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Modeling host genetic regulation of influenza pathogenesis in the collaborative cross.

Ferris MT, Aylor DL, Bottomly D, Whitmore AC, Aicher LD, Bell TA, Bradel-Tretheway B, Bryan JT, Buus RJ, Gralinski LE, Haagmans BL, McMillan L, Miller DR, Rosenzweig E, Valdar W, Wang J, Churchill GA, Threadgill DW, McWeeney SK, Katze MG, Pardo-Manuel de Villena F, Baric RS, Heise MT - PLoS Pathog. (2013)

Bottom Line: Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations.Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population.We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.

View Article: PubMed Central - PubMed

Affiliation: Carolina Vaccine Institute, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, United States of America. mtferris@email.unc.edu

ABSTRACT
Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.

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A novel Mx1 allele differentially impacts host response to influenza.The founder strain alleles at Mx1 were grouped based on their phenotypic effects into three functionally distinct classes corresponding to domesticus (dom: A/J, C57BL6/J, 129s1/SvImJ, NOD/HiLtJ and WSB/EiJ), castaneus (cast: CAST/EiJ) and musculus (mus: PWK/PhJ and NZO/ShILtJ). Points shown are individual pre-CC animals with these haplotypes, mean bars are shown for each class. These functionally distinct classes were separable based upon differences in (A) D4 weight and (B) Log titer, with the heterozygous classes showing intermediate phenotypes. Across the pre-CC population, homozygous dom animals had severe weight loss and high titers. Homozygous mus animals showed little weight loss and low titers. Homozygous cast animals showed little weight loss, but had intermediate viral titers. Brackets between groups represent significant differences (* = p<0.05, ** = p<0.003) based on Tukey's HSD. We found no difference by qPCR (C) in expression of Mx1 at 2 days post-infection following influenza infection in a strain from each of these three functional classes. By sequencing Mx1 (D), we were able to identify five haplotypes across the eight founder strains (Haplotype 1 = A/J, C57BL/6J, 129S1/SvImJ, NOD/HiLtJ; Haplotype 2 = WSB/EiJ; Haplotype 3 = PWK/PhJ; Haplotype 4 = NZO/HiLtJ; Haplotype 5 = CAST/EiJ). Arrows indicate locations of polymorphisms, with small arrows indicating non-coding changes, and large arrows indicating coding changes. Colors correspond to the founder strains having those polymorphisms (brown = multiple strains possess mutation). Grey exons indicate those not transcribed due to either deletion and frameshift, or insertion and early stop codon.
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ppat-1003196-g004: A novel Mx1 allele differentially impacts host response to influenza.The founder strain alleles at Mx1 were grouped based on their phenotypic effects into three functionally distinct classes corresponding to domesticus (dom: A/J, C57BL6/J, 129s1/SvImJ, NOD/HiLtJ and WSB/EiJ), castaneus (cast: CAST/EiJ) and musculus (mus: PWK/PhJ and NZO/ShILtJ). Points shown are individual pre-CC animals with these haplotypes, mean bars are shown for each class. These functionally distinct classes were separable based upon differences in (A) D4 weight and (B) Log titer, with the heterozygous classes showing intermediate phenotypes. Across the pre-CC population, homozygous dom animals had severe weight loss and high titers. Homozygous mus animals showed little weight loss and low titers. Homozygous cast animals showed little weight loss, but had intermediate viral titers. Brackets between groups represent significant differences (* = p<0.05, ** = p<0.003) based on Tukey's HSD. We found no difference by qPCR (C) in expression of Mx1 at 2 days post-infection following influenza infection in a strain from each of these three functional classes. By sequencing Mx1 (D), we were able to identify five haplotypes across the eight founder strains (Haplotype 1 = A/J, C57BL/6J, 129S1/SvImJ, NOD/HiLtJ; Haplotype 2 = WSB/EiJ; Haplotype 3 = PWK/PhJ; Haplotype 4 = NZO/HiLtJ; Haplotype 5 = CAST/EiJ). Arrows indicate locations of polymorphisms, with small arrows indicating non-coding changes, and large arrows indicating coding changes. Colors correspond to the founder strains having those polymorphisms (brown = multiple strains possess mutation). Grey exons indicate those not transcribed due to either deletion and frameshift, or insertion and early stop codon.

Mentions: We grouped the eight founder alleles at HrI1 by their estimated effects on each phenotype [49], [54], [55]. Alleles from five strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ and WSB/EiJ) affected the host response similarly and were associated with decreased influenza resistance (i.e. higher titers, higher weight loss, more pathology), increased module K and decreased expression of modules B and C. A/J, C57BL/6J and WSB/EiJ had previously been identified [27], [56] as having nonfunctional Mx1 alleles. In contrast, the NZO/HILtJ and PWK/PhJ alleles within the pre-CC population shared similar effects and increased influenza resistance. Previously, CAST/EiJ was characterized as having a full length Mx1 allele, based on analysis of portions of the Mx1 locus [56]. Despite the presence of a full length transcript, the effect of the CAST/EiJ allele across the pre-CC population was intermediate in conferring resistance in our QTL models. Pre-CC mice with the CAST/EiJ allele showed low-to-intermediate weight loss. In contrast, these animals had viral titers that were intermediate between animals with nonfunctional Mx1 alleles and those with a NZO/HILtJ or PWK/PhJ allele (Figure 4). These three functional groups held true when considering animals from the eight founder strains. Importantly, animals from the CAST/EiJ strain showed intermediate weight loss, but had viral titers no different than founder strains with nonfunctional Mx1 alleles (Figure 1). We conclude that three Mx1 alleles segregate in the pre-CC population, with the CAST/EiJ allele being functionally distinct from the classical protective Mx1 allele, where this allele confers limited protection from viral replication, but does protect from virus-induced weight loss. We found no significant differences in Mx1 mRNA gene expression in the lung at two days post-infection using one strain from each allele group (C57BL/6J, CAST/EiJ and PWK/PhJ, Figure 4). C57BL/6J had the highest mean level of up-regulation, with CAST/EiJ intermediate and PWK/PhJ having the lowest level of expression. This suggests that the differences between the CAST/EiJ and PWK/PhJ alleles are due to coding changes within the gene and not variation in gene expression.


Modeling host genetic regulation of influenza pathogenesis in the collaborative cross.

Ferris MT, Aylor DL, Bottomly D, Whitmore AC, Aicher LD, Bell TA, Bradel-Tretheway B, Bryan JT, Buus RJ, Gralinski LE, Haagmans BL, McMillan L, Miller DR, Rosenzweig E, Valdar W, Wang J, Churchill GA, Threadgill DW, McWeeney SK, Katze MG, Pardo-Manuel de Villena F, Baric RS, Heise MT - PLoS Pathog. (2013)

A novel Mx1 allele differentially impacts host response to influenza.The founder strain alleles at Mx1 were grouped based on their phenotypic effects into three functionally distinct classes corresponding to domesticus (dom: A/J, C57BL6/J, 129s1/SvImJ, NOD/HiLtJ and WSB/EiJ), castaneus (cast: CAST/EiJ) and musculus (mus: PWK/PhJ and NZO/ShILtJ). Points shown are individual pre-CC animals with these haplotypes, mean bars are shown for each class. These functionally distinct classes were separable based upon differences in (A) D4 weight and (B) Log titer, with the heterozygous classes showing intermediate phenotypes. Across the pre-CC population, homozygous dom animals had severe weight loss and high titers. Homozygous mus animals showed little weight loss and low titers. Homozygous cast animals showed little weight loss, but had intermediate viral titers. Brackets between groups represent significant differences (* = p<0.05, ** = p<0.003) based on Tukey's HSD. We found no difference by qPCR (C) in expression of Mx1 at 2 days post-infection following influenza infection in a strain from each of these three functional classes. By sequencing Mx1 (D), we were able to identify five haplotypes across the eight founder strains (Haplotype 1 = A/J, C57BL/6J, 129S1/SvImJ, NOD/HiLtJ; Haplotype 2 = WSB/EiJ; Haplotype 3 = PWK/PhJ; Haplotype 4 = NZO/HiLtJ; Haplotype 5 = CAST/EiJ). Arrows indicate locations of polymorphisms, with small arrows indicating non-coding changes, and large arrows indicating coding changes. Colors correspond to the founder strains having those polymorphisms (brown = multiple strains possess mutation). Grey exons indicate those not transcribed due to either deletion and frameshift, or insertion and early stop codon.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585141&req=5

ppat-1003196-g004: A novel Mx1 allele differentially impacts host response to influenza.The founder strain alleles at Mx1 were grouped based on their phenotypic effects into three functionally distinct classes corresponding to domesticus (dom: A/J, C57BL6/J, 129s1/SvImJ, NOD/HiLtJ and WSB/EiJ), castaneus (cast: CAST/EiJ) and musculus (mus: PWK/PhJ and NZO/ShILtJ). Points shown are individual pre-CC animals with these haplotypes, mean bars are shown for each class. These functionally distinct classes were separable based upon differences in (A) D4 weight and (B) Log titer, with the heterozygous classes showing intermediate phenotypes. Across the pre-CC population, homozygous dom animals had severe weight loss and high titers. Homozygous mus animals showed little weight loss and low titers. Homozygous cast animals showed little weight loss, but had intermediate viral titers. Brackets between groups represent significant differences (* = p<0.05, ** = p<0.003) based on Tukey's HSD. We found no difference by qPCR (C) in expression of Mx1 at 2 days post-infection following influenza infection in a strain from each of these three functional classes. By sequencing Mx1 (D), we were able to identify five haplotypes across the eight founder strains (Haplotype 1 = A/J, C57BL/6J, 129S1/SvImJ, NOD/HiLtJ; Haplotype 2 = WSB/EiJ; Haplotype 3 = PWK/PhJ; Haplotype 4 = NZO/HiLtJ; Haplotype 5 = CAST/EiJ). Arrows indicate locations of polymorphisms, with small arrows indicating non-coding changes, and large arrows indicating coding changes. Colors correspond to the founder strains having those polymorphisms (brown = multiple strains possess mutation). Grey exons indicate those not transcribed due to either deletion and frameshift, or insertion and early stop codon.
Mentions: We grouped the eight founder alleles at HrI1 by their estimated effects on each phenotype [49], [54], [55]. Alleles from five strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ and WSB/EiJ) affected the host response similarly and were associated with decreased influenza resistance (i.e. higher titers, higher weight loss, more pathology), increased module K and decreased expression of modules B and C. A/J, C57BL/6J and WSB/EiJ had previously been identified [27], [56] as having nonfunctional Mx1 alleles. In contrast, the NZO/HILtJ and PWK/PhJ alleles within the pre-CC population shared similar effects and increased influenza resistance. Previously, CAST/EiJ was characterized as having a full length Mx1 allele, based on analysis of portions of the Mx1 locus [56]. Despite the presence of a full length transcript, the effect of the CAST/EiJ allele across the pre-CC population was intermediate in conferring resistance in our QTL models. Pre-CC mice with the CAST/EiJ allele showed low-to-intermediate weight loss. In contrast, these animals had viral titers that were intermediate between animals with nonfunctional Mx1 alleles and those with a NZO/HILtJ or PWK/PhJ allele (Figure 4). These three functional groups held true when considering animals from the eight founder strains. Importantly, animals from the CAST/EiJ strain showed intermediate weight loss, but had viral titers no different than founder strains with nonfunctional Mx1 alleles (Figure 1). We conclude that three Mx1 alleles segregate in the pre-CC population, with the CAST/EiJ allele being functionally distinct from the classical protective Mx1 allele, where this allele confers limited protection from viral replication, but does protect from virus-induced weight loss. We found no significant differences in Mx1 mRNA gene expression in the lung at two days post-infection using one strain from each allele group (C57BL/6J, CAST/EiJ and PWK/PhJ, Figure 4). C57BL/6J had the highest mean level of up-regulation, with CAST/EiJ intermediate and PWK/PhJ having the lowest level of expression. This suggests that the differences between the CAST/EiJ and PWK/PhJ alleles are due to coding changes within the gene and not variation in gene expression.

Bottom Line: Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations.Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population.We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.

View Article: PubMed Central - PubMed

Affiliation: Carolina Vaccine Institute, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, United States of America. mtferris@email.unc.edu

ABSTRACT
Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.

Show MeSH
Related in: MedlinePlus