A previous study using a panel of reassortant duck viruses containing human N2 NAs suggested that influenza virus NA specific activity decreased markedly between 1957 and 1968 [30
]. We have confirmed and extended this observation using a set of human viruses generated by reverse genetics that contain N2 NAs representative of viruses circulating from 1957 to 2005. NA activity decreased from 1957 to 1968, and increased again from 1968 to 1997. This trend in NA activities correlates with the observed historic influenza mortality from H3N2 viruses which was highest in 1957, decreased over the following decade, but increased again in the 1990s (). Low NA activity in the 1968 virus is consistent with lower mortality from this pandemic compared to the 1957 pandemic or H3N2 epidemics during the 1990s. It has been suggested that conservation of the N2 NA in the 1968 virus resulted in relatively low pandemic mortality, but this does not explain why later viruses with no antigenic shift in HA or NA caused higher mortality. The 1997 N2 NA had the second highest activity, and this virus caused the highest epidemic mortality since the 1957 pandemic [2
Comparison of NA activity with excess mortality from influenza.
Although the correlation we report here between NA activity, secondary bacterial pneumonia in our model, and historical excess mortality in humans is intriguing, other factors must be involved. A large proportion of excess deaths related to influenza are coded as cardiovascular disease, cerebrovascular disease, or diabetes [36
] and most of these are presumably not related to bacterial super-infection. Virus specific factors contribute to excess morbidity and mortality and differences in these virulence factors account for differences from season to season. Our data suggest that NA activity is one of these factors, and that its impact can be seen in secondary bacterial pneumonia deaths. Other virulence factors such as antigenic novelty of the HA, modulation of interferon [37
] or cytokine expression by NS1 [38
], or interactions between several genes may be involved, and the NA may have effects on the host unrelated to sialic acid cleavage as suggested by its ability to activate TGF-beta [39
Our previous studies indicated that pharmacological inhibition of the viral NA improves survival from secondary pneumococcal pneumonia following influenza [13
]. However, these studies were done using a single virus and could not establish whether a dose effect based on the activities of different viruses might exist. In this study, we created a set of recombinant viruses that differ from each other only in NA activity. A hierarchy of support for bacterial adherence and secondary bacterial pneumonia could be seen when recombinant viruses were compared in cell culture and animal models of viral-bacterial interactions. The NA of highest activity (from the 1957 pandemic influenza strain) was capable of inducing more pneumococcal adherence to cultured respiratory epithelial cells and higher mortality from secondary pneumococcal pneumonia in mice than an NA with 2-fold less activity (Syd97). Similar weight loss and lung viral titers in the influenza phase of the infections indicated that differences in viral kinetics were not interfering in the comparisons. A virus with the NA of the recent Fuj02 strain, with NA specific activity comparable to that of the Syd97 NA, more effectively supported secondary bacterial pneumonia than a virus with another 2-fold decrease in relative activity (Mem72) when tested on the same background (paired with the Fuj02 HA). The Fujian virus caused high epidemic mortality during the most reason influenza season (2003−2004) and contributed to a number of well-publicized deaths from methicillin-resistant Staphylococcus aureus
]. The activity of the 1918 NA has not been reported, and activities of N1 NAs from before 1957 have not been studied in a comprehensive fashion. Our data provide direct evidence that influenza virus NA activity is a predictor of mortality from secondary bacterial pneumonia.
These results, together with our previous data showing that oseltamivir treatment prevents secondary pneumococcal pneumonia in mice even when administered late in the viral infection [13
], warrant clinical studies of prevention and treatment of bacterial complications of influenza with NA inhibitors. Effectiveness studies of these drugs in treatment of influenza have already shown decreases in complications, although the populations studied have most often not included those most vulnerable to secondary infections due to underlying illnesses or extremities of age [41
]. Pandemic planning must take into account the possibility that many of the deaths during the next pandemic will be from bacterial complications of influenza, and an increased focus on the NA including stockpiling of NA inhibitors is therefore essential.