Detection of evolutionarily distinct avian influenza a viruses in antarctica

Abstract

ABSTRACT Distinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adélie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra- and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment. IMPORTANCE Avian influenza viruses (AIVs) are typically maintained and spread by migratory birds, resulting in the existence of distinctly different viruses around the world. However, AIVs have not previously been detected in Antarctica. In this study, we characterized H11N2 viruses sampled from Adélie penguins from two geographically different sites in Antarctica and show that the segmented AIV genome diverged between 49 and 80 years ago from other AIVs, with several genes showing similarity and shared ancestry with H3N8 equine influenza viruses. This study provides the first insight into the ecology of AIVs in Antarctica and highlights the potential risk of an introduction of highly pathogenic AIVs into the continent.

FIG 1

Map of sampling sites in Antarctica. Adélie penguins were sampled from two locations in Antarctica: Admiralty Bay, King George Island; and Kopaitik Island, Rada Covadonga, Antarctic Peninsula. Inset: An Adélie penguin. The map is courtesy of Marco Villarroel, reproduced with permission.

FIG 2

Phylogenetic tree of H11 (A) and N2 (B) sequences. Sequences from the viruses isolated from Adélie penguins in Antarctica are highly diverged from other circulating AIVs. The HA phylogenetic tree was constructed using maximum likelihood methodology and shows ancestry with Eurasian avian H11 viruses, while the NA sequence is highly diverged from all other clades but with shared ancestry with North American N2 viruses. Detailed phylogenetic trees with virus designations can be found in Fig. S2 in the supplemental material.

FIG 3

Phylogenetic trees of the internal gene sequences of avian, equine, and mammalian influenza A viruses. Major avian influenza clades are broadly classified into Western, Eastern, or global based on the geographic location of sample collection. Phylogenetic trees were constructed using maximum likelihood methodology. AIV sequences from the penguins are marked as “Antarctica,” with branches colored red. Blue branches indicate South American avian influenza sequences, green branches indicate avian influenza viruses from other regions, and orange branches indicate equine influenza viruses. Detailed phylogenetic trees with virus designations can be found in Fig. S3 in the supplemental material.

FIG 4

HA structural homology modeling. The structures of penguin and a related sandpiper H11 virus were modeled based on the HA crystal structures of H13 (PDB ID number 4kps), H16 (PDB ID number 4f23), and H2 (PDB ID number 2wr3) in complex with an α2,3Gal-linked sialic acid analogue (avian-like receptor). The receptor binding pocket residues for both H11s are typically avian, with E190, G225, Q226, and G228 (H3 numbering), suggesting α2,3-linked SA receptor (avian-like) binding (purple and green balls). Comparison of the receptor binding pocket in the A/peng/Ant/178/13 with that in A/sharp tailed sandpiper/Australia/10/2004 reveals differences at I155, A186, and T187 (red or blue sticks for penguin or sandpiper H11, respectively), which may influence preferences for different variants of 2,3-linked glycans. Several major antigenic sites also differ among the H11s; for example, epitope Sb (at the top of the 190 helix) and upper parts of Sa (the area around the 160 loop). Another unique structural feature distinguishing the penguin H11 from the nearest known H11 sequences and the H13 and H16 structures are different indel patterns around G133 (yellow circle).