[Molecular characterization of influenza A viruses – epidemiology and genetics]. (Doctoral thesis)

Original title: Molekulare Charakterisierung von Influenza A Viren - Epidemiologie und Genetik.
Influenza A viruses are distinguished into various subtypes based on the composition of their surface proteins with 16 different variants of hemagglutinin (H) and 9 different variants of neuraminidase (N) genes. Almost all combinations were identified in avian species. In humans, only subtypes H1N1 and H3N2 are present, causing annual epidemics and rarely pandemics. Due to the segmented genome, two unique mechanisms thrive the evolution of influenza A viruses - antigenic shift or the exchange of gene segments or reassortments and antigenic drift, the continuous change of antigenicity of the surface proteins, which will be the focus of this work.
A major change in the cleavage site of the hemagglutinin (HA) protein results in huge morbidity and mortality in infected poultry, named highly pathogenic avian influenza (HPAI). After initial introduction of these viruses in Africa, in 2006 HPAI spread from Nigeria to neighboring countries. In the first study, complete genome sequencing of HPAI (H5N1) viruses from 2007 and subsequent phylogenetic analysis revealed that all gene sequences were more closely related to the first strains of sublineage A and C found in Nigeria in 2006 than to any strain found outside of the country. Six out of eight viruses had evolved by at least three reassortment events from previously identified sublineages A and C viruses. Our results suggested that HPAI (H5N1) viruses initially imported into Nigeria in 2006 have been gradually replaced by various reassortments. Interestingly, in all reassortants nonstructuralprotein genes were derived from sublineage C with two characteristic amino acids (compared to sublineage A). If the high prevalence of reassortants was typical for West-Africa in 2007, the absence of such reassortments anywhere else suggests that reintroductions of H5N1 from Africa into Eurasia must be a rare event. Further, the results indicated that despite extensive eradication campaigns HPAI (H5N1) continued to circulate in Nigeria, allowing various reassortment events between viruses from diverse genetic lineages.
Human influenza viruses are one of the major pathogens worldwide causing respiratory diseases with significant morbidity and mortality in risk groups. Effective treatment against influenza viruses is available; however, assessment of viral drug susceptibility remains important. During the season 2007-2008, new H1N1 virus variants emerged containing a mutation in their neuraminidase (NA) gene, which conferred resistance to the neuraminidase inhibitor oseltamivir. These viruses spread efficiently and eventually became the prevailing variant of H1N1 viruses. To study if their emergence was associated with treatment with oseltamivir, we investigated clinical and epidemiological data of patients infected with oseltamivir-resistant in comparison to drug-sensitive viruses.
Human cases of oseltamivir-resistant influenza A H1N1 emerging in 2007-2008 in Luxembourg were not associated with treatment, prophylaxis or stockpiling of oseltamivir. Following initial local seeding, resistant strains spread synchronously to sensitive strains causing a similar epidemiology and clinical symptoms. However, phylogenetic analysis revealed genomic markers segregating oseltamivir-resistant from oseltamivir-sensitive viruses in NA and polymerase basic protein 2 (PB2) gene sequences. Specific mutations were present in resistant viruses but absent in drug-sensitive variants indicating that drug-resistant variants that contained these mutations most probably resulted in enhanced fitness compared to resistant virus variants without these additional mutations.
Finally, with emergence of a new influenza A virus of swine-origin in 2009, serological studies to assess antibody protection levels in humans became of interest. Serological studies on swine influenza viruses (SIVs) in humans with occupational exposure to pigs have only been reported from the Americas, but not from Europe. Thus in this study, we analyzed neutralizing antibodies against the pandemic H1N1 2009 influenza virus and an avian-like, enzootic H1N1 SIV in swine contacts in Luxembourg compared to a matched general population. We showed that professional swine contacts in Western Europe elicited more frequently neutralizing antibodies against both H1N1 viruses than controls.
Part of the general population, however, also tested positive against either one or both viruses, while exposure to them was unlikely. Sequential infections with variants of human seasonal H1N1 viruses may have increased the chance of serological cross-reaction with the antigenically distinct H1N1 viruses tested for, and we assume that these crossreactive antibodies may provide some level of cross-protection. Further studies are required to determine to what extent the serological responses correlate with infection.
In conclusion, this work advances the knowledge about influenza A virus’ molecular epidemiology and complex mechanisms of virus evolution