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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 [1]. Seasonal influenza epidemics cause high economic loss, morbidity and deaths every year [2]. 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 [8].
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 [21]. 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 [22]).
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
We infected 53 recombinant inbred strains of the BXD population plus the parental strains C57BL/6J, DBA/2J and B6D2F1 mice with mouse-adapted H1N1 virus (PR8M, H1N1 [18]) 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 (Figure 1, Additional file 1: Table S1). Similarly, survival rates (Additional file 2: Figure S1A, B) and mean time to death (MTTD; Additional file 2: Figure S1C) were highly variable between the different BXD strains. Furthermore, the trait percent survival showed a strain-dependent progression over time.
Additional file 1. Table S1. Body weight loss from day 1 until day 7 for all strains showing the mean values, SEM per day and number of mice per strain analyzed.
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Additional file 2. Figure S1. Differential susceptibility to influenza A infection among different BXD strains, parental strains and F1 generation. Rank-ordered strain distribution pattern illustrating the percentage of surviving mice per strain after influenza A infection for 53 BXD, parental DBA/2J and C57BL/6J strains and F1 (B6D2F1) mice for day 7 (A, trait ID: 13001) and day 8 (B, trait ID: 13002). Rank-ordered strain distribution pattern showing mean time to death for 53 BXD, parental DBA/2J and C57BL/6J strains and F1 (B6D2F1) mice (C, trait ID: 12996).
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Figure 1. BXD strains exhibit variable kinetics of weight loss and survival after infection with Influenza A virus. Mice from 53 BXD and parental strains were infected intra-nasally with 2 × 103 FFU of PR8 virus. Weight loss and survival of infected mice was followed over a period of 13 days. Mortality includes mice that were sacrificed because they had lost more than 25% of body weight. Three phenotypic groups can be distinguished: in the first group (A), all infected mice within a given BXD strain survived, in the second group (B) less than 50% of infected mice within a given BXD strain died, in the third group (C), more than 50% of infected mice within a given BXD strain died. From the weight loss curve of the second and third group it also becomes obvious that non-surviving mice were all approaching the 75% body weight loss endpoint before dying.
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.
Principal Component Analysis (PCA) of strain mean body weight loss from day 1 until day 7 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, PC1 that explains most of the weight loss among strains (more than 80%, see Additional file 3: Figure S2). This component corresponds to differences in loss from day 3 to 7 p.i. (Figure 2). In contrast, PC2 accounts for only about 10% of the variance in weight loss (Additional file 3: Figure S2). This component corresponds only to very early weight loss after virus inoculation, on days 1 and 2 p.i.(Figure 2), well before any appreciable virus replication has occurred. Thus, PC2 is highly likely to be a technical, but still interesting effect associated with the stressful procedure of intranasal inoculation and full anesthesia. PC1 most likely reflects the biologically relevant variation among strains in response to viral replication.
Additional file 3. Figure S2. Principal component analysis of all body weight traits. Percent contribution of principal components to the total variance for body weight traits from day 1 until day 7 p.i. PC1 contributes about 80% and PC2 about 10% to the total variance.
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Figure 2. Principal component analysis of all body weight traits reveals two distinct groups. PCA loading plot of principal components PC1 and PC2 (Supplement Figure 2) for body weight traits form day 1 to day 7 p.i. shows two distinct groups. The variation of body weight loss at day 3 to day 7 (ID 13007 to ID 13011) are mainly represented by factor 1. On the other hand, variation of body weight loss at day 1 (ID 13005) and 2 (ID 13006) are most likely explained by factor 2.
Analysis of body weight loss revealed a significant QTL on chromosome 5 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 (Figure 1B, C). After day 7, surviving mice started to gain weight (Figure 1A, B). For this reason, we limited our analyses of body weight traits to the period of day 1 to day 7 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 5 that we named 'QTL for influenza virus resistance on chromosome 5' (Qivr5) adopting the nomenclature proposed by [12]. This QTL exhibited genome-wide significance on days 5 (LRS: 19.0, effect size: 30%) and 6 (LRS: 19.4, effect size: 31%, Figure 3). However, the effect of Qivr5, although weaker, can also be detected on days 2, 3, and 4 and 7 (Figure 3). Most interestingly; the resistance allele at Qivr5 is inherited from the nominally sensitive DBA/2J parental strain, illustrating the genetic complexity of the influenza response. Suggestive loci (LRS values between 10 and 15) map to chromosomes 2, 6, 9, proximal and distal 16, and chromosome 17. The effect of these loci was also time-dependent (Figure 3A-G).
Figure 3. Genome-wide linkage analysis for body weight loss after H1N1 infection identifies time-dependent QTLs. Analysis of body weight from day 1 (A), day 2 (B), day 3 (C), day 4 (D), day 5 (E), day 6 (F) and day 7 (G) after PR8 infection revealed time-dependent QTLs. A significant QTL is located on chromosome 5 and suggestive QTLs are observed on chromosomes 2, 6, 9, 10, 16 and 17. Dead or euthanized mice were included for the analysis by manually setting their body weight to 75%. Interval mapping is shown across the entire genome as blue line representing the LRS scores; green line: DBA/2J alleles increase trait values; red line: C57BL/6J alleles increase trait values. Upper x-axis shows mouse chromosomes, lower x-axis shows physical map in mega bases for each chromosome, y-axis represents LRS score. The horizontal lines mark the genome-wide significant thresholds at p<0.05 (red line) and suggestive thresholds at p<0.37 (gray line).
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 [24]. 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).
Additional file 4. Figure S3. Body weight changes in mock-infected C57BL/6J and DBA/2J mice. Female DBA/2J (n=4) and C57BL/6J (n=3) mice were intranasally inoculated with 25 μl PBS under anesthesia. Body weight changes for each group of treated mice at various days p.i. is shown with reference to the starting weight (% body weight). Data represent mean values +/- SEM.
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We also performed a QTL analysis for the PC1 and PC2 described above. PC1 detects a significant QTL on chromosome 5 (LRS: 19, effect size: 30%, Figure 4A) as well as suggestive QTL peaks on proximal chromosome 16 and on chromosome 17 (Figure 4A). Thus, the PC1 confirmed the significant QTL on chromosome 5 found with the daily body weight loss traits. Interval mapping of PC2 detected no significant QTL and one suggestive QTL on chromosome 13 (Figure 4B) which was not seen in any other trait.
Figure 4. Genome-wide linkage scan for PC1 and PC2 body weight loss. Interval mapping of principle component PC1 (A) for body weight loss traits at day 1 to 7 after PR8 infection shows significant QTLs on chromosome 5 and suggestive QTLs on chromosomes 16 and 17. Interval mapping of PC2 (B) for body weight loss traits reveals no significant QTLs and a suggestive QTL on chromosome 13. Interval mapping is shown across the entire genome as blue line representing the LRS scores; green line: DBA/2J alleles increase trait values; red line: C57BL/6J alleles increase trait values. Upper x-axis shows mouse chromosomes, lower x-axis shows physical map in mega bases for each chromosome, y-axis represents LRS score. The horizontal lines mark the genome-wide significant thresholds at p<0.05 (red line) and suggestive thresholds at p<0.37 (gray line).
Survival rate and mean time to death traits confirmed the QTL on chromosome 5 and detected another significant QTL on chromosome 19
The analysis of survival rate traits on days 7, 8 and 11 p.i. revealed significant peaks on chromosomes 5 and 19 and suggestive peaks on chromosomes 1, 2, 3, 10, 16 and 17 (Figure 5A-C). The effects of these QTLs were again time-dependent. Qivr5 was significant at day 7 (LRS: 24.8, effect size: 37%) and day 8 p.i. (LRS: 19.7, effect size: 31% Figure 5A, B) and its effect was still evident from day 9 until 11 p.i. (Additional file 5: Figure S4 and Figure 5C). Similarly, another significant QTL with time-dependent effects was observed on chromosome 19 (Qivr19) at day 8 (LRS: 19.4; effect size: 29%) that could also be detected at day 7 and days 9–11 (Figure 5A-C and Additional file 5: Figure S4). Qivr5 and Qivr19 resulted from a positive influence of DBA/2J on survival, whereas Qivr16 (distal locus) and Qivr17-2 resulted from a positive influence of C57BL/6J alleles to increase survival. Furthermore, MTTD analysis confirmed Qivr5 as a significant QTL (LRS: 20.5, effect size: 32%), and Qivr19 was almost significant for this trait (Figure 5D). In addition, suggestive QTLs were found on chromosomes 2 and 17 for the MTTD trait.
Additional file 5. Figure S4. Genome-wide linkage analysis for survival after H1N1 infection at days 9 and 10 p.i. Analysis of survival from day 9 (A) and day 10 (B) after PR8 infection revealed suggestive QTLs on chromosomes 5, 16, 17 and 19. Interval mapping is shown across the entire genome as blue line representing the LRS scores; green line: DBA/2J alleles increase trait values; red line: C57BL/6J alleles increase trait values. Upper x-axis shows mouse chromosomes, lower x-axis shows physical map in mega bases for each chromosome, y-axis represents LRS score. The horizontal lines mark the genome-wide significant thresholds at p0.05 (red line) and suggestive thresholds at p0.37 (gray line).
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Figure 5. Genome-wide linkage scan for survival rate and mean time to death confirms QTLs on chromosomes 5 and 19. Interval mapping of survival rates (in %) after PR8 infection at day 7 (A), day 8 (B) and day 11 (C) show significant QTLs on chromosomes 5 and 19. Suggestive QTLs are observed on chromosomes 2, 3, 10, 16 and 17. Interval mapping using mean time to death confirmed the significant QTL on chromosome 5 and revealed suggestive QTLs on chromosomes 2, 17 and 19 (D). Interval mapping is shown across the entire genome as blue line representing the LRS scores; green line: DBA/2J alleles increase trait values; red line: C57BL/6J alleles increase trait values. Upper x-axis shows mouse chromosomes, lower x-axis shows physical map in mega bases for each chromosome, y-axis represents LRS score. The horizontal lines mark the genome-wide significant thresholds at p<0.05 (red line) and suggestive thresholds at p<0.37 (gray line).
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
The strongest QTLs map to chromosomes 5 and 19. 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 Qivr19, the linkage to distal chromosome 16 increased and became nearly significant for both survival at day 8 and MTTD (Figure 6A, B). The linkage to chromosome 2 increased slightly. However, control for the contribution of Qivr5 did not reveal any additional loci linked to survival or MTTD traits (Figure 6C, D).
Figure 6. Composite interval mapping indicates interactions of QTLs on chromosomes 5 and 19. The influence of the markers rs13483633 on chromosome 19 (A, B) and rs13478587 on chromosome 5 (C, D) was factored out and the residual LRS were calculated. Two different traits, survival day 8 (A, C) and mean time of death (B, D) were tested by using the web tool of GeneNetwork. Interval mapping is shown across the entire genome as blue line representing the LRS scores; green line: DBA/2J alleles increase trait values; red line: C57BL/6J alleles increase trait values. Upper x-axis shows mouse chromosomes, lower x-axis shows physical map in mega bases for each chromosome, y-axis represents LRS score. The horizontal lines mark the genome-wide significant thresholds at p<0.05 (red line) and suggestive thresholds at p<0.37 (gray line).
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 (Trait Variance = Q1 + Q2 + Q1 x Q2), we found two potential interactions between Qivr5 and a new locus on chromosome 9 as well as between Qivr5 and Qivr19, respectively, for the survival trait at day 8 (Figure 7A). The inclusion of an interaction term increases the LRS by 23 (Additional file 6: Figure S5). For both Qivr5 - chromosome 9 interaction (Figure 7B) and Qivr5 – Qivr19 interaction (Figure 7C), the allele combination DBA/2J / DBA/2J showed highest and the combination C57BL/6J / C57BL/6J lowest survival scores. All differences were highly significant.
Additional file 6. Figure S5. Pair Scan analysis of trait 13002. The table provides the breakdown of trait 13002 as an example for a pair scan analysis study that compares the fit of the alternative models. An analysis of this type can be regenerated rapidly on any of the traits presented in our manuscript.
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Figure 7. Interactions are observed between several QTLs. The graph displays pair-scan results for the trait percent survival at day 8 p.i. (ID13002) (A). The upper left half of the plot highlights any epistatic interactions, the lower right half provides a summary of LRS of the full model, representing cumulative effects of linear and non-linear terms based on the model 'Trait Variance = Q1 + Q2 + Q1 x Q2 '. Subsequently, BXD lines were grouped according to their allele combinations and trait values were compared between groups (B, C). The largest differences are indicated by asterisks. They are highly significant between the respective groups (p < 0.0001). For the various loci, trait values for the following markers were used: Qivr5: rs13478587, chromosome 9: rs6191976, Qivr19: rs13483633. Number of lines and p-values for Qivr5 – chromosome 9: D2/D2: n = 17, D2/B6: n = 17, B6/D2: n = 6, B6/B6 n = 12; D2/D2 vs B6/B6: p < 0.0001, D2/B6 vs B6/B6: p = 0.003. Number of lines and p-values for Qivr5 – Qivr19: D2/D2: n = 18, D2/B6: n = 16, B6/D2: n = 4, B6/B6: n = 14; p-values: D2/D2 vs B6/B6: p < 0.0001, D2/B6 vs B6/B6: p = 0.0008. Abbreviations: D2: DBA/2J, B6: C57BL/6J.
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.
For the candidate gene searches, we defined the critical regions of the QTLs manually by considering peak height, its shoulder and bootstraps as guidance (Figure 8). We then investigated these Qivr intervals for potential candidate genes that may be causal for the studied traits (Additional file 7: Figure S6). We first used the QTLminer tool in GeneNetwork [25] 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 1 and day 60 after infection of C57BL/6J mice with PR8M virus (the latter data set is derived from a separate study (Pommerenke et al., PLoS ONE, in press). Subsequently, these genes were further characterized for the following attributes: genes that were up- or up-regulated in infected lungs by at least 1.5-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 [26], and genes that were differentially expressed by at least 1.5-fold in C57BL/6J and DBA/2J mice in infected lungs between day 1 and day 8 p.i. [23] 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 S6, and the final results are presented in Table 1. 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 31 genes as the most likely candidates to regulate the traits controlled by Qivr2-2, Qivr5, Qivr16, Qivr17-2 and Qivr19 (listed in Table 2). These genes were further evaluated based on their known function from the literature and phenotypes in knock-out mouse mutants (see discussion).
Additional file 7. Figure S6. Scheme to identify potential candidate genes in the analyzed QTL intervals. The detailed steps are described in the main manuscript.
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Additional file 8. Table S2. List of genes in the QTL intervals that were studied in detail. The QTLminer tool in GeneNetwork was to identify all genes in the QTL intervals and to obtain associated GO terms and KEGG pathways. All annotated genes within the QTL intervals that were expressed between day 1 and day 60 after infection of C57BL/6J mice with PR8M virus were further analyzed for the following attributes: genes that were up- or down-regulated in infected lungs 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 in C57BL/6J and DBA/2J mice in infected lungs between day 1 and day 8 p.i. Abbreviations: FS: frame shift in coding region, SNP: single nucleotide polymorphism, cis-eQTL: cis-regulated expression QTL, B6: C57BL/6J, D2: DBA/2J, ns: non-synonymous codons, stop_G: stop codon gained, stop_L: stop codon lost, DEL: deletion, INS: insertion; ns (…): number of non-synonymous codons. Legends: Expression d0-d60, log2 ≥8 at any day during infection; No: log2 8; ?: no expression data available. Regulation during infection (d0-d60): + - up or down regulation, genes that exhibited at least a 2-fold difference in changes of the expression levels in lungs of infected mice compared to non-infected controls; (+)(-): up-or down-regulation, genes that exhibited at least a 1.5-fold difference in changes of the expression levels in lungs of infected mice compared to the non-infected controls; no: no regulation. SNP: ns (.) number of non-synonymous codons; STOP_L: Stop codon lost, STOP_G: Stop codon gained; no: no SNPs between B6 and D2. Indel: DEL: Deletion; INS: Insertion; no: no INDEL. cisQTL: yes (…): LRS value has to be greater than ≥18; no: no QTL or LRS 18. diff expressed (B6-D2) during infection: Genes were defined as differentially expressed (log2 ≥8) at any day in infected lungs of C57BL/6J and DBA/2J; + -: Genes were defined as differentially expressed exhibiting at least a 2.0 difference in changes of the expression levels in infected lungs of C57BL/6J and DBA/2J; (+)(-) differentially expressed: Genes were defined as differentially expressed exhibiting at least a 1.5 difference in changes of the expression levels in infected lungs of C57BL/6J and DBA/2J; no: no regulation or not expressed; Score: the different attributes were counted.
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Figure 8. Detailed maps of QTLs contributing to host susceptibility and resistance to PR8 influenza A infection. Interval mapping was performed for chromosomes 2 (A), 5 (B), 16 (C), 17 (D) and 19 (E). The critical intervals were selected based on the peak shapes and the bootstrap signals: Qivr2-1 (56–68 Mb), Qivr5 (140–153 Mb), Qivr16 (64–78 Mb), Qivr17-2 (30–44 Mb) and Qivr19 (37–45 Mb). The Qivr intervals are delineated by red arrows. The yellow bars represent the frequencies of peak LRS using bootstrap analysis. The multicolored chatters along the top of the graph are hyperlinks to sites with additional genetic and sequence information. The orange chatter along the x-axis indicates the density of SNPs present in the BXD strains. Interval mapping is shown across the entire genome as blue line representing the LRS scores; green line: DBA/2J alleles increase trait values; red line: C57BL/6J alleles increase trait values. Upper x-axis shows mouse chromosomes, lower x-axis shows physical map in mega bases for each chromosome, y-axis represents LRS score. The horizontal lines mark the genome-wide significant thresholds at p<0.05 (red line) and suggestive thresholds at p<0.37 (gray line).
Table 1. Candidate genes in mapped QTL intervals
Table 2. GO-terms and functions observed in knock-out mice of 31 potential candidates from QTL intervals
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 [12]. 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 [12] 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 [12] of a total of 26 susceptible strains. Furthermore, five strains that were resistant in our study were susceptible in the study by [12]. 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 [33]. 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 [34]. 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 [26], differences in expression between DBA/2J and C57BL/6J [23], 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 [35]. 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 [12]. 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 [38]. 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 [39]. 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 [40]. 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 [48]. 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) [49]. It is involved in modulating pro-inflammatory responses in macrophages [50].
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 [51]. 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 [33]. Pla2g7 expression levels in susceptible A/J mice were higher than in resistant C57BL/6J mice after infection with H3N2 virus [33]. 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 [26]. Tnfrsf21 which was identified by [33] as potential candidate of Qivr17-2 also exhibits a cis-eQTL in non-infected BXD mice [26] 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 [70].
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, [18] 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 webcite). In order to test for batch effects or other co-factors, we visualized the data using multidimensional scaling based on the Sammon mapping method [71]. 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
QTL mapping was performed using the web-based complex trait analysis available on the GeneNetwork website (http://www.genenetwork.org webcite) 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 webcite 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 [72]. 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 [25]. 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 webcite) and the Jackson Laboratory's MGI Mouse Genome Database project (http://www.informatics.jax.org/ webcite) to identify potential candidate genes. The GeneRIF database (http://www.ncbi.nlm.nih.gov/projects/GeneRIF/GeneRIFhelp.html webcite) 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 [73]. All of these sequence data are available at http://ucscbrowser.genenetwork.org/ webcite, the GeneNetwork Variant Browser (http://www.genenetwork.org/webqtl/main.py?FormID=snpBrowser webcite), 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 webcite.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TN, MH and KS conceived and designed the experiments. TN and MH infected the mice and collected the data. HK, RA, RW and KS performed and interpreted the QTL analysis. FK performed the analysis of environmental and experimental factors. SS and LL provided BXD mice for experimental studies. HK, RW and KS wrote the manuscript. All authors read and approved the final manuscript.
Authors' information
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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Ovsyannikova IG, Vierkant RA, Pankratz VS, Jacobson RM, Poland GA: Extended LTA, TNF, LST1 and HLA gene haplotypes and their association with rubella vaccine-induced immunity.
PLoS ONE 2010, 5(7):e11806. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Schiller C, Nitschke MJ, Seidl A, Kremmer E, Weiss EH: Rat monoclonal antibodies specific for LST1 proteins.
Hybridoma (Larchmt) 2009, 28(4):281-286. Publisher Full Text
Mulcahy H, O'Rourke KP, Adams C, Molloy MG, O'Gara F: LST1 and NCR3 expression in autoimmune inflammation and in response to IFN-gamma, LPS and microbial infection.
Immunogenetics 2006, 57(12):893-903. PubMed Abstract | Publisher Full Text
Kilding R, Iles MM, Timms JM, Worthington J, Wilson AG: Additional genetic susceptibility for rheumatoid arthritis telomeric of the DRB1 locus.
Arthritis Rheum 2004, 50(3):763-769. PubMed Abstract | Publisher Full Text
Lavi S, McConnell JP, Rihal CS, Prasad A, Mathew V, Lerman LO, Lerman A: Local production of lipoprotein-associated phospholipase A2 and lysophosphatidylcholine in the coronary circulation: association with early coronary atherosclerosis and endothelial dysfunction in humans.
Circulation 2007, 115(21):2715-2721. PubMed Abstract | Publisher Full Text
Kim JY, Hyun YJ, Jang Y, Lee BK, Chae JS, Kim SE, Yeo HY, Jeong TS, Jeon DW, Lee JH: Lipoprotein-associated phospholipase A2 activity is associated with coronary artery disease and markers of oxidative stress: a case–control study.
Am J Clin Nutr 2008, 88(3):630-637. PubMed Abstract | Publisher Full Text
Kostopanagiotou G, Avgerinos E, Costopanagiotou C, Arkadopoulos N, Andreadou I, Diamantopoulou K, Lekka M, Smyrniotis V, Nakos G: Acute lung injury in a rat model of intestinal ischemia-reperfusion: the potential time depended role of phospholipases A(2).
J Surg Res 2008, 147(1):108-116. PubMed Abstract | Publisher Full Text
Sutton BS, Crosslin DR, Shah SH, Nelson SC, Bassil A, Hale AB, Haynes C, Goldschmidt-Clermont PJ, Vance JM, Seo D, et al.: Comprehensive genetic analysis of the platelet activating factor acetylhydrolase (PLA2G7) gene and cardiovascular disease in case–control and family datasets.
Hum Mol Genet 2008, 17(9):1318-1328. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wang Q, Hao Y, Mo X, Wang L, Lu X, Huang J, Cao J, Li H, Gu D: PLA2G7 gene polymorphisms and coronary heart disease risk: a meta-analysis.
Thromb Res 2010, 126(6):498-503. PubMed Abstract | Publisher Full Text
Sadegh-Nasseri S, Chen M, Narayan K, Bouvier M: The convergent roles of tapasin and HLA-DM in antigen presentation.
Trends Immunol 2008, 29(3):141-147. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Turnquist HR, Petersen JL, Vargas SE, McIlhaney MM, Bedows E, Mayer WE, Grandea AG 3rd, Van Kaer L, Solheim JC: The Ig-like domain of tapasin influences intermolecular interactions.
J Immunol 2004, 172(5):2976-2984. PubMed Abstract | Publisher Full Text
Garbi N, Tiwari N, Momburg F, Hammerling GJ: A major role for tapasin as a stabilizer of the TAP peptide transporter and consequences for MHC class I expression.
Eur J Immunol 2003, 33(1):264-273. PubMed Abstract | Publisher Full Text
Williams AP, Peh CA, Purcell AW, McCluskey J, Elliott T: Optimization of the MHC class I peptide cargo is dependent on tapasin.
Immunity 2002, 16(4):509-520. PubMed Abstract | Publisher Full Text
Raghuraman G, Lapinski PE, Raghavan M: Tapasin interacts with the membrane-spanning domains of both TAP subunits and enhances the structural stability of TAP1 x TAP2 Complexes.
J Biol Chem 2002, 277(44):41786-41794. PubMed Abstract | Publisher Full Text
Rufer E, Leonhardt RM, Knittler MR: Molecular architecture of the TAP-associated MHC class I peptide-loading complex.
J Immunol 2007, 179(9):5717-5727. PubMed Abstract | Publisher Full Text
Fukuda A, Tokonabe S, Hamada M, Matsumoto M, Tsukui T, Nogi Y, Hisatake K: Alleviation of PC4-mediated transcriptional repression by the ERCC3 helicase activity of general transcription factor TFIIH.
J Biol Chem 2003, 278(17):14827-14831. PubMed Abstract | Publisher Full Text
Kim YK, Bourgeois CF, Pearson R, Tyagi M, West MJ, Wong J, Wu SY, Chiang CM, Karn J: Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency.
EMBO J 2006, 25(15):3596-3604. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Briggs FB, Goldstein BA, McCauley JL, Zuvich RL, De Jager PL, Rioux JD, Ivinson AJ, Compston A, Hafler DA, Hauser SL, et al.: Variation within DNA repair pathway genes and risk of multiple sclerosis.
Am J Epidemiol 2010, 172(2):217-224. PubMed Abstract | Publisher Full Text
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Sammon JW: A nonlinear mapping for data structure analysis.
Ljungberg K, Holmgren S, Carlborg O: Simultaneous search for multiple QTL using the global optimization algorithm DIRECT.
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Wang X, Agarwala R, Capra J, Chen Z, Church D, Ciobanu D, Li Z, Lu L, Mozhui K, Mulligan M, et al.: High-throughput sequencing of the DBA/2J mouse genome.
BMC Bioinformatics 2010, 11(Suppl 4):O7. BioMed Central Full Text


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Daniel Neafsey
Daniel Neafsey leads the Malaria Genome Sequencing and Analysis Group at the Broad Institute. In this capacity, he oversees projects aimed at decoding and analyzing the genomes of the parasites that causes malaria as well as the mosquitoes that transit them to humans. Malaria carries a heavy global burden: It affects millions of people each year, killing more than 800,000, mostly children under the age of five living in sub-Saharan Africa.
Neafsey's work, together with a network of collaborators stretching from the Broad Institute, the Harvard School of Public Health and other institutions worldwide, focuses on mapping the variation within and across both parasite and mosquito genomes. The knowledge flowing from this work offers a deeper understanding of the biology and evolution of malaria as well as the genomic basis of key traits that impact human health, such as drug and insecticide resistance, parasite virulence, and the effectiveness of vaccines.
Neafsey, who works in the Broad's Genome Sequencing and Analysis Program, first joined the Institute in 2004 after earning his Ph.D. in biology from Harvard University and a brief postdoctoral fellowship, also at Harvard.Select Publications
Neafsey DE et al. SNP genotyping defines complex gene-flow boundaries among African malaria vector mosquitoes. Science 2010 Oct 22;330(6003):514-7.
Neafsey DE et al. Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence. Genome Biology 2008;9(12):R171.
Carlton JM, Escalante AA, Neafsey D, Volkman SK. Comparative evolutionary genomics of human malaria parasites. Trends in Parasitology 2008 Dec;24(12):545-50.

subset: commoncrawl


text: I just noticed that the stackless module has a an attribute "stackless" that points to itself. This is highly irregular. I noticed this because some of the tests in stackless/test do from stackless import * and then proceed to do stuff such as r = stackless.channel() I'd like to remove this module self reference if no one objects. K -------------- next part -------------- An HTML attachment was scrubbed... URL: <http://www.stackless.com/pipermail/stackless/attachments/20100326/627d16e3/attachment.htm>


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What is REVERSE 911®?REVERSE 911® is the most nationally recognized emergency notification solution for public safety. Hundreds of law enforcement operations, state and local governments, schools, non-profit organizations and government agencies rely on REVERSE 911® from DCC (Dialogic Communications Corp.) for immediate interaction with targeted audiences every day. REVERSE 911® uses a patented combination of database and GIS mapping technologies to deliver outbound notifications. This includes map-based communication where users can quickly target a precise geographic area and saturate it with thousands of calls per hour. The system's interactive technology provides immediate communication with recipients and aids in rapid response to specific needs. List-based notification allows users to create a list of individuals with common characteristics (e.g., Neighborhood Crime Watch group, emergency responder teams) and contact them with useful information as needed.About REVERSE 911®Time and again, REVERSE 911® plays a key role in ensuring effective routine and critical communication to alert local residents and mobilize personnel. Communities across North America are safer because their leaders use the REVERSE 911® system.The REVERSE 911® system has a wide variety of potential uses in many organizations, such as:
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NIST works to assure that the U.S. realization of the basic and derived measurement units is consistent with the realization in other nations. NIST Laboratories engage in a number of international activities to support trade and global science, and to promote the international acceptance of U.S. measurement standards.
International Metrology
Web Page last modified: Friday, March 21, 2008
Web Page created: Wednesday, June 20, 2001


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Down with the Underweight Down Under. ~ Matas Vilgalys
A little bit of photoshop adbusting in Berlin.
There's Beauty in the Breakdown.
The discussion over slenderness within the modeling industry has been ongoing since Twiggy popularized the waiflike look decades ago. Since the noughties began, this discussion has been able to push many magazines and designers to extend their casting beyond the usual frail 14-year-olds that traditionally line the runways. Featured "larger" models in editorials, as well as entire issues devoted to shape have been more than enough to push this issue into the spotlight. A sea change for the artificial representations constructed within the beauty industry is now pending, thanks to work like the internationally infamous Dove Campaign for Real Beauty, and it seems Australia might be the first nation to impose a significant new protocol for how these portrayals go to press.
Australian Youth Minister Kate Ellis has taken this issue on as a federal concern towards national health and happiness. The program would reward considerate designers, stores, magazines and advertisers with a seal that declares them body-image friendly, similar to the way USDA organic foods are acknowledged with their own seal. The following criteria will determine those who gain this positive symbol of change:
> Disclose when images have been retouched and refrain from enhancing photographs in a way that changes a person's body shape, for example, lengthening their legs or trimming their waist, or removing freckles, lines and other distinguishing marks.
> Only use models aged 16 or older to model adult clothes – both on catwalks and in print.
> Refrain from using models who are very thin – or male models who are excessively muscular.
> Stocking clothing in a wide variety of sizes in shops to reflect the demand from customers.
> Using a broad range of body shapes, sizes and ethnicities in editorial and advertising.
> Not promoting rapid weight loss, cosmetic surgery, excessive exercising or any advertisements or editorial content that may promote a negative body image.
Matas Vilgalys is a senior at CU Boulder pursuing double degrees in Art and Advertising. He likes cheese, working on projects, loving the mountains, running, and talking about North Carolina, the greatest state in the union and coincidentally where he is from.
Incorrect source, offensive, or found a typo? Email us (please put title in subject bar of email so we'll be able to fix). Or do you want to write for Elephant?
{Waylon H. Lewis C Enterprises 2015: Use Rights in perpetuity. Ownership remains with author.}


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Combat harsh, cold winters while hitting the slopes for new adventures. Ski down the highest altitudes with the peace of mind that your skin is protected from the blistering cold as you rocket to base camp.
With unique outerwear styles for men, women, boys and girls, Academy Sports + Outdoors carries apparel that'll warm you from head-to-toe during those nippy winter months. It's a fact that the majority of body heat escapes from the head; so retain your body heat with stylish headwear, like the Seirus Men's Fine Solid Quick Draw Hat or the 180s Men's Tec Fleece Ear Warmer.
Plus, snow can seriously damage skin and can even cause frostbite, so make sure to protect your hands top-of-the-line heated gloves from reliable brands like Seirus and Manzella.


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Peter Thomas History Fund Supports Spirit of 45 Mission
The mission of Spirit of '45 Day is to remind us of the Greatest Generation's courage, shared sacrifice, and commitment to service so that their example of national unity and community will inspire future generations of Americans, especially our nation's youth.
To fulfill this mission, the Peter Thomas History Fund was established and will be announced at the 4th Annual Naples Spirit of '45 Greatest Generation Breakfast at the Naples Hilton, Saturday, August 10th, from 8 am - 11 am,
The Peter Thomas History Fund will support Collier County History Fair students, grades 6-12, who advance from the local competition to the 19 year-old Florida History Fair with a chance to advance to the 36 year old, National History Day competition. The Fund will be housed at the Holocaust Museum of Southwest Florida, Inc, located in Naples.
The 2012 Greatest Generation Breakfast saw the launch of two local history awards and one state-wide award. The Hodges Heritage Award and the 'One More Hill Award' were in honor of WWII Veterans, Earl Hodges, long time Naples resident and Dr. Franklyn Johnson, university professor and author of required military school reading, 'One More Hill'. These awards support the 5 year-old, Collier County History Fair, started by Lois Bolin, co-founder of Naples Spirit of '45, Judge Lynn Hixon-Holley (RET), Mike Peppe, Jeffery Hirsch, Keith Basik, and Lavigne Ann Kirkpatrick.
A state-wide history award, the Spirit of '45 Award, was started at the Florida History Fair competition in honor WWII Veteran and Home Front workers and Peter's Thomas' work to keep alive the memories and legacy of WWII Veterans. It is supported by the Museum of Military Memorabilia and Naples Spirit of 45. It is open to all students in all Florida counties, who advance to the state competition and is awarded to an outstanding entry about a person, event, or innovation that relates to Florida's role in WWII.
Donations to the Peter Thomas History fund can be made to the SWFL Holocaust Museum, Inc., located in Naples noting an earmark for the Peter Thomas History Fund. For more information, contact Amy Snyder, Executive Director of the
The Greatest Generation Breakfast is free, due to generous sponsors, but seating is limited and reservations are required. To make a reservation, call 239-649-2300.
This story is contributed by a member of the community and is neither endorsed nor affiliated with Naples Daily News.
Subscribe to the Naples Daily News
Need Help? Call us at 1-888-878-6842.
Monday-Friday: 6am-6pm / Saturday: 7am-3pm / Sunday: 7am-2pm


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DALLAS – The Sooners arrive at the Cotton Bowl on Saturday morning with a loss, and Texas arrives with a quarterback. Who saw either one coming?
Oklahoma Football: Why David Ash better be braced for the Sooner blitz
David Ash's emergence as an effective QB has restored Longhorn confidence and changed the way OU has to play this game.
Two years in a row, the Sooners played Texas knowing 'Horn quarterbacks — Garrett Gilbert, Case McCoy, a rookie Ash — couldn't beat them. Now the Sooners know different.
Which means Mike Stoops knows what he has to do. Here comes the Oklahoma blitz.
Stoops dialed up blitzes repeatedly last week in Lubbock, and it made life miserable for veteran Texas Tech quarterback Seth Doege. Expect the same in the Cotton Bowl.
"What we did the other night was we made the quarterback think and adjust," Stoops said. "You don't want the quarterback to get comfortable. That's the worst thing you can do."
OU got to Ash and McCoy last year, with a school record eight sacks, helping lead to five Texas turnovers and three defensive Sooner touchdowns.
But that was a mismatch. This is not. Ash has displayed the development that he can hit open receivers, often quickly. He's not the skittish freshman who completed just 11 of 20 passes for 107 yards against OU last season, with two interceptions and a lone TD, scored with 2:31 left in the game.
Ash's numbers are impressive: 19-of-23 for 326 yards and four touchdowns against Ole Miss; 30-of-37 for 304 yards and three TDs against OSU; 22-of-29 for 269 yards and a TD against West Virginia. Only one interception total all season.
"Again, you have to keep the quarterback off balance," Stoops said. "He's the most important player on any team. You have to threaten him all the time. Make him make decisions quickly and not let him get comfortable."
Continue reading this story on the...


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The Mountain Oasis Electronic Music Summit pulled 6,000-7,000 people each night of its 2013 edition, but still lost money. Producer Ashley Capps said the event will not return this year.
- Filed Under
Concert promoter Ashley Capps says he's still gung-ho on booking concerts in Asheville, despite the cancellation of his Mountain Oasis Electronic Music Summit festival.
The Halloween electronic music gathering was an artistic success, he said, but it was a money loser and he could not find a way to make it grow. Before creating Mountain Oasis in 2013, he produced the similar Moogfest celebration for three years. Moogfest is now under a different promoter and will return April 23-27 with a workshop and concert format.
"The four years that we did (the festivals), they made money one year, ...


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Mumford & Sons to Headline Jersey Shore Music Festival
Spring is on the horizon and things are already looking up at the Jersey shore! This June folk rockers Mumford & Sons will headline a music two-day music festival.
Spring is on the horizon and things are already looking up at the Jersey shore! This June folk rockers Mumford & Sons will headline a music two-day music festival.
What would think if your dog went missing seventeen months ago?
A federal audit of Sandy aid money used to produce "Stronger than the Storm" TV commercials was described as "routine and standard" by Gov. Christie's office Monday. The Democratic congressman who sought the audit says otherwise.
By a 22-point margin yesterday, Gov. Chris Christie got the landslide win he was craving.
It feels like only yesterday.....
The Hurricane Sandy New Jersey Relief Fund has partnered with Jersey Cares to mobilize 5,000 volunteers in meaningful service projects to continue clean-up and rebuilding efforts throughout Atlantic, Monmouth, Ocean and Bergen Counties on the anniversary of Hurricane Sandy. Here's how to partner with thousands of your neighbors on Sandy Service Day!
When I think back to the night Hurricane Sandy slammed into New Jersey, I think darkness. I remember pulling an all-nighter (and then some) here at the radio station, the Superstorm impacting all around outside. The fact that the eye of the weather system was making landfall under the cover of night made the situation all that more ominous. So as the first anniversary of Hurricane Sandy approaches I'm looking forward to standing with you on Brigantine Beach to light up the night on October 29th.
In an effort to repair broken homes due to Hurricane Sandy, tragedy struck.
Already instrumental in 'restoring the shore' with his songwriting with 'Because We Can', Jon Bon Jovi returned to his hometown to give more.....
Looking for a tasty way to help Restore the Shore? Head to Northfield Community School on Saturday for a pancake breakfast fundraiser!
Atlantic City's oldest casino now has a new entertainment attraction, and Governor Chris Christie believes it is a symbol that the shore is back.


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Male pattern baldness is a sex-linked characteristic that is passed from mother to child. A man can more accurately predict his chances of developing male pattern baldness by observing his mother's father than by looking at his own father.
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christian bale
Christian Bale Wins Best Supporting Actor for 'The Fighter' (VIDEO)
While he did open his speech with the expression, "Bloody hell!" he promised to keep it at least cleaner than his co-star did. "I'm not going to drop the f-bomb like she did," he said of Leo. "I've done that plenty before."
SAG Award Winners: 'King's Speech,' 'Fighter,' 'Modern Family' Among the Honored (VIDEO)
'The Fighter' won two of its four nominations, as Melissa Leo and Christian Bale received statuettes. Dickie Eklund, the man whom Bale played in the biopic, surprised him onstage. "How do you like me now?" Eklund joked.
In television, notable wins included Betty White for female actor in a comedy series ('Hot in Cleveland'), and Alec Baldwin's fifth consecutive SAG victory for '30 Rock.'
Killing Justin Bieber on 'South Park' (VIDEO)
On the new episode (Wed., 10PM ET on Comedy Central), we follow the continuing adventures of Eric Cartman as "The Coon." Cartman is a self-styled "Batman-esque" caped crusader, and like the Dark Knight, Eric has very clear-cut ideas about what is right and what is wrong.
As Cartman narrates in full-on, hyperbolic Christian Bale-mode, we get the full info about the evil forces that he opposes. "The Coon's" greatest enemies include the following: "hippies," trendy supermarket chain 'Whole Foods' -- and floppy-haired teen sensation Justin Bieber. Now, Cartman must "take out" his greatest enemy. But is he strong enough to stop Mr. Bieber?
Tracy Jordan goes all Christian Bale on TGS set
It's pretty funny, though it probably goes on a little bit too long. Jordan is ranting at the cameraman because he dares to film him while he's trying to act. They should actually show this at the end of one of the episodes, or maybe as an extra on the next DVD set. At one point you can even hear Tracy make a reference to TMZ.
It could be a little NSFW, though all of the bad words are bleeped out. For the record, Bale has effin' apologized.
Right now on Cinematical
- So, if in The Curious Case of Benjamin Button, Brad Pitt starts off as a tiny old-man, why wouldn't he end up as a giant baby? Cinematical has the answer.
- When we were in high school, my friends and I got together with a VHS camcorder and made Scream 4. It was pretty amazing. What's weird is that even though we didn't get a film deal, Scream 4 is heading to theaters. Neve Campbell needs a job, people.
- Granted, I've missed a lot of movies in the theaters this year, but I still think The Dark Knight is one of the best films of the year. What do you think? Check out Cinematical's top 10 list.
- Hey, want to see something really creepy? Jerry O'Connell and his wife, Rebecca Romijn, are putting her maternity leave to good use by making Mystique and Me, a new Funny or Die video. It's quite funny and quite disturbing.
- As much as I enjoyed The Dark Knight, I don't think Maggie Gyllenhaal and Christian Bale had a ton of chemistry -- at least not enough to make it onto the Cinematical Seven's Best On-Screen Chemistry of 2008 list.
Journeyman fans might want to see the new Terminator flick
Did you even know there was going to be a fourth film in the Terminator series? Yup, and it's going to star Christian Bale, Sam Worthington, and Anton Yelchin (Huff and Chekov in the new Star Trek movie). Sorry, Arnold Schwarzenegger is busy being the Governor of California, but there another name added to the cast, and what a cool name it is.
Moon Bloodgood, who played Livia in Journeyman and Rita in the short-lived Daybreak, is about to sign on to the new sci-fi movie, Terminator Salvation: The Future Begins. The film will be directed by McG (he directed the Charlie's Angels big screen movie) and is the first film in a planned trilogy. Bale will play John Connor (currently played by Thomas Dekker on FOX's The Sarah Connor Chronicles)., fighting evil robots after most of mankind has been destroyed. I have no idea if this plot will make sense with what is currently going on in the FOX show (or in the previous movies, for that matter), but thinking too much about all of it will give you a headache anyway, so just accept whatever happens.
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5 Movies Interview
Vanessa Hudgens interview: 'I learned about a prostitute's entire life'
Vanessa Hudgens continues to break free from the sweet shackles of High School Musical by playing teen prostitute Cindy in the compelling new thriller The Frozen Ground, which is based on shocking real events.
Digital Spy met the luminous actress to discuss the movie and its electrifying reunion of Con Air's Nicolas Cage and John Cusack as, respectively, a kind state trooper and a serial killer stalking Cindy.
Hudgens also reveals what we can expect from her turn in Machete Kills. But first, the small matter of a naughty reindeer called Jack who caused havoc in one memorable scene...
> Nicolas Cage, Vanessa Hudgens in 'The Frozen Ground' trailer
> Nicolas Cage interview: "It's not all cobras and flaming skulls"
The Frozen Ground is out in UK cinemas now.


text: Brass Voit VR1 Mar 5, 2013 15:20:17 GMT -8 Quote Select PostDeselect PostLink to PostMemberGive GiftBack to Top Post by surflung on Mar 5, 2013 15:20:17 GMT -8 - I have this VR1 project in the works and thought I'd share this photo. The cans need repair of the 4 extra holes that were drilled in the face. I don't know if I can repair/fill the corrosion pits. - I stripped the whole regulator for re-plating. Removed the chrome with muriatic acid. Removed the underlying nickel with Brownells room temperature nickel stripper. Then buffed off the residue with a scotch bright pad... It's kind of beautiful!- I'm planning to plug the holes with brass screws. Fill with copper brazing rod, and grind the surface flat. I'm hoping I can fill corrosion pits with brazing rod, too.


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Gulf Coast Cowboy Hour June 13th Playlist
Welcome to another Gulf Coast Cowboy Hour!
Our playlist for this week is:
- Weenies and Bikinis / Brent Burns
- Lazy Haze / Todd Sparks
- Mexico / Jimmy Buffett
- Tropical Vacation / Kelly McGuire
- Breachfront Town / Larry Joe Taylor
- The Gulf of Mexico / Steve Earle
- I'm a Trop Rock Junkie / Trop Rock Junkies
- I Can't Spell Caribbean / Living Soul
- Sand In Her Shoes / Thom Shepherd
- Knee Deep / Zac Brown Band featuring Jimmy Buffett
- A Pirate Looks at Forty / Roger Creager
- Blame It On the Margaritas / Howard Livingston
We'll be kicking off this week's show with the aptly named "Weenies and Bikinis" from Brent Burns. Ragtops and Flipflops is where you can find this tune!
"Lazy Haze" is a new song on our program from Todd Sparks. Todd and his Shrimpboat Cowboys released this song on their latest album, The Gulf of Mexico.
"Mexico" is a Jimmy Buffett song we've featured a handful of times on the show. It comes from the Barometer Soup album.
Kelly McGuire's "Tropical Vacation" is one of our favorite songs from his award winning Boat in Belize CD!
Larry Joe Taylor gave us "Beachfront Town" on his 2007 Times album. This guy always knows how to make you feel like you're on the coast!
"The Gulf of Mexico" is a Steve Earle song that he wrote in the aftermath of the British Petroleum oil spill disaster. This song, and more like it, can be found on his I'll Never Get Out of this World Alive CD.
The Trop Rock Junkies, a band we've only recently discovered, gave us this tune on their Live at the Hideaway Cafe CD.
Something about Living Soul is really special! "I Can't Spell Caribbean" is the title track of their first album.
"Sand In Her Shoes" is a great Thom Shepherd song we like to play as often as we can get away with. Check out his Tropicalifragilisticexpialidocious CD for more tunes like it!
"Knee Deep" is another spectacular song we love from the Zac Brown Band. Featuring Jimmy Buffet himself, you don't get much better than this! This hit was on the best-selling You Get What You Give record.
Some covers are terrible mockeries of the original songs they are trying to emulate, but luckily this is not the case at all with Roger Creager's version of "A Pirate Looks at Forty"!