Serological studies confirmed that A/Beijing/262/95 (H1N1) and A/Sydney/5/97 (H3N2) were markedly different from previous vaccine strains. The data were used by the USPHS and WHO to recommend inclusion of an two new strains in inactivated influenza vaccines for the 1998-1999 season. Our laboratory produced reference reagents for these strains to facilitate qualification and release of approximately 80 million doses of influenza vaccine for the United States. In addition, as a reference strain for production purposes, we cloned a reassortant influenza virus strain (RESVIR 13) with increased ability to replicate in eggs, and this strain was distributed to WHO Influenza Centers, national laboratories and manufacturers. In response to the appearance of a new avian H5 subtype of influenza A virus in man, we prepared for the possibility of a pandemic if global spread of the strain should occur. We have produced a reference antiserum against this and similar avian strains. Efforts are ongoing in our laboratory to produce high growth reassortants of these viruses which could be used for manufacturing purposes. In the process of this work, we have found incompatibilities between the avian H5 strains and the human high growth donor strains which interfere with gene transfer between the avian and human strains. In our reassorting system, this is partly a function of the markedly reduced time required for maximum growth of the avian strain as compared to the human strain in eggs. However, the relative difficulty of reassorting in vitro may reflect the probability of reassorting occurring (or not) in nature, and could be useful in focusing attention on certain influenza A subtypes as part of pandemic preparations. We also examined the replication role affected by interaction between M1 protein and ribonucleocapsid (RNP) complex. We compared the binding capacity of M1 proteins and RNP complexes from the high-growth strain, PR8, with the wild-type strain, A/Nanchang/933/95 (H3N2). The M1 protein of PR8 with RNP complexes are more sensitive to salt or low pH disruption compared to that of the Nanchang strain under the same conditions. These studies suggest that efficient dissociation of M1 protein from RNP complexes increases viral replication. To explore the mechanism of the association between M1 protein and RNP complexes, we cloned the M1 gene and modified it by substitution and deletion mutations. The altered M1 proteins were expressed and characterized with monoclonal antibodies to M1 protein. Association of the altered M1 proteins with purified RNP complexes were carried out by in vitro reconstitution. The results showed that the basic amino acid residues located in one of the RNA binding domains (AA 101-105 with a sequence of RKLKR) contribute to the binding and that the association of M1 and RNP in this region is sensitive to salt disruption. A second RNP-binding site, resistant to salt disruption, was mapped to the N-terminal region of the M1 protein.
