96 Pathogenesis and Immunology

Where does the primary replication occur?

Cellular proteases are often required to cleave viral proteins to form the mature infectious virus particle. Thus, additional factors to entry receptors can determine the site of viral replication. In humans, the replication of the influenza virus is generally restricted to the epithelial cells of the upper and lower respiratory tract. This is because of the limited expression of serine protease, tryptase Clara, secreted by nonciliated Clara cells of the bronchial epithelia. The purified enzyme cleaves the polypeptide HA chain precursor HA0 of extracellular particles and activates HA in virions rendering them infectious. Some highly virulent avian influenza strains, however, contain genetic insertions at the cleavage site of HA leading to processing by ubiquitous proteases. This may cause altered tropism and additional sites of replication in animals and humans (Gamblin 2004). Tissue tropism of avian influenza (H5N1) in humans is not well defined. In one case, viral RNA was detected in lung, intestine, and spleen by a reverse transcription polymerase chain reaction but posi- tive-stranded viral RNA, indicating virus replication, was confined exclusively to the lung and intestine (Uiprasertkul 2005). Thus, H5N1 viral replication in humans may be restricted to the respiratory and intestinal tract in contrast to disseminated infections documented in other mammals and birds.

How does the infection spread in the host?

Once influenza has efficiently infected respiratory epithelial cells, replication occurs within hours and numerous virions are produced. Infectious particles are preferentially released from the apical plasma membrane of epithelial cells into the airways by a process called budding. This favors the swift spread of the virus within the lungs due to the rapid infection of neighboring cells.

Alterations in the HA cleavage site by naturally occurring mutants can dramatically influence the tropism and pathogenicity of influenza. As a result, it can be recognized by other cellular proteases. This would explain why many of the individuals infected with avian influenza (H5N1) in Hong Kong had gastrointestinal, hepatic, and renal, as well as respiratory symptoms and why viruses from these patients were neurovirulent in mice (Park 2002). Whether these symptoms result from hematogenic spread or reflect non-pulmonal means of viral entry into the host remains unclear. However, mutation in NA may also, in part, explain the pantropic nature of influenza. For example, the laboratory-derived WSN/33 strain of influenza, a variant of the first human influenza virus ever isolated, unlike most human influenza strains, can replicate in vitro in the absence of added trypsin. In this virus an in-frame deletion that removes the glycolization site at residue 46 of NA allows neuraminidase to bind and sequester plasminogen. This leads to higher local concentrations of this ubiquitous protease precursor and thus to increased cleavage of the HA. These findings suggest a means by which influenza A viruses, and perhaps other viruses as well, could become highly pathogenic in humans. (Goto & Kawaoka 1998). Interestingly, studies with the genetically reconstructed 1918 Spanish influenza pandemic virus (H1N1) revealed additional mechanisms of NA-mediated HA cleavability that may be relevant to the replication and virulence of that virus (Tumpey 2005).

Finally, animal studies have revealed that the site of inoculation can determine the pathway of spread of the influenza virus in the host. For example, the neutrotropic