To model host factors in the context of influenza virus infection, the lethal dose of a highly pathogenic H5N1 virus (A/Hong Kong/213/03) in C57BL/6J and DBA/2J mice was determined and genetic elements associated with survival after infection were identified. Gene mapping with recombinant inbred BXD strains revealed five loci located on chromosomes 2, 7, 11, 15, and 17 associated with resistance to H5N1 virus.
66 BXD RI strains were inoculated with an intermediate dose (104EID_50) of HK213 virus in 30 l PBS, and morbidity and mortality were monitored for 30 days. On average, 7.2 female mice (range, 4 to 21) were inoculated per BXD RI strain. Survival time (measured in days) and mortality rate (percentage of mice that succumbed to infection) were calculated for each BXD RI strain. Lungs from B6, D2, BXD43, BXD68, BXD73, BXD67, and BXD97 mice infected with 104EID_50 were obtained 3 days after infection and immediately homogenized. HK213 virus-infected lungs were harvested on days 2, 4, and 7 after infection and stored. Immunohistochemistry was performed on formalin-fixed lung tissue harvested from HK213 virus-infected or uninfected D2 mice and B6 mice. The amounts of chemokine (C-C motif) ligand 2 (CCL2) and tumor necrosis factor alpha (TNFalpha) present in the lung homogenates of infected animals were determined.
Mapping of quantitative trait loci (QTL) was performed using the WebQTL module of GeneNetwork (www.genenetwork.org) using NCBI Build 36 coordinates. Mapping was done with 66 different BXD RI strains for survival time (measured in days) after infection with highly pathogenic H5N1 virus and for mortality rate. Statistical analysis of differences in survival after H5N1 virus infection was performed by a log-rank test, and P values of <0.01 were considered significant.
Genome-wide linkage analysis with disease parameters, survival time and mortality rate identified three QTL that were significantly associated with resistance to infection:
QTL Ifvrq1 (influenza virus resistance QTL 1) mapped to Chromosome 2 between 33 and 52 Mb, p<0.0001 with a LOD score of more than 3.8 [Fig 2] and accounted for 7% of trait variance.
QTL Ifvrq2 (influenza virus resistance QTL 2) mapped to Chromosome 7 between 100 and 114 Mb, p<0.0001 with a LOD score of more than 3.8 [Fig 2] and accounted for 5% of trait variance.
QTL Ifvrq3 (influenza virus resistance QTL 3) mapped to Chromosome 17 between 68 and 84 Mb, p<0.0001 with a LOD score of more than 3.8 [Fig 2] and accounted for 9% of trait variance.
Two suggestive QTL mapped to Chromosomes 11 (101-107 Mb) and 15 (51-57 Mb) accounting for 1% and 5% of trait variance respectively.
The B6 allele had positive effect on survival and mortality at all 5 loci.
RNA expression profiling was performed on several BXD RI lines infected with HK213 virus for 72 h. The four susceptible BXD lines (-67, -73, -83, and -97) had an average survival time of 10.5 days, and all mice succumbed to infection. Resistant BXD RI lines (-43, -68, and -98) had an average survival time of 29.3 days, and their mortality rate was less than 2%.
Comparison of RNA expression between resistant and susceptible mice, including the parental D2 and B6 strains, identified many genes whose expression differed significantly.
Table two lists several candidate genes whose functions are potentially linked to infection or inflammation for all 5 QTL.
Genes Gsn, Hc and St6galnac4 are candidate genes for QTL Ifvrq1
Genes A530023O14Rik, EG667823, Fam168a, Myo7a, Phca, Plekhb1, Prkrir, Rassf10, Rnf121, Rps3, Slco2b1, Trim12, Trim34 and Trpc2 are potential candidate genes for QTL Ifvqr2.
Genes Ndufaf7, Cdc42ep3, Dlgap1, Mta3, Prkd3, and Qpct are candidate genes for Ifvqr3.