Influenza A virus preparedness: Characterization of NS gene reassortants and the antiviral efficacy of tannin-rich plant extracts. (Doctoral thesis)
- Clinical and Applied Virology
Seasonal influenza virus epidemics causing up to 500 000 deaths each year represent a substantial public health burden. In addition to seasonal epidemics, influenza A virus (IAV) can cause global pandemics, as evidenced by the swine-origin influenza virus in 2009. The recent creation of a highly pathogenic avian influenza H5 subtype virus that efficiently transmits between ferrets in the laboratory increases concerns about the acquisition of humanto- human transmission of highly pathogenic strains. Avian H7N9 recently emerged in China and infected more than 130 people in 2013. Such examples highlight the need for effective IAV preparedness, including IAV surveillance, prevention, risk management strategies and sufficient treatment options.
A prerequisite for effective IAV preparedness is a thorough characterization of past and circulating IAV strains, since it facilitates a prompt reaction to newly emerging strains. In the first part of the present study, pandemic H1N1 (pH1N1) NS gene reassortants were constructed by reverse genetics and characterized. pH1N1 emerged as a pandemic IAV in 2009 and continues to circulate nowadays as a seasonal strain. Besides infecting humans, pH1N1 has also been detected in swine or birds. Thus, there is a considerable risk of developing new reassortants with other co-circulating avian, swine or human strains. The viral non-structural protein 1 (NS1) is a key player in inhibiting the antiviral immune response and a known virulence factor. By reverse genetics, eight reassortants carrying NS genes of human, avian or swine strains in the genetic background of pH1N1 were constructed. pH1N1 was highly permissive to NS genes from various host species, showing only minor fitness losses in 6 out of 8 reassortants on A549 cells. However, introduction of NS from highly pathogenic avian influenza virus (HPAIV) H5N1 attenuated the virus on A549 and DF-1 cells and in vivo. NS1 sequence comparisons revealed a five amino acid deletion in position 80-84 that is also found in most contemporary H5N1 strains, but hardly ever in non-H5 subtypes. Deletion of positions 80-84 in pH1N1 NS1 attenuated viral replication in vitro, possibly explaining the absence of this deletion in virtually all naturally occurring non-H5 strains. Mechanistically, NS1 from pH1N1 allowed higher general host gene expression than NS1 from HPAIV H5N1. Importantly, a previously unknown role in the regulation of the general host gene expression was attributed to the deletion of amino acids 80-84. This regulation occurs possibly at the level of pre-mRNA maturation. The high viral fitness of several pH1N1 NS reassortants created in Part 1 of the study, as well as naturally occurring reassortments and point mutations show the high variability of IAV strains and therefore their propensity to antiviral resistance. Thus, new safe and effective antiviral drugs are needed. Two classes of drugs are currently licensed for the treatment of IAV infections, namely neuraminidase and matrix protein inhibitors, preventing release of new virions from the infected host cell or viral uncoating, respectively. Development of antivirals targeting a different step of the viral life cycle would be especially advantageous. Therefore in the second part of this study, a tannin-rich extract from Pelargonium sidoides DC, EPs® 7630 (Umckaloabo®), which is licensed to treat acute bronchitis, was investigated for its antiviral effects. EPs® 7630 showed dose-dependent activity against several IAV strains. It inhibited an early step of influenza infection and impaired viral hemagglutination as well as neuraminidase activity. EPs® 7630 was not virucidal, as virus preincubation (unlike cell preincubation) did not influence infectivity. Importantly, EPs® 7630 showed no propensity to resistance development in vitro. Condensed tannins and pseudotannins were identified as the active principle and structure-activity relations were investigated. Chain length influenced antiviral activity, as monomers and dimers were less effective than oligoand polymers. Importantly, gallocatechin and its stereoisomer epigallocatechin exert antiviral activity also in their monomeric form. In addition, EPs® 7630 administered by inhalation significantly improved survival and illness of influenza-infected mice, demonstrating the benefit of EPs® 7630 in treatment of influenza. These data have confirmed and specified the antiviral activity of tannin-rich plant extracts and selected tannins. However, different classes and molecular weights of tannins are often found together in plant extracts, and may differ in their antiviral activities. Nevertheless, there are only few systematic comparisons of their anti-IAV structure-activity relations. A better understanding of the antiviral activity of different tannin structures against IAV is warranted to optimize plant-based antivirals. In the third part of this study, Hamamelis virginiana L. was chosen as a model plant, since it is rich in different tannins that have been previously well characterized. We compared the anti-IAV effect of Hamamelis virginiana bark extract, fractions enriched in tannins of different molecular weights and individual tannins of defined structures, including pseudotannins. The bark extract was active against different IAV strains, including the recently emerged avian H7N9 strain. Fractions enriched in tannins of different molecular weights were produced by a collaborator using ultrafiltration, a simple, reproducible and easily upscalable method. A highly potent fraction enriched in high molecular weight condensed tannins was identified as the best performing antiviral candidate. This ultrafiltration concentrate and the bark extract inhibited early and, to a minor extent, later steps in the IAV life cycle. Interesting mechanistic differences between tannin structures were observed: high molecular weight tannin containing extracts and tannic acid (1702 g/mol) inhibited both IAV receptor binding and neuraminidase activity. In contrast, the tested low molecular weight compounds (< 500 g/mol) inhibited neuraminidase but not hemagglutination. Average molecular weight of the compounds seemed to positively correlate with receptor binding (but not neuraminidase) inhibition. In general, neuraminidase inhibition seemed to contribute little to the antiviral activity. Importantly, antiviral use of the ultrafiltration fraction enriched in high molecular weight condensed tannins and, to a lesser extent, the unfractionated bark extract was preferable over individual isolated compounds. In summary, this study contributes to different aspects of IAV preparedness, namely to the characterization of current and possibly emerging IAV strains and to the development and optimization of antivirals. Briefly, a previously unknown role of a naturally occurring NS1 five amino acid deletion in the regulation of the antiviral host response was identified. Also, the finding that pH1N1 reassorted with most of the tested human, avian and swine NS gene segments without a major loss in fitness highlights the need for IAV surveillance of NS reassortants. Finally, antiviral activity of EPs® 7630, a tannin-rich plant extract already licensed for acute bronchitis treatment, was demonstrated in vitro and in vivo. The established antiviral structure-activity relations of tannins and pseudotannins from Pelargonium sidoides and Hamamelis virginiana are of interest for developing and improving plant-based antivirals.