Host genetic variation affects resistance to infection with a highly pathogenic H5N1 influenza A virus in mice

Abstract

Despite the prevalence of H5N1 influenza viruses in global avian populations, comparatively few cases have been diagnosed in humans. Although viral factors almost certainly play a role in limiting human infection and disease, host genetics most likely contribute substantially. To model host factors in the context of influenza virus infection, we determined the lethal dose of a highly pathogenic H5N1 virus (A/Hong Kong/213/03) in C57BL/6J and DBA/2J mice and identified genetic elements associated with survival after infection. The lethal dose in these hosts varied by 4 logs and was associated with differences in replication kinetics and increased production of proinflammatory cytokines CCL2 and tumor necrosis factor alpha in susceptible DBA/2J mice. Gene mapping with recombinant inbred BXD strains revealed five loci or Qivr (quantitative trait loci for influenza virus resistance) located on chromosomes 2, 7, 11, 15, and 17 associated with resistance to H5N1 virus. In conjunction with gene expression profiling, we identified a number of candidate susceptibility genes. One of the validated genes, the hemolytic complement gene, affected virus titer 7 days after infection. We conclude that H5N1 influenza virus-induced pathology is affected by a complex and multigenic host component.

FIG. 1.

Increased susceptibility to H5N1 virus infection of DBA/2J (D2) mice. (A) D2 mice (triangles), B6 mice (circles), and B6D2F1 mice (squares) were inoculated with a 10-fold serial dilution of HK213 H5N1 virus in 30 μl PBS (n = 5 mice per dilution). The mortality rate was monitored for 30 days, and the average mortality from two independent experiments is presented. (B) B6 mice (filled circles) and D2 mice (open circles) were inoculated with 10⁴ EID₅₀ of HK213 virus in 30 μl PBS. The lungs were isolated at multiple time points after infection, and the virus titer was determined. The average virus titer ± standard deviation (log₁₀ 50% tissue culture infective doses [TCID₅₀]/ml) at 8 and 24 h postinfection represents one experiment with five mice per group; the average virus titers on days 2, 4, and 7 postinfection represent multiple experiments with at least four mice per group. *, P < 0.001. (C) B6 mice (gray bars) and D2 mice (black bars) were inoculated with 10² or 10³ EID₅₀ of HK213 virus in 30 μl PBS. Forty-eight hours later, the lungs were isolated, and the virus titer was determined. *, P < 0.001. (D) Immunohistochemical staining of viral nucleoprotein (brown) in lung tissue from B6 mice and D2 mice infected with 10⁴ EID₅₀ of HK213 virus.

FIG. 2.

Genome-wide linkage analysis for survival after H5N1 virus infection. Sixty-six different BXD RI mouse strains (derived from D2 and B6 strains) were infected with 10⁴ EID₅₀ of HK213 virus in 30 μl PBS. Mortality rate (measured in %) and survival (measured in days) were recorded. (A) The average survival (±standard error of the mean) is shown for each strain (gray bars) and the parental B6 and D2 strains (black bars). (B and C) QTL analysis (www.genenetwork.org) was performed on survival data (B) or mortality rate data (C). QTL associated with influenza virus resistance (Qivr) were located on chromosomes 2, 7, and 17, as indicated by an LOD score of more than 3.8 (pink horizontal line in QTL plot). Survival after H5N1 infection was also associated with a B6 allele on chromosome 11 and 15 with suggestive LOD scores (between pink and gray horizontal lines).

FIG. 3.

RNA expression analysis in lung tissue obtained from uninfected or H5N1 virus-infected D2 mice and B6 mice. RNA expression levels were determined and analyzed. (A) Genes with an adjusted full-model P value of less than 0.01 (approximately 10% of the total number of probes) were selected and separated according to the three-model factor P values. “Strain” (red circle) indicates all genes with a significant difference (adjusted P value, <0.001) between DBA/2J mice and C57BL/6J mice. “Infection” (green circle) indicates all genes with a significant increase or decrease (adjusted P value, <0.001) in RNA expression level as a result of H5N1 virus infection. “Interaction” (yellow circle) indicates all genes with a change in RNA expression level that is dependent on the mouse strain (adjusted P value, <0.001). (B) All genes with an adjusted full-model P value of <0.01 (as shown in panel A) located within the five Qivr are shown.

FIG. 4.

Effect of hemolytic complement on mortality after infection with highly pathogenic H5N1 influenza A virus. (A) Hemolytic complement (Hc)-deficient mice (B10.D2.Hc⁰, solid symbols) and Hc-sufficient mice (B10.D2.Hc¹, open symbols) were infected with 10⁴ EID₅₀ (circles) or 10⁵ EID₅₀ (squares) of a highly pathogenic H5N1 influenza A virus (A/Hong Kong/213/03 [HK213]), and survival was determined for 21 days postinfection. (B) At 4 and 7 days post-infection with 10⁴ EID₅₀ of HK213 virus, lungs were isolated from B10.D2.Hc⁰ mice and B10.D2.Hc¹ mice. The viral load on day 7 in the Hc-deficient lungs was significantly higher than that in the Hc-sufficient lungs. TCID₅₀, 50% tissue culture infective dose.