Project Tycho datasets contain case counts for reported disease conditions for countries around the world. The Project Tycho data curation team extracts these case counts from various reputable sources, typically from national or international health authorities, such as the US Centers for Disease Control or the World Health Organization. These original data sources include both open- and restricted-access sources. For restricted-access sources, the Project Tycho team has obtained permission for redistribution from data contributors. All datasets contain case count data that are identical to counts published in the original source and no counts have been modified in any way by the Project Tycho team. The Project Tycho team has pre-processed datasets by adding new variables, such as standard disease and location identifiers, that improve data interpretabilty. We also formatted the data into a standard data format.

Each Project Tycho dataset contains case counts for a specific condition (e.g. measles) and for a specific country (e.g. The United States). Case counts are reported per time interval. In addition to case counts, datsets include information about these counts (attributes), such as the location, age group, subpopulation, diagnostic certainty, place of aquisition, and the source from which we extracted case counts. One dataset can include many series of case count time intervals, such as "US measles cases as reported by CDC", or "US measles cases reported by WHO", or "US measles cases that originated abroad", etc.

Depending on the intended use of a dataset, we recommend a few data processing steps before analysis:

• Analyze missing data: Project Tycho datasets do not inlcude time intervals for which no case count was reported (for many datasets, time series of case counts are incomplete, due to incompleteness of source documents) and users will need to add time intervals for which no count value is available. Project Tycho datasets do include time intervals for which a case count value of zero was reported.
• Separate cumulative from non-cumulative time interval series. Case count time series in Project Tycho datasets can be "cumulative" or "fixed-intervals". Cumulative case count time series consist of overlapping case count intervals starting on the same date, but ending on different dates. For example, each interval in a cumulative count time series can start on January 1st, but end on January 7th, 14th, 21st, etc. It is common practice among public health agencies to report cases for cumulative time intervals. Case count series with fixed time intervals consist of mutually exxclusive time intervals that all start and end on different dates and all have identical length (day, week, month, year). Given the different nature of these two types of case count data, we indicated this with an attribute for each count value, named "PartOfCumulativeCountSeries".

Influenza A viruses (IAV) circulate endemically among many wild aquatic bird populations that seasonally migrate between wintering grounds in southern latitudes to breeding ranges along the perimeter of the circumpolar arctic. Arctic and subarctic zones are hypothesized to serve as ecologic drivers of the intercontinental movement and reassortment of IAVs due to high densities of disparate populations of long distance migratory and native bird species present during breeding seasons. Iceland is a staging ground that connects the East Atlantic and North Atlantic American flyways, providing a unique study system for characterizing viral flow between eastern and western hemispheres. Using Bayesian phylodynamic analyses, we sought to evaluate the viral connectivity of Iceland to proximal regions and how inter-species transmission and reassortment dynamics in this region influence the geographic spread of low and highly pathogenic IAVs. Findings demonstrate that IAV movement in the arctic and subarctic follows seabird migration around the perimeter of the circumpolar north, favoring short-distance flights between proximal regions rather than long distance flights over the polar interior. Iceland connects virus movement between mainland Europe and North America, particularly due to the westward migration of wild birds from mainland Europe to Northeastern Canada and Greenland. Though virus diffusion rates were similar among avian taxonomic groups in Iceland, gulls act as recipients and not sources of IAVs to other avian hosts prior to onward migration. These data identify patterns of virus movement in northern latitudes and inform future surveillance strategies related to seasonal and emergent IAVs with pandemic potential. Methods Field sample collection From May 2010 through February 2018, we obtained IAV isolates from various species of seabirds, shorebirds, and waterfowl as well as environmental sampling of avian fecal material from locations throughout Iceland (capture and swab data can be found here: https://doi.org/10.5066/XX (Dusek et al. 202X)). Live sampled birds were captured using a 18m x 12m cannon-propelled capture net, noose pole, or hand capture. Birds found dead or moribund were also sampled. Hunter-harvested waterfowl and fisheries-bycatch seabirds were sampled as available. All birds were identified to species and, for live birds, individually marked with metal bands. Age characteristics were determined and age was documented for each bird according to the following schemes adapted from U.S. Geological Survey year classification codes: hatched in same calendar year as sampling (1CY), hatched previous calendar year (2CY), hatched previous calendar year or older, exact age unknown (2CY+), hatched three calendar years prior to sampling (3CY), hatched four calendar years prior to sampling (4CY), hatched more than four calendar years prior to sampling (4CY+), or unknown if age could not be determined (U) (Olsen KM, 2004; Prater, Marchant, & Vuorinen, 1977; USGS, 2020). Due to species specific differences, not all aging categories could be applied to all species sampled. All live birds were immediately released following completion of sampling. To sample for IAV, a single polyester-tipped swab was used to swab the cloaca only (2010-2013) or to first swab the oral cavity then the cloaca (2014-2017). Opportunistic environmental sampling of fecal material was also conducted using a direct swabbing method (2018). Each swab sample was immediately placed in individual cryovials containing 1.25 ml viral transport media (Docherty & Slota, 1988). Vials were held on ice for up to 5 hours prior to being stored in liquid nitrogen or liquid nitrogen vapor. Samples were shipped on dry ice from Iceland to Madison, Wisconsin, USA by private courier with dry ice replenishment during shipping. Once received in the laboratory, samples were stored at -80o C until analysis. Virus extraction, RT-PCR, virus isolation Viral RNA was extracted from swab samples using the MagMAXTM-96 AI/ND Viral RNA Isolation Kit (Ambion, Austin, TX) following the manufacturer’s procedures. Real-time RT-PCR was performed using previously published procedures, primers, and probes (Spackman et al., 2002) designed to detect the IAV matrix gene. RT-PCR assays utilized reagents provided in the Qiagen OneStep® RT-PCR kit. Virus isolation was performed in embryonating chicken egg culture on all swab samples exhibiting positive Ct values from RT-PCR analysis (Woolcock, 2008), with a primary cut off value of 45 on primary screen and 22 on secondary screen. All virus isolates were screened for the presence of H5 and H7 IAV subtypes using primers and probes specific for those subtypes (Spackman et al., 2002). Egg-grown virus isolates were sequenced using multiple standard methods including Sanger, Roche 454, and Illumina (HiSeq 2000 and MiSeq) sequencing (Dusek et al., 2014; Guan et al., 2019; Hall et al., 2014). Datasets for phylodynamic analyses Global dataset: The PB2 segment was selected as the basis for phylodynamic analysis. Advantages of focusing on PB2 - the largest internal segment of the IAV genome - include maximizing the number of nucleotides in the analysis (>2000 nts) and investigating transmission dynamics without targeting a specific subtype. All available avian and marine mammal IAV PB2 genes sequenced between 2009 and 2019 globally were downloaded from the National Center for Biotechnology Information Influenza Virus Resource database (NCBI IVR) (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) on February 12, 2020, resulting in 13,469 sequences. Duplicate sequences (based on collection date, location, and nucleotide content) and sequences with less than 75% unambiguous bases were removed, and all vaccine derivative and laboratory-synthesized recombinant sequences were excluded. Sequences in the dataset were only included if isolation dates, location, and host species were available, resulting in 7,245 remaining sequences. Downsampling of taxa: The downsampling strategy aimed to reduce the number of sequence taxa for computation and mitigate sampling bias while maintaining the genetic diversity in the dataset. Four variables were considered important for explaining genetic diversity in the IAV sequence dataset: geographic region, host taxa, sampling year, and hemagglutinin (HA) subtype. Geographic regions included North America, Europe, Iceland, Asia, Africa, and South America (Australia and Antarctica were removed due to insufficient sequence counts). HA subtypes included H1, H2, H3, H4, H6, H8, H10, H11, H12, H13, H14, H15, H16, and pooled H5/7/9. H5, H7, and H9 were combined, as these were over-represented in the global dataset. Host categories included Anseriformes, Charadriiformes, Galliformes, and Other, which comprised all other avian taxa and marine mammals. To inform the downsampling strategy and evaluate if any of the four variables were correlated, a multiple correspondence analysis (MCA) was performed (JMP Pro v.14.0.0 (JMP Version 14.0.0, 1989-2019)). The MCA uses categorical data as input, which for this study included the sampling metadata associated with each sequence (region, host taxa, year, and HA subtype). Through representation of the variables in two-dimensional Euclidean space, significant clustering of HA subtypes with host taxa was detected (Supplementary Fig. 1), indicative of host-specific subtypes that are a well-known feature of influenza. These findings confirmed by previously published data on species-specificity of HA subtypes (Byrd-Leotis, Cummings, & Steinhauer, 2017; Long, Mistry, Haslam, & Barclay, 2019; Verhagen et al., 2015) led us to downsample the dataset stratifying taxa by two non-overlapping variables: geographic region and HA subtype. Data were downsampled to maintain 21-75 taxa per geographic region category and 6-30 per HA subtype category, resulting in a total of 301 sequences (outgroup). This step was performed to mitigate sampling bias resulting in over-representation of species or viral strains, while accounting for genetic diversity in the dataset. Next, to ensure relative evenness of geographic state groupings for discrete trait analyses, virus sequences from Iceland (n=93) were downsampled by stratifying taxa by HA subtype and maintaining 1-15 sequences per category, resulting in 63 sequences (ingroup). These 63 sequences were used for global and local discrete trait analyses and reflected the composition of diverse subtypes by host for the full Iceland sequence dataset. The resulting dataset reflected the underlying composition of host-specific subtypes present in this localized system. To assist with rooting and time-calibration of the tree, historical avian sequences from NCBI IVR were downloaded for the years 1979-2008. These were downsampled by year to ensure one sequence per year, resulting in 30 historic sequences. The total downsampled dataset, including the outgroup (n=301), ingroup (n=63), and historic sequences (n=30) resulted in a total of 394 sequences. Europe-Iceland-North America Datasets: To elucidate viral dynamics between significant source regions and Iceland and within-Iceland phylodynamics, a second analysis was performed at a restricted scale to Europe, Iceland, and North America. The cleaned global dataset described above (n=7245) was downsampled to include significant source regions of North America (n=3222) and Europe (n=407), totaling 3629 sequences. To identify at lower spatial resolution the source/sink locations relevant to Iceland, a K-means cluster analysis was performed (JMP Pro v.14.0.0 (JMP Version 14.0.0, 1989-2019)) using latitude/longitude coordinates for each of the 3629 sequences (obtained by extracting sampling location from the strain name of each sequence and searching in www.geonames.org). A total of 20 intraregional clusters resulted in highest support. Identified clusters with <50 sequences were combined with geographically proximal

The introduction of HPAI H5N1 clade 2.3.4.4b viruses to North America in late 2021 resulted in avian influenza outbreaks in poultry, mortality events in many wild bird species, and spillover into many mammalian species. Reassortment events with North American low pathogenic virus were identified as early as February 2022 and over 100 genotypes have been characterized. Such diversity increases the complexity and time required for monitoring virus evolution. Here, we performed ordination and clustering analyses on sequence data from H5N1 viruses identified in North America between January 2020 to December 2023 to visualize virus genotypic diversity in poultry and wildlife populations. Our results reveal that ordination and cluster-based approaches can complement traditional phylogenetic analyses specifically for the preliminary assignment of H5N1 viruses to genotypic groups or to identify novel genotypes. Our study expands current knowledge on genotype diversity of H5N1 viruses in North America and describes a rapid approach for early virus genotype assignment.

Highly pathogenic avian influenza virus (HPAIV) has caused extensive mortalities in wild birds, with a disproportionate impact on raptors since 2021. The population-level impact of HPAIV can be informed by telemetry studies that track large samples of initially healthy, wild birds. We leveraged movement data from 71 rough-legged hawks (Buteo lagopus) across all major North American migratory bird flyways concurrent with the 2022–2023 HPAIV outbreak and identified a total of 29 mortalities, of which 11 were confirmed, and an additional ~9 were estimated to have been caused by HPAIV. We estimated a 28% HPAIV cause-specific mortality rate among rough-legged hawks during a single year concurrent with the HPAIV outbreak in North America. Additionally, the overall annual mortality rate during the HPAIV outbreak (47%) was significantly higher than baseline annual mortality rates (3–17%), suggesting that HPAIV-caused deaths were additive above baseline mortality levels. HPAIV mortalities were c..., , # Increased mortality rates caused by highly pathogenic avian influenza virus in a migratory raptor

Dataset DOI: 10.5061/dryad.n2z34tn92

Description of the data and file structure

We leveraged movement data from GPS-tracked rough-legged hawks Buteo lagopus that coincided with the HPAIV panzootic in North America to determine its effect on annual mortality. All missing and unavailable data represented as NAÂ

Files and variables

File: AnnualMortality.csv

Description:Â spreadsheet used to analyze the HPAIV effect on annual mortality.

Variables

• index: index number
• tagid: transmitter ID (unique to an individual hawk)
• year: 12-month study period. 2020 = 1-Mar-2020 to 28-Feb-2021, etc...
• date_begin: date within the 12-month study period that tracking began
• date_end:Â date within the 12-month study period that tracking ended
• duration: tracking duration (number of days)
• fate: individual fate during the 12-month study period...,

Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both breeds in the COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken breeds. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both breeds of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.MethodsViruses. All procedures using infectious material were reviewed and approved by the Institutional Biosafety Committee of US National Poultry Research Center (USNPRC), US Department of Agriculture-Agricultural Research Service, Athens, GA. The HPAI virus isolate A/turkey/Indiana/22-003707-003/2022 H5N1 (TK/IN/22) was provided by Dr. Mia Torchetti, National Veterinary Services Laboratories, US Department of Agriculture-Animal and Plant Health Inspection Service, Ames, IA. The A/Vietnam/1203/2004 H5N1 HPAI virus (Viet/04), A/Whooper Swan/Mongolia/244/2005 H5N1 (WS/Mongolia/05) HPAI virus, and A/Flycatcher/CA/14875-1/1994 H7N1 low pathogenic avian influenza virus isolates were provided by the repository at the USNPRC. Virus isolates were propagated and titrated in SPF embryonating chicken eggs using standard procedures [1]. Titers were determined using the Reed-Muench method [2].Vaccines. Two commercial rHVT-H5 vaccines were selected because they are licensed in the US (and may be licensed elsewhere) and were supplied by the manufacturers: 2.2-HVT (Vectormune HVT AIV, Ceva Animal Health LLC, Lenexa, KS) (serial 395-134); and COBRA-HVT (Vaxxitek HVT+IBD+H5, Boehringer-Ingelheim Animal Health USA, Ridgefield, CT) (serial EW003). The amino acid similarity between the vaccine antigens and the challenge virus HA1 was 91.7% (COBRA-HVT) and 91.2% (2.2-HVT).Challenge study design. All animal work was reviewed and approved by the USNPRC Institutional Animal Care and Use Committee. Mixed sex, SPF WL chickens (Gallus gallus domesticus) were obtained at hatch from in-house flocks. Broiler chicken eggs were obtained from a commercial hatchery at 18 days of incubation prior to administration of any in ovo vaccines and were hatched on-site. All birds were randomly assigned to vaccine groups based on breed. Vaccine groups are shown in Table 1. All vaccines were prepared and administered on the day of hatch by the subcutaneous route at the nape of the neck in accordance with the manufacturer’s instructions (0.2ml per chicken). Serum was collected from all chickens 25 days post vaccination to evaluate the antibody response to the vaccines.Four weeks post vaccination (four weeks of age) chickens were challenged with a target dose 6.0log10 50% egg infectious doses (EID50) per bird of TK/IN/22 in 0.1ml by the intrachoanal route (titration of the challenge virus after dilution confirmed the challenge dose to be 6.7log10 EID50 per bird). Oropharyngeal and CL swabs were collected from all birds at 2-, 4-, and 7-days post challenge (DPC). Swabs were also collected from dead and euthanized birds.To evaluate antibody-based DIVA-VI tests, serum was collected at 7-, 10- and 14DPC. Mortality and morbidity were recorded for 14DPC. Surviving birds were euthanized at 14DPC. If birds were severely lethargic or presented with neurological signs, they were euthanized and were counted as mortality at the next observation time for mean death time calculations. Euthanasia was performed by cervical dislocation in accordance with American Veterinary Medical Association guidelines.Quantitative rRT-PCR (qRRT-PCR). RNA was extracted from OP and CL swabs using the MagMax magnetic bead extraction kit (Thermo Fisher Scientific, Waltham, MA) with the wash modifications as described by Das et al., [3]. Quantitative real-time RT-PCR was conducted as described previously [4] on a QuantStudio 5 (Thermo Fisher Scientific) instrument. A standard curve was generated from a titrated stock of TK/IN/22 and was used to calculate titer equivalents using the real time PCR instrument’s software.Hemagglutination inhibition assay. Hemagglutination inhibition (HI) assays were run in accordance with standard procedures [5]. All pre-challenge sera collected at 25 days post vaccination were tested against the challenge virus and the closest isolates available to the vaccine antigens. The serum from the 2.2-HVT group was tested against WS/Mongolia/05 (99.3% similarity) and the serum from the COBRA-HVT group was tested against Viet/04 (98.2% similarity). Titers of eight or below were considered negative.Commercial ELISA. A commercial AIV antibody ELISA (AI Ab Test, IDEXX laboratories, Westbrook, ME) was used in accordance with the manufacturer’s instructions. Sera were tested to detect anti-NP antibodies pre-challenge (25days pos-vaccination) and at 7-, 10- and 14DPC.Enzyme-linked lectin assay (ELLA) for detection of neuraminidase inhibition (NI) antibody. The ELLA was performed as previously described with minor modifications [6, 7]. Briefly, the NA activity of a beta-propiolactone inactivated H7N1 virus (A/Flycatcher/CA/14875-1/1994) was quantified to determine the effective concentration (EC) of antigen. The 98% EC (EC98) of antigen was subsequently used for the ELLA-NI assays. For ELLA-NI assay, the antigen and serum mixture was incubated overnight (approximately18hr) at 37°C and the NA activity was determined following the procedure as described in Spackman et al. [7]. The average background absorbance value was subtracted from the sample absorbance value then that value was divided by the average values of wells with only NA antigen. This value was multiplied by a factor of 100 to calculate the percent NA activity. The percent NI activity of individual serum samples was determined by subtracting the percent NA activity from 100%. A cut-off value for positive NI activity was determined by adding three standard deviations to the mean NI activity of pre-challenge sera (i.e., NA antibody negative sera) of each corresponding group of chickens at 7-, 10- and 14DPC. Each serum was tested at dilutions of 1:20 and 1:40.References.1. Spackman E, Killian ML. Avian Influenza Virus Isolation, Propagation, and Titration in Embryonated Chicken Eggs. Methods Mol Biol. 2020;2123:149-64. Epub 2020/03/15.2. Reed LJ, Muench H. A simple method for estimating fifty percent endpoints. American Journal of Hygiene. 1938;27:493-7.3. Das A, Spackman E, Pantin-Jackwood MJ, Suarez DL. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR. Journal of Veterinary Diagnostic Investigation. 2009;21(6):771-8.4. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. Journal of Clinical Microbiology. 2002;40(9):3256-60.5. Spackman E, Sitaras I. Hemagglutination Inhibition Assay. Methods Mol Biol. 2020;2123:11-28. Epub 2020/03/15.6. Bernard MC, Waldock J, Commandeur S, Strauss L, Trombetta CM, Marchi S, et al. Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study. Front Immunol. 2022;13:909297. Epub 20220617.7. Spackman E, Suarez DL, Lee CW, Pantin-Jackwood MJ, Lee SA, Youk S, Ibrahim S. Efficacy of inactivated and RNA particle vaccines against a North American Clade 2.3.4.4b H5 highly pathogenic avian influenza virus in chickens. Vaccine. 2023. Epub 20231104.