Molecular basis for the generation in pigs of influenza A viruses with pandemic potential

Abstract

Genetic and biologic observations suggest that pigs may serve as "mixing vessels" for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.

FIG. 1

Comparison of lectin staining in duck intestine (colon) and pig trachea. The M. amurensis lectin specific for NeuAcα2,3Gal (designated α2,3; detected with fluorescein isothiocyanate-labeled anti-DIG antibody) bound to both duck intestinal epithelium and pig tracheal epithelium, whereas S. nigra lectin specific for NeuAcα2,6Gal (designated α2,6; detected with rhodamine-labeled anti-DIG antibody) bound only to the latter. Blue staining in the connective tissue represents autofluorescence. Magnification, ×300.

FIG. 2

Phylogenetic tree of influenza A virus H1 HA genes. Nucleotide residues 1 to 1731 of each H1 HA were analyzed by the neighbor-joining method (34). Horizontal distances are proportional to the minimum number of nucleotide differences required to join nodes and H1 HA sequences. Vertical lines are for spacing branches and labels. Bootstrap values (1,000 replications) are presented for each node. The node shown by the arrow at A indicates the hypothetical introductory virus in pigs that originated from birds. The HA nucleotide sequences represent USSR77 (A/USSR/90/77), KIEV79 (A/Kiev/59/79), SWHOK81 (A/swine/Hokkaido/2/81), SWWIS61 (A/swine/Wisconsin/1/61), SWIW30 (A/swine/Iowa/15/30), DKALB (A/duck/Alberta/35/76), TYMN81 (A/turkey/Minnesota/1661/81), MALTN85 (A/mallard/Tennessee/11464/85), DKAUS80 (A/duck/Australia/749/80), DKBAV (A/duck/Bavaria/1/77), SWNED80 (A/swine/Netherlands/3/80), SWGER91 (A/swine/Germany/8533/91), SWSHOL (A/swine/Schleswig-Holstein/1/92), SWVIR (A/swine/Italy-Vir/671/87), SWBEL83 (A/swine/Belgium/1/83), SWNED85 (A/swine/Netherlands/12/85), and SWGER81 (A/swine/Germany/2/81) from this study; LENING54 (A/Leningrad/1/54), reported by Beklemishev et al. (2); WSN33 (A/WSN/33), reported by Hiti et al. (19); PR8-34 (A/Puerto Rico/8/34), reported by Winter et al. (41); SWNJ76 (A/swine/New Jersey/11/76), reported by Both et al. (5); and SWNEB92 (A/swine/Nebraska/1/92) reported by Olsen et al. (30).

FIG. 3

The HA1 sequences of avian and European avian-like swine influenza viruses. The HA1 portions, which form the receptor-binding sites, are compared, using the SWARN79 sequence as a baseline. Abbreviations are given in the legend to Fig. 2.

FIG. 4

Globular head of the influenza virus HA molecule (ribbon, cyan), illustrating the location of Ser145 (space-filled, purple) relative to that of bound sialic acid (ball and stick, red). Inset shows the entire molecule. This figure is based on the H3 HA structure of A/Aichi/2/68 (H3N2) complexed with sialic acid as determined by Weis et al. (40) and is not intended to represent the actual three-dimensional structure of the H1 molecule.

FIG. 5

Models for the generation of pandemic influenza virus strains in pigs. (A) In the classical genetic reassortment model, avian and human viruses bind, respectively, to NeuAcα2,3Gal and NeuAcα2,6Gal (α2,3 and α2,6) linkages in pig trachea, setting the stage for the emergence of a reassortant that infects a large fraction of the human population. The segments in the center of each particle represent the viral genome. The reassortant HA gene (black) is derived from an avian virus. (B) In this adaptation model, avian viruses acquire the ability to replicate efficiently in humans during adaptation to the NeuAcα2,6Gal linkage in pigs. This change is mediated by a mutation in the HA gene. (C) Alternatively, an avian influenza virus is transmitted directly to humans where it reassorts with a human virus, or (D) it acquires the ability to recognize the NeuAcα2,6Gal linkage after direct introduction from birds, leading to efficient replication in humans.