this work?
Please cite FluGenome H5N1 tool in your publications as follows:
Lu, Davis, Rowley, and Donis. "A Web-based tool for the clade designation of highly pathogenic avian influenza H5N1 viruses" in Options for the Control of Influenza VI. J.M. Katz, N. Cox & A.W. Hampson (Eds.) London: Blackwell, 2007, ##-##.