Distinct gene loci control the host response to influenza H1N1 virus infection in a time-dependent manner
© Nedelko et al.; licensee BioMed Central Ltd. 2012
Received: 10 April 2012
Accepted: 10 August 2012
Published: 20 August 2012
There is strong but mostly circumstantial evidence that genetic factors modulate the severity of influenza infection in humans. Using genetically diverse but fully inbred strains of mice it has been shown that host sequence variants have a strong influence on the severity of influenza A disease progression. In particular, C57BL/6J, the most widely used mouse strain in biomedical research, is comparatively resistant. In contrast, DBA/2J is highly susceptible.
To map regions of the genome responsible for differences in influenza susceptibility, we infected a family of 53 BXD-type lines derived from a cross between C57BL/6J and DBA/2J strains with influenza A virus (PR8, H1N1). We monitored body weight, survival, and mean time to death for 13 days after infection. Qivr5 (quantitative trait for influenza virus resistance on chromosome 5) was the largest and most significant QTL for weight loss. The effect of Qivr5 was detectable on day 2 post infection, but was most pronounced on days 5 and 6. Survival rate mapped to Qivr5, but additionally revealed a second significant locus on chromosome 19 (Qivr19). Analysis of mean time to death affirmed both Qivr5 and Qivr19. In addition, we observed several regions of the genome with suggestive linkage. There are potentially complex combinatorial interactions of the parental alleles among loci. Analysis of multiple gene expression data sets and sequence variants in these strains highlights about 30 strong candidate genes across all loci that may control influenza A susceptibility and resistance.
We have mapped influenza susceptibility loci to chromosomes 2, 5, 16, 17, and 19. Body weight and survival loci have a time-dependent profile that presumably reflects the temporal dynamic of the response to infection. We highlight candidate genes in the respective intervals and review their possible biological function during infection.
Influenza A virus represents a major health threat to humans. The 1918 H1N1 pandemic caused at about 30 to 50 million deaths . Seasonal influenza epidemics cause high economic loss, morbidity and deaths every year . The course and outcome of an influenza A virus infection is influenced by viral and host factors. Host risk factors, like obesity or pregnancy, became evident during the recent swine flu pandemics [3, 4]. Furthermore, genetic factors in humans associated with a higher susceptibility to influenza infections and severe disease outcome have been suspected for the 1918 pandemics, as well as the H5N1 human infections [5–7]. Recently, the importance of IFITM3 as a crucial factor for host susceptibility has been demonstrated in mice and humans .
The importance of host factors to host susceptibility and resistance has been demonstrated clearly in animal models. We and others have shown in mouse infection models that the susceptibility of the host to influenza A infection strongly depends on the genetic background [9–17]. In particular, DBA/2J mice are highly susceptible to many influenza A virus subtypes, including those that were directly derived from human isolates without prior adaptation to the mouse [9, 13, 16–18]. In contrast, C57BL/6J mice are more resistant. After infection with mouse-adapted H1N1 (PR8M virus), DBA/2J mice loss weight very rapidly and die within 5–7 days post infection (p.i.), whereas C57BL/6J mice loss weight until days 6–8 after infection and regain their initial weight by 14 days p.i. [9, 18]. Viral load in the lungs of DBA/2J infected mice is much higher and lung pathology is very severe compared to infected C57BL/6J mice. Also, the production of chemokines and cytokines is much higher in DBA/2J mice [9, 18].
However, the genomic regions that are responsible for the differential response after infection with H1N1 have not been determined. Therefore, we used a large family of BXD type recombinant inbred strains generated by crossing C57BL/6J (resistant) to DBA/2J (susceptible) to map genetic loci that modulate disease severity. The BXD genetic reference population (GRP) is made up of a set of progeny strains, each with a defined and fixed genetic architecture. It is one of the largest families of strains, consisting of about 80 fully inbred strains [19, 20] available from the Jackson Laboratory and a new set of 80 additional lines that are still in production at the University of Tennessee. Individuals within each single strain are essentially isogenic (except for the sex chromosomes) and genotypes for the entire family, including most of the new strains, are known . Genetic variation among this family has be exploited extensively in the past to systematically study the genetics of many traits (for examples of phenotypes see the GeneNetwork database ).
Here, we infected over 50 of the BXD strains with influenza A H1N1 virus and monitored body weight, survival, and mean time to death for the following 13 days post infection. We identified two significant and several suggestive loci peaks for all three traits. All showed a time-dependent appearance. Data mining of the intervals revealed several candidate genes, several of which may be important for the host response to influenza A virus infection.
Susceptibility to influenza A virus after experimental infection of BXD mouse strains is highly variable
For body weight loss and survival, three different phenotypic response groups can be defined (Figure 1). In the first (Figure 1A), all infected mice within a strain survived, in the second (Figure 1B) a majority but not all individuals within a strain survived, and in the third group (Figure 1C), a majority died. Most remarkably, in the first group four strains—BXD9, BXD13, BXD43, BXD97—were highly resistant indicating that the infection may not cause any major pathology (Figure 1A). In contrast, BXD28 belonging to the third group, lost body weight much more rapidly than even the highly susceptible DBA/2J parent (Figure 1C). These results illustrate a large variation of responses within the BXD family.
By day 7 p.i., all infected mice had succumbed to infection in 11 strains, whereas 14 others exhibited more limited mortality (Additional file 2: Figure S1A). The incidence of mortality increased in some strains from day 8 p.i. through day 11 but not thereafter (Additional file 2: Figure S1B). For the MTTD phenotype, 17 strains showed no mortality after infection, similar to the resistant C57BL/6J parent. Three strains—BXD28, BXD18, BXD103—exhibited a MTTD that was even shorter than for the susceptible DBA/2J parent (Additional file 2: Figure S1C). Although this study was conducted over a period of approximately three years, and although mice were received from different sources and different experimenters performed the infection experiments, we did not note any significant influence of these potential confounds and cofactors.
Analysis of body weight loss revealed a significant QTL on chromosome 5 and several suggestive QTLs with time-dependent effects
Another suggestive QTL peak was found on chromosome 10 on day 1 (LRS: 14.1, effect size: 23%, Figure 3A). Its effect is lower at day 2 and not apparent at later days. These observations are in accordance with the PCA (Figure 2) that reveals two separate time-dependent influences on body weight variance among strains. The QTL appeared at an early time point after infection, when virus replication has just begun and strong inflammatory host responses are not yet evident [9, 18, 23]. These observations indicate that the effect is most likely related to the experimental protocol, namely the stress to anesthesia and intra-nasal application as well as treatment recovery. Treatment-dependent QTLs were described previously . It is worth to note that mock-infection of the parental DBA/2J and C57BL/6J mice did not lead to a lasting body weight loss over a longer time interval except for a slight drop in body weight on day 1 p.i. (Additional file 4: Figure S3).
Survival rate and mean time to death traits confirmed the QTL on chromosome 5 and detected another significant QTL on chromosome 19
In conclusion, survival and MTTD traits confirmed the significant QTL on chromosome 5, revealed an additional significant QTL on chromosome 19 and several suggestive QTLs. All QTLs showed a time-dependent effect.
Composite interval and pair-scan mapping indicates various interactions of QTLs
Analysis of QTL regions identified several candidate genes that may contribute to host susceptibility or resistance
In total, the mapping studies revealed five QTLs on chromosomes 2, 5, distal 16, 17 and 19 (Qivr2-2, Qivr5, Qivr16, Qivr17-2 and Qivr19) that did merit further analysis because they were consistently observed in at least two traits and exerted an effect on at least two different days p.i.
Candidate genes in mapped QTL intervals
Allele increasing survival
No of genes in interval
No of genes expressed during infection
No of genes with Indels (FS in coding region)
No of genes with SNPs (non-synonymous codons / stop codons)
Cis-eQTLs in non-infected lung
Differentially expressed btw B6/D2
4 / 0
9 / 0
5 / 0
58 / 4c-f
37– 45 Mb
14 / 1h
GO-terms and functions observed in knock-out mice of 31 potential candidates from QTL intervals
Type polymorphism in ORF
integrin beta 6
Integrin-mediated signaling pathway, inflammatory response, cell-matrix adhesion
Baldness associated with macrophage infiltration of skin, exaggerated pulmonary inflammation, impaired mucosal mast cell response to nematode infection.
interferon induced with helicase C domain 1
Response to virus, innate immune response, regulation of apoptosis, RIG-I-like receptor signaling pathway
Increased virus-associated morbidity and mortality, decreased cytokine response to several viral infection.
eukaryotic translation initiation factor 3, subunit B
Translation, translation initiation
sidekick homolog 1 (chicken)
eukaryotic translation initiation factor 2 alpha kinase 1
Negative regulation of translation, response to stress, negative regulation of cell proliferation, regulation of eIF2 alpha phosphorylation by heme
Enlarged heart size, abnormal red blood cell development, morphology, physiology with macrocytic anemia.
ring finger protein (C3H2C3 type) 6
Ubiquitin-dependent protein catabolic process, positive regulation of transcription, DNA-dependent
roundabout homolog 1 (Drosophila)
Cell differentiation, axon guidance, chemotaxis
Neonatal death, aphagia, delayed lung maturation and bronchial hyperplasia.
Nuclear receptor interaacting protein 1
Regulation of transcription
Female infertility due to ovulation failure. Male and female mice are smaller than wild-type littermates.
ubiquitin specific peptidase 25
Ubiquitin-dependent protein catabolic process
crystallin, alpha A
Negative regulation of apoptosis, negative regulation of caspase activity, lens fiber cell morphogenesis
Small lenses that develop progressive opacity beginning in the nucleus.
Negative regulation of transcription from RNA polymerase II promoter, regulation of cell differentiation, protein kinase cascade
membrane-associated ring finger (C3HC4) 2
Endocytosis, biological process
TAP binding protein
Antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent; defense response
Reduced and thermolabile MHC class I surface expression due to impaired peptide loading with stabilizing peptides, impaired T cell selection, altered NK repertoire, lower CD8+ T cell numbers, impaired responses to select class I-restricted antigens.
histocompatibility 2, O region alpha locus
Antigen processing and presentation of peptide or polysaccharide antigen via MHC class II, regulation of T cell differentiation, Graft-versus-host disease, viral myocarditis
Abnormal antigen presentation via MHC class II, enhanced selection of CD4+ single positive thymocytes. Mice homozygous for a different knock-out allele show increased serum IgG1 levels.
histocompatibility 2, class II, locus DMa
Antigen processing and presentation of exogenous peptide antigen via MHC class II, positive regulation of T cell differentiation, positive regulation of immune response, Graft-versus-host disease, viral myocarditis
Impaired antigen presenting cell function, poor IgG responses to T-dependent antigens, reduced numbers of mature CD4+ T cells, increased susceptibility to Leishmania major infection.
transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)
Antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent; positive regulation of T cell mediated cytotoxicity, protection from natural killer cell mediated cytotoxicity
No CD8+ T cells, although numbers of CD4+ T cells and B cells are normal.
histocompatibility 2, O region beta locus
Antigen processing and presentation of peptide or polysaccharide antigen via MHC class II, Graft-versus-host disease, viral myocarditis
histocompatibility 2, class II antigen A, beta 1
Antigen processing and presentation of peptide or polysaccharide antigen via MHC class II, Graft-versus-host disease, viral myocarditis
Depletion of mature CD4+ T cells, deficiency in cell-mediated immune responses, increased susceptibility to viral infections.
histocompatibility 2, class II antigen A, alpha
Antigen processing and presentation of exogenous peptide antigen via MHC class II, positive regulation of T cell differentiation, Graft-versus-host disease, viral myocarditis
Lack of cell surface expression of MHC class II molecules on macrophages, decreased CD4-positive T cell number, increased CD8-positive T cell number, thymus hyperplasia, enlarged lymph nodes, altered splenocyte response to staphylococcal enterotoxin B.
leukocyte specific transcript 1
Negative regulation of lymphocyte proliferation, immune response, cell morphogenesis
ns (1) deletion
general transcription factor II H, polypeptide 4
Regulation of transcription, DNA-dependent
histocompatibility 2, T region locus 23
Antigen processing and presentation of peptide antigen via MHC class I, Graft-versus-host disease, viral myocarditis
CD4+ T cells have enhanced responses after infection or immunization, are resistant to suppressor activity mediated by a subset of CD8+ T cells, but are more susceptible to NK cell lysis.
histocompatibility 2, blastocyst
Antigen processing and presentation
ns (4) stop_L
ribonuclease P 21 subunit (human)
ns (1) stop_G
tripartite motif protein 26
phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma)
Inflammatory response, lipid catabolic process
cytochrome P450, family 39, subfamily a, polypeptide 1
Lipid metabolic process, oxidation reduction
sorbin and SH3 domain containing 1
Transport, focal adhesion assembly, positive regulation of establishment of protein localization in plasma membrane
Decreased triglyceride levels, altered glucose homeostasis, decreased white blood cells and resistance to developing glucose intolerance induced by a high fat diet.
tectonic family member 3
ns (1) insertion (2)
Hermansky-Pudlak syndrome 1 homolog (human)
Positive regulation of natural killer cell activation, secretion of lysosomal enzymes
Hypopigmentation and increased bleeding time. Impaired natural killer cell function, reduced secretion of kidney lysosomal enzymes, abnormal retinofugal neuronal projections characterize some alleles.
dynamin binding protein
Intracellular signaling cascade, regulation of Rho protein signal transduction
DBA/2J and C57BL/6J mice have been shown previously to differ largely in their susceptibility to H1N1 (PR8M) influenza A virus [9, 18]. Here, we expanded these studies and utilized the BXD recombinant inbred set of mouse strains to map the genomic regions that are responsible for differences in these two mouse strains. We monitored three phenotypic traits, body weight over time, survival over time and mean time to death to identify quantitative trait for influenza resistance. Two significant QTLs, Qivr5 and Qivr19, were found on chromosomes 5 and 19, respectively. Furthermore several suggestive QTLs, Qivr2-2, Qivr16 and Qivr17-2 were observed in at least two traits and at two days on chromosomes 2, 16 and 17, respectively. Composite mapping revealed an additional almost significant QTL at distal chromosome 16, Qivr16.
A similar analysis for host resistance to influenza has been performed previously after infecting 66 BXD strains with H5N1 influenza virus. This study reported three significant QTLs on chromosomes 2, 7, and 17 . Thus, none of these significant QTLs overlaps with the QTLs identified in our analysis. Five of the strains that were resistant (all infected mice survived) in our study were also resistant in the study of  where a total of 14 strains were found to be resistant. Five strains that were highly susceptible in our study (100% of infected mice died) were also highly susceptible in the study by  of a total of 26 susceptible strains. Furthermore, five strains that were resistant in our study were susceptible in the study by . Thus, there is also not much overlap between the two studies with respect of susceptible and resistant strains. The differences between the two studies are most likely explained by the use of two different influenza virus subtypes. The H1N1 virus from our study represents a subtype with a monobasic hemagglutinin (HA) cleavage site, whereas the H5N1 which was used in the study by Boon et al. is a subtype with a polybasic HA cleavage site. The cellular tropism of these two subtypes for virus replication and processing is quite different, because monobasic viruses are dependent on cell-specific proteases for the processing of the HA whereas polybasic subtypes can be processed by more ubiquitously expressed host proteases, e.g.[27–32]. Therefore, the contribution of host factors to susceptibility may be different between H1 and H5 containing virus subtypes.
Another study described the genetic mapping of susceptibility and resistance factors after infecting a panel of 29 AxB / BxA congenic strains with a mouse-adapted H3N2 influenza virus . The AxB / BxA congenic strains were generated from a cross of susceptible A/J and resistant C57BL/6J parental mouse strains. The authors found three major QTLs on chromosomes 2, 6 and 17. The QTL on chromosome 17 overlaps with the Qivr17-2 locus which we found in our study. Furthermore, the candidate gene Pla2g7 that was identified in their study was also detected as candidate gene in our analysis (see below).
The influence of genetic factors determining the host response to H1N1 influenza virus infections was also examined in mice of the pre-Collaborative Cross collection . In this study, gene expression levels in extreme responders were used to identify expression QTLs (eQTL). One gene that exhibited a cis-eQTL, Sik1 (salt inducible kinase 1), was located in the Qivr17-2 interval from our study, and we also identified it as potential quantitative trait gene (Table 2). This gene is associated with the GO terms ‘negative regulation of transcription from RNA polymerase II promoter, regulation of cell differentiation, and protein kinase cascade’. However, no specific infection-related functions have been yet described for this gene.
One of the most interesting findings in our study was the time-dependent effect of QTLs which we observed in the body weight and survival traits. The peak QTLs for the two significant QTLs, Qivr5 and Qivr19, were found at different times p.i., day 6 and day 8, respectively. In addition, the effects of both QTLs were not only evident at the times p.i. where they exerted the significant peak QTL signals but also several days before and after the peak. Furthermore, for the suggestive QTLs, also time-dependent effects were observed. These results suggest that the causal genes underlying different QTLs act at different time points of the host defense.
Most interestingly, Qivr5 as well as Qivr19 represent a positive influence on body weight, survival and MTTD from the DBA/2J haplotype, the susceptible strain. These findings indicate that genomic regions from the susceptible parent are able to increase resistance when combined with the resistant parental genome. We are now analyzing several BXD strains that were more resistant than the parental C57BL/6J mice in more detail. One possible mechanism to explain such an effect may be that an activator (secreted ligand or transcription factor) is expressed in susceptible DBA/2J mice but the corresponding target (receptor or regulated gene) is mutated. On the other hand, in C57BL/6J mice, the target but not the activator may be mutated. If the wild type alleles are now coming together in a BXD strain, the functional activator finds its functional target and thereby an increased resistance state is achieved.
Both composite and interaction mapping revealed many genetic interactions between C57BL/6J and DBA/2 J alleles. Thus, many genomic regions from the parental strains are able to contribute to the host response and this effect depends strongly on the allele combinations in the respective QTLs. These observations may be studied further in double congenic mouse lines.
We subsequently analyzed the five QTL intervals, Qivr2-2, Qivr5, Qivr16, Qivr17-2 and Qivr19 in more detail to identify genes that may be causal for resistance or susceptibility. In total, 830 genes are located in these intervals. We narrowed down the total number of genes to 31 candidates (Table 2) by using additional information, such as temporal expression after PRM8 infection (Pommerenke et al., PLoS ONE, in press), cis-eQTLs in non-infected lungs , differences in expression between DBA/2J and C57BL/6J , and sequence variants in the coding regions.
Qivr5 contains the candidate gene Eif2ak1 (eukaryotic translation initiation factor 2 alpha kinase 1) that is a member of eIF2alpha kinases which have been associated with anti-viral host responses . Boon et al. described another eIF2alpha kinase, Eif2ak2 / Pkr (eukaryotic translation initiation factor 2-alpha kinase 2), in the Qirv17 locus after infection with influenza H5N1 . Eif2ak2 plays a critical role in modulating immunoglobulin expression during RSV infection. In addition Eif2ak2 knock-out mouse mutants are more susceptible to influenza infections [36, 37]. We have initiated the generation of a congenic mouse lines for the chromosome 5 interval to verify and further characterize the effect of this region for resistance to influenza infection.
Qivr2-2 contains two candidate genes, Itgb6 (integrin beta 6) and Ifih1 (interferon induced with helicase C domain 1), with known functions in the host defense to viral infections. Itgb6 mouse knock-out mutants exhibit severe pneumonia and an increase in granulocyte recruitment to the lung . The protease-activated receptor 1-mediated enhancement of Itgb6- dependent TGF-beta activation has been proposed to represent one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury . The Ifih1 gene is also known as MDA5 (Melanoma Differentiation-Associated protein 5). IFIH1 is part of the RIG-I-like receptor (RLR) family, which function as pattern recognition receptors and are activated upon binding of virus dsRNA . IFIH1 functions as cytosolic receptor that leads to the selective activation of type I IFN genes and is indispensable for sustained expression of IFN in response to paramyxovirus infection [41, 42]. Ifih1 mutant knock-out mice exhibit an impaired response to different viral pathogens [43, 44].
Qivr16 contains two potential genes with known functions in the host defense and lung function, Robo1 (roundabout homolog 1 (Drosophila)) and Nrip1 (nuclear receptor interacting protein 1). DBA/2J mice carry a frame shift mutation in the Robo1 gene which might lead to an impaired function of the encoded protein. Robo1 has been described to be involved in guidance and migration of axons, myoblasts, and leukocytes in vertebrates (e.g.[45–47]) but is also expressed in the developing lung . Robo1 knock-out mutants exhibit a delayed lung maturation and bronchial hyperplasia. The latter results suggest that Robo1 may be involved in maintaining proper lung function and it may become essential when lung epithelium is destroyed during an influenza infection. Nrip1/Rip140 functions as a co-activator for cytokine gene promoter activity via direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP) . It is involved in modulating pro-inflammatory responses in macrophages .
Qivr17-2 represents a positive influence of the C57BL/6 J genotype on body weight, survival and MTTD. This QTL is located in a gene-rich region which carries many genes that are involved in the host immune response, in particular the H2 histocompatibility genes which are involved in antigen presentation . Therefore, many candidate genes are found in the Qivr17-2 region. The Lst1 (leukocyte specific transcript 1) gene is of special interest because the DBA/2J allele mice carries a single nucleotide deletion in the first exon resulting in a frame shift of the open reading frame. This mutation most likely results in a non-functional Lst1 protein in DBA/2 J mice. We confirmed the presence of the deletion by sequencing the parental DBA/2J and some BXD strains carrying the DBA/2J allele. The wild type allele was confirmed in C57BL/6J mice and in some BXD strains carrying the C57BL/6J allele. In humans, LST1 plays a role in the regulation of the immune response to inflammatory diseases such as rheumatoid arthritis, microbial infection or Rubella vaccine-induced immunity [52–55]. Also, Lst1 is up-regulated after influenza A infection in C57BL/6J mice starting at day 2 and exhibits a strong peak of expression at day 8 p.i. Pommerenke et al., 2012 (Pommerenke, C., E. Wilk, B. Srivastava, A. Schulze, N. Novoselova, R. Geffers, and K. Schughart. 2012. Global transcriptome analysis in influenza-infected mouse lungs reveals the kinetics of innate and adaptive host immune responses. PLoS ONE. 7:e41169.). Thus, the expression profile and known functions of Lst1 fit well with a possible critical role for the host defense to influenza A virus. We initiated the generation of knock-out mice to evaluate the role of Lst1 in more detail. In addition, a second, most interesting candidate, Pla2g7 (phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma)) was identified in the Qivr17-2 interval. In humans, increased activities of certain variants of PLA2G7 were associated with early coronary atherosclerosis and with endothelial dysfunction, but the gene may also exert an anti-inflammatory function [56–60]. The Pla2g7 gene was also identified as a potential candidate gene for susceptibility against infections with H3N2 influenza virus . Pla2g7 expression levels in susceptible A/J mice were higher than in resistant C57BL/6J mice after infection with H3N2 virus . We also showed previously that Pla2g7 exhibits a cis-eQTL between C57BL/6J and DBA/2J in non-infected lungs where the DBA/2J allele shows high levels of expression . Tnfrsf21 which was identified by  as potential candidate of Qivr17-2 also exhibits a cis-eQTL in non-infected BXD mice  but was not found to be regulated in C57BL/6 mice after infection (data not published). Tapbp (TAP binding protein) plays a major role in the antigen processing and MHC class I presentation by stabilizing the TAP peptide transporter, e.g.[61–65]. Also, Tap2 (transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)) gene is involved in antigen processing and presentation [63, 66]. Gtf2h4 (general transcription factor II H, polypeptide 4) encodes a general transcription factor. Recruitment and activation of Gtf2h4 represents a rate-limiting step for the emergence of HIV from latency and sequence variants have been associated with multiple sclerosis [67–69].
Within the Qivr19 interval, only one gene, Hps1 (Hermansky-Pudlak syndrome 1 homolog (human)), has been associated with the host responses to infection. Mice carrying a natural mutation in the Hps1 gene showed an increased inflammatory response in alveolar macrophages after intranasal challenge with LPS .
The GeneNetwork database allows searching for other phenotypic traits that exhibit a genome-wide significant (LRS ≥ 18) within the Qivr intervals identified by our study. Two phenotypic traits, related to neuronal responses (trait ID 11285) and body weight changes (trait ID 12838), are located to the Qivr16 locus. Also, the Qivr17-2 interval contained significant QTLs for other traits. Two traits are related to host infectious diseases, ‘Ectromelia virus survival’ (ID 12672) and ‘Chlamydia psittaci (6BC) infection response’ (ID 11025) and four traits are associated with immune cell responses (ID 10201, 10466, 10238, 10236). In addition two traits described seizure responses (ID 10388, 10507), and one trait has not been disclosed yet (ID 13920). Within the early time chromosome 10 interval, three other traits exhibit their most significant QTLs: ‘3a,5a-THDOC in blood plasma 3 days after cycle 5 of chronic intermittent air vapor’ (ID13027) and two non-disclosed traits. The first trait may relate to stress responses in the central nervous system (ID 13292 and 13846).
The mapping of resistance and susceptibility loci in the BXD population revealed several new QTLs and potential gene candidates that may be critical for the host defense against influenza A virus infection. Body weight and survival QTLs showed a time-dependent profile indicating that the genetic factors in these QTLs are important for the host response in a temporal dynamic fashion. Five QTL regions were examined in detail, and we identified several possible candidate genes that may be critical for the host response to influenza A infections in humans.
The mouse inbred strains C57BL/6J, DBA/2J and B6D2F1 were delivered from Janvier, France. Recombinant inbred mouse strains BXD were purchased from three different sources: The Jackson Laboratory, the University of Tennessee Health Science Center (Memphis, TN) and from Harlan, The Netherlands. For the analysis, mice were transferred to the animal facility in Braunschweig and adapted for at least two weeks to the new environment before starting experiments. Animals were maintained under specific pathogen free conditions. All experiments in mice were approved by an external committee and according to the national guidelines of the animal welfare law in Germany (‘Tierschutzgesetz in der Fassung der Bekanntmachung vom 18. Mai 2006 (BGBl. I S. 1206, 1313), das zuletzt durch Artikel 20 des Gesetzes vom 9. Dezember 2010 (BGBl. I S. 1934) geändert worden ist.’). The protocol used in these experiments has been reviewed by an ethics committee and approved by the ‘Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany’, according to the German animal welfare law (Permit Numbers: 33.42502/04-108/06, 33.9.42502-04-051/09).
Virus and infection of mice
The mouse-adapted influenza strain A/Puerto Rico/8/34 (H1N1; PR8M,  and references therein) was used for all infection studies. Stocks were prepared by infection of 10-day-old embryonated chicken eggs. After mice were anesthetized by intra-peritoneal injection of Ketamin-Xylazine solution in sterile NaCl (50 mg/ml Ketamine, Invesa Arzneimittel GmbH, Freiburg; 2% Xylazine, Bayer Health-Care, Leverkusen) with a dose adjusted to the individual body weight, mice were infected intranasally with 2 × 103 FFU of PR8M in 20 μl of sterile phosphate-buffered saline. Mice were assayed daily for body weight (determined as % of initial weight at day 0) and mortality during 13 days p.i. We used death as the end point for survival. Mice were sacrificed if body weight loss exceeded 25%. It should be noted that for mice that did not show any signs of body weight loss over the entire time period after infection, we do not have additional parameters to verify that they have indeed been infected. However, these cases were very few. In addition, we have ample experience with this infection method and the failure rate, for example with the DBA/2J strain, is less that 5%.
Data handling and statistical analysis
In total, 283 BXD mice and 127 mice from the parental strains or F1 generation were used for the infection experiments. In total 53 BXD strains were infected with an average of 5 mice per strain. We performed all primary calculations using simple features and functions of Microsoft Excel. Three sets of analysis were performed for the following variables: (1) body weight in percentage from starting weight (day 0) using the strain medians to exclude outliers; (2) survival by calculating the survival rate of each strain from day 7 to 13, (3) mean time to death in days. For statistical analyses, tests and visualization we used R, a free software environment for statistical computing and graphics (http://www.R-project.org). In order to test for batch effects or other co-factors, we visualized the data using multidimensional scaling based on the Sammon mapping method . No clusters with respect to any of the co-factors age, weight at day 0, experimenter, time of infection, or source of mice could be found in the visualizations.
QTL mapping was performed using the web-based complex trait analysis available on the GeneNetwork website (http://www.genenetwork.org) and the mapping module to analyze phenotypes in context of mouse genotypic differences. Interval mapping evaluates potential QTLs at regular intervals and estimates the significance at each location with a graphical representation of the likelihood ratio statistics (LRS) using 2000 permutation tests [19, 22]. LRS values may be converted to LOD scores by dividing by 4.61. For the two locus model the following equation was used: Var = Q1 + Q2 + Q1xQ2 + e, where Var = the between-strain mean variance in the trait, Q1 and Q2 are makers tightly linked to the loci, Q1XQ2 is the ‘additive-by-additive’ epistatic interaction term, and e is the residual error. The original data sets can be obtained at http://www.genenetwork.org with the following identification numbers: body weight: 13005 to 13017; survival: 13000 to 13004 and mean time to death: 12996. We performed full genome scans for epistatic interactions using the pair-scan module that is implemented in GeneNetwork. This module exploits the direct global optimization algorithm developed by . The code compares the fit (as measured by LRS scores) for a purely additive model, a purely epistatic model, and the full model. The code also implements a permutation test (n = 500) and this enabled us to estimate the empirical p value of the alternative models.
Candidate gene discovery
The QTL region analysis was initially performed using the QTLminer which has been implemented in GeneNetwork . By using the automatic function of GeneNetwork we identified significant cis-QTLs with LRS higher than 18 at a genome-wide p-value of < 0.05. Additionally the genes mapped within the analyzed QTLs were surveyed by the National Center for Biotechnology Information (NCBI) Entrez Gene website (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) and the Jackson Laboratory's MGI Mouse Genome Database project (http://www.informatics.jax.org/) to identify potential candidate genes. The GeneRIF database (http://www.ncbi.nlm.nih.gov/projects/GeneRIF/GeneRIFhelp.html) was used as a primary source to search for known gene functions and corresponding citations.
All sequence variants between B6 and D2 parental genomes (SNPs, indels) were extracted by using a comparative analysis that relies on approximately 100x whole genome shot gun of DBA/2J . All of these sequence data are available at http://ucscbrowser.genenetwork.org/, the GeneNetwork Variant Browser (http://www.genenetwork.org/webqtl/main.py?FormID=snpBrowser), and the NCBI Short Read Archive (18 files total, e.g., SRX037575, SRX013980, SRX013299, SRX012582, SRX012581, SRX012580); http://www.biomedcentral.com/1471-2105/11/S4/O7.
Tatiana Nedelko and Heike Kollmus contributed equally as first authors.
Robert W. Williams and Klaus Schughart contributed equally as senior authors.
This work was supported by intra-mural grants from the Helmholtz-Association (Program Infection and Immunity) and a research grant FluResearchNet (No. 01KI07137) from the German Ministry of Education and Research to KS and the virtual institute ‘GeNeSys’ funded by the Helmholtz Association. The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Mice for these experiments were maintained by the animal caretakers of the Central Animal Facilities at the HZI. We wish to thank Christin Fricke for excellent technical assistance.
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