Characterization of the 1918 “Spanish” influenza virus neuraminidase gene

Analysis of the genetic sequence of the 1918 influenza has been undertaken to elucidate its possible origin and the reasons for its virulence (17). The NA gene is important both because of its functional role in promoting the dissemination of the virus during infection and because, like HA, it is a principal target of the human immune system.

In many ways, analyses of the 1918 NA sequences give results similar to that of the 1918 HA study (7). In both cases, phylogenetic analyses show that of all mammalian isolates, the 1918 sequences are the most closely related to avian isolates, but also suggest that the 1918 sequences share enough characteristics with mammalian isolates to distinguish them from the avian clade. The placement of the Brevig/18 NA nucleotide sequence in the phylogenetic trees is usually within and near the root of the mammalian clade, suggesting that the 1918 NA is very similar to the ancestor of all subsequent swine and human isolates. The number of differences between the Brevig/18 sequence and early human strains, or between the Brevig/18 sequence and swine strains, is also consistent with its being the ancestor of all subsequent mammalian N1s (Fig. 3). At the same time, and in contrast to the results with HA, phylogenetic analyses of the NA protein place Brevig/18 within the avian clade. In these cases, branch lengths are very short, and bootstrap values are low, suggesting that there are not enough differences among the sequences to place them unambiguously.

The phylogenetic results suggest that Brevig/18 sequence is intermediate between avian and mammalian sequences, and are consistent with the idea that pandemic viruses acquire their surface proteins directly (with little modification) from avian viruses. Nevertheless, Brevig/18 NA differs at 26 amino acids from its nearest known avian relative (A/Dk/Alberta/76). By contrast, the 1957 pandemic N2 and the N1 from the 1997 Hong Kong H5N1 outbreak differ by only 18 and 2, respectively, from their nearest avian relatives. These results can be interpreted in different ways. Either Brevig/18 NA would more closely resemble a 1918 avian N1, but avian sequences have drifted away from that ancestral sequence over the past 80 years, or the 1918 sequence had acquired mammalian specific changes in a mammalian host in the years preceding the 1918 pandemic. That the ultimate source of the 1918 NA was avian is supported by the phylogenetic analyses, but the precise path of the gene from its avian source to its pandemic form cannot be determined by its sequence alone.

As with HA, the functional and antigenic sites of Brevig/18 NA closely resemble avian isolates. The 15 conserved amino acids making up the active site of the molecule are retained, as are the seven glycosylation sites found in all avian strains. Twenty-two amino acids have been identified as antigenic in the N2 subtype (3). Of the homologous amino acids in N1, 15 have shown variation in human strains. Brevig/18 matches the avian consensus at 14 of the 15 residues, suggesting little or no antigenic pressure on the protein before 1918. Human strains from the 1930s show drift at these sites, with PR/34 differing at 5 of the 15 and WS/33 at 8 of the 15 amino acids.

Several early human strains have deletions of 11 to 16 amino acids in the stalk region of NA (18) that may affect the activity of the protein (19). It has been suggested that the stalk deletions in the earliest human strains, PR/34 and WS/33, may have been inherited from the 1918 pandemic strain (20). However, PR/34 and WS/33 have different stalk deletions, Weiss/43 has a full-length stalk, FM/47 has yet a third distinct deletion, and all subsequent human isolates have full-length stalks. Sw/Iowa/30, shown phylogenetically to be a descendant of the 1918 virus, does not have a stalk deletion. Stalk deletions are common in N1 strains found in chickens (21) but were not found in the five wild-type avian N1s sequenced for this study. The 1918 strain does not have a stalk deletion, suggesting that the various deletions found in early human strains are likely to be artifacts of their extensive culture in various hosts, although naturally occurring stalk deletions in these early human viruses cannot be ruled out.

The amino acid residue 354 in the NA protein is of potential interest. This residue is Asp in Brevig/18. An extensive search of GenBank (in December, 1999) demonstrated that this position is occupied by Gly in nearly all other NA proteins, regardless of subtype. Three exceptions were found, all of which have Glu at this position: A/Equine/Prague/1/56 (H7N7), A/Equine/Cornell/16/74 (H7N7), and A/Swine/England/191973/92 (H1N7). Position 354, which is buried internally in the globular head of NA (as aligned to the N9 crystal structure), is located between two blocks of amino acids that are well conserved in all NA subtypes, and the N-terminal block contains one of the amino acids known to be an active site residue. The change from Gly to Asp (or Glu) at this position could conceivably result in a conformational change in this region of the protein, with consequences affecting NA activity and possibly virulence.

The minimal changes, if any, necessary to allow an avian N1 to function in a mammalian host have not been determined. Differences between Brevig/18 NA and its nearest avian relative are not all necessarily related to mammalian adaptation. Some may reflect differences between the current nearest avian relative and the pandemic's actual 1918 avian ancestor, and some could reflect random mutations that did not diminish the fitness of the virus. One way to distinguish among these possibilities is to compare the 1918 sequence with that of Sw/England/92, a swine-adapted H1N1 that resulted from the independent introduction of an avian H1N1 into European swine in the late 1970s. The H1N1 lineage represented by Sw/England/92, although it was detected in pigs as early as 1979, remains distinctly avian phylogenetically (Fig. 2). Nevertheless, it must by definition have the minimal adaptations necessary to allow it to function in a mammalian host. As shown in Table 2, five amino acids have changed in two separate introductions of avian N1s into mammals (Sw/England/92 and Brevig/18); they may represent the minimal changes necessary for mammalian adaptation. Two of these positions have replacements that are either chemically similar (285) or identical (344) in Brevig/18 and Sw/England/92, suggesting that these two sites may be particularly important.

One of the characteristics of the 1918 pandemic was its unusual virulence, reflected in the heightened severity of illness and the prevalence of pneumonic complications. The virulence of influenza viruses is a complicated function of genetic characteristics of the virus itself, the immune status of the infected individual, and the dose and route of transmission. Pandemic viruses generally exhibit higher virulence than interpandemic strains, probably in large part because of their antigenic novelty. If both HA and NA are replaced, as occurred in 1957, the pandemic strain will likely be more virulent than if only one of these antigenic targets is replaced, as occurred in 1968 when only HA was replaced. The severity of the 1918 pandemic suggests that both HA and NA were antigenically novel and that the virus had not circulated widely in the human population before the spring of 1918. This is supported by sequence and phylogenetic analyses of both 1918 HA (7) and NA.

The relationship between virulence and the genetic structure of the virus is complex. There are a few examples of simple changes in a single gene resulting in dramatic changes in virulence. One of these is the insertion of basic amino acids in the HA cleavage site, which allows the virus to grow in many tissues outside its normal host cells. Although this change has been found in the H5 and H7 subtypes in birds, it was not found in the 1918 HA (7, 22). In the NA gene, the loss of a glycosylation site at amino acid 146 in WSN/33 (15) contributes to making the virus exceptionally virulent as well as neurotropic in mice (23, 24). This change also was not found in the 1918 strains. Future research may reveal other single gene changes with a dramatic effect on virulence.

It is clear that successful virus replication and spread depends on tightly coordinated interactions among the virus' genes. Reassortment is an important source of variation in influenza viruses, but not all gene combinations result in functional virus, and several genes appear to contribute to the adaptation of a virus to a new host. A pandemic virus must, by definition, be both well adapted to replication in and spread among humans and antigenically novel. In 1957 and 1968, this dual requirement was met by the combination of novel surface protein(s) and PB1 from avian sources with other internal proteins from the previously circulating human-adapted virus (25–27). Analysis of the surface proteins of the 1918 virus suggests that it also acquired its HA and NA from avian viruses, although it is possible that these genes may have been adapting in a mammalian host for some time before emerging in pandemic form.

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.100140097.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.100140097

We are grateful to Dr. Johan V. Hultin and the residents of Brevig Mission, AK, for their generosity in supporting this project. We also thank Drs. Edwin D. Kilbourne, Richard D. Slemons, and Ian H. Brown for their contributions of strains and sequences. This project was supported by grants from the American Registry of Pathology, the Department of Veterans Affairs, and intramural funds of the Armed Forces Institute of Pathology.