Highly pathogenic avian influenza A (H5N1) in marine mammals and seabirds in Peru

Abstract

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses (lineage 2.3.4.4b) are rapidly invading the Americas, threatening wildlife, poultry, and potentially evolving into the next global pandemic. In November 2022 HPAI arrived in Peru, triggering massive pelican and sea lion die-offs. We report genomic characterization of HPAI/H5N1 in five species of marine mammals and seabirds (dolphins, sea lions, sanderlings, pelicans and cormorants). Peruvian viruses belong to lineage 2.3.4.4b, but they are 4:4 reassortants where 4 genomic segments (PA, HA, NA and MP) position within the Eurasian lineage that initially entered North America from Eurasia, while the other 4 genomic segments (PB2, PB1, NP and NS) position within the American lineage (clade C) that circulated in North America. These viruses are rapidly accruing mutations, including mutations of concern, that warrant further examination and highlight an urgent need for active local surveillance to manage outbreaks and limit spillover into other species, including humans.

Fig. 1. Collection sites and animals sampled.

A Map of Peru showing collection sites representative of the northern, central and southern regions. Mass die-offs of both birds and mammals occurred along the entire coast of Peru, but map only shows positive locations of animals samples in this study. B Photographic record of animals sampled, including common dolphin, South American sea lion, sanderling, Guanay cormorant, Peruvian booby and Peruvian pelican.

Fig. 2. Global H5N1 phylogenies for all eight genome segments.

Phylogenetic trees for AIVs collected globally and submitted to GISAID since January 1, 2021, inferred using ML methods. Branches shaded by AIV lineage; tips shaded by AIV subtype and location. H5N1 viruses from the 2021–2023 Western hemisphere outbreak are shaded yellow, South American H5N1 viruses (except Peru) are shaded green, and Peruvian H5N1 viruses are shaded pink. Cartoon animals denote notable outbreaks in mink in Spain, sea lions in Peru, and seals in Maine, USA. A detailed tree is provided for the PB2 segment, including bootstrap values, labels, and a box around the R6 reassortant clade that is presented in greater detail in Fig. 3A. Smaller trees are provided for the other 7 segments. Raw tree files for each genome segment are available at GitHub (https://github.com/mostmarmot/Peru_AIV/).

Fig. 3. H5N1 genotypes identified in the Americas, 2021–2023.

Each genotype box represents one of eight genome segments (1–8), shaded by lineage: grey = Eurasian lineage; pink = American lineage (clade A); blue = American lineage (clade B); green = American lineage (clade C); orange = American lineage (clade D).

Fig. 4. Spread of reassortant “R6” viruses.

A Time-scaled maximum clade credibility (MCC) tree inferred for the PB2 segment using Bayesian approaches for 98 representative H5N1 viruses (R6 genotype) belonging to the reassortant clade C in the American lineage (see Fig. 2). Branches shaded by location. South American virus names shaded by country or Peruvian region. Posterior probabilities provided for key nodes. Estimated timing of R6 virus entry into Peru/Chile is provided. Diamonds indicate the 3 viruses with PB2:D701N mutations. Raw MCC tree available on GitHub (https://github.com/mostmarmot/Peru_AIV/). B Discrete phylogeographic transition history of R6 viruses at four time points. Red lines with arrows indicate interhemispheric transitions from North America to South America. C Proportion of H5N1 viruses sequenced in the Americas between 2021–2023 that belong to different genotypes (Fig. 2) over time.

Fig. 5. SNP and mutational analysis of Peruvian HPAI a/H5N1 viruses (shown in bold).

Additional reference sequences used are available through GenBank and GISAID. A total of 22 variable sites across genomic segments were identified relative to the original A/H5N1 goose/Guangdong reference from 1996, the A/Vietnam/1203/2004 reference used to annotate amino acid positions in the CDC inventory, plus two additional more recent references representing R6 reassortants from 2022 identified in birds (chicken/wyoming/2022) and mammals (skunk/washington/22-019274-001/2022) (shaded in grey). Mutation PB2 D701N (shown with an *) has been previously linked to mammalian host adaptation and enhanced transmission^,. The remaining 21/22 sites have not been previously characterised, and 4 may warrant special consideration (shown in bold) as they are concentrated in recent mammalian samples (Supplement Table 2), including 2 (PB2 D701N and PA M86I) that later show up in the genome sequenced from the human case in Chile (A/Chile/25946/2023). Dots represent amino acids identical to those present in annotation reference A/Goose/Guangdong/1/96; colours have been randomly assigned to amino acids simply to aid in identification of differences; X indicates there is no genomic information available for that position; - - indicates a deletion in the sequence.