Clinical sample processing and animal ethics
We received five free-range layer duck carcasses (12 weeks old) from a single farm in Alappuzha district (Village: Cheruthana, Block: Haripad; Latitude: 9.34139; Longitude: 76.4424) in Kerala State, India, on November 3, 2022. The region lies within the Kuttanad delta region on the west coast of Kerala, where rice is cultivated below sea level.¹² It is home to the Vembanad-Kol wetland (Ramsar site), where many migratory waterfowl from the Central Asian Flyway visit during winter migration (October-March). The birds had a history of corneal opacities, torticollis, and unusual mortality. An autopsy was performed, and organs were collected from all birds. Approximately 100 mg of pooled organ sample was triturated in 900 µL of 1X PBS (pH 7.2) using 2.2 mm bead and tissue homogenizer iRUPT24P (Neuation). The homogenizer runs at 3000 rpm for 10 cycles, with a run time and pause time of 15 seconds each and an agitation speed of 5 m/s. The tissue triturate was then centrifuged at 3000 rpm for 5 minutes. Animal experimentation and sample processing were carried out in the animal and laboratory wings, respectively, of the BLS III containment laboratory in accordance with the Animal Ethics Committee of the ICAR-National Institute of High Security Animal Diseases, Bhopal (M.P.), under approval No. 131/IAEC/NIHSAD/22.

RNA extraction and real-time RT-PCR
Viral RNA was extracted from the supernatant of the tissue triturate using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. Influenza A virus typing and HA and NA subtyping were performed using the GoTaq® Probe 1-Step RT-qPCR System (Promega, USA).^5,13,14

Virus isolation
Isolation of the virus was performed in both 9 day-old specific pathogen-free (SPF) chicken eggs and 12 day-old duck eggs as described earlier.¹⁵ The supernatant of the tissue triturate was used as an inoculum for virus propagation in eggs. A total of 800 µL of inoculum was prepared by treating the supernatant with 2X antibiotics (Antimycotic mix and Gentamicin). The eggs were then inoculated with 100 µL of inoculum each via the allantoic cavity route. The eggs were kept in a humid incubator at 37 °C till the death of the embryo. All the eggs were screened every 12 hours, and the dead eggs were chilled at 4 °C. The infected allantoic fluid was harvested aseptically and stored in 1 mL aliquots at -80 °C until further use. Hemagglutination inhibition (HI) assay with H5-subtype-specific sera was used to confirm the viral subtype.¹⁵

Intravenous pathogenic index
Intravenous pathogenicity index (IVPI) of the virus (harvested from chicken eggs) was estimated using 6 week-old SPF chickens as per the WOAH guidelines.¹⁵

RNA extraction, sequencing and analysis
The RNA was extracted from infected allantoic fluid collected from duck eggs using the QIAamp® Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The cDNA library was prepared separately using Protoscript® First Strand cDNA synthesis Kit (New England Biolabs, USA) with random hexamer and U12 primer,¹⁶ which were mixed after preparation. The complete genome sequencing, using Sanger’s chemistry, has been determined as described previously.¹⁷ The sequences have been deposited in GenBank under accession numbers PP340455-PP340462. Representative avian influenza virus sequences were taken from public database/ GenBank for comparison. The nucleotide sequence alignment was performed using BioEdit (ver 7.7.1),¹⁸ and phylogenetic relationships were estimated using MEGA (ver 7.0.26).¹⁹ Neighbor-joining trees were constructed for all 8 genes [polymerase basic (PB) 2, PB1, polymerase acidic (PA), HA, nucleoprotein (NP), NA, matrix (M), and non-structural (NS)] by using Tamura-Nei model of nucleotide substitution available in MEGA software. Trees were statistically evaluated using the bootstrap method (with 1000 resampling datasets).

Polyclonal sera and antigenic analysis
Polyclonal antisera were raised in chickens as described previously.²⁰ The H5N1 virus [A/duck/India/11CA04/2022 (dk/11CA04/22)] harvested from the chicken eggs used in the antigenic analysis was inactivated by β-propiolactone at a final concentration of 0.1%. Three SPF chickens were inoculated with the inactivated virus along with the adjuvant. The first inoculation used Freund’s complete adjuvant, followed by a booster dose on the 14^th day, with Freund’s incomplete adjuvant. The serum was collected on 21^st day, aliquoted, and stored at -40 °C until further use. Other serum and viruses, A/Duck/India/04CA05/2024(H5N1) and A/Chicken/India/03CL488/2011(H5N1) used in this study were obtained from the repository of ICAR-NIHSAD, Bhopal. Antigenic analysis was performed using the hemagglutination inhibition assay.¹⁵