Males and females from resistant Fayoumi and susceptible Leghorn chicken lines were either challenged with a lentogenic strain of Newcastle Disease virus or given a mock infection at 3 weeks of age. The lung transcriptomes generated by RNA-sequencing were studied using contrasts across the challenged and non-challenged birds, the two lines, and three time points (2,6, and 10 days post-infection) using Weighted Gene Co-expression Network Analysis (WGNCA). The data can be retrieved by navigating to https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5859/. Click the Download button to access the Sample and data relationship dataset file.

To determine the gender impact on the immune response of chickens, the mRNA was isolated and sequenced from the lungs of 48 chickens of 2 lines as three time-points post-infection (2,6, and 10 days post-infection), and in two treatment groups. The data can be retrieved by navigating to https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5859/.

To determine the gender impact on the immune response of chickens, the mRNA was isolated and sequenced from the lungs of 48 chickens of 2 lines as three time-points post-infection (2,6, and 10 days post-infection), and in two treatment groups. The data can be retrieved by navigating to https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5859/. Click the Download button to access the Sample and data relationship dataset file.

,Treatment groups of ducks were exposed to different virus doses (2, 4, 6 log10 50% egg infectious doses) and by different routes (contact or intrachoanal). The experimental setting was a laboratory with animal care as approved by the institutional animal care and use committee as appropriate for the species and age of bird. Data are the virus titers shed by the oral and cloacal route for individual mallard ducks exposed to H5N1 highly pathogenic avian influenza virus by day post exposure. Samples were collected through 11 days post exposure. Virus titer equivalents were determined by quantitative real-time RT-PCR. Serological data are serum antibody titers to the challenge virus as determined by hemagglutination inhibition assay (reciprocal of the log2 dilution) determined with serum collected 10 or 11 days post exposure.,,

,Tabulated individual data points for data reported in the associated publication: 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 Nov 30;41(49):7369-7376. doi: 10.1016/j.vaccine.2023.10.070. Epub 2023 Nov 4. PMID: 37932132.,The highly pathogenic avian influenza virus (HPAIV) isolate A/turkey/Indiana/22-003707-003/2022 H5N1 (TK/IN/22) and A/Gyrfalcon/Washington/41088/2014 H5N8 (GF/WA/14) isolate were each propagated and titrated in embryonating specific pathogen free (SPF) chicken eggs using standard procedures and titers were determined using the Reed-Muench method.,An in-house vaccine was produced by de novo synthesizing the HA gene of TK/IN/22 that was modified to be low pathogenic (LP) and placing it in a PR8 backbone using rg methods as described . The vaccine (SEP-22-N9) contained 6 genes from PR8 and a de novo synthesized N9 NA from A/blue winged teal/Wyoming/AH0099021/2016 (H7N9). The rg virus was inactivated by treatment with 0.1% beta-propiolactone. Vaccines were produced with Montanide ISA 71 VG (Seppic Inc., Fairfield, NJ) adjuvant at ambient temperature in a L5M-A high shear mixer (Silverson Machines, Inc., East Longmeadow, MA) for 30sec at 1,000rpm, then for 3min at 4,000rpm using an emulsifying screen in accordance with the adjuvant manufacturer’s instructions.,Sham vaccine was prepared in-house using sterile phosphate buffered saline as described above.,Commercial vaccines were supplied by the manufacturers. The commercial inactivated vaccine (1057.R1 serial 590088) (rgH5N1) (Zoetis Inc., Parsippany, NJ) was produced with the GF/WA/14 (clade 2.3.4.4c HA gene) and the remaining 7 gene segments including the NA from PR8 (1). The Sequivity vaccine (serial V040122NCF) (RP) (Merck and Co. Inc., Rahway, NJ) is an updated version of their replication restricted alphavirus vector vaccine that expresses the TK/IN/22 H5 HA (modified to be low pathogenic LP).,Three-week-old, mixed sex, SPF white leghorn chickens (Gallus gallus domesticus) were obtained from in-house flocks and were randomly assigned to vaccine groups.,All vaccines were administered by the subcutaneous route at the nape of the neck. Commercial vaccines were given at the volumes instructed by the manufacturer (0.5ml each). In-house vaccine was given at a dose of 512 hemagglutination units per bird in 0.5ml. Three weeks post vaccination chickens were challenged with 6.7 log10 50% egg infectious doses (EID50) of TK/IN/22 in 0.1ml by the intrachoanal route.,Oropharyngeal (OP) and cloacal (CL) swabs were collected from all birds at 2-, 4-, and 7-days post challenge (DPC). Swabs were also collected from dead and euthanized sham vaccinates at 1DPC.,To evaluate antibody-based DIVA-VI tests, blood for serum was collected from the RP and SEP-22-N9 vaccinated groups at 7, 10 and 14DPC because the SEP-22-N9 vaccine does not elicit antibodies to N1 and the RP vaccine does not elicit antibodies to the N1 or NP proteins.,Mortality and morbidity were recorded for 14DPC after which time the remaining birds were euthanized. If birds were severely lethargic or had neurological signs they were euthanized and were counted as mortality at the next observation time for mean death time calculations.,To determine if there was a difference in antibody levels based on the order of vaccination with the RP vaccine and an inactivated vaccine, groups of 20 chickens (hatch-mates of the chickens in the challenge study) were given one dose of each vaccine three weeks apart (Supplementary Table 1). The first dose was administered at three weeks of age using the RP or SEP-22-N9 vaccine as described above. Then a second dose of either the same vaccine or the other vaccine was administered three weeks later (six weeks of age). All birds were bled for serum three weeks after the second vaccination (nine

Data from influenza A virus (IAV) infected ferrets (Mustela putorius furo) provides invaluable information towards the study of novel and emerging viruses that pose a threat to human health. This gold standard animal model can recapitulate many clinical signs of infection present in IAV-infected humans, supports virus replication of human and zoonotic strains without prior adaptation, and permits evaluation of virus transmissibility by multiple modes. While ferrets have been employed in risk assessment settings for >20 years, results from this work are typically reported in discrete stand-alone publications, making aggregation of raw data from this work over time nearly impossible. Here, we describe a dataset of 333 ferrets inoculated with 107 unique IAV, conducted by a single research group (NCIRD/ID/IPB/Pathogenesis Laboratory Team) under a uniform experimental protocol. This collection of ferret tissue viral titer data on a per-individual ferret level represents a companion dataset to ‘An aggregated dataset of serially collected influenza A virus morbidity and titer measurements from virus-infected ferrets’. However, care must be taken when combining datasets at the level of individual animals (see PMID 40245007 for guidance in best practices for comparing datasets comprised of serially-collected and fixed-timepoint in vivo-generated data). See publications using and describing data for more information: Kieran TJ, Sun X, Tumpey TM, Maines TR, Belser JA. 202X. Spatial variation of infectious virus load in aggregated day 3 post-inoculation respiratory tract tissues from influenza A virus-infected ferrets. Under peer review. Kieran TJ, Sun X, Maines TR, Belser JA. 2025. Predictive models of influenza A virus lethal disease: insights from ferret respiratory tract and brain tissues. Scientific Reports, in press. Bullock TA, Pappas C, Uyeki TM, Brock N, Kieran TJ, Olsen SJ, Davis CD, Tumpey TM, Maines TR, Belser JA. 2025. The (digestive) path less traveled: influenza A virus and the gastrointestinal tract. mBio, in press. Kieran TJ, Sun X, Maines TR, Beauchemin CAA, Belser JA. 2024. Exploring associations between viral titer measurements and disease outcomes in ferrets inoculated with 125 contemporary influenza A viruses. J Virol98: e01661-23. https://doi.org/10.1038/s41597-024-03256-6 Related dataset: Kieran TJ, Sun X, Creager HM, Tumpey TM, Maine TR, Belser JA. 2025. An aggregated dataset of serial morbidity and titer measurements from influenza A virus-infected ferrets. Sci Data, 11(1):510. https://doi.org/10.1038/s41597-024-03256-6 https://data.cdc.gov/National-Center-for-Immunization-and-Respiratory-D/An-aggregated-dataset-of-serially-collected-influe/cr56-k9wj/about_data Other relevant publications for best practices on data handling and interpretation: Kieran TJ, Maines TR, Belser JA. 2025. Eleven quick tips to unlock the power of in vivo data science. PLoS Comput Biol, 21(4):e1012947. https://doi.org/10.1371/journal.pcbi.1012947 Kieran TJ, Maines TR, Belser JA. 2025. Data alchemy, from lab to insight: Transforming in vivo experiments into data science gold. PLoS Pathog, 20(8):e1012460. https://doi.org/10.1371/journal.ppat.1012460

,To enhance the efficacy of the current Newcastle disease vaccine, we have tested two potential adjuvants (Imiquimod and ODN-1826) in chickens. Birds were treated with PBS, Imiquimod or ODN-1826 (50 µg/bird) or vaccinated intranasally with live LaSota strain with or without Imiquimod or ODN-1826 (50 µg/bird). Two weeks after vaccination, birds were challenged with virulent New-castle disease virus (chicken/CA/212676/2002). The experimental setting was a laboratory with animal care as approved by the institutional animal care and use committee as appropriate for the species and age of bird. Data are the serum antibody titers to the vaccine or challenge virus as determined by hemagglutination inhibition assay. Virus shedding titers by the oral and cloacal route for individual birds exposed to virulent Newcastle disease virus were determined by quantitative real-time RT-PCR. Expression of antiviral genes from tissues collected at 1 and 3 days after treating 2 week old SPF chickens with adjuvant and/or vaccines were determined by quantitative PCR.,Resources in this dataset:,

Raw sequencing data as generated by the five different methods used are provided for each of the three samples used in the comparison. The files are in FASTQ format as exported from the Oxford Nanopore’s MK1C using MinION flowcells. Files are labeled according to the method (as described in the paper) and the Sample ID). The MK1C exports data in blocks of 6000 reads per FASTQ file and all the FASTQ files from each method and sample are grouped in a common folder.

,Data are the individual group values for oral and cloacal virus shedding and antibody titers for reach treatment group from: Mo et al., The pathogenicity and transmission of live bird market H2N2 avian influenza viruses in chickens, Pekin ducks, and guinea fowl. Vet Mic 260:109180, 2021. https://doi.org/10.1016/j.vetmic.2021.109180,Methods: Six H2N2 low pathogenic avian influenza viruses from US LBMs were selected based on recency and to represent the different genotypes present in the live birds markets during the time period (i.e., the presence or absence of a NA stalk deletion): A/duck/PA/14-030488-5/2014 (Dk/PA/14), A/chicken/NY/16-032621-2/2016 (Ck/NY/16), A/chicken/CT/17-008911-4/2017 (Ck/CT/17), A/chicken/NY/18-002471-4/2018 (CK/NY/02471/18), A/chicken/NY/18-042097-3/2018 (Ck/NY/042097/18) and A/chicken/NY/19-012787-1/2019 (Ck/NY/19). Isolates were evaluated in White Leghorn chickens (Gallus gallus), guinea fowl (Numida meleagris) and Pekin ducks (Anas platyrhynchos). Chickens and guinea fowl were challenged at 4 weeks of age and Pekin ducks were challenged at 2 weeks of age with 6log10 of virus by the intra-choanal route. “Contact” birds, which were hatch-mates of the inoculated birds, were co-housed with the inoculated birds 24hrs post inoculation to evaluate transmission. Viral loads in OP and CL swabs collected at 2, 4, 7, 10, and 14 days post inoculation were determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). RNA was extracted from swabs using the MagMAX96 Viral RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) and the KingFisher Flex Magnetic Particle Processing System (Thermo Fisher Scientific), with an additional wash step to remove inhibitors (Das et al., 2009). The qRT‐PCR for AIV detection was conducted based on the standard USDA M gene AIV qRT‐PCR procedure (Spackman et al., 2002) using an Applied Biosystems® 7500 Fast Real‐Time PCR system (Thermo Fisher Scientific). Cycle threshold (Ct) values were determined by the 7500 Fast Software v2.3. For relative quantification, Ct values were converted to titer equivalents based on the standard curve method (Larionov et al., 2005). Values were established from ten-fold dilutions of the same titrated stock of the virus used to challenge the birds. The limit of detection was determined to be 0.8Log10 per reaction. Serological testing for antibodies to the virus utilized the hemagglutination inhibition (HI) assays using homologous antigens were performed to quantify antibody responses with serum collected from chickens, guinea fowl and Pekin ducks at 14 dpi based on the standard protocol (OIE, 2019). HI titers were reported as reciprocal log2 titers, and titers greater than 3 log2 (1:8) were considered positive.,,