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An H5N1 avian influenza A virus was transmitted to humans in Hong Kong in 1997. Although the virus causes systemic infection and is highly lethal in chickens because of the susceptibility of the hemagglutinin to furin and PC6 proteases, it is not known whether it also causes systemic infection in humans. The clinical outcomes of infection in Hong Kong residents ranged widely, from mild respiratory disease to multiple organ failure leading to death. Therefore, to understand the pathogenesis of influenza due to these H5N1 isolates, we investigated their virulence in mice. The results identified two distinct groups of viruses: group 1, for which the dose lethal for 50% of mice (MLD50) was between 0.3 and 11 PFU, and group 2, for which the MLD50 was more than 103 PFU. One day after intranasal inoculation of mice with 100 PFU of group 1 viruses, the virus titer in lungs was 107 PFU/g or 3 log units higher than that for group 2 viruses. Both types of viruses had replicated to high titers (>106 PFU/g) in the lungs by day 3 and maintained these titers through day 6. More importantly, only the group 1 viruses caused systemic infection, replicating in nonrespiratory organs, including the brain. Immunohistochemical analysis demonstrated the replication of a group 1 virus in brain neurons and glial cells and in cardiac myofibers. Phylogenetic analysis of all viral genes showed that both groups of Hong Kong H5N1 viruses had formed a lineage distinct from those of other viruses and that genetic reassortment between H5N1 and H1 or H3 human viruses had not occurred. Since mice and humans harbor both the furin and the PC6 proteases, we suggest that the virulence mechanism responsible for the lethality of influenza viruses in birds also operates in mammalian hosts. The failure of some H5N1 viruses to produce systemic infection in our model indicates that multiple, still-to-be-identified, factors contribute to the severity of H5N1 infection in mammals. In addition, the ability of these viruses to produce systemic infection in mice and the clear differences in pathogenicity among the isolates studied here indicate that this system provides a useful model for studying the pathogenesis of avian influenza virus infection in mammals.

When expressed in vitro, the neuraminidase (NA) of A/WSN/33 (WSN) virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the viral hemagglutinin. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses whose NAs lacked plasminogen-binding activity because of a mutation at the C terminus, from Lys to Arg or Leu. In the presence of trypsin, these mutant viruses replicated similarly to wild-type virus in cell culture. By contrast, in the presence of plasminogen, the mutant viruses failed to undergo multiple cycles of replication while the wild-type virus grew normally. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brain. Furthermore, another mutant WSN virus, possessing an NA with a glycosylation site at position 130 (146 in N2 numbering), leading to the loss of neurovirulence, failed to grow in cell culture in the presence of plasminogen. We conclude that the plasminogen-binding activity of the WSN NA determines its pathogenicity in mice.Influenza A viruses possess two virion surface glycoproteins, a hemagglutinin (HA) and a neuraminidase (NA). The HA binds to cell surface receptors and mediates fusion between the endosomal membrane and the viral envelope. The latter event requires cleavage of the HA into HA1 and HA2 subunits, thereby exposing the N-terminal hydrophobic region, which is thought to interact with the host membrane and trigger membrane fusion (32). Thus, influenza A viruses cannot infect host cells unless the HA is proteolytically cleaved (14,15).Although the virulence of influenza A viruses is controlled polygenically, the HA plays a pivotal role in determining the severity of infection in avian strains (8,11,26). The HA cleavage site sequences in virulent and avirulent avian influenza viruses differ; the former possess a series of basic amino acids at this site, while the latter do not (10, 13). The ubiquitous host proteases furin and PC6, which specifically recognize these multiple basic residues, cleave the HAs of virulent viruses, leading to systemic infection (12,27). By contrast, the HAs of avirulent viruses are not cleaved by these proteases because they lack the requisite series of basic residues at their cleavage sites. Instead, they are susceptible to proteases that are presumably localized in the respiratory and/or intestinal tract, thus leading to localized viral infection.All mammalian influenza viruses, excluding equine H7N7 viruses, have a single Arg residue at the HA cleavage site. Thus, the HAs cannot be cleaved by ubiquitous furin or PC6 protease, resulting in a localized infection. However, a mouseadapted human isolate, A/WSN/33 (WSN; H1N1), which is recognized as a neurovirulent strain, causes systemic infection when inoculated intranasally into mice (3). Studies with WSN-A/Hong Kong/68 (H3N2) reassortant viruses indicated that the NA gene determines WSN neurovirulence in mice by facilitating HA cleavage (24). Only a si...

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.