IntroductionHuman host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1.MethodsWe profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene.ResultsIncreased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1β), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-γ) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-α, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL-6, IL-8 and PaO2 in critical patients.ConclusionsWhile infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness.

The density and utility of the molecular genetic linkage map of the widespread use of RFLP markers and maps in suncultivated sunflower (Helianthus annuus L.) has been greatly inflower has been restricted by a lack of public RFLP creased by the development and mapping of several hundred simple sequence repeat (SSR) markers. Of 1089 public SSR markers de-probes, consequent lack of a dense public RFLP map, scribed thus far, 408 have been mapped in a recombinant inbred line and low-throughput nature of RFLP markers. The diffi-(RIL) mapping population (RHA280 ϫ RHA801). The goal of the culties posed by the historic lack of public, single-copy present research was to increase the density of the sunflower map by DNA markers were only weakly offset by the emerconstructing a new RIL map (PHA ϫ PHB) based on SSRs, adding gence of facile, universal DNA markers, e.g., RAPDs loci for newly developed SSR markers to the RHA280 ϫ RHA801 RIL (Williams et al., 1990, 1993) and AFLPs (Vos et al., map, and integrating the restriction fragment length polymorphism 1995). RAPDs have primarily been used for tagging (RFLP) and SSR maps of sunflower. The latter was accomplished by phenotypic loci in sunflower, e.g., rust (Puccinia helianadding 120 SSR marker loci to a backbone of 80 RFLP marker loci thi Schw.) and Orobanche cumana Wallr. resistance on the HA370 ϫ HA372 F 2 map. The map spanned 1275.4 centimorgans (cM) and had a mean density of 6.3 cM per locus. The genes (Lawson et al., 1998; Lu et al., 2000). While RAPD PHA ϫ PHB SSR map was constructed from 264 SSR marker loci, and AFLP markers have a multitude of uses, both are spanned 1199.4 cM, and had a mean density of 4.5 cM per locus. The dominant, multicopy, and often nonspecific in nature RHA280 ϫ RHA801 map was constructed by adding 118 new SSR and, as a whole, unsatisfactory for establishing a geand insertion-deletion (INDEL) marker loci to 459 previously nome-wide framework of DNA markers for anchoring mapped SSR marker loci. The 577-locus map spanned 1423.0 cM and cross referencing genetic linkage maps. Single-copy, and had a mean density of 2.5 cM per locus. The three maps were codominant DNA markers, e.g., SSRs, are preferred for constructed from 1044 DNA marker loci (701 unique SSR and 89 such purposes and, until recently, have been lacking unique RFLP or INDEL marker loci) and supply a dense genomein sunflower. wide framework of sequence-based DNA markers for molecular breeding and genomics research in sunflower.

Immune responses during infection with pandemic H1N1 2009 influenza A virus (2009-H1N1) are still poorly understood. Using an experimental infection model in ferrets, we examined the pathological features and characterized the host immune responses by using microarray analysis, during infection with 2009-H1N1 A/California/07/2009 and seasonal A/Brisbane/59/2007. Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of interferon-stimulated genes were expressed early, correlated to lung pathology, and abruptly decreased expression on day 7 following infection of A/California/07/2009. Interestingly, the drop in innate immune gene expression was replaced by a significant increase of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-influenza antibodies were first observed on day 7, commensurate with the viral clearance. We propose that lung pathology in humans occurs during the innate phase of host immunity and a delay or failure to switch to the adaptive phase may contribute to morbidity and mortality during severe 2009-H1N1 infections.

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.

Wild biotypes of cultivated sunflower ( Helianthus annuus L.) are weeds in corn ( Zea mays L.), soybean ( Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides-imidazolinones (IMIs) or sulfonylureas (SUs)-have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes ( AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 ( Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 ( AHAS3), 6 ( AHAS2), and 9 ( AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant x wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.

Broomrape (Orobanche cumana Wallr.) is a root parasite of sunflower that is regarded as one of the most important constraints of sunflower production in the Mediterranean region. Breeding for resistance is the most effective method of control. P-96 is a sunflower line which shows dominant resistance to broomrape race E and recessive resistance to the very new race F. The objective of this study was to map and characterize quantitative trait loci (QTL) for resistance to race E and to race F of broomrape in P-96. A population from a cross between P-96 and the susceptible line P-21 was phenotyped for broomrape resistance in four experiments, two for race E and two for race F, by measuring different resistance parameters (resistance or susceptibility, number of broomrape per plant, and proportion of resistant plants per F(3) family). This population was also genotyped with microsatellite and RFLP markers. A linkage map comprising 103 marker loci distributed on 17 linkage groups was developed, and composite interval mapping analyses were performed. In total, five QTL ( or1.1, or3.1, or7.1 or13.1 and or13.2) for resistance to race E and six QTL ( or1.1, or4.1, or5.1, or13.1, or13.2 and or16.1) for resistance to race F of broomrape were detected on 7 of the 17 linkage groups. Phenotypic variance for race E resistance was mainly explained by the major QTL or3.1 associated to the resistance or susceptibility character ( R(2)=59%), while race F resistance was explained by QTL with a small to moderate effect ( R(2) from 15.0% to 38.7%), mainly associated with the number of broomrape per plant. Or3.1 was race E-specific, while or1.1, or13.1 and or13.2 of were non-race specific. Or13.1, and or13.2 were stable across the four experiments. Or3.1, and or7.1 were stable over the two race E experiments and or1.1 and or5.1 over the two race F experiments. The results from this study suggest that resistance to broomrape in sunflower is controlled by a combination of qualitative, race-specific resistance affecting the presence or absence of broomrape and a quantitative non-race specific resistance affecting their number.

The seed oil concentrations of large‐seeded, low‐oil and small‐seeded, high‐oil sunflower (Helianthus annuus L.; x = 17) cultivars differ by 180 to 280 g kg−1 We identified quantitative trait loci (QTL) for seed oil and other seed traits in a low‐ × high‐oil (RHA280 × RHA801) recombinant inbred line (RIL) mapping population segregating for apical branching (B), phytomelanin pigment (P), and hypodermal pigment (Hyp) loci. B, Hyp, and P mapped to linkage groups 10, 16, and 17, respectively. The seed oil concentrations of RHA280 and RHA801 were 254 and 481 g kg−1, respectively. Composite interval mapping (CIM) identified 40 QTL for seed oil concentration, 100‐seed weight, seed length, width and depth, kernel and pericarp weight, and kernel‐to‐pericarp weight ratio in 14 DNA marker intervals on 10 of 17 linkage groups. Twenty‐four of the QTL were tightly linked to B, P, and Hyp and may have been partly or wholly caused by the pleiotropic effects of B, P, and Hyp Multilocus QTL analyses were performed using B, P, Hyp, and four DNA marker loci as independent variables in mixed linear models. Seventy percent of the additive effects (39/56) and 42% of the additive × additive and additive × additive × additive effects (189/448) were significant (p < 0.05). The linked, pleiotropically acting, and epistatically interacting QTL identified for seed traits in RHA280 × RHA801 were presumably targeted by selection in the transition from large‐seeded, low‐oil to small‐seeded, high‐oil cultivars in sunflower.

A detailed linkage map of Helianthus annuus was constructed based on segregation at 234 RFLP loci, detected by 213 probes, in an F2 population of 289 individuals (derived from a cross between the inbred lines HA89 and ZENB8). The genetic markers covered 1380 centiMorgans (cM) of the sunflower genome and were aranged in 17 linkage groups, corresponding to the haploid number of chromosomes in this species. One locus was found to be unlinked. Although the average interval size was 5.9 cM, there were a number of regions larger than 20 cM that were devoid of markers. Genotypic classes at 23 loci deviated significantly from the expected ratios (1∶2∶1 or 3∶1), all showing a reduction in the ZENB8 homozygous class. The majority of these loci were found to map to four regions on linkage groups G, L and P.