The severity of influenza A virus infections is modulated by the expression of virulence factors such as PB1-F2. In contrast to the PB1-F2-encoding viruses that caused the three pandemics of the 20th century, the H1N1 virus responsible for the 2009 pandemic does not express PB1-F2 and is associated with a relatively mild disease phenotype. In order to better understand the consequences of potential genetic change in the pandemic Cal/09 influenza virus, we conducted experiments designed to characterize the disease phenotype associated with a PB1-F2-expressing version of Cal/09.
By developing a reverse-genetics system for the Cal/09 virus, we were able to rescue three Cal/09 viruses that have identical PB1 protein sequences: the wild-type virus and variants that encode either the 66N or the 66S PB1-F2 protein (Fig. ). We examined the growth kinetics for all three Cal/09 viruses and observed enhanced replication by the 66S virus in the human epithelial cell line A549, leading to peak titers roughly 1 log unit higher than those for the other Cal/09 viruses. This effect was not observed in the canine MDCK cell line. Since influenza viruses replicate very efficiently in MDCK cells, it is likely that the rather mild contribution to virulence by the PB1-F2 protein in the Cal/09 background is simply not detectable in this cell system.
Our in vivo
experiments were conducted in two different mouse strains. We first tested the effect of PB1-F2 on the virulence of Cal/09 virus infection in BALB/c mice. Expression of PB1-F2 in the Cal/09 virus had no significant effect on disease pathogenesis (Fig. ). We observed only transient morbidity, with no mortality, following infection with any of the Cal/09 viruses. In addition, peak viral titers in the lungs were similar for the Cal/09 rWT, 66N, and 66S virus groups. Analysis of cytokine/chemokine levels in the supernatants of lung homogenates revealed increased expression of proinflammatory genes (e.g., MCP-1, MIP-1β, and RANTES), which are essential for the recruitment and activation of immune cells in infected tissues (6
). These proinflammatory proteins have also been shown to prevent the apoptosis of alveolar macrophages in the context of influenza virus infection in vivo
). Accordingly, we detected more-severe histopathology in the lung tissues of mice infected with the PB1-F2-expressing viruses, especially the 66S mutant virus, than in those of rWT-infected mice. In summary, the introduction of a functional PB1-F2 ORF did not enhance the mortality associated with infection with the Cal/09 virus in the BALB/c mouse system. PB1-F2 expression did, however, result in a modulated host immune response characterized by increased expression of proinflammatory genes.
The data obtained in our BALB/c mouse study suggested that in a more susceptible mouse strain, such as the DBA/2 mouse (2
), we would observe more-pronounced differences between the disease phenotype caused by the PB1-F2-expressing Cal/09 viruses and that caused by the wild-type virus. However, under the experimental conditions used, the results of our studies of DBA/2 mice were very similar to the results of our BALB/c mouse studies. Expression of the 66S PB1-F2 protein was correlated with enhanced expression of the proinflammatory genes IL-1β and RANTES. Additionally, we observed that expression of PB1-F2 did not affect the susceptibility of BALB/c mice to secondary bacterial infections.
Taken together, our mouse studies suggest that a genetic change resulting in the presence of a functional PB1-F2 ORF would not significantly increase the virulence of primary infection caused by the Cal/09 virus or the susceptibility of a host to secondary bacterial infection with Streptococcus pneumoniae.
The fact that the expression of the PB1-F2 protein in the Cal/09 virus did not have a significant impact on primary viral infection or secondary bacterial infection in mice is intriguing, given that the prototype PB1-F2 proteins have been shown to affect both viral and bacterial infections (5
). As previously mentioned, the sequence of the Cal/09 PB1-F2 protein is unique. Even though Cal/09 PB1-F2 is predicted to contain known functional regions, such as the mitochondrial targeting sequence, the protein seems to have a lesser effect on viral virulence than prototype PB1-F2 proteins, at least in the context of the Cal/09 virus. This supports findings that enhanced viral virulence conferred by PB1-F2 expression is a strain-specific phenomenon (17
The results of our studies on weight loss and viral replication in ferrets paralleled the results of our mouse studies. Expression of PB1-F2 was not statistically significantly associated with enhanced virulence, but ferrets infected with the 66S variant did demonstrate slightly more weight loss (without mortality) than those infected with the 66N or wild-type Cal/09 virus. PB1-F2 expression did cause significant dysregulation of peripheral leukocyte counts, including lymphopenia and elevated neutrophil levels. Infection with the reassortant PR8-PB1-F2 Cal/09 virus caused the most severe disease phenotype of all viruses studied in ferrets; importantly, however, the PR8 PB1 protein may contribute to the observed phenotype.
In summary, we found that mutations enabling the production of PB1-F2 by the Cal/09 influenza virus do not have a significant impact on the virulence of the virus in mice or in ferrets. These preliminary observations, however, invite further investigations into the virulence of other PB1-F2 mutant viruses and of possible reassortants of the Cal/09 virus with current viruses. The present findings enhance our understanding of PB1-F2 as a virulence factor and provide new insights into the impact that genetic changes may have on the virulence of the 2009 pandemic virus.