Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes

Abstract

The influenza A virus pandemic of 1918-1919 resulted in an estimated 20-40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Figure 1

Sequence of the 1918 NS segment. (A) Diagram of the NS gene segment showing the positions of the two overlapping ORFs. Nucleotides 1–26 and 862–890 are noncoding. (B) The sequence of the Brevig Mission/1/18 NS gene segment. The coding sequences start at nucleotide 27, and both NS1 and NEP share the initial 10 codons. The NEP translation product is shown above the line of nucleotides, whereas the NS1 product is below the line. The numbering of the gene is aligned to A/WSN/33 (GenBank no. M12597) and refers to the sequence in the sense (mRNA) orientation.

Figure 2

Phylogenetic tree of influenza A NS1 genes. The tree is a NJ tree using p distance. Nine groups are indicated: groups 1, 3, 6, 7, and 9 are avian, groups 2 and 8 are equine, group 4 is human, and group 5 is swine. A/Brevig Mission/1/18 (arrow) is near the root of group 5. Bootstrap values are given for selected nodes, and a distance bar is shown under the tree.

Figure 3

Construction of viruses that express either the 1918 NS1 protein or the control PR8 NS1 protein. Coding strategy and RT-PCR analysis of the 1918 NS1–2A-NEP and PR8 NS1–2A-NEP viruses. In the NS1–2A-NEP constructs, the NS1 coding sequences do not overlap the NEP coding sequences. The NS1 and NEP coding sequences are separated by the foot-and-mouth disease virus 2A autoprotease and are expressed as a single polyprotein. The polyprotein is cleaved into two peptides, an NS1 with 16 additional 2A-derived amino acids at its carboxy terminus and an NEP with a single 2A-derived proline at its amino terminus. RT-PCR was performed to amplify the NS1 ORFs. The primer-binding sites (sequences available on request) are indicated by arrows.

Figure 4

Percent survival of mice infected with PR8 NS1–2A-NEP or 1918 NS1–2A-NEP viruses. Mice were infected intranasally with 1 × 10⁴ pfu of wild-type WSN virus (diamonds), PR8 NS1–2A-NEP virus (squares) or 1918 NS1–2A-NEP virus (triangles), or mock infected with PBS (not shown). Each group had six mice. The percentage of mice surviving on each day is indicated. All PBS-treated mice survived.

Figure 5

Replication of wild-type WSN and 1918 NS viruses in tissue culture and in mice. (A) Multicycle growth in MDCK cells of wild-type WSN virus (diamonds) or either of two independently isolated 1918 NS viruses (1918 A, squares or 1918 B, triangles). Cells were infected at a multiplicity of infection = 0.001. Viruses were titered by plaque assay on MDCK cells. (B) Survival of mice after infection with wild-type WSN virus or 1918 NS viruses. Mice were infected intranasally with 1 × 10⁴ pfu. Eight mice were infected with each virus or were mock infected with PBS. The percent of mice surviving on each day is indicated. All PBS-treated mice survived. (C) Average lung titers of mice infected with wild-type WSN or with 1918 NS viruses on days 2, 4, and 6 after infection. The average titers on each day are reported. Three mice were used for each wild-type WSN time point and six mice were used for each 1918 NS virus time point. Viruses were titered by plaque assay on MDCK cells.