Pathogenic 1997 H5N1 viruses are lethal for chickens and humans in nature, and in laboratory experiments they are lethal for mice and ferrets (5
; this study). These H5N1 viruses were highly virulent despite the fact that their encoded NS1 proteins contain a defect in one of their major functions directed at countering the host antiviral (IFN) response, namely, the binding of CPSF30, which causes the suppression of the production of mature cytoplasmic cellular mRNAs, including IFN-β mRNA and presumably other antiviral mRNAs (6
). Strong binding of CPSF30 requires the presence of F and M at positions 103 and 106, respectively, in the NS1 protein (56
). Instead of these two amino acids, the NS1 proteins of pathogenic 1997 H5N1 viruses contain L and I, respectively, at positions 103 and 106 and as a consequence bind CPSF30 nonoptimally. Changing these two amino acids in the HK97 NS1 protein to the post-2003 consensus residues in the NS1 proteins of H5N1 viruses (F and M at 103 and 106, respectively) strengthens CPSF30 binding and enhances virus replication in tissue culture (56
). In the present study, we demonstrated that changing these two NS1 amino acids to the consensus amino acids leads to a very dramatic (300-fold) increase in the lethality of the virus in mice.
Our results indicate that this enhanced virulence of HK97G2+ is likely due to its earlier and more efficient replication and systemic spread. After intranasal inoculation with HK97G2+ but not wt HK97, virus was readily detected in blood within 24 h of infection. Consistent with this rapid establishment of viremia, HK97G2+ also reached much higher titers in the spleen by 2 days and in the brain by 4 days. The total amount of HK97G2+ virus at days 2 and 4 in the lung, spleen, and brain substantially exceeds the total amount of the wt HK97 virus (), showing that the HK97G2+ virus replicated considerably more rapidly than the wt HK97 virus. However, the titer of the HK97G2+ virus in the lung was only slightly higher than that of the wt HK97 virus. It is likely that this relatively smaller difference in titers reflects the fact that the HK97G2+ virus not only replicates more rapidly in the lungs but also spreads more rapidly from the lungs. Such a rapid dissemination from the lung would explain why the HK97G2+ virus caused less damage and cell death in the lungs and attracted fewer infiltrating inflammatory cells into the lungs than did the wt HK97 virus. In addition, the host cytokine response in the lung was essentially the same as the cytokine response in the lung to wt HK97 virus infection from 2 to 6 days postinfection.
In contrast, the cytokine and chemokine levels in the spleen and brain, as measured by both direct protein assays and microarray analysis, were much higher in HK97G2+-infected mice than in wt-HK97-infected mice. These differences largely mirrored the time course of accumulation of the two viruses in these two organs. On day 2 postinfection, the HK97G2+ virus achieved a considerably higher titer than the wt HK97 virus in the spleen, and the HK97G2+ virus induced higher chemokine and cytokine levels in the spleen on this day. Similarly, the HK97G2+ virus spread to the brain faster than the wt HK97 virus, where it replicated faster, and the chemokine and cytokine levels in the brain at 4 and 6 days postinfection were much larger after HK97G2+ virus infection. We interpret these results to indicate that the increased levels of the cytokines and chemokines produced in the spleen and brain after HK97G2+ virus infection represent mostly the responses of local uninfected cells, including trafficking immune cells, to increased viral loads in these two organs.
Consistent with the more rapid spread and replication of the HK97G2+ virus in the brain, mice infected with HK97G2+ but not with wt HK97 exhibited extensive brain damage by 6 days postinfection, at which point most mice infected with HK97G2+ succumbed to infection. Our results fit a model in which the lung is the site of initial enhanced replication by the HK97G2+ virus, but lethality results from rapid dissemination to other organs, particularly the brain, where severe pathology occurred. We conclude that changing the NS1 amino acids at positions 103 and 106 to F and M, respectively, enables the 1997 H5N1 virus to replicate more rapidly and to spread throughout the body more efficiently, particularly to the brain, dramatically increasing its virulence. wt HK97 also spreads to the brain, albeit much more slowly. Spread of wt HK97 virus to the brain was also observed in previous studies (44
). However, a recent study has provided evidence that the wt HK97 virus kills mice by rapid replication in the lungs that overcomes the host immune response (17
In contrast to the H5N1 HK97 NS1 protein, CPSF30 binding by the NS1 protein of H1N1 viruses appears to be less critical for optimal suppression of the host antiviral response. For example, the NS1 protein of the 2009 H1N1 virus does not bind CPSF30, because the consensus binding site is blocked by other NS1 amino acids. Removal of this block, leading to the establishment of CPSF30 binding, has only a minimal effect on IFN production, virus replication, and mouse virulence (14
). An important issue is therefore why strong CPSF30 binding by the NS1 protein of the H5N1 HK97 virus is required for optimal suppression of the host antiviral response. One possibility is that the suppression of the activation of IRF3 and IFN-β transcription by the NS1 protein of H5N1 viruses is actually not as effective as the suppression mediated by the NS1 protein of H1N1 viruses and that this difference has not yet been detected by the methods that have so far been employed. It has already been established that the NS1 proteins of different influenza A virus subtypes differ in their ability to suppress the activation of IRF3 and IFN-β transcription (28
). The NS1 proteins of human H2N2 and H3N2 strains do not inhibit the activation of IRF3 and IFN-β transcription, whereas the NS1 proteins of currently circulating H1N1 strains do inhibit these activations (28
). Perhaps the NS1 proteins of H1N1 and H5N1 viruses also differ, specifically in the extent to which they inhibit the activation of IRF3 and IFN-β transcription.
It will be important to elucidate the mechanism by which the HK97G2+ virus rapidly disseminates from the lung and enters the brain. For some H5N1 viruses, neurotropic spread via the vagus nerve to the brain has been demonstrated (21
). In the present case, an alternate possibility is suggested by our finding that the HK97G2+ virus but not the wt HK97 virus was readily detected in blood within 24 h of infection, indicating that HK97G2+ might increase pulmonary vascular permeability, thereby establishing viremia at very early times of infection. At day 1 postinfection, MCP-1 and TNF-α were increased in HK97G2+-infected lungs compared to levels in wt-HK97-infected lungs. These two cytokines are known to increase pulmonary vascular permeability (29
), which has previously been implicated in influenza virus pathogenesis (60
). This early viremia would enable HK97G2+ to rapidly seed peripheral organs, such as the spleen, and subsequently spread to the brain, where cytokinemia may also increase the permeability of the blood-brain barrier, whereas the wild-type virus may be restricted to slower neurotropic spread through peripheral nerves.
Previous studies have shown that humans (10
), birds (53
), mice (2
), and ferrets and martens (11
) infected with certain strains of H5N1 influenza viruses develop severe brain infection, whereas other viral strains lead to more striking pulmonary damage (26
). Our results with the HK97G2+ virus, coupled with a recent study of the wt HK97 virus (17
), indicate that a two-amino-acid change in the NS1 protein likely leads to a dramatic change in the site of severe pathology induced by the HK97 virus, from the lung to the brain.