Comparative genomics analysis of influenza H7N9 viruses

Transcription

1 Comparative genomics analysis of influenza H79 viruses Objectives Upon completion of this exercise, you will be able to use the Research Database (IRD; to: Search for virus sequences and view detailed information about these sequences in IRD Save selected sequences as a working set in your private Workbench space Build a phylogenetic tree on a set of sequences to infer their evolutionary relationships Use the Meta-CAS tool to identify positions that significantly differ between groups of virus sequences Perform a multiple sequence alignment to observe sequence conservation and variations Determine if significant positions are located in viral protein Features and examine Feature Variant ype reports Motivation H79 viruses have a remarkable 36% case fatality rate. Since the first outbreak of H79 viruses in 03, the H79 viruses have experienced five waves. As of 6 July 07, a total of 58 laboratory-confirmed cases of human infection with avian influenza A(H79) viruses, including 60 deaths (FAO). Most human cases are exposed to the A(H79) virus through contact with infected poultry or contaminated environments, including live poultry markets. Although the virus has not acquired the ability to effectively transmit between humans, it is critically important to monitor the evolution of this highly pathogenic virus. Potentially it may become a long-term threat to public health. Question Explore the evolution of human H79 viruses in the past five flu seasons. Analysis workflow Search for sequences and save sequences into working sets: - search for H79 HA nucleotide sequences from human - save them as a working set Construct nucleotide phylogenetic tree: - construct a phylogenetic tree using nucleotide sequences saved in the working set - visualize the tree and color nodes to reveal seasonal branching patterns Run Metadata-driven Comparative Analysis ool (Meta-CAS): - input protein sequences associated with the working set into Meta-CAS - group the sequences into two sets based on phylogeny: 03-0 and identify positions that are significantly different between the two sets - convert the analysis coordinates into H77 HA coordinates Visualize multiple sequence alignment: - examine the HA protein alignment used in Meta-CAS - observe the variant positions in the alignment Determine if the significant positions are located in Features: - follow the Feature hyperlink on the Meta-CAS report - examine Features containing the significant positions

2 ext format convention. Bold text action buttons or options on the website that need to be clicked or selected.. text in double quotes text that needs to be entered into an input box on the website. Walkthrough steps. Search for H79 HA sequences and save matching sequences into a working set a. Open a browser and go to > Search Data > Search s > ucleotide s. b. he ucleotide Search page allows you to search for sequences based on data type, virus type, subtype, clade classification, strain name, segment, complete genome/sequence or not, host, geographical region, country, date range and more. For this exercise, we are going to search for HA segment sequences from human H79 isolates. Select the following criteria. Data ype: S Genome Segments Select Segments: S HA Complete? S HA Virus ype: S A Host: S Human Subtype: H79 Advanced Options: S Exclude Laboratory Strains c. IRD shows instant count of search results (in red) to help you search quickly and efficiently. When you select search criteria on search pages, you will instantly know how many records match your search criteria without actually running the search. SEARCH DAA AALYZE & VISUALIZE WORBECH SUBMI DAA HELP Home ucleotide Search ucleotide Search Search for influenza sequences, proteins, and strains using two types of searches. Use the advanced search to allow you to refine your search with the more fine grained search, and you can pick your viewing options. Results matching your criteria: 3 DAA YPE COMPLEE GEOME HOS GEOGRAPHIC GROUPIG Genome Segments Protein Strain VIRUS YPE A B C Provisional D (PMID:595369) SUBYPE Complete Genome Only SELEC SEGMES Complete? All PB PB PA HA P A MP S Human Choose a Geographic... COURY Choose a Country... H79 * Use comma to separate multiple entries. Ex: H, H7, H3. SRAI AME CLADE CLASSIFICAIO one * Use comma to separate multiple entries. Ex: A/chicken/Israel/055/008, A/chicken/Laos/6/008. Global H Clade (SOP) US H Clade (SOP) H5 Clade (SOP) 009 ph Similarity DAE RAGE (SOP) From: YYYY o: YYYY o add month to search, see Advance Options: Month Range ADVACED OPIOS Show All ip: o select multiple or deselect, Ctrl click (Windows) or Cmd click (MacOS) Select Advanced Option LABORAORY SRAIS Include laboratory strains Exclude laboratory strains Laboratory strains only Laboratory strains are those flagged as "LAB" in the country field or "lab" in the host field in GenBank records. Remove Add Another Advanced Option Clear Search d. Click the orange Search button to run the query. e. he search results will be displayed. Here you can: i. Convert the nucleotide display to the corresponding strain or protein display by clicking Show Strain Display or Show Protein Display. ii. Store selected sequences as a working set in the Workbench so that you can save the dataset and run various analyses on the dataset in the future.

3 iii. Save the search query to your Workbench and rerun the search again later. iv. Select records and run an analysis on the selected records by mousing-over Run Analysis and clicking a desired analysis option. v. Download the sequences (gene, CDS, protein) by clicking Download. vi. View the details for any item in the results table by clicking View. f. ow click the Collection Date header in the results table to sort records by date. g. ext, click Display Settings above the table. In the light blue box, change Items Per Page to 00 and click Apply. h. Please note that all sequences in IRD undergo an auto curation pipeline and any potential sequence artifacts are displayed in the last column named Curation Report. A complete description of the auto-curation process is described in a standard operating procedure (SOP) document linked from the column header. i. o analyze these sequences, we will save these records into a working set so that we can retrieve the data from the Workbench later and run About Us various Community Announcements analyses Links Resources on the Support same Sign Out dataset. o do so, tick the checkbox above the table and then click the Add to working set button above the table. SEARCH DAA AALYZE & VISUALIZE WORBECH SUBMI DAA HELP Home ucleotide Search Results ucleotide Search Results IRD uses an automated pipeline to detect potential sequence artifacts or poor quality by aligning virus nucleotide sequences submitted to the resource to a curated profile of like sequences. he pipeline sets flags indicating the category and location of artifacts, or the type of poor quality sequence. hese flags are summarized in sequence search results and working sets as follows: Flag CR (issues only in non coding regions); Flag CDS (issues in CDS, possibly also in CR); Pass (no issues); Ambig Seq (excessive s or ambiguity symbols, or insufficient similarity to the profile). See SOP for further details. yun.zhang@jcvi.org Your Selected Items: 3 items selected Deselect All Show Strain Display Show Protein Display Add to Working Set Save Search Run Analysis Download Your search returned 3 segments. Search Criteria Displaying 50 records per page, sorted by Strain ame in Display Settings ascending order. Select all 3 segments 3 ext > Page: of 3 More columns were returned than can be displayed without scrolling. Use scroll bars at top and bottom of display to move right and left or reduce the number of columns displayed by using the Display Settings link above. Segment Protein ame Accession Complete Genome Segment Length Subtype * Collection Date Flu Host Species Country State/Province Season (SOP) Strain ame HA CY8768 Yes 73 H79 03 Human China /A /A A/Anhui/ DEWH730/03 HA CY853 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 0/03 HA CY85 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 0/03 HA CY859 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 03/03 j. You ll be prompted HA CY8537 Yes to log 73 in H79 to your 03 Workbench Human China /A account A/Anhui/DEWH7 0/03 in order to save data to a working set. If you don t HA CY855 Yes 73 H79 03 Human /A /A A/Anhui/DEWH7 05/03 have an account HA CY8553 already, Yes 73 simply H79 register 03 Humanfor Chinaan account /A /A for A/Anhui/DEWH7 06/03 free by choosing the Register for a new account HA CY856 Yes 73 H79 03 option and following the prompts. HA CY8569 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 07/03 Human China /A /A A/Anhui/DEWH7 08/03 k. A lightbox HA of CY8577 Add to Yes Working 73 H79 Set 03 will Humanpop China up. /A ow /A create A/Anhui/DEWH7 09/03 a new working set and name it human H79 J76633 Yes 683 0/6/03 Human China /A /A A/Beijing//03 HA. Click HAAdd J766 to Working Yes 683 H79Set 0/6/03 to save Human the China sequences /A /A A/Beijing//03 to a working set. l. HA J7669 Yes 683 H79 0/6/03 Human China /A /A A/Beijing/3/03 Access your Workbench by clicking the Workbench tab. You will see the newly created working set at the HA F096 Yes 70 H79 03//03 Human China /A /A *A/Changsha//03(H79) HA F097 Yes top of the content list. 70 H79 0/9/03 Human China /A /A *A/Changsha//03(H79) HA MF3706 Yes 683 H79 0/0/07 *Human China /A /A A/Changsha/6/07 HA MF3705 o 683 H79 03//07 *Human China /A /A A/Changsha//07 HA MF37055 o 683 H79 03//07 *Human China. Construct an HA HA MF37059 segment o 683 phylogenetic H79 03/7/07 *Humantree China /A /A A/Changsha//07 /A /A A/Changsha/58/07 a. HA MF37063 o 683 H79 0/3/07 *Human China /A A/Changsha/70/07 ow we will HA construct C9953 o a 683 phylogenetic H79 0//03 Human tree China using /A the /A HA A/Fujian//03 segment sequences from human H79 isolates. In Research Database - ucleotide Search Results 8/3/07 HA Y867 Yes 683 H79 0/3/05 Human China /A /A A/Fujian/S03/05 the Workbench table, click View for the new working set - human H79 HA. HA J03067 o 7 H79 /5/03 *Human China /A /A A/Guangdong/03/03 Loading About Research Us Community Database... Announcements Links Resources Support Sign Out HA J0307 o 7 H79 /6/03 *Human China /A /A A/Guangdong/0/03 b. he working set details page displays the sequence records saved in the working set. Select all records by HA J0308 o 7 H79 /7/03 *Human China /A /A *A/Guangdong/05/03(H79) yun.zhang@jcvi.org SEARCH DAA AALYZE HA & VISUALIZE F6693 WORBECH Yes 683 SUBMI H79 DAA 08/0/03 HELP *Human China /A /A *A/Guangdong//03(H79) clicking the checkbox above the table. hen go to Run Analysis > Generate Phylogenetic ree. Home My Workbench Working Set HA(human X H79 HA07083) Yes 683 H79 0/03 Human China /A /A A/Guangdong/6/03 Working Set - human H79 HA07083-Segment HA X53685 o 683 H79 /03 Human China /A /A A/Guangdong/7/03 Data ype: Segment Created: 08/3/07 Modified: 08/3/07 Access: Private Description: Edit Working Set Details IRD uses an automated pipeline to detect potential sequence artifacts or poor quality by aligning virus nucleotide sequences submitted to the resource to a curated profile of like sequences. he pipeline sets flags indicating the category and location of artifacts, or the type of poor quality sequence. hese flags are summarized in sequence search results and working sets as follows: Flag CR (issues only in non coding regions); Flag CDS (issues in CDS, possibly also in CR); Pass (no issues); Ambig Seq (excessive s or ambiguity symbols, or insufficient similarity to the profile). See SOP for further details. Your Selected Items: 3 items selected Deselect All Remove Copy to Working Set Edit Working Set Using ree Convert Working Set Run Analysis Download Identify Similar s (BLAS) Displaying 50 records per page, sorted by Strain ame in ascending order. Align s (MSA) Display Settings Visualize Aligned s Select all 3 segments 3 ext > Page: of 3 Generate Phylogenetic ree More columns were returned than can be displayed without scrolling. Use scroll bars at top and bottom of display to move right and left or reduce the number of columns displayed by using the Display Settings link above. Analyze Variation (SP) Segment Protein ame Accession Complete Genome Segment Length Subtype * Collection Date Metadata driven Flu Comparative Analysis ool Host Species Country State/Province Season Strain ame (SOP) Format Conversion HA CY8768 Yes 73 H79 03 Human China /A PCR Primer /A Design A/Anhui/ DEWH730/03 HA CY853 Yes 73 H79 03 Human China /A Flu Profile /A esting A/Anhui/DEWH7 0/03 ool with Entire Working Set HA CY85 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 0/03 HA CY859 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 03/03 HA CY8537 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 0/03 HA CY855 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 05/03 HA CY8553 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 06/03 HA CY856 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 07/03 HA CY8569 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 08/03 HA CY8577 Yes 73 H79 03 Human China /A /A A/Anhui/DEWH7 09/03 HA J76633 Yes 683 H79 0/6/03 Human China /A /A A/Beijing//03 HA J766 Yes 683 H79 0/6/03 Human China /A /A A/Beijing//03 3

4 c. In the Select ype light box, choose ucleic Acid (CDS). d. On the Generate Phylogenetic ree page, choose Quick ree, Label ree ips With Strain ame and Date, and then click Build ree. Generate Phylogenetic ree utorial he "Quick ree" option uses PhyML [ Guindon, S. and Gascuel, O., (003) Syst Biol. 5: ] and IRD defined settings to infer phylogenies based on sequences for datasets of at most 000 sequences. he "Custom ree" option offers a choice between the PhyML or RaxML [Stamatakis, A. et al. (005) Bioinformatics : 56 63] algorithms, and the ability to define parameter settings. he Custom ree option, using RaxML, must be used for datasets exceeding 000 sequences. Click here to view a tutorial on generating a phylogenetic tree using IRD tools. AALYSIS AME REE GEERAIO Quick ree (Let IRD set all parameters view all parameters) Custom ree ( for setting of custom parameters and for large datasets ) SOURCE OF SEQUECES O BE AALYZED * IPU 3 SEGMES SELECED FOR REE ype: CDS with HA LABEL REE IPS (EDS) WIH Strain ame Specify custom format of tip label (max ) Strain ame Accession umber Date Country USA State Segment Protein Symbol Season Subype Host Species 009 ph like Phenotype Markers US Swine H Clade Global Swine H Clade H5 Clade Clear Build ree e. If you are logged into your Workbench and a very large dataset is submitted to the tree, a recommendation for running the job in the CIPRES high performance computing environment will appear. If you choose the CIPRES option, your selected sequences will be automatically transferred to CIPRES for tree calculation. Once the analysis is completed, the analysis result will be sent back to IRD and saved to your personal Workbench. f. On the Processing page, you can save the tree upon completion by providing an analysis name, e.g, human H79 HA Fastme and then clicking Save to Workbench. g. Once the analysis is finished, a View Phylogenetic ree page will be loaded. Here you can save the phylogenetic file in ewick or PhyloXML format to your computer. h. In the Enhanced ree Viewer section, choose Archaeopteryx-js and click View ree. View Phylogenetic ree Save Analysis ewick File PhyloXml File Phylip File ree Parameters PhyML Log ree Build Parameters Click the "View ree" button below to launch the tree viewer software in a new window. If you prefer other viewing software, the tree data is available for download in ewick or PhyloXml format using the buttons above. Due to security concerns, certain browsers (e.g. Safari and Firefox) have disabled Java plug ins by default. If the ree Viewer takes a long time to load, please test your browser's Java plug in to make sure it can display Java Applets properly. If Java applets are not displayed properly, please read the following document: "Enabling Java" for instructions on how to fix this. If your tree is very large, it may reach the display limit of the Archaeopteryx viewer. he exact limit depends in part on your computer. If Archaeopteryx is unsatisfactory, download your data as a ewick or PhyloXml file and use other viewing software. EHACED REE VIEWER he IRD team provides software that allows 'decoration' of your tree by features such as host species, year, country, and subtype. his custom software is based on Archaeopteryx. In the tree viewer, use the drop down menu for basic decoration or advanced decoration to select the feature for coloring. he decorated tree and corresponding legend can be exported using options in the File drop down menu. A user's guide is available. How to create a publication quality tree image Viewer Mode: View ree i. he Archaeopteryx tree viewer will be displayed in a new window. i. Many tree visualization options exist including: reroot the tree, collapse/expand/display subtree, swap descendants, decorate (color) the tree leaves by any associated metadata (e.g. host, country or year of isolation, HA or A subtype, etc.), resize the tree, zoom in/out, fit the tree to window, change the font size, etc.

5 ii. In the Visualizations panel, choose Year in the Label Color dropdown list. he Label Color Year legend will be displayed at the top of the viewer. Change decoration colors as needed. iii. Further explore the tree by looking at the collection date field in the sequence label and how date is associated with the tree topology. iv. Reroot the tree or swap descendants as needed. o do so, click an internal node and then click the desired option in the pop-up box. v. You can export the tree image in PG, SVG (vector image), ewick, or phyloxml format. o do so, in the Download section at the bottom of the control panel, choose the desired format in the dropdown list and then clicking Download j. he tree shows that the H79 virus has evolved in the past five years. here are two major lineages: () 03-0, and () which includes all but one 07 isolates. A separate bootstrapped tree analysis shows 00% support value for these two lineages. Within the cluster, A/aiwan/- CGMH/0 and A/aiwan/-CGMH/0 are two passages of the same 0 isolate from a patient who traveled to China. 5

6 3. Metadata-driven Comparative Analysis ool (Meta-CAS) Metadata-driven Comparative Analysis ool for s (Meta-CAS) A unique comparative genomics analysis tool in IRD to identify nucleotide/amino acid positions that significantly differ between two or more groups of virus sequences. Meta-CAS consists of three parts: a multiple sequence alignment (using MUSCLE), a chi-square goodness of fit test to identify positions (columns) of the multiple sequence alignment that significantly differ from the expected (random) distribution of residues between all metadata groups, and a Pearson's chi-square test to identify the specific pairs of metadata groups that contribute to the observed statistical difference. Picket BE, et al. (03) "Metadata-driven Comparative Analysis ool for s (meta-cas): an Automated Process for Identifying Significant Variations Dependent on Differences in Viral Metadata." Virology, 7(-):5-5. doi: 0.06/j.virol a. ow we will use Meta-CAS to identify amino acid positions that significantly differ between 03-0 isolates and isolates. Go to Workbench > View for working set - human H79 HA. b. On the working set details page, select all records by clicking the checkbox above the table. hen go to Run Analysis > Metadata-driven Comparative Analysis ool. c. In the Select ype light box, choose Amino Acid (AA) - HA. d. he Meta-CAS tool setting page will be displayed. In the Grouping section, choose Auto Grouping. ext, type in 0 in the Provide year break points text box. hen click Continue. Metadata-driven Comparative Analysis ool (meta-cas) he meta CAS tool provides the capability to perform customized comparative genomics analyses with minimal manual manipulation. You can perform a statistical analysis on sequences assigned to up to 0 different groups to determine which residues significantly correlate with one or more metadata fields. he meta CAS tool looks for positions that significantly differ between user defined groups of sequences. However, biological biases due to covariation, codon biases, and differences in genotype, geography, time of isolation, or others may affect the robustness of the underlying statistical assumptions. Click here to view meta CAS tutorial. See the SOP for a detailed description of how meta CAS functions. utorial ote: An asterisk (*) = required field AALYSIS AME SEQUECE GROUPIG Manual Grouping Auto Grouping P VALUE HRESHOLD he P value threshold is used as the maximum probablity level for the likelihood that the position is different among the groups simply by chance. Enter threshold value: 0.05 Provide year break points: 0 * Ex: 989, 00 o get 3 groups of 989 & before, , after 00 IPU SEQUECES * 3 records were previously selected from search results FORMA OF SEQUECES PROVIDED * Unaligned FASA Aligned FASA exus Clustal Clear Continue e. his will separate the sequences into two groups: <=0 and >=05. Click Run. 6

7 Metadata-driven Comparative Analysis ool (meta-cas) - Setup Subset Mouse click on the sequences to select them, Ctrl and/or shift key enable the multiple selection. After selecting the sequences, click on "Add" to move into the group. "Remove" will send the selected sequences back to the main list. Auto grouping on: Year Group: <=0 9 Group: >=05 0 MAI LIS OF SEQUECES LIS OF SEQUECES GROUP Add Remove HA /A RJ9D5 A/anjing//03 AGJ73503 HA /A R9YJJ0 A/anchang//03 AGO80 HA /A RGX5 A/Zhejiang/HZ/03 AGM6 HA /A SVZ7 A/Wuxi//03(H79) AG697 HA /A SUZS9 A/Wuxi//03(H79) AG696 HA /A R A/Shanghai/0/03 AGL38 HA /A U3MV9 A/Changsha//03(H79) AGS06 LIS OF SEQUECES GROUP Add Remove HA /A A0A0H3WB3 A/Quzhou//05 AI833 HA /A A0A0H3W3E A/Quzhou//05 AI85 HA /A A0A07APH8 A/Huai'an/00/05 AJU5335 HA /A A0A07AWG3 A/Huai'an/00/05 AJU5339 HA /A A0AL6VE A/Fujian/S03/05 APR7385 HA /A A0AS6RB6 A/Qingyuan/GIRD0/07 AQW37 HA /A A0AU9WX7 A/Guangdong/SP0/07 AQY sequences 0 sequences Clear Run f. Once the analysis is finished, a Metadata-driven Comparative Analysis Report page will be loaded. he report has two parts: a Chi-square est of Independence result table listing the positions that have a significant non-random distribution between your specified groups, and a Pearson's chi-square test result table listing the specific pairs of groups that contribute to the observed statistical difference. Since this analysis only deals with two groups of sequences, the results in the two tables are identical. i. Review the Chi-square test results to see the positions that differ significantly between the two lineages. he residue diversity column lists the counts for each residue within a group. ow sort the results by the p-square value to push the most different positions to the top of the table. What is the position number with the highest p-square value? ii. Examine positions hree isolates from group (05-07 lineage) have an insertion of RRA. iii. o compare the variant positions identified by Meta-CAS with H7 positions without this insertion, we will convert the numbering of the current analysis to H77 HA numbering by selecting H77 A/etherlands/9/03/H77 from the Reference Coordinate dropdown menu. his numbering conversion is based on the structure-based mapping of Burke and Smith (PLoS One. 0, 9():e30). iv. Save the analysis result to your Workbench by clicking Save Analysis. 7

8 . Visualize protein sequence alignment a. ow we are going to view the protein sequence alignment to confirm the Meta-CAS results and to verify clade relationships inferred from the phylogenetic analysis. From the Meta-CAS report page, click Visualize Aligned s at the top of the page. b. In the Label By lightbox, choose Strain ame > Run. c. he alignment is presented in the JalView visualization window. Scroll right up to the region of he insertion is observed in three isolates from 07, but absent from the other H79 isolates. d. ext, examine position Q is observed in the same set of 07 isolates. e. We can change the View Option to Conserved vs. reference such that only the first sequence shows full characters; for the remaining sequences, only the nucleotides/residues differing from the reference sequence are shown as full characters. f. You can download the input sequences or alignment in various formats, or save the alignment to your Workbench. 5. Determine if the significant positions are located in Features Features (SFs) are defined as interesting protein regions with known structural or functional properties. hey are curated from literature or obtained from other databases and validated by domain experts. Once a Feature region has been defined, the number of distinct amino acid sequences observed in the sequence database are determined and each defined as a unique variant type. he reference strain is always Variant ype (V). he Feature (SF) column in the Meta-CAS table provides a convenient linkout to a list of all Features that contain that amino acid position. a. Return to the Meta-CAS report page. Click View SF for position 3. What Feature(s) are mapped to this position? Feature Variant ypes It is possible that the numbered positions from the custom alignment and the numbered positions in the Featuare Reference Strain are different even though they refere to the equivalent sequence posiiton. Below is a table showing how the custom position relates to the homologous position in the reference strain. Custom Amino Acid Position Amino Acid Position in SF Reference Strain Your Selected Items: 0 items selected Download Save Search Your search returned 3 record. Search Criteria Displaying 50 records per page, sorted by Feature Identifier in Display Settings ascending order. Select all 3 results Feature Identifier Feature ame Variant Feature Category ypes Length Amino Acid Position Evidence Comments A_H7_SF3 A_H7_SF35 A_H7_SF0 A_H7_experimentallydeterminedepitope_36(7) A_H7_experimentallydeterminedepitope_336(7) A_H7_determinant ofvirulence_333(0) 7 96 epitope 36(30 HA) 3 IEDB: /A 7 7 epitope 336(30 HA) 35 IEDB:67 /A 0 80 functional 333(35 HA) 3 PubMed:80077 A polybasic cleavage site (pcs) in the hemagglutinin (HA) is required for high pathogenicity. Introduction of three different pcs motifs (PEIPRRRR/GLF from A/chicken/Germany/R8/003(H77), PEIPGSRVRR/GLF from A/chicken/Italy/5/999(H7), and PEIPRHRGR/GLF from A/chicken/England/635/05(H77)) into three different low pathogenic H77 viruses (A/turkey/Germany/R/00, A/chicken/Germany/R36/0, and A/turkey/Germany/R53/03) conferred increased virulence in chickens. 8

9 SEARCH DAA AALYZE & VISUALIZE WORBECH SUBMI DAA HELP Home My Workbench Analysis... Feature Results Feature Details ( A_H7_determinant of virulence_333(0)) Feature Details (SOP) 5//07 Research Database - Feature Details Report For more information about using SFVs, click here. For a detailed description of the development and application of the SFV approach for the study of influenza virus, please read this scientific article: oronha JM, Liu M, Squires RB, Pickett BE, Hale BG, Air GM, Galloway SE, akimoto, Schmolke M, Hunt V, lem E, García Sastre A, McGee M, Scheuermann RH. (0) Feature Variant ype (Flu SFV) analysis: evidence for a role of S in influenza host range restriction. J Virol, 86: b. Click doi: 0.8/JVI View PMID: for SF0 to get Loading to the About Research Us Community Database... Announcements Feature Links (SF) Resources Details Support page. Sign Out SEQUECE FEAURE DEFIIIO Protein ame HA yun.zhang@jcvi.org SEARCH DAA Feature ame AALYZE & VISUALIZE WORBECH A_H7_determinant of virulence_333(0) SUBMI DAA HELP Home My Workbench Feature Analysis... ID Feature Results A_H7_SF0 Feature Details ( A_H7_determinant of virulence_333(0)) Reference Strain A/turkey/Italy/058/00(H73) Reference Feature Accession Details (SOP) AY58609 Reference Position 333(35 HA) 3 For more information about using SFVs, click here. For a detailed description of the development and application of the SFV approach for the study of influenza virus, please read this scientific article: oronha JM, Liu M, Squires RB, Pickett BE, Hale BG, Air GM, Galloway SE, akimoto, Schmolke M, Hunt V, lem E, García Sastre A, McGee M, Scheuermann SOURCE SRAI(S) RH. (0) Feature Variant ype (Flu SFV) analysis: evidence for a role of S in influenza host range restriction. J Virol, 86: doi: 0.8/JVI PMID: V Source Source Strain umber Position SEQUECE FEAURE DEFIIIO A/chicken/Germany/R8/03(H77) V Protein ame 3 Feature ame Feature ID Reference Strain Reference Accession Reference Position SOURCE SRAI(S) Source Strain V umber Source Position 3D Source Evidence Protein Publication Comment Accession Codes Structure AJ60350 /A PubMed:80077 EXP A polybasic cleavage site (pcs) in the HA hemagglutinin (HA) is required for high pathogenicity. Introduction of three different A_H7_determinant of virulence_333(0) pcs motifs (PEIPRRRR/GLF from A_H7_SF0 A/chicken/Germany/R8/003(H77), PEIPGSRVRR/GLF from A/turkey/Italy/058/00(H73) A/chicken/Italy/5/999(H7), and AY58609 PEIPRHRGR/GLF from A/chicken/England/635/05(H77)) into 333(35 HA) 3 three different low pathogenic H77 viruses (A/turkey/Germany/R/00, A/chicken/Germany/R36/0, and A/turkey/Germany/R53/03) conferred 3D increased virulence in chickens. Source Evidence Protein Publication Comment Accession Codes Structure A/chicken/Germany/R8/03(H77) i. his SF is V 5 determinants 333 AJ60350 /A of virulence. PubMed:80077 A EXP A polybasic cleavage cleavage site (pcs) in thesite (pcs) in the hemagglutinin (HA) is 3 hemagglutinin (HA) is required for high pathogenicity. Introduction of three different required for high pathogenicity. Introduction pcs of motifs three (PEIPRRRR/GLF from pcs motifs into three different low A/chicken/Germany/R8/003(H77), PEIPGSRVRR/GLF from pathogenic H77 viruses conferred increased virulence in chickens. A/chicken/Italy/5/999(H7), and PEIPRHRGR/GLF from ii. What is the reference strain used to define A/chicken/England/635/05(H77)) the position coordinates into of this SF? What is the position three different low pathogenic H77 viruses (A/turkey/Germany/R/00, range on the reference strain? A/chicken/Germany/R36/0, and A/turkey/Germany/R53/03) conferred increased virulence chickens. iii. Which Variant ype carries 338RRA3? Click strain count for the V. VARIA YPES Excel Download FASA Download View Phylogenetic ree Find a V(s) Strain Count Variant ype 3 V 736 V 87 V 3 05 V 79 V 5 7 V 6 60 V 7 58 V 8 36 V 9 7 V 0 V 6 V 9 V 3 7 V V 5 8 V 6 7 V 7 6 V 8 6 V 9 8 V 0 6 V 5 V 5 V 3 5 V 5 V 5 5 V 6 5 V 7 V 8 V 9 5 V 30 5 V 3 V 3 V 33 3 V 35 3 V 36 Variation P E I P G R G L F H L L V P.[SRVR] P R[R] S H[QLHHMR] P L.[SRHR] R[RR] [RE] A A H[QLHHMR] L R D[RSRHRR] D[RRHRR] Q[AYRRM] [CSPLSRCRE] [RR] P S [R] Q V R A E R [R] [] R R[RA] otal Variations 3 V 37 c. At step 3.d, V 38 the 35Q substitution is [CSPLSRCR] observed in the 07 Guangdong isolates. ow search for 3 V 39 R[R] V 0 Q[AYQQM] 9 Feature(s) that cover this position. Go to Search Data > Feature Variant ypes. V [DRSRHRRI] V S I H[QLHHMR] d. On the V 3 Feature Variant ypes R[] landing page, choose the 3 following criteria and then click Search. V R[R] 5 V 5 Q[AYQRM] 9 Virus ype: V 6 S A R R[R] / Subtype for HA and A: H7 Select Segments: S HA Feature type: S Functional Amino Acid Coordinates: Start

10 Feature Variant ypes utorial his component of IRD provides data on specific characteristic regions and/or sub regions termed ' Features' (SF) defined for all influenza virus proteins. he SFs and their metadata are derived from scientific literature and/or public domain databases. Variant types (V) of SFs are computed by multiple sequence alignments of all relevant protein sequences in IRD. Variant types that carry a mutation(s) that has been experimentally determined to give rise to a phenotype, such as increased virulence are denoted as a Phenotype Variant ype (PV). hese PV annotations are only available for a subset of the influenza virus subtypes. For more information about these PV annotations, click here. ote: V is not always a functional epitope. For more information about using SFVs, click here. For a detailed description of the development and application of the SFV approach for the study of influenza virus, please read this scientific article: oronha JM, Liu M, Squires RB, Pickett BE, Hale BG, Air GM, Galloway SE, akimoto, Schmolke M, Hunt V, lem E, García Sastre A, McGee M, Scheuermann RH. (0) Feature Variant ype (Flu SFV) analysis: evidence for a role of S in influenza host range restriction. J Virol, 86: doi: 0.8/JVI PMID: Go to Feature List Results matching your criteria: VIRUS YPE A B C SEQUECE FEAURE YPE SUBYPE FOR HA AD A H7 * Use comma to separate multiple entries. Ex: H, H3, H7,,. AMIO ACID COORDIAES Start: 35 o: SELEC SEGMES EYWORD SEARCH * Use comma to separate multiple entries. Ex: alpha helix, Beta strand, IEDB: Clear Search e. he Feature search results will be displayed. Feature Variant ypes Your Selected Items: 0 items selected Download Save Search Your search returned record. Search Criteria Displaying 50 records per page, sorted by Feature Identifier in Display Settings ascending order. Select all results More columns were returned than can be displayed without scrolling. Use scroll bars at top and bottom of display to move right and left or reduce the number of columns displayed by using the Display Settings link above. Feature Identifier Feature Variant Feature Category Amino Acid Position Evidence Comments ame ypes Length A_H7_SF6 A_H7_SF7 A_H7_SF8 A_H7_determinantof receptorbinding_9() A_H7_determinantof receptorbinding_35() A_H7_determinantof receptor binding_99(6) 7 functional 9( HA),38(0 HA) PubMed:08086 Structural results suggest a novel mechanism by which residues Arg and Arg9 (H3 numbering) are use to compensate for the deletion of th 0 loop and form interactions with t receptor analogs. 8 functional 35(7 HA),37(9 HA) PubMed: wo specific mutations; Q6L and G8S in glycan receptor binding si of H7 HA substantially increase its binding affinity to human receptor functional 99(8 HA) 59 PubMed:356 Solid phase receptor binding assay are used to characterize receptorbinding profiles of H7 influenza virus isolated from aquatic birds, land bas poultry, and horses in Eurasia and America A_H7_SF0 A_H7_determinantofvirulence_333(0) 0 80 functional 333(35 HA) 3 PubMed:80077 A polybasic cleavage site (pcs) in th hemagglutinin (HA) is required for hig pathogenicity. Introduction of three different pcs motifs (PEIPRRRR/GLF from A/chicken/Germany/R8/003(H77 PEIPGSRVRR/GLF from A/chicken/Italy/5/999(H7), an PEIPRHRGR/GLF from A/chicken/England/635/05(H7 into three different low pathogenic H77 viruses (A/turkey/Germany/R/00, A/chicken/Germany/R36/0, an A/turkey/Germany/R53/03) conferred increased virulence in chickens. f. Click View for SF7. i. Examine this Feature following instructions described in 5.b. ii. Q35L (6 in H3 coordinates) enabled the H79 viruses to have a mixed alpha-,3/alpha-,6 receptor preference, which increased binding to mammalian-like receptors in the human upper airway. he very early strains of the 03 outbreak including A/Shanghai//03 and A/Shanghai/JS0/03 carry Q at this position. Later human H79 isolates from 03 through 05 all carry L, suggesting that these isolates have already evolved to fit human receptors. he 3 human isolates from Guangdong from 07 harbor Q instead of L, suggesting that they may have originated from an avian source. 0

11 Discussion. Has the H79 virus evolved over the seasons? he H79 virus has evolved over time. During the first wave of the outbreak in 03, all viruses form a single lineage. In fall 0, two separate lineages emerged. One of them evolved into the 0-07 lineage.. Can you infer the possible origin of the 07 human isolates? Based on the human H79 tree and the unique substitutions found in the 07 Guangdong cluster, these 07 human isolates may have originated from a different introduction event. References. oronha JM et al. virus sequence feature variant type analysis: evidence of a role for S in influenza virus host range restriction. J Virol. 0, 86(0): Pickett BE et al. Metadata-driven comparative analysis tool for sequences (meta-cas): an automated process for identifying significant sequence variations that correlate with virus attributes. Virology. 03, 7(-): FAO. H79 situation update, 6 July 07. Retrieved from: Zmasek CM et al. AV: Display and manipulation of annotated phylogenetic trees. Bioinformatics. 00, 7: