Despite their uniform ability to bind to oligosaccharide-containing terminal sialic acids, influenza A viruses show differences in receptor specificity. To test whether agglutination of erythrocytes from different animal species could be used to assess the receptor specificity of influenza A viruses, we determined the agglutinating activities of a range of virus strains, including those with known receptor specificities, using erythrocytes from seven animal species. All equine and avian viruses, including those known to recognize N-acetyl and N-glycolyl sialic acid linked to galactose by the alpha2,3 linkage (NeuAc alpha2,3Gal and NeuGc alpha2,3Gal), agglutinated erythrocytes from all of the animal species tested (chickens, ducks, guinea pigs, humans, sheep, horses, and cows). The human viruses, including those known to preferentially recognize NeuAc alpha2,6Gal, agglutinated all but the horse and cow erythrocytes. Fluorescence-activated cell sorting analysis of erythrocytes using linkage-specific lectins [Sambucus nigra agglutinin for sialic acid (SA) alpha2,6Gal and Maackia amurensis agglutinin for SA alpha2,3Gal] showed that both cow and horse erythrocytes contain a large amount of SA alpha2,3Gal-, but virtually no SA2,6Gal-specific lectin-reactive oligosaccharides on the cell surface, while human and chicken erythrocytes contained both types of oligosaccharides. Considering that the majority (>93%) of sialic acid in horse and cow erythrocytes is of the N-glycolyl type, our results suggest that viruses able to agglutinate these erythrocytes (i.e., avian and equine viruses) recognize NeuGc alpha2,3Gal. These findings also show that agglutinating assays with erythrocytes from different animal species would be useful in characterizing the receptor specificity of influenza A viruses.

Influenza A viruses can be isolated from a variety of animals, but their range of hosts is restricted. For example, human influenza viruses do not replicate in duck intestine, the major replication site of avian viruses in ducks. Although amino acids at positions 226 and 228 of hemagglutinin (HA) of the H3 subtype are known to be important for this host range restriction, the contributions of specific amino acids at these positions to restriction were not known. Here, we address this issue by generating HAs with site-specific mutations of a human virus that contain different amino acid residues at these positions. We also let ducks select replication-competent viruses from a replication-incompetent virus containing a human virus HA by inoculating animals with 1010.5 50% egg infectious dose of the latter virus and identified a mutation in the HA. Our results showed that the Ser-to-Gly mutation at position 228, in addition to the Leu-to-Gln mutation at position 226 of the HA of the H3 subtype, is critical for human virus HA to support virus replication in duck intestine.

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Glycogen synthase kinase-3␤ (GSK-3␤) is a serine/threonine kinase with a broad array of cellular targets, such as cytoskeletal proteins and transcription factors. Recent studies with GSK-3␤-null mice showed impaired NFB-mediated survival responses. Because NFB serves a dual role as a key regulator of cytokine-induced inflammatory gene expression and apoptosis, we investigated whether modulation of GSK-3␤ expression affects cytokine-induced and NFB-mediated inflammatory gene expression. We observed that tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤) treatment of primary cultures of human microvascular cells reduced net endogenous active GSK-3␤ protein levels while inducing inflammatory cytokine (IL-6 and monocyte chemoattractant protein-1 (MCP-1)) expression. Interestingly, inhibition of GSK-3␤ by antisense oligonucleotides or pharmacological agent (10 mM lithium) potentiated TNF-induced expression of IL-6 and MCP-1 by 2-6-fold suggesting that inhibition of GSK-3␤ under inflammatory conditions (exposure to TNF-␣ and IL-1␤) may contribute to enhanced cytokine expression. Overexpression of GSK-3␤ in endothelial cells, in contrast, significantly inhibited (by 70%, p < 0.01) both TNF-␣ and IL-1␤-induced expression of IL-6, MCP-1, and vascular cell adhesion molecule-1. Using adenoviruses in lipopolysaccharide-stimulated mice, overexpression of GSK-3␤ significantly decreased TNF-␣ expression in lung and heart tissues (38 and 15%, respectively), further confirming the anti-inflammatory role of GSK-3␤. Overexpression of GSK-3␤ did not affect the TNF-␣-induced nuclear translocation of NFB but reduced the nuclear half-life of TNF-␣-induced NFB considerably (by as much as 9 h) and enhanced phosphorylation (by as much as 33%). Interestingly, neither endothelial cell survival nor NFB-mediated expression of anti-apoptotic genes was affected by GSK-3␤ overexpression. We conclude that GSK-3␤ selectively regulates NFB-mediated inflammatory gene expression by controlling the flow of NFB activity between transcription of inflammatory and survival genes. GSK-32 was originally identified in 1980 as a serine/threonine kinase involved in glycogen metabolism (1-7). Two isomers, GSK-3␣ and -3␤, sharing an 85% overall sequence homology have been isolated in mammals. The isomers have been implicated in multiple biological processes including cell proliferation, cell differentiation, embryonic development, cell motility, cell cycle, transcription, and metabolism. Unique to GSK-3␤ is its reported involvement in NFB-mediated cell survival (8). GSK-3␤ is also responsible for the phosphorylation of a variety of proteins, including transcription factors (c-Jun, c-Myc, and CCAAT/enhancer-binding protein), translation eukaryotic initiation factor 2, and cytoskeletal proteins. GSK-3␤ activity is down-regulated by phosphorylation.In vivo, the function of GSK-3␤ is not clear. Data from knock-out mice studies show GSK-3␤ to be necessary for cell survival and NFB activity (8). Knock-out mice deficient in components involved in the NFB signa...

In 1991 and 1992, H1N2 influenza A viruses were isolated from the lungs of pigs with overt signs of respiratory disease at farms in the Chiba and Kanagawa prefectures of Japan. To determine the genetic origin of these isolates, we phylogenetically analyzed partial nucleotide sequences of their genes. The results indicate that influenza viruses possessing the N2 of human influenza virus and seven other gene segments of classical H1N1 swine influenza virus, which were first isolated in 1980, have become established in Japanese pigs.