Influenza virus inoculum volume is critical to elucidate age-dependent mortality in mice

Abstract

The elderly exhibit increased mortality to influenza viral infection for unclear reasons. Mice are frequently used to model how aging impacts disease. Several studies have shown that aged mice exhibit an increased mortality to influenza virus, but two recent studies demonstrated the opposite. These two studies administered the virus intranasally in 20 µL, whereas the other studies used a viral inoculum in at least 30 µL. To determine whether the volume of the inoculum could explain the conflicting reports, we infected young and aged mice via intranasal instillation of 40 µL or 20 µL containing 1 x 10⁴ plaque-forming units (PFU) of H1N1 influenza virus. We found that intranasal administration of 40 µL but not 20 µL of inoculum resulted in age-dependent mortality in mice. Compared to aged mice infected with 40 µL inoculum, those infected with 20 µL inoculum showed reduced levels of live virus and IFN-β in the lung 3 days postinfection. Furthermore, aged mice administered 40 µL of Evans blue intranasally displayed increased dye retention in their bronchoalveolar lavage fluid compared to those administered 20 µL of Evans blue. Our data demonstrate that the inoculating volume of virus is critical for adequate delivery of influenza virus to the lung and thus for efficient infection of aged mice. These findings shed light on discrepant results in the literature regarding aged mice and influenza infection, and establish that mice can be used to examine how aging impacts the response to this biomedically important infection.

Figure 1

Volume of viral inoculum is critical for eliciting an age‐dependent mortality during IAV infection. Young (2–4 months of age) and aged (18 months of age) C57BL/6 male (a, b) or female (c, d) mice were infected i.n. with either 20 µL or 40 µL inoculum of virus containing 1 x 10⁴ PFU of PR8 strain IAV, and mortality was monitored. In both sexes, an age‐dependent increase in mortality was only noted with the 40 µL inoculum. *p < 0.05, **p < 0.01 (Gehan–Breslow–Wilcoxon test). n = 10–12/group (males), 10/group (females)

Figure 2

Increase in the volume of viral inoculum in aged mice via the i.n. route leads to higher viral load and increased IFN‐β levels. Increase in the inoculating volume of dye in noninfected aged mice leads to increased dye detection within bronchoalveolar lavage (BAL). Female young (2–4 months of age) and aged (18‐month old) C57BL/6 mice were infected i.n. with 1 x 104 PFU in either 20 µL or 40 µL, then live virus within lung tissue was measured 3 days postinfection by plaque assay (a) and concentration of IFN‐β measured in the lung lysate 3 days postinfection via ELISA (b). Young and aged noninfected female mice were administered 63 picomoles of Evans blue in either 20 µL or 40 µL, and dye was detected in BAL by colorimetry (c). *p < 0.05, **p < 0.01 (two‐way analyses corrected for multiple comparisons, see Supporting Information Appendix S1). Each data point represents a biological replicate, and error bars ±95% confidence interval