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Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi. © 2017 The Author(s).

Pandemic influenza A H1N1 (pH1N1) virus emerged in 2009. In the subsequent 4 years, it acquired several genetic changes in its hemagglutinin (HA). Mutations may be expected while virus is adapting to the human host or upon evasion from adaptive immune responses. However, pH1N1 has not displayed any major antigenic changes so far. We examined the effect of the amino acid substitutions found to be most frequently occurring in the pH1N1 HA protein before 1 April 2012 on the receptor-binding properties of the virus by using recombinant soluble HA trimers. Two changes (S186P and S188T) were shown to increase the receptor-binding avidity of HA, whereas two others (A137T and A200T) decreased binding avidity. Construction of an HA protein tree revealed the worldwide emergence of several HA variants during the past few influenza seasons. Strikingly, two major variants harbor combinations of substitutions (S186P/A137T and S188T/A200T, respectively) with opposite individual effects on binding. Stepwise reconstruction of the HA proteins of these variants demonstrated that the mutations that increase receptor-binding avidity are compensated for by the acquisition of subsequent mutations. The combination of these substitutions restored the receptor-binding properties (avidity and specificity) of these HA variants to those of the parental virus. The results strongly suggest that the HA of pH1N1 was already optimally adapted to the human host upon its emergence in April 2009. Moreover, these results are in agreement with a recent model for antigenic drift, in which influenza A virus mutants with high and low receptor-binding avidity alternate.

Genome analysis is no longer a field reserved for specialists and experimental laboratories are doing groundbreaking research using genome sequencing and analysis. In this new era, it is essential that data, analysis and results are shared between scientists. But this can be a challenge, even more so with no computational specialist. Here we present a setup for analysis and publication of genome data of 70 species of Aspergillus fungi. The platform is based on R, Python and uses the RShiny framework to create interactive web‐applications. It allows all participants to create interactive analysis which can be shared with the team and in connection with publications. We present analysis for investigation of genetic diversity, secondary and primary metabolism and general data overview. The platform, the Aspergillus Mine, is a collection of analysis tools based on data from collaboration with the Joint Genome Institute. The Aspergillus Mine is not intended as a genomic data sharing service but instead focuses on creating an environment where the results of bioinformatic analysis is made available for inspection. The data and code is public upon request and figures can be obtained directly from the web‐app. This resource will be of great benefit to the Aspergillus community which is in a rapid development in regards to genome sequencing and analysis. At the moment, the service includes analysis of more than 70 genomes, and is expected to double in the next 6 months, with the final goal of the project is the analysis of 300 Aspergillus species.

The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo . While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.

The global presence, high potential for industrial applications, and medical significance of the ascomycete fungal genus Aspergillus have made this one of the best studied group of fungi with a scientific community that is in size second only to yeast in fungal research. Genomic resources for Aspergillus were among the first to become available in fungal research and after a period of little development have recently again been pushed to the forefront of fungal genomics.

Influenza A viruses (IAVs) are important pathogens of animals and man. In humans, IAV is the cause of seasonal epidemics and occasional pandemics. The pandemics are caused by suddenly appearing IAVs from animal reservoirs, to which most humans have no immunity. The hemagglutinin (HA) protein plays crucial roles in the early stages of virus infection as it binds to sialic acid receptors, which results in endocytic uptake of the virions, and is responsible for the low pH-induced fusion of viral and cellular membranes. Because of its role in sialic acid receptor binding, it is the most important determinant of virus tropism. In addition, the HA protein is also the major immunogen. Antibody levels against HA correlate with protection against infection and disease. This thesis focuses on these two key features of the HA protein. The HA-receptor interactions were studied by using recombinant soluble stable HA trimers, which are secreted from cells, by replacing the transmembrane anchor of HA with a GCN4 trimerization motif. First we studied to what extent the expression system used, and thus the glycosylation state of the recombinant proteins, interfered with HA-receptor interactions. The results show that this is indeed the case. Differences in receptor specificity between different HA proteins could be demonstrated using recombinant proteins produced in mammalian cells, but not with the HA proteins made in insect cells. This technology was subsequently applied to compare the receptor-binding properties of the HA protein from the swine origin new pandemic H1N1 virus with a closely related H1 protein derived from a swine virus. While the new pandemic H1 protein exhibited hardly any binding to different substrates, the swine protein efficiently bound to a number of sialylated glycans. The two amino acid residues responsible for this difference were identified, which provided new insight into HA-receptor interactions. Also the vaccine potential of the recombinant soluble trimeric HA proteins was evaluated. Immunization of chickens, mice ferrets or swine with the HA trimer preparations was shown to protect against a homologous (lethal) challenge with IAV, including the highly pathogenic avian influenza virus H5N1. Vaccination with the trimers induced strong antibody responses and diminished virus replication and excretion upon challenge and decreased clinical effects of infection. In addition, we investigated to what extent the glycosylation state of the HA preparations would affect their immunogenicity. The results consistently demonstrate that HA proteins carrying terminal mannose residues, such as insect-cell produced HA proteins, induce significantly lower antibody titers against HA when compared to fully glycosylated HA preparations. In summary, the research described in this thesis demonstrates the potential of recombinant trimeric soluble HA for the study of HA-receptor interactions and for vaccination purposes.

Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated N-linked glycans have been reported to be essential for IAV entry [Chu VC, Whittaker GR (2004) Proc Natl Acad Sci USA 102:18153–18158], thereby implicating the requirement for proteinaceous receptors in IAV entry. Here we show, using different N -acetylglucosaminyl transferase 1 (GnT1)-deficient cells, that N-linked sialosides can mediate, but are not required for, entry of IAV. Entry into GnT1-deficient cells was fully dependent on sialic acid. Although macropinocytic entry appeared to be affected by the absence of sialylated N-glycans, dynamin-dependent entry was not affected at all. However, binding of HA to GnT1-deficient cells and subsequent entry of IAV were reduced by the presence of serum, which could be reversed by back-transfection of a GnT1-encoding plasmid. The inhibitory effect of serum was significantly increased by inhibition of the viral receptor-destroying enzyme neuraminidase (NA). Our results indicate that decoy receptors on soluble serum factors compete with cell surface receptors for binding to HA in the absence of sialylated N-glycans at the cell surface. This competition is particularly disturbed by the additional presence of NA inhibitors, resulting in strongly reduced IAV entry. Our results indicate that the balance between HA and NA is important not only for virion release, but also for entry into cells.