Serological studies confirmed that inlfuenza A H1N1 and influenza A H3N2 viruses have undergone marked antigenic changes from the previous vaccine strain. The data were used by the USPHS and WHO to recommend inclusion of two new strains (A/New Caledonia/20/99 H1N1 and A/Panama/2007/99 H3N2) in inactivated influenza vaccines for the 2000-2001 season. Our laboratory produced reference reagents for these strains to facilitate qualification and release of approximately 75 million doses of influenza vaccine for the United States. In addition, as reference for production purposes, we cloned two new H3N2 influenza A reassortant virus strains (RESVIR 16 and RESVIR 17) with increased ability to replicate in eggs; these strains were distributed to WHO Influenza Centers, national laboratories and manufacturers, and the RESVIR 17 A/Panama/2007/99 strain was selected for use in vaccine production. In response to the appearance of avian influenza A virus subtypes in man, we have continued preparations for the possibility of a pandemic. We have produced new reference antiserum against an H9 influenza subtype hemagglutinin, and anticipate producing additional reference antisera for other subtype hemagglutinins as we are able to obtain and prepare materials. We previously demonstrated that the binding activities of matrix protein (M1) to ribonucleoprotein (RNP) complexes of influenza virus are central to viral replication and virulence. To further examine these binding activities, we have studied the effect of M1 on RNP formation and nuclear export, which are important stages during viral replication and morphogenesis. We have established a method to reconstitute RNP complexes by using in vitro translated nucleoprotein (NP), M1 and in vitro transcribed viral RNA (vRNA). Our results indicated that, in absence of M1, NP and vRNA only formed mini-RNPs. Whereas, in presence of M1, these mini-RNPs formed a higher order of the structure which resembles the native RNP from virus. To examine the requirement of M1 for nuclear export of RNP, a cell-line expressing RNP was established in our laboratory. M1 was co-expressed in the cell by transfection of plasmid encoding for M1. Without expression of M1 in such cells, RNP remained in the nucleus. However, RNP was transported from nucleus to the cytosol when M1 protein was also expressed in the same cells. Our research results from these studies suggest that M1 may promote the formation of RNP by association with the oligomers of vRNA/NP in the infected cell. With this association, M1 will transport RNP from nucleus to cytosol for the final virus assembly.
