BACKGROUND
influenza a virus represents a major health threat to humans.
the  <dig> h1n <dig> pandemic caused at about  <dig> to  <dig> million deaths  <cit> .
seasonal influenza epidemics cause high economic loss, morbidity and deaths every year  <cit> .
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  <cit> .
furthermore, genetic factors in humans associated with a higher susceptibility to influenza infections and severe disease outcome have been suspected for the  <dig> pandemics, as well as the h5n <dig> human infections  <cit> .
recently, the importance of ifitm <dig> as a crucial factor for host susceptibility has been demonstrated in mice and humans  <cit> .
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  <cit> .
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  <cit> .
in contrast, c57bl/6j mice are more resistant.
after infection with mouse-adapted h1n <dig> , dba/2j mice loss weight very rapidly and die within 5–7 days post infection , whereas c57bl/6j mice loss weight until days 6– <dig> after infection and regain their initial weight by 14 days p.i.
<cit> .
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  <cit> .
however, the genomic regions that are responsible for the differential response after infection with h1n <dig> have not been determined.
therefore, we used a large family of bxd type recombinant inbred strains generated by crossing c57bl/6j  to dba/2j  to map genetic loci that modulate disease severity.
the bxd genetic reference population  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  <dig> fully inbred strains  <cit>  available from the jackson laboratory and a new set of  <dig> additional lines that are still in production at the university of tennessee.
individuals within each single strain are essentially isogenic  and genotypes for the entire family, including most of the new strains, are known  <cit> .
genetic variation among this family has be exploited extensively in the past to systematically study the genetics of many traits .
here, we infected over  <dig> of the bxd strains with influenza a h1n <dig> 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.
RESULTS
susceptibility to influenza a virus after experimental infection of bxd mouse strains is highly variable
we infected  <dig> recombinant inbred strains of the bxd population plus the parental strains c57bl/6j, dba/2j and b6d2f <dig> mice with mouse-adapted h1n <dig> virus  and followed body weight and survival over the next 13 days.
mice that lost more than 25% of their starting weight were sacrificed and also recorded as dead.
after infection, body weight changes were highly variable between the bxd strains over the period of 13 days post infection .
similarly, survival rates  and mean time to death  were highly variable between the different bxd strains.
furthermore, the trait percent survival showed a strain-dependent progression over time.
for body weight loss and survival, three different phenotypic response groups can be defined .
in the first , all infected mice within a strain survived, in the second  a majority but not all individuals within a strain survived, and in the third group , a majority died.
most remarkably, in the first group four strains—bxd <dig>  bxd <dig>  bxd <dig>  bxd97—were highly resistant indicating that the infection may not cause any major pathology .
in contrast, bxd <dig> belonging to the third group, lost body weight much more rapidly than even the highly susceptible dba/2j parent .
these results illustrate a large variation of responses within the bxd family.
by day  <dig> p.i., all infected mice had succumbed to infection in  <dig> strains, whereas  <dig> others exhibited more limited mortality .
the incidence of mortality increased in some strains from day  <dig> p.i.
through day  <dig> but not thereafter .
for the mttd phenotype,  <dig> strains showed no mortality after infection, similar to the resistant c57bl/6j parent.
three strains—bxd <dig>  bxd <dig>  bxd103—exhibited a mttd that was even shorter than for the susceptible dba/2j parent .
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.
principal component analysis  of strain mean body weight loss from day  <dig> until day  <dig> was carried out to reduce the number and redundancy of related measurements, as well as to evaluate whether this set of measurements can be broken down into statistically and genetically independent processes.
this analysis revealed one major component, pc <dig> that explains most of the weight loss among strains .
this component corresponds to differences in loss from day  <dig> to  <dig> p.i.
.
in contrast, pc <dig> accounts for only about 10% of the variance in weight loss .
this component corresponds only to very early weight loss after virus inoculation, on days  <dig> and  <dig> p.i., well before any appreciable virus replication has occurred.
thus, pc <dig> is highly likely to be a technical, but still interesting effect associated with the stressful procedure of intranasal inoculation and full anesthesia.
pc <dig> most likely reflects the biologically relevant variation among strains in response to viral replication.
analysis of body weight loss revealed a significant qtl on chromosome  <dig> and several suggestive qtls with time-dependent effects
we mapped body weight loss following infection day by day.
for purposes of analysis, body weights of mice that had died or that were euthanized were assigned a weight equal to 75% of their initial weight.
it should be noted that mice which died continuously lost weight and were close to 75% body weight loss before they were found dead .
after day  <dig>  surviving mice started to gain weight .
for this reason, we limited our analyses of body weight traits to the period of day  <dig> to day  <dig> p.i.
in order to avoid mixing data from dead with recovering mice.
interval mapping of body weight loss detected a significant time-dependent locus on chromosome  <dig> that we named ‘qtl for influenza virus resistance on chromosome 5’  adopting the nomenclature proposed by  <cit> .
this qtl exhibited genome-wide significance on days  <dig>  and  <dig> .
however, the effect of qivr <dig>  although weaker, can also be detected on days  <dig>   <dig>  and  <dig> and  <dig> .
most interestingly; the resistance allele at qivr <dig> is inherited from the nominally sensitive dba/2j parental strain, illustrating the genetic complexity of the influenza response.
suggestive loci  map to chromosomes  <dig>   <dig>   <dig>  proximal and distal  <dig>  and chromosome  <dig>  the effect of these loci was also time-dependent .
another suggestive qtl peak was found on chromosome  <dig> on day  <dig> .
its effect is lower at day  <dig> and not apparent at later days.
these observations are in accordance with the pca  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  <cit> .
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  <cit> .
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  <dig> p.i.
.
we also performed a qtl analysis for the pc <dig> and pc <dig> described above.
pc <dig> detects a significant qtl on chromosome  <dig>  as well as suggestive qtl peaks on proximal chromosome  <dig> and on chromosome  <dig> .
thus, the pc <dig> confirmed the significant qtl on chromosome  <dig> found with the daily body weight loss traits.
interval mapping of pc <dig> detected no significant qtl and one suggestive qtl on chromosome  <dig>  which was not seen in any other trait.
survival rate and mean time to death traits confirmed the qtl on chromosome  <dig> and detected another significant qtl on chromosome 19
the analysis of survival rate traits on days  <dig>   <dig> and  <dig> p.i.
revealed significant peaks on chromosomes  <dig> and  <dig> and suggestive peaks on chromosomes  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> .
the effects of these qtls were again time-dependent.
qivr <dig> was significant at day  <dig>  and day  <dig> p.i.
and its effect was still evident from day  <dig> until  <dig> p.i.
.
similarly, another significant qtl with time-dependent effects was observed on chromosome  <dig>  at day  <dig>  that could also be detected at day  <dig> and days 9– <dig> .
qivr <dig> and qivr <dig> resulted from a positive influence of dba/2j on survival, whereas qivr <dig>  and qivr17- <dig> resulted from a positive influence of c57bl/6j alleles to increase survival.
furthermore, mttd analysis confirmed qivr <dig> as a significant qtl , and qivr <dig> was almost significant for this trait .
in addition, suggestive qtls were found on chromosomes  <dig> and  <dig> for the mttd trait.
in conclusion, survival and mttd traits confirmed the significant qtl on chromosome  <dig>  revealed an additional significant qtl on chromosome  <dig> and several suggestive qtls.
all qtls showed a time-dependent effect.
composite interval and pair-scan mapping indicates various interactions of qtls
the strongest qtls map to chromosomes  <dig> and  <dig>  we therefore performed composite interval mapping in which the contributions of these strong qtls were factored out to reveal possible secondary qtls.
when we controlled for qivr <dig>  the linkage to distal chromosome  <dig> increased and became nearly significant for both survival at day  <dig> and mttd .
the linkage to chromosome  <dig> increased slightly.
however, control for the contribution of qivr <dig> did not reveal any additional loci linked to survival or mttd traits .
finally, we analyzed the joint additive effects of qtls described above as well as possible epistatic interactions among these qtls.
when using a full two-locus model , we found two potential interactions between qivr <dig> and a new locus on chromosome  <dig> as well as between qivr <dig> and qivr <dig>  respectively, for the survival trait at day  <dig> .
the inclusion of an interaction term increases the lrs by  <dig> .
for both qivr <dig> - chromosome  <dig> interaction  and qivr <dig> – qivr <dig> interaction , the allele combination dba/2j / dba/2j showed highest and the combination c57bl/6j / c57bl/6j lowest survival scores.
all differences were highly significant.
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  <dig>   <dig>  distal  <dig>   <dig> and  <dig>  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.
for the candidate gene searches, we defined the critical regions of the qtls manually by considering peak height, its shoulder and bootstraps as guidance .
we then investigated these qivr intervals for potential candidate genes that may be causal for the studied traits .
we first used the qtlminer tool in genenetwork  <cit>  to identify all genes in the qtl intervals and to obtain associated go terms.
next, we select all annotated genes within the qtl intervals that were expressed between day  <dig> and day  <dig> after infection of c57bl/6j mice with pr8m virus .
subsequently, these genes were further characterized for the following attributes: genes that were up- or up-regulated in infected lungs by at least  <dig> -fold in c57bl/6j after pr8m infection, genes carrying an insertion or deletion or a non-synonymous nucleotide change in the open reading frame, genes exhibiting a cis-expression qtl in the lung  <cit> , and genes that were differentially expressed by at least  <dig> -fold in c57bl/6j and dba/2j mice in infected lungs between day  <dig> and day  <dig> p.i.
<cit>  and pommerenke et al., plos one, in press).
the strategy of the qtl mining is shown as flow chart in the additional file 7: figure s <dig>  and the final results are presented in table  <dig>  furthermore, the attributes of all genes located in the qtl regions are listed in detail in the additional file 8: table s2).
using these combined attributes, we identified  <dig> genes as the most likely candidates to regulate the traits controlled by qivr2- <dig>  qivr <dig>  qivr <dig>  qivr17- <dig> and qivr <dig> .
these genes were further evaluated based on their known function from the literature and phenotypes in knock-out mouse mutants .
up- or down-regulated genes were defined as genes that exhibited at least a  <dig>  difference change in expression levels in lungs of infected mice compared to the non-infected controls.
genes were defined as differentially expressed exhibiting at least a  <dig>  difference in expression changes in infected lungs of c57bl/6j and dba/2j.
genes with sequence variations: arobo <dig>  blst <dig>  cbrd <dig>  dh2-ab <dig>  eh2-bl, frpp <dig>  gtctn <dig>  hdhdpsi.
abbreviations: fs: frame shift in coding region, snp: single nucleotide polymorphism, cis-eqtl: cis regulated expression qtl, b6: c57bl/6j, d2: dba/2j.
the genotype of c57bl/6j was used as reference for the sequence polymorphisms.
the knockout mutant phenotype was identified in the mgi mouse genome database.
gene fign  fulfilled most criteria but was found to be expressed only at late times p.i.
and was therefore omitted from the candidate gene list.
abbreviations: ko: knockout mutant, na: not analyzed, snp: single nucleotide polymorphism, ns: non-synonymous codons, orf: open reading frame, stop_g: stop codon gained, stop_l: stop codon lost.
discussion
dba/2j and c57bl/6j mice have been shown previously to differ largely in their susceptibility to h1n <dig>  influenza a virus  <cit> .
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, qivr <dig> and qivr <dig>  were found on chromosomes  <dig> and  <dig>  respectively.
furthermore several suggestive qtls, qivr2- <dig>  qivr <dig> and qivr17- <dig> were observed in at least two traits and at two days on chromosomes  <dig>   <dig> and  <dig>  respectively.
composite mapping revealed an additional almost significant qtl at distal chromosome  <dig>  qivr <dig>
a similar analysis for host resistance to influenza has been performed previously after infecting  <dig> bxd strains with h5n <dig> influenza virus.
this study reported three significant qtls on chromosomes  <dig>   <dig>  and  <dig>  <cit> .
thus, none of these significant qtls overlaps with the qtls identified in our analysis.
five of the strains that were resistant  in our study were also resistant in the study of  <cit>  where a total of  <dig> strains were found to be resistant.
five strains that were highly susceptible in our study  were also highly susceptible in the study by  <cit>  of a total of  <dig> susceptible strains.
furthermore, five strains that were resistant in our study were susceptible in the study by  <cit> .
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 h1n <dig> virus from our study represents a subtype with a monobasic hemagglutinin  cleavage site, whereas the h5n <dig> 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.
<cit> .
therefore, the contribution of host factors to susceptibility may be different between h <dig> and h <dig> containing virus subtypes.
another study described the genetic mapping of susceptibility and resistance factors after infecting a panel of  <dig> axb / bxa congenic strains with a mouse-adapted h3n <dig> influenza virus  <cit> .
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  <dig>   <dig> and  <dig>  the qtl on chromosome  <dig> overlaps with the qivr17- <dig> locus which we found in our study.
furthermore, the candidate gene pla2g <dig> that was identified in their study was also detected as candidate gene in our analysis .
the influence of genetic factors determining the host response to h1n <dig> influenza virus infections was also examined in mice of the pre-collaborative cross collection  <cit> .
in this study, gene expression levels in extreme responders were used to identify expression qtls .
one gene that exhibited a cis-eqtl, sik <dig> , was located in the qivr17- <dig> interval from our study, and we also identified it as potential quantitative trait gene .
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, qivr <dig> and qivr <dig>  were found at different times p.i., day  <dig> and day  <dig>  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, qivr <dig> as well as qivr <dig> 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  is expressed in susceptible dba/2j mice but the corresponding target  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- <dig>  qivr <dig>  qivr <dig>  qivr17- <dig> and qivr <dig> in more detail to identify genes that may be causal for resistance or susceptibility.
in total,  <dig> genes are located in these intervals.
we narrowed down the total number of genes to  <dig> candidates  by using additional information, such as temporal expression after prm <dig> infection , cis-eqtls in non-infected lungs  <cit> , differences in expression between dba/2j and c57bl/6j  <cit> , and sequence variants in the coding regions.
qivr <dig> contains the candidate gene eif2ak <dig>  that is a member of eif2alpha kinases which have been associated with anti-viral host responses  <cit> .
boon et al.
described another eif2alpha kinase, eif2ak <dig> / pkr , in the qirv <dig> locus after infection with influenza h5n <dig>  <cit> .
eif2ak <dig> plays a critical role in modulating immunoglobulin expression during rsv infection.
in addition eif2ak <dig> knock-out mouse mutants are more susceptible to influenza infections  <cit> .
we have initiated the generation of a congenic mouse lines for the chromosome  <dig> interval to verify and further characterize the effect of this region for resistance to influenza infection.
qivr2- <dig> contains two candidate genes, itgb <dig>  and ifih <dig> , with known functions in the host defense to viral infections.
itgb <dig> mouse knock-out mutants exhibit severe pneumonia and an increase in granulocyte recruitment to the lung  <cit> .
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  <cit> .
the ifih <dig> gene is also known as mda <dig> .
ifih <dig> is part of the rig-i-like receptor  family, which function as pattern recognition receptors and are activated upon binding of virus dsrna  <cit> .
ifih <dig> 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  <cit> .
ifih <dig> mutant knock-out mice exhibit an impaired response to different viral pathogens  <cit> .
qivr <dig> contains two potential genes with known functions in the host defense and lung function, robo <dig> ) and nrip <dig> .
dba/2j mice carry a frame shift mutation in the robo <dig> gene which might lead to an impaired function of the encoded protein.
robo <dig> has been described to be involved in guidance and migration of axons, myoblasts, and leukocytes in vertebrates  but is also expressed in the developing lung  <cit> .
robo <dig> knock-out mutants exhibit a delayed lung maturation and bronchial hyperplasia.
the latter results suggest that robo <dig> may be involved in maintaining proper lung function and it may become essential when lung epithelium is destroyed during an influenza infection.
nrip1/rip <dig> 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 -binding protein   <cit> .
it is involved in modulating pro-inflammatory responses in macrophages  <cit> .
qivr17- <dig> 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 h <dig> histocompatibility genes which are involved in antigen presentation  <cit> .
therefore, many candidate genes are found in the qivr17- <dig> region.
the lst <dig>  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 lst <dig> 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, lst <dig> plays a role in the regulation of the immune response to inflammatory diseases such as rheumatoid arthritis, microbial infection or rubella vaccine-induced immunity  <cit> .
also, lst <dig> is up-regulated after influenza a infection in c57bl/6j mice starting at day  <dig> and exhibits a strong peak of expression at day  <dig> p.i.
pommerenke et al.,  <dig> .
thus, the expression profile and known functions of lst <dig> 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 lst <dig> in more detail.
in addition, a second, most interesting candidate, pla2g <dig> ) was identified in the qivr17- <dig> interval.
in humans, increased activities of certain variants of pla2g <dig> were associated with early coronary atherosclerosis and with endothelial dysfunction, but the gene may also exert an anti-inflammatory function  <cit> .
the pla2g <dig> gene was also identified as a potential candidate gene for susceptibility against infections with h3n <dig> influenza virus  <cit> .
pla2g <dig> expression levels in susceptible a/j mice were higher than in resistant c57bl/6j mice after infection with h3n <dig> virus  <cit> .
we also showed previously that pla2g <dig> exhibits a cis-eqtl between c57bl/6j and dba/2j in non-infected lungs where the dba/2j allele shows high levels of expression  <cit> .
tnfrsf <dig> which was identified by  <cit>  as potential candidate of qivr17- <dig> also exhibits a cis-eqtl in non-infected bxd mice  <cit>  but was not found to be regulated in c57bl/ <dig> mice after infection .
tapbp  plays a major role in the antigen processing and mhc class i presentation by stabilizing the tap peptide transporter, e.g.
<cit> .
also, tap <dig> ) gene is involved in antigen processing and presentation  <cit> .
gtf2h <dig>  encodes a general transcription factor.
recruitment and activation of gtf2h <dig> represents a rate-limiting step for the emergence of hiv from latency and sequence variants have been associated with multiple sclerosis  <cit> .
within the qivr <dig> interval, only one gene, hps <dig> ), has been associated with the host responses to infection.
mice carrying a natural mutation in the hps <dig> gene showed an increased inflammatory response in alveolar macrophages after intranasal challenge with lps  <cit> .
the genenetwork database allows searching for other phenotypic traits that exhibit a genome-wide significant  within the qivr intervals identified by our study.
two phenotypic traits, related to neuronal responses  and body weight changes , are located to the qivr <dig> locus.
also, the qivr17- <dig> interval contained significant qtls for other traits.
two traits are related to host infectious diseases, ‘ectromelia virus survival’  and ‘chlamydia psittaci  infection response’  and four traits are associated with immune cell responses .
in addition two traits described seizure responses , and one trait has not been disclosed yet .
within the early time chromosome  <dig> interval, three other traits exhibit their most significant qtls: ‘3a,5a-thdoc in blood plasma 3 days after cycle  <dig> of chronic intermittent air vapor’  and two non-disclosed traits.
the first trait may relate to stress responses in the central nervous system .
CONCLUSIONS
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.

 influenza a virus represents a major health threat to humans. the h1n pandemic caused at about to 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. furthermore, genetic factors in humans associated with a higher susceptibility to influenza infections and severe disease outcome have been suspected for the pandemics, as well as the h5n human infections. recently, the importance of ifitm 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. 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. in contrast, c57bl/6j mice are more resistant. after infection with mouse-adapted h1n, dba/2j mice loss weight very rapidly and die within 5–7 days post infection, whereas c57bl/6j mice loss weight until days 6– after infection and regain their initial weight by 14 days p.i.. 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. however, the genomic regions that are responsible for the differential response after infection with h1n have not been determined. therefore, we used a large family of bxd type recombinant inbred strains generated by crossing c57bl/6j to dba/2j to map genetic loci that modulate disease severity. the bxd genetic reference population 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 fully inbred strains available from the jackson laboratory and a new set of additional lines that are still in production at the university of tennessee. individuals within each single strain are essentially isogenic 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. here, we infected over of the bxd strains with influenza a h1n 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 we infected recombinant inbred strains of the bxd population plus the parental strains c57bl/6j, dba/2j and b6d2f mice with mouse-adapted h1n virus and followed body weight and survival over the next 13 days. mice that lost more than 25% of their starting weight were sacrificed and also recorded as dead. after infection, body weight changes were highly variable between the bxd strains over the period of 13 days post infection. similarly, survival rates and mean time to death were highly variable between the different bxd strains. furthermore, the trait percent survival showed a strain-dependent progression over time. for body weight loss and survival, three different phenotypic response groups can be defined. in the first, all infected mice within a strain survived, in the second a majority but not all individuals within a strain survived, and in the third group, a majority died. most remarkably, in the first group four strains—bxd bxd bxd bxd97—were highly resistant indicating that the infection may not cause any major pathology. in contrast, bxd belonging to the third group, lost body weight much more rapidly than even the highly susceptible dba/2j parent. these results illustrate a large variation of responses within the bxd family. by day p.i., all infected mice had succumbed to infection in strains, whereas others exhibited more limited mortality. the incidence of mortality increased in some strains from day p.i. through day but not thereafter. for the mttd phenotype, strains showed no mortality after infection, similar to the resistant c57bl/6j parent. three strains—bxd bxd bxd103—exhibited a mttd that was even shorter than for the susceptible dba/2j parent. 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. principal component analysis of strain mean body weight loss from day until day was carried out to reduce the number and redundancy of related measurements, as well as to evaluate whether this set of measurements can be broken down into statistically and genetically independent processes. this analysis revealed one major component, pc that explains most of the weight loss among strains. this component corresponds to differences in loss from day to p.i.. in contrast, pc accounts for only about 10% of the variance in weight loss. this component corresponds only to very early weight loss after virus inoculation, on days and p.i., well before any appreciable virus replication has occurred. thus, pc is highly likely to be a technical, but still interesting effect associated with the stressful procedure of intranasal inoculation and full anesthesia. pc most likely reflects the biologically relevant variation among strains in response to viral replication. analysis of body weight loss revealed a significant qtl on chromosome and several suggestive qtls with time-dependent effects we mapped body weight loss following infection day by day. for purposes of analysis, body weights of mice that had died or that were euthanized were assigned a weight equal to 75% of their initial weight. it should be noted that mice which died continuously lost weight and were close to 75% body weight loss before they were found dead. after day surviving mice started to gain weight. for this reason, we limited our analyses of body weight traits to the period of day to day p.i. in order to avoid mixing data from dead with recovering mice. interval mapping of body weight loss detected a significant time-dependent locus on chromosome that we named ‘qtl for influenza virus resistance on chromosome 5’ adopting the nomenclature proposed by. this qtl exhibited genome-wide significance on days and. however, the effect of qivr although weaker, can also be detected on days and and. most interestingly; the resistance allele at qivr is inherited from the nominally sensitive dba/2j parental strain, illustrating the genetic complexity of the influenza response. suggestive loci map to chromosomes proximal and distal and chromosome the effect of these loci was also time-dependent. another suggestive qtl peak was found on chromosome on day. its effect is lower at day and not apparent at later days. these observations are in accordance with the pca 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. 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 p.i.. we also performed a qtl analysis for the pc and pc described above. pc detects a significant qtl on chromosome as well as suggestive qtl peaks on proximal chromosome and on chromosome. thus, the pc confirmed the significant qtl on chromosome found with the daily body weight loss traits. interval mapping of pc detected no significant qtl and one suggestive qtl on chromosome which was not seen in any other trait. survival rate and mean time to death traits confirmed the qtl on chromosome and detected another significant qtl on chromosome 19 the analysis of survival rate traits on days and p.i. revealed significant peaks on chromosomes and and suggestive peaks on chromosomes and. the effects of these qtls were again time-dependent. qivr was significant at day and day p.i. and its effect was still evident from day until p.i.. similarly, another significant qtl with time-dependent effects was observed on chromosome at day that could also be detected at day and days 9–. qivr and qivr resulted from a positive influence of dba/2j on survival, whereas qivr and qivr17- resulted from a positive influence of c57bl/6j alleles to increase survival. furthermore, mttd analysis confirmed qivr as a significant qtl, and qivr was almost significant for this trait. in addition, suggestive qtls were found on chromosomes and for the mttd trait. in conclusion, survival and mttd traits confirmed the significant qtl on chromosome revealed an additional significant qtl on chromosome and several suggestive qtls. all qtls showed a time-dependent effect. composite interval and pair-scan mapping indicates various interactions of qtls the strongest qtls map to chromosomes and we therefore performed composite interval mapping in which the contributions of these strong qtls were factored out to reveal possible secondary qtls. when we controlled for qivr the linkage to distal chromosome increased and became nearly significant for both survival at day and mttd. the linkage to chromosome increased slightly. however, control for the contribution of qivr did not reveal any additional loci linked to survival or mttd traits. finally, we analyzed the joint additive effects of qtls described above as well as possible epistatic interactions among these qtls. when using a full two-locus model, we found two potential interactions between qivr and a new locus on chromosome as well as between qivr and qivr respectively, for the survival trait at day. the inclusion of an interaction term increases the lrs by. for both qivr - chromosome interaction and qivr – qivr interaction, the allele combination dba/2j / dba/2j showed highest and the combination c57bl/6j / c57bl/6j lowest survival scores. all differences were highly significant. 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 distal and 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. for the candidate gene searches, we defined the critical regions of the qtls manually by considering peak height, its shoulder and bootstraps as guidance. we then investigated these qivr intervals for potential candidate genes that may be causal for the studied traits. we first used the qtlminer tool in genenetwork to identify all genes in the qtl intervals and to obtain associated go terms. next, we select all annotated genes within the qtl intervals that were expressed between day and day after infection of c57bl/6j mice with pr8m virus. subsequently, these genes were further characterized for the following attributes: genes that were up- or up-regulated in infected lungs by at least -fold in c57bl/6j after pr8m infection, genes carrying an insertion or deletion or a non-synonymous nucleotide change in the open reading frame, genes exhibiting a cis-expression qtl in the lung, and genes that were differentially expressed by at least -fold in c57bl/6j and dba/2j mice in infected lungs between day and day p.i. and pommerenke et al., plos one, in press). the strategy of the qtl mining is shown as flow chart in the additional file 7: figure s and the final results are presented in table  furthermore, the attributes of all genes located in the qtl regions are listed in detail in the additional file 8: table s2). using these combined attributes, we identified genes as the most likely candidates to regulate the traits controlled by qivr2- qivr qivr qivr17- and qivr. these genes were further evaluated based on their known function from the literature and phenotypes in knock-out mouse mutants. up- or down-regulated genes were defined as genes that exhibited at least a difference change in expression levels in lungs of infected mice compared to the non-infected controls. genes were defined as differentially expressed exhibiting at least a difference in expression changes in infected lungs of c57bl/6j and dba/2j. genes with sequence variations: arobo blst cbrd dh2-ab eh2-bl, frpp gtctn hdhdpsi. abbreviations: fs: frame shift in coding region, snp: single nucleotide polymorphism, cis-eqtl: cis regulated expression qtl, b6: c57bl/6j, d2: dba/2j. the genotype of c57bl/6j was used as reference for the sequence polymorphisms. the knockout mutant phenotype was identified in the mgi mouse genome database. gene fign fulfilled most criteria but was found to be expressed only at late times p.i. and was therefore omitted from the candidate gene list. abbreviations: ko: knockout mutant, na: not analyzed, snp: single nucleotide polymorphism, ns: non-synonymous codons, orf: open reading frame, stop_g: stop codon gained, stop_l: stop codon lost. discussion dba/2j and c57bl/6j mice have been shown previously to differ largely in their susceptibility to h1n influenza a virus. 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, qivr and qivr were found on chromosomes and respectively. furthermore several suggestive qtls, qivr2- qivr and qivr17- were observed in at least two traits and at two days on chromosomes and respectively. composite mapping revealed an additional almost significant qtl at distal chromosome qivr a similar analysis for host resistance to influenza has been performed previously after infecting bxd strains with h5n influenza virus. this study reported three significant qtls on chromosomes and. thus, none of these significant qtls overlaps with the qtls identified in our analysis. five of the strains that were resistant in our study were also resistant in the study of where a total of strains were found to be resistant. five strains that were highly susceptible in our study were also highly susceptible in the study by of a total of 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 h1n virus from our study represents a subtype with a monobasic hemagglutinin cleavage site, whereas the h5n 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.. therefore, the contribution of host factors to susceptibility may be different between h and h containing virus subtypes. another study described the genetic mapping of susceptibility and resistance factors after infecting a panel of axb / bxa congenic strains with a mouse-adapted h3n 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 and the qtl on chromosome overlaps with the qivr17- locus which we found in our study. furthermore, the candidate gene pla2g that was identified in their study was also detected as candidate gene in our analysis. the influence of genetic factors determining the host response to h1n 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. one gene that exhibited a cis-eqtl, sik, was located in the qivr17- interval from our study, and we also identified it as potential quantitative trait gene. 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, qivr and qivr were found at different times p.i., day and day 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, qivr as well as qivr 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 is expressed in susceptible dba/2j mice but the corresponding target 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- qivr qivr qivr17- and qivr in more detail to identify genes that may be causal for resistance or susceptibility. in total, genes are located in these intervals. we narrowed down the total number of genes to candidates by using additional information, such as temporal expression after prm infection, cis-eqtls in non-infected lungs, differences in expression between dba/2j and c57bl/6j, and sequence variants in the coding regions. qivr contains the candidate gene eif2ak that is a member of eif2alpha kinases which have been associated with anti-viral host responses. boon et al. described another eif2alpha kinase, eif2ak / pkr, in the qirv locus after infection with influenza h5n. eif2ak plays a critical role in modulating immunoglobulin expression during rsv infection. in addition eif2ak knock-out mouse mutants are more susceptible to influenza infections. we have initiated the generation of a congenic mouse lines for the chromosome interval to verify and further characterize the effect of this region for resistance to influenza infection. qivr2- contains two candidate genes, itgb and ifih, with known functions in the host defense to viral infections. itgb 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 ifih gene is also known as mda. ifih is part of the rig-i-like receptor family, which function as pattern recognition receptors and are activated upon binding of virus dsrna. ifih 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. ifih mutant knock-out mice exhibit an impaired response to different viral pathogens. qivr contains two potential genes with known functions in the host defense and lung function, robo ) and nrip. dba/2j mice carry a frame shift mutation in the robo gene which might lead to an impaired function of the encoded protein. robo has been described to be involved in guidance and migration of axons, myoblasts, and leukocytes in vertebrates but is also expressed in the developing lung. robo knock-out mutants exhibit a delayed lung maturation and bronchial hyperplasia. the latter results suggest that robo may be involved in maintaining proper lung function and it may become essential when lung epithelium is destroyed during an influenza infection. nrip1/rip 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 -binding protein. it is involved in modulating pro-inflammatory responses in macrophages. qivr17- 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 h histocompatibility genes which are involved in antigen presentation. therefore, many candidate genes are found in the qivr17- region. the lst 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 lst 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, lst plays a role in the regulation of the immune response to inflammatory diseases such as rheumatoid arthritis, microbial infection or rubella vaccine-induced immunity. also, lst is up-regulated after influenza a infection in c57bl/6j mice starting at day and exhibits a strong peak of expression at day p.i. pommerenke et al.,. thus, the expression profile and known functions of lst 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 lst in more detail. in addition, a second, most interesting candidate, pla2g ) was identified in the qivr17- interval. in humans, increased activities of certain variants of pla2g were associated with early coronary atherosclerosis and with endothelial dysfunction, but the gene may also exert an anti-inflammatory function. the pla2g gene was also identified as a potential candidate gene for susceptibility against infections with h3n influenza virus. pla2g expression levels in susceptible a/j mice were higher than in resistant c57bl/6j mice after infection with h3n virus. we also showed previously that pla2g exhibits a cis-eqtl between c57bl/6j and dba/2j in non-infected lungs where the dba/2j allele shows high levels of expression. tnfrsf which was identified by as potential candidate of qivr17- also exhibits a cis-eqtl in non-infected bxd mice but was not found to be regulated in c57bl/ mice after infection. tapbp plays a major role in the antigen processing and mhc class i presentation by stabilizing the tap peptide transporter, e.g.. also, tap ) gene is involved in antigen processing and presentation. gtf2h encodes a general transcription factor. recruitment and activation of gtf2h represents a rate-limiting step for the emergence of hiv from latency and sequence variants have been associated with multiple sclerosis. within the qivr interval, only one gene, hps ), has been associated with the host responses to infection. mice carrying a natural mutation in the hps 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 within the qivr intervals identified by our study. two phenotypic traits, related to neuronal responses and body weight changes, are located to the qivr locus. also, the qivr17- interval contained significant qtls for other traits. two traits are related to host infectious diseases, ‘ectromelia virus survival’ and ‘chlamydia psittaci infection response’ and four traits are associated with immune cell responses. in addition two traits described seizure responses, and one trait has not been disclosed yet. within the early time chromosome interval, three other traits exhibit their most significant qtls: ‘3a,5a-thdoc in blood plasma 3 days after cycle of chronic intermittent air vapor’ and two non-disclosed traits. the first trait may relate to stress responses in the central nervous system.  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. 
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. analysis of multiple gene expression data sets and sequence variants in these strains highlights about strong candidate genes across all loci that may control influenza a susceptibility and resistance. we have mapped influenza susceptibility loci to chromosomes and body weight and survival loci have a time-dependent profile that presumably reflects the temporal dynamic of the response to infection. qivr was the largest and most significant qtl for weight loss. there is strong but mostly circumstantial evidence that genetic factors modulate the severity of influenza infection in humans. we monitored body weight, survival, and mean time to death for 13 days after infection.
 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 bxd-type lines derived from a cross between c57bl/6j and dba/2j strains with influenza a virus. we monitored body weight, survival, and mean time to death for 13 days after infection. qivr was the largest and most significant qtl for weight loss. the effect of qivr was detectable on day post infection, but was most pronounced on days and survival rate mapped to qivr but additionally revealed a second significant locus on chromosome. analysis of mean time to death affirmed both qivr and qivr 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 strong candidate genes across all loci that may control influenza a susceptibility and resistance.  we have mapped influenza susceptibility loci to chromosomes and 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. 