Highly Pathogenic Avian Influenza A(H5N1) Virus–Induced Mass Death of Wild Birds, Caspian Sea, Russia, 2022

In May 2022, we observed a substantial die-off of wild migratory waterbirds on Maliy Zhemchuzhniy Island in the Caspian Sea, Russia. The deaths were caused by highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b virus. Continued surveillance of influenza viruses in wild bird populations is needed to predict virus spread over long distances.


Figures 10).
As a result, it has been shown that flight routes passing through the Caspian Sea region cover not only the territory of Eurasia but also parts of Africa.They also pass through countries in Europe and the Middle East.Those results enables us to assume that the influenza virus spreads through avian spring migration to the Caspian Sea, either directly from Israel and the Levant or through Europe (likely via Romania, as indicated by phylogenetic analysis).In addition, analysis of flight routes and migration corridors (notable for their diverse destinations) indicates the significance of the Caspian region in the spread of avian influenza virus and early detection of new variants.
Maliy Zhemchuzhniy Island is located in the northern part of the Caspian Sea near the maritime boundary of the Republic of Kalmykia and the Astrakhan region (latitude 45°02′ N, longitude 48°19′ E).This flat, low-lying island, composed of sand and clam shells, was formed during a regression of the Caspian Sea in the 1930s on the site of an underwater shoal.It is constantly exposed to wind and waves, which cause changes in its configuration, and areas are either completely or partially lacking terrestrial vegetation.During 2016-2022, the size of the island remained relatively constant.In 2021, the island was estimated to be 26.33 ha and had a length of ≈2 km and maximum width of ≈0.3 km.

Samples
In May 2022, we collected 10 samples from Caspian terns on the island during a mass die-off of wild birds.In all of those samples, the matrix protein gene segment of AIV A and the hemagglutinin gene segment of the H5Nx subtype were detected by using multiplex real-time PCR (AmpliSens Influenza virus А H5N1-FRT PCR kit; InterLabService Ltd, https://en.interlabservice.ru).All analyzed viruses were isolated from 10-day-old chicken embryonated eggs by using chicken embryo inoculation.All viruses caused the death of chicken embryos within 2 days.Isolates were shown to be H5 positive by using real-time PCR.

Genome Sequencing and Phylogenetic Analysis
Next-generation sequencing of complete genomes was performed by using the Illumina MiSeq platform (https://www.illumina.com)and associated reagent kits according to the manufacturer's methodology.RNA was extracted by using the QIAamp Viral RNA Mini Kit (https://www.qiagen.com).Whole-genome amplification was performed by using a modified protocol (6).DNA libraries were prepared by using a Nextera DNA Flex Library Prep kit (Illumina) and sequenced by using MiSeq Reagent Kit v3 (600-cycle) (Illumina).Consensus sequences were generated by using Bowtie software (https://www.bowtiepro.com).
Nucleotide sequences were deposited in the GISAID database under accession nos.EPI_ISL_16020401-405. Multiple alignments were performed by using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle); editing, which included translating the nucleotide sequences into amino acid sequences, was performed by using BioEdit (https://bioedit.software.informer.com)and UGENE (http://www.ugene.net)software.Initial maximum-likelihood phylogenies for each of the gene segments were generated with RAxML (7) by using the general time-reversible nucleotide substitution model.Final dendrograms were generated and visualized by using MEGA5 (8).Bootstrap support values were generated by using 1,000 rapid bootstrap replicates.
The challenge study and all experiments with live viruses were conducted in a Biosafety Level 3 facility.

andAppendix 1 Figure 6 .
Romania closely related to the Caspian Sea strains.Closed triangles indicate Egyptian-like virus strains from Russia isolated in 2020.Sequences were obtained from the GISAID EpiFlu database (https://www.gisaid.org).Scale bar indicates nucleotide substitutions per site.HPAI, high pathogenicity avian influenza; LPAI, low pathogenicity avian influenza.Phylogenetic analysis of polymerase acidic protein gene segments of avian influenza viruses.Trees were constructed by using the maximum-likelihood method.Red asterisks indicate low pathogenicity viruses found within highly pathogenic virus groups that were related to the Caspian Sea strains (presumably, highly pathogenic viruses appeared because of reassortment with low pathogenicity viruses).Red box indicates virus strains from Romania closely related to the Caspian Sea strains.Closed circles indicate the highly pathogenic avian influenza H5N1 virus strains isolated from dead Caspian terns on Maliy Zhemchuzhniy Island, Caspian Sea, Russia in May 2022.Closed triangles indicate Egyptian-like virus strains from Russia isolated in 2020.Sequences were obtained from the GISAID EpiFlu database (https://www.gisaid.org).Scale bar indicates nucleotide substitutions per site.HPAI, high pathogenicity avian influenza; LPAI, low pathogenicity avian influenza.