Influenza A viruses (IAVs) cause substantial morbidity and mortality in yearly epidemics, which result from the ability of the virus to alter the antigenicity of its envelope proteins. Despite the rapid replication of this virus and its ability to infect a wide variety of cell types, viremia is rare and the infection is generally limited to the upper respiratory tract. The preimmune host defense response against IAV is generally, therefore, successful. We have previously provided (and summa-

Normal bovine and mouse sera contain a component, termed ( inhibitor, that . The association of (3 inhibitor resistance with loss of this carbohydrate side chain suggested that (3 inhibitors may be lectins. In support of this hypothesis, treatment of the 13 inhibitorsensitive parent virus Mem71H-BelN with periodate converted it to the resistant state. Furthermore, the inhibitory activity of both bovine and mouse sera for the parental virus was abrogated by D-mannose. We conclude that the 13 inhibitors in bovine and mouse sera are mannose-binding lectins that inhibit hemagglutination and neutralize virus infectivity by binding to carbohydrate at the tip of the HA spike, blocking access of cell-surface receptors to the receptor-binding site on HA.

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Collagenous lectins (collectins) present in mammalian serum and pulmonary fluids bind to influenza virus and display antiviral activity in vitro, but their role in vivo has yet to be determined. We have used early and late isolates of H3N2 subtype influenza viruses that differ in their degree of glycosylation to examine the relationship between sensitivity to murine serum and pulmonary lectins in vitro and the ability of a virus to replicate in the respiratory tract of mice. A marked inverse correlation was found between these two parameters. Early H3 isolates (1968 to 1972) bear 7 potential glycosylation sites on hemagglutinin (HA), whereas later strains carry 9 or 10. Late isolates were shown to be much more sensitive than early strains to neutralization by the mouse serum mannose-binding lectin (MBL) and rat lung surfactant protein D (SP-D) and bound greater levels of these lectins in enzyme-linked immunosorbent assays and Western blot analyses. They also replicated very poorly in mouse lungs compared to the earlier strains. Growth in the lungs was greatly enhanced, however, if saccharide inhibitors of the collectins were included in the virus inoculum. The level of SP-D in bronchoalveolar lavage fluids increased on influenza virus infection. MBL was absent from lavage fluids of normal mice but could be detected in fluids from mice 3 days after infection with the virulent strain A/PR/8/34 (H1N1). The results implicate SP-D and possibly MBL as important components of the innate defense of the respiratory tract against influenza virus and indicate that the degree or pattern of glycosylation of a virus can be an important factor in its virulence.

Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca 2؉ -dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.