Evolution of drug resistance in influenza A viruses

Abstract

Influenza A viruses are important pathogens of humans, other mammals and birds. Swine are considered to be the ‘mixing vessel’ for influenza viruses because of their susceptibility to infection with not only swine influenza viruses but also human and avian influenza viruses. After infection of pigs with different influenza viruses, reassortment events between genomic RNA segments and point mutations can take place which can result in novel influenza virus strains capable of causing human pandemics. To combat infections, vaccination is available in many countries for humans, but not typically used in pigs. However, anti-influenza drugs have been used to treat livestock, and mutations conferring drug resistance occur in circulating strains. The mechanisms responsible for the emergence and spread of drug resistant mutations against amantadine and oseltamivir have been studied previously but often gave conflicting results. Therefore, this PhD thesis focused on resolving the mechanisms responsible for this rapid drug resistance spread. In chapter one I examine the extent of reassortment events in swine influenza A viruses by analysing within subtype reassortment and extrapolating the results for the between subtype reassortment. Reassortment is one of the mechanisms that can be responsible for mutations, conferring resistance to drugs, to spread between strains, and thus spread in the host population. The findings of this chapter show that the genomic segments most prone to reassortment code for a polymerase (PB1) and both glycoproteins, within all three subtypes studied. Since particular mutations in the matrix protein (MP) segment cause resistance to amantadine, my study focused on MP compared to other segments and revealed moderate level of reassortment. MP reassorts well with polymerases, both within and between subtype, while nonstructural (NS) is least likely to reassort. Chapter two of this thesis aimed at resolving the origin and spread of the most common drug resistance conferring mutation in swine influenza viruses which causes amantadine resistance. I show first that this mutation occurred in swine influenza viruses and was therefore not transmitted from the recently ancestral avian influenza strains, and second that the prevalence of resistance in swine influenza viruses is due to functional linkage of mutations at other sites and not by direct drug pressure. In chapter three I examine the mechanisms responsible for the rapid rise and spread of oseltamivir resistance in human influenza H1N1 viruses which arose in the absence of drug use. The primary mutation lies in the neuraminidase glycoprotein but because of the close functional interaction I focus on changes in haemagglutinin that occurred in association with resistance. The results showed several mutations in haemagglutinin were associated with resistance suggesting selection acting on haemagglutinin in order to balance the activity of both glycoproteins. Overall these results show the importance of functional linkage between segments as a mechanism for the occurrence of drug resistance conferring mutations, and reassortment as a means of spreading these mutations into newly emerging strains.