FIGURE 2 | Schematic diagram of the derivation of the H5N1 reference vaccine strain, NIBRG-14, by reverse genetics.

The haemagglutinin (HA) and neuraminidase (NA) genome segments of the wild-type H5N1 virus were cloned into 'rescue' plasmids under the control of an RNA polymerase I promoter (green). During cloning, the HA segment is modified to delete the stretch of basic amino acids that confer the highly pathogenic phenotype of the H5N1 virus (red spot on the HA rescue plasmid). These were transfected into Vero cells together with rescue plasmids containing the remaining six viral genome segments from PR8 (blue) and four expression plasmids that encode the viral replicase machinery of the PR8 virus (yellow). Genetically, the rescued NIBRG-14 virus is a reassortant containing six segments from PR8 (blue) and two from the wild-type H5N1 (green), with the HA segment containing a deletion (red spot) compared with its parent virus. The phenotype of NIBRG-14 is expression of the H5N1 coat proteins, good growth in eggs (a PR8 trait), likelihood of attenuation for humans (another PR8 trait) and lack of pathogenicity (owing to the HA deletion).

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