An influenza virus pandemic is a looming danger for a naïve population. Since H5N1 influenza viruses continue to circulate asymptomatically in duck and geese populations, there needs to be a system in place to rapidly generate a vaccine to control a potential pandemic influenza virus outbreak. Novel avian virus strains have not been extensively manipulated in laboratory settings and often grow less efficiently in vaccine substrates than laboratory-adapted strains. By combining pandemic influenza virus genes with genes derived from a high-growth donor strain, such as PR8, reassortant viruses can be generated that will facilitate the rapid production of the pandemic vaccines in a timely manner. It is known that the contribution of the M gene from the PR8 vaccine donor strain is responsible for some of the high-growth characteristics of the PR8 strain (12
). Recent evidence links the polymerase genes to the high level of virulence of H5N1 in mammalian species (21
). It also has been shown that not all laboratory-adapted versions of PR8, which have been maintained in various ways in laboratory settings since its isolation in 1934, have the same growth characteristics (7
). Although the incorporation of the internal genes from a laboratory-adapted strain, PR8, increases avian virus yield in eggs, the total protein yield of reassortant viruses may not be optimal. In this paper, we addressed these issues by delineating regions of the PR8 NA gene that can be used to modify the pandemic strain NA gene to increase total viral protein yield.
Since the NA genes of both VN1203 and PR8 are N1 subtypes, they have a high degree of identity at the amino acid level throughout a large portion of the protein. In contrast, the stalk regions, which hold the globular head domain off of the surface of the viral particle, have a much lower percentage of identical amino acids between these two strains. We postulated that the stalk region, either its length or sequence, is a critical component in generating a VN1203 vaccine strain with higher growth or increased protein yield. To accomplish this, we created a panel of NA constructs in which sections of the stalk were deleted or contained regions of the PR8 NA gene in place of the VN1203 NA gene. This was accomplished by either swapping out just the VN1203 NA stalk region for the PR8 stalk region or by substituting the entire amino terminus region, including the internal cytoplasmic tail, the transmembrane domain, and the stalk region. These NA constructs then were studied using reassortant viruses of two different backgrounds: first, a panel of 7:1 reassortant viruses were created that contained NA as the only gene contributed by the H5N1 strain, with the seven other genes being derived from PR8, or second, 6:2 reassortant viruses with HA from the H5N1 strain paired with the various NA constructs, and the remaining six genes coming from PR8.
Several properties of these viruses were analyzed in this study to characterize growth and antigen yield. While all 10 of the strains showed overall high levels of replication in eggs (with EID50
titers between 108
[Table ]), the overall total protein yields in eggs varied significantly between the various strains (Fig. ). In fact, the five reassortant viruses that contained HA from VN1203 (JV19, JV20, JV21, JV23, and JV24) grew to EID50
titers comparable to those of viruses containing PR8 HA, but they had significantly lower protein yields. This goes counter to the thought that high infectivity in eggs directly correlates with high protein yield and an increase in the expected number of doses of vaccine per egg. This might indicate that in eggs, the system used for this study, the presence of the VN1203 HA did not hinder, and in fact might aid, the infectivity of the virus. Previous work has shown that deletions in the stalk region lead to the inability of the NA to properly function with the cognate HA and with a concomitant decrease in replication in some cell types or eggs (15
). This phenomenon likely is strain dependent, though many short-stalk influenza virus strains replicate in tissue culture to levels equivalent to those of the parent virus (5
Two of the strains that were tested contained a 33-amino-acid deletion of the NA stalk. Short-stalk mutants displayed moderate to severe growth defects. For example, JV17 did not form plaques on MDCK cells (Fig. ). This inability to form plaques reinforces the idea that the balance of HA and NA is critical for efficient exit from and entry into the MDCK cells (17
). Since the EID50
results for JV17, at 108
per ml, indicate a moderate level of replication, it is obvious that the deletion made in the stalk still is partially compatible with the H5 HA glycoprotein, but it is unable to infect neighboring cells to form plaques on MDCK monolayers. When the stalk-deleted NA was paired with the VN1203 HA, there was an increase in compatibility between the glycoproteins, and the JV21 strain formed pinpoint plaques on MDCK cells.
The JV24 virus, a 6:2 reassortant containing VN1203 HA, and a chimeric form of NA with the PR8 N terminus on the VN1203 NA, grew robustly in eggs and represents a candidate for use in vaccine production. The JV24 strain contained the carboxy terminus of the VN1203 NA, which includes the external epitopes that are present in the native VN1203 NA protein. The chimeric NA also provided the PR8 internal portion of the NA gene, which may allow for the more productive interactions with the other influenza virus proteins, including M1 protein from the PR8 strain. The percentage of NA and HA proteins in JV24 was compared to that in JV20 by Western blotting using antibody to VN1203 NA or PR8 HA antigen. The results indicated that JV24 contained slightly more NA protein (approximately 15% more) than JV20, but there were not differences in the percentages of HA proteins between JV24 and JV20 (data not shown), suggesting that a chimeric form of NA with the PR8 N terminus increases the incorporation of NA into the virus with the PR8 background and enhances the release of viral particles from cells. It has been suggested that the transmembrane domains and cytoplasmic tails of HA and NA play a role in association with M1 protein (2
), which also interacts with ribonucleoproteins (3
). The interaction of glycoproteins with M1 protein, therefore, may enhance the incorporation of ribonucleoprotein cores into viral particles and subsequently may increase the viral replication rate.
Both the PR8 and VN1203 NA genes are from the N1 subtype, although they demonstrate significant amino acid divergence in the stalk region. The molecular engineering of improved NA genes should not be limited to H5 strains only, and we plan to apply this to other clades of H5 and to H7N7 and H9N2 prepandemic strains as well. Preliminary alignments indicate that H7N7 (such as A/chicken/Netherlands/2586/2003) and H9N2 (A/chicken/Israel/1953/2004) isolates have significant divergence from PR8 NA and a longer stalk region than that of either the VN1203 or the PR8 NA protein (data not shown). Annual influenza virus strains that do not grow well in eggs also could have their yield significantly boosted by swapping in the PR8 NA N terminus for the original N terminus of the NA gene. This would leave the native NA antigenic region intact, but it could increase growth by efficiently complementing the six internal genes that come from PR8 when using reverse genetics to engineer reassortant viruses with the desired growth properties to rapidly initiate the manufacture of influenza virus vaccines.