Swine Influenza Viruses – Evolution and Zoonotic Potential

Abstract

Influenza A virus (IAV) is an important respiratory pathogen with a broad host range. The natural reservoir for IAV is waterfowls, but both human and swine are considered natural hosts. During the past century IAV has caused severe pandemics as well as seasonal epidemics in the human population. In pigs, swine influenza virus (SIV) is endemic worldwide and is associated with economic losses for the farmer due to the impact on pig health causing lowered production. Swine has been shown to be susceptible to infection with IAVs of different host origin and has hence been considered as potential mixing vessels of new IAVs. Furthermore, transmission of IAVs from swine to human and vice versa has been documented on several occasions and further classifies this virus as a highly important zoonosis. This aspect enhances the possibility of the formation and establishment of new and potentially more virulent viruses with the capacity to cause severe pandemics. Therefore, it is important to gain a deeper understanding of the evolution of SIVs, their zoonotic potential as well as host-range characteristics and this PhD project aimed at elucidating parts of these important points. The PhD thesis begins with a presentation of the aims and a brief introduction of the situation of SIV in Denmark. In the background section an extensive review on IAVs with emphasis on SIV is provided. The results obtained during the PhD are presented in two complete manuscripts and one work in progess, followed by a joint discussion of the main results. Manuscript I analyze the genetic and antigenic evolution of two of the most prevalent SIVs circulating in Denmark. In total, 78 sequences of the H1N1 and H1N2 subtypes, collected in the period 2003-2012, was analyzed. The genetic analysis was based on several computational methods for estimation of phylogeny, selection pressure, evolutionary rates and time of most recent common ancestor for the surface glycoproteins, HA and NA. The antigenic relationship of the Danish H1 SIVs was determined by antigenic cartography. High evolutionary rates of HA and NA compared with low evolution suggests that evolution is primarily controlled by purifying selection. Further, a high level of genetic relatedness and of low evolution was observed for the Danish H1 sequences, this observation was supported by both phylogeny and antigenic cartography. Antigenic cartography also revealed few antigenic outliers that potentially indicated drift away from current H1 viruses. The time of most recent common ancestor for H1 was estimated to be markedly earlier than previously suggested. Phylogenetic analysis of the Danish N2 gene revealed that two different lineages are circulating in Denmark. Manuscript II describes the biological characterization of four different Danish SIVs and includes an experimental pathogenesis study performed in ferrets, which are regarded as the most appropriate small animal model for human IAV infections. The viruses chosen for this study were two enzootic SIVs (H3N2 and H1N2) and two new SIV reassortants (H1avN2hu and H1pdmN2sw). The two reassortants were detected for the first time in 2011 and have since then become established and are now circulating in Danish pigs. Viral replication in nasal wash samples and viral load in respiratory organs were determined. Growth kinetics of the four SIVs were determined in vitro using respiratory swine and human cell lines. The affinity of HA of the four SIVs for a2,3- and a2,6-receptors were assessed as well as receptor kinetics and antiviral susceptibility of NA. This study showed that all four SIVs were able to infect and transmit efficiently and to high titers via direct contact and H3N2 was found also to transmit efficiently via the airborne route. H3N2 and H1pdmN2sw were found to induce the most severe lung lesions, consistent with these two viruses expressing the highest viral load in lung tissue samples. Growth kinetics demonstrated that all four SIVs were able to infect and replicate to high titers in both swine and human respiratory cell lines. Receptor studies showed a high preference for binding to a2,6-receptors for the Danish SIVs. NA kinetics revealed a high enzyme activity for H1pdmN2sw compared to the remaining viruses, suggesting that NA activity alone is not sufficient for the observed airborne transmission of H3N2. Furthermore, it was revealed that the Danish SIVs were found to be sensitive to all of the neuraminidase inhibitors tested. Based on the findings in this study it was proposed that viruses with a human-like HA play a more significant role in transmission compared to viruses with only a humanlike NA. Furthermore, this study also underlined the importance of continued surveillance of SIVs in order to detect new reassortants as well as the necessity of assessing their zoonotic potentials. Manuscript III describes the establishment of a reverse genetics system based on a backbone from the Danish H1N2 SIV, which is one of the two most prevalent subtypes in Denmark. Recently, a variant of a North American swine H3N2 virus containing a pandemic M gene was transmitted to humans in the US and on few occasions human-to-human transmission was observed. These events underline the need for a reverse genetics system to be used for an analysis of the behavior of a pandemic M gene in a Danish SIV.