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Glycosylation is a prevalent, yet heterogeneous post translational modification of proteins with a broad range of implications in molecular biology. The heterogeneity and non-template driven nature of glycans and glycoconjugates makes them challenging to study. However, the advent of techniques like glycomics, glycoproteomics and glycoengineering have revolutionized the field of glycobiology as a fast-growing and promising research area. Emerging biochemical glycoengineering and enrichment tools, developments in MS fragmentation methods and detection software continuously open new possibilities to study glycans and glycoconjugates and make glycan research more available for the broader research community. In this dissertation we combined existing and newly developed glycomics, glycoproteomics and glycoengineering techniques to study and engineer cell-surface N-glycosylation in the context of genetic glycosylation defects and the evolution of receptor recognition in influenza A/H3N2 viruses.

Glycosylation is a prevalent, yet heterogeneous post translational modification of proteins with a broad range of implications in molecular biology. The heterogeneity and non-template driven nature of glycans and glycoconjugates makes them challenging to study. However, the advent of techniques like glycomics, glycoproteomics and glycoengineering have revolutionized the field of glycobiology as a fast-growing and promising research area. Emerging biochemical glycoengineering and enrichment tools, developments in MS fragmentation methods and detection software continuously open new possibilities to study glycans and glycoconjugates and make glycan research more available for the broader research community. In this dissertation we combined existing and newly developed glycomics, glycoproteomics and glycoengineering techniques to study and engineer cell-surface N-glycosylation in the context of genetic glycosylation defects and the evolution of receptor recognition in influenza A/H3N2 viruses.

Glycosylation is a prevalent, yet heterogeneous post translational modification of proteins with a broad range of implications in molecular biology. The heterogeneity and non-template driven nature of glycans and glycoconjugates makes them challenging to study. However, the advent of techniques like glycomics, glycoproteomics and glycoengineering have revolutionized the field of glycobiology as a fast-growing and promising research area. Emerging biochemical glycoengineering and enrichment tools, developments in MS fragmentation methods and detection software continuously open new possibilities to study glycans and glycoconjugates and make glycan research more available for the broader research community. In this dissertation we combined existing and newly developed glycomics, glycoproteomics and glycoengineering techniques to study and engineer cell-surface N-glycosylation in the context of genetic glycosylation defects and the evolution of receptor recognition in influenza A/H3N2 viruses.

We have previously shown that 11 patients became naturally coinfected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during coinfection of host animals and in vitro ; however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two coinfecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally coinfected patients. IMPORTANCE Reassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural coinfection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen.

Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans. DOI: http://dx.doi.org/10.7554/eLife.12217.001, eLife digest Influenza viruses, commonly called flu, infect millions of people and animals every year and occasionally causes pandemics in humans. The immune system can neutralise flu viruses by recognising the proteins on the virus surface, generically referred to as antigens. These antigens change as flu viruses evolve to escape detection by the immune system. These changes tend to be relatively small such that exposure to one flu virus generates immunity that is still effective against other related flu viruses. However, over time, the accumulation of these small changes can result in larger differences such that prior infections no longer provide protection against the new virus. Influenza A viruses infect a wide variety of birds and mammals. Viruses can also transmit from one species to another, which may result in the introduction of viruses with antigens that are new to the recipient species and which have the potential to cause substantial outbreaks. Pig flu viruses have long been considered to be a potential risk for human pandemic viruses and were the source of the 2009 pandemic H1N1 virus. Importantly, humans often transmit flu viruses to pigs. Understanding the dynamics and consequences of this two-way transmission is important for designing effective strategies to detect and respond to new strains of flu. Influenza A viruses of the H1 and H3 subtypes circulate widely in pigs. However, it was poorly understood how closely related swine and human viruses circulating in different regions were to one another and how much the antigens varied between the different viruses. Lewis, Russell et al. have now analysed the antigenic variation of hundreds of H1 and H3 viruses from pigs on multiple continents. The antigenic diversity of recent swine flu viruses resembles the diversity of H1 and H3 viruses observed in humans over the last 40 years. A key factor driving the diversity of the H1 and H3 viruses in pigs is the frequent introduction of human viruses to pigs. In contrast, only one flu virus from a bird had contributed to the observed antigenic diversity in pigs in a substantial way. Once in pigs, human-derived flu viruses continue to evolve their antigens. This results in a tremendous diversity of flu viruses that can be transmitted to other pigs and also to humans. These flu viruses could pose a serious risk to public health because they are no longer similar to the current human flu strains. These findings have important implications not only for developing flu vaccines for pigs but also for informing the development of more-effective surveillance and disease-control strategies to prevent the spread of new flu variants. DOI: http://dx.doi.org/10.7554/eLife.12217.002

The evolutionary interaction between influenza A virus and the human immune system, manifest as ‘antigenic drift’ of the viral haemagglutinin, is one of the best described patterns in molecular evolution. However, little is known about the genome-scale evolutionary dynamics of this pathogen. Similarly, how genomic processes relate to global influenza epidemiology, in which the A/H3N2 and A/H1N1 subtypes co-circulate, is poorly understood. Here through an analysis of 1,302 complete viral genomes sampled from temperate populations in both hemispheres, we show that the genomic evolution of influenza A virus is characterized by a complex interplay between frequent reassortment and periodic selective sweeps. The A/H3N2 and A/H1N1 subtypes exhibit different evolutionary dynamics, with diverse lineages circulating in A/H1N1, indicative of weaker antigenic drift. These results suggest a sink–source model of viral ecology in which new lineages are seeded from a persistent influenza reservoir, which we hypothesize to be located in the tropics, to sink populations in temperate regions. Influenza is one of the most important respiratory infections of humans, responsible for 250,000 to 500,000 deaths annually 1 .O f the three types of influenza virus, type A is the most virulent and is associated with seasonal (winter) epidemics in temperate regions, more persistent transmission in the tropics 2 , and occasional largescale global pandemics characterized by increased morbidity and mortality.

Background One pathway through which pandemic influenza strains might emerge is reassortment from coinfection of different influenza A viruses. Seasonal influenza vaccines are designed to target the circulating strains, which intuitively decreases the prevalence of coinfection and the chance of pandemic emergence due to reassortment. However, individual-based analyses on 2009 pandemic influenza show that the previous seasonal vaccination may increase the risk of pandemic A(H1N1) pdm09 infection. In view of pandemic influenza preparedness, it is essential to understand the overall effect of seasonal vaccination on pandemic emergence via reassortment. Methods and Findings In a previous study we applied a population dynamics approach to investigate the effect of infection-induced cross-immunity on reducing such a pandemic risk. Here the model was extended by incorporating vaccination for seasonal influenza to assess its potential role on the pandemic emergence via reassortment and its effect in protecting humans if a pandemic does emerge. The vaccination is assumed to protect against the target strains but only partially against other strains. We find that a universal seasonal vaccine that provides full-spectrum cross-immunity substantially reduces the opportunity of pandemic emergence. However, our results show that such effectiveness depends on the strength of infection-induced cross-immunity against any novel reassortant strain. If it is weak, the vaccine that induces cross-immunity strongly against non-target resident strains but weakly against novel reassortant strains, can further depress the pandemic emergence; if it is very strong, the same kind of vaccine increases the probability of pandemic emergence. Conclusions Two types of vaccines are available: inactivated and live attenuated, only live attenuated vaccines can induce heterosubtypic immunity. Current vaccines are effective in controlling circulating strains; they cannot always help restrain pandemic emergence because of the uncertainty of the oncoming reassortant strains, however. This urges the development of universal vaccines for prevention of pandemic influenza.