MOLECULAR MECHANISMS OF SERUM RESISTANCE OF HUMAN INFLUENZA H3N2 VIRUS AND THEIR INVOLVEMENT IN VIRUS ADAPTATION IN A NEW HOST

H3N2 human influenza viruses that are resistant to horse, pig, or rabb it serum possess unique amino acid mutations in their hemagglutinin (H A) protein. To determine the molecular mechanisms of this resistance, we characterized the receptor-binding properties of these mutants by m easuring their affinity for total serum protein inhibitors and for sol uble receptor analogs. Pig serum-resistant variants displayed a marked ly decreased affinity for total pig serum sialylglycoproteins (which c ontain predominantly 2-6 linkage between sialic acid and galactose res idues) and for the sialyloligosaccharide 6'-sialyl(N-acetyllactosamine ). These properties correlated with the substitution 186S --> I in HAI . The major inhibitory activity in rabbit serum was found to be a beta inhibitor with characteristics of mannose-binding lectins. Rabbit ser um-resistant variants exhibited decreased sensitivity to this inhibito r due to the loss of a glycosylation sequon at positions 246 to 248 of the HA. In addition to a somewhat reduced affinity for 6'-sialyl (N-a cetyllactosamine)-containing receptors, horse serum-resistant variants lost the ability to bind the viral neuraminidase-resistant CO-acetyla ted sialic acid moieties of equine alpha(2)-macroglobulin because of t he mutation 145N --> K/D in their HA1. These results indicate that inf luenza viruses become resistant to serum inhibitors because their affi nity for these inhibitors is reduced. To determine whether natural inh ibitors play a role in viral evolution during interspecies transmissio n, we compared the receptor-binding properties of H3N8 avian and equin e viruses, including two strains isolated during the 1989 to 1990 equi ne influenza outbreak, which was caused by an avian virus in China. Av ian strains bound 4-O-acetylated sialic acid residues of equine alpha( 2)-macroglobulin, whereas equine strains did not. The earliest avian-l ike isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian viruses; a later isolate, ho wever, displayed binding properties more similar to those of classical equine strains. These data suggest that the neuraminidase-resistant s ialylglycoconjugates present in horses exert selective pressure on the receptor-binding properties of avian virus HA after its introduction into this host.