1 INTRODUCTION
Influenza viruses remain important pathogens that cause respiratory
diseases in humans and animals. Human seasonal influenza A and B viruses
annually cause severe morbidity and economic losses worldwide. The
Centers for Disease Control (CDC) estimates around 23,000 flu-related
deaths in the United States each year.[1] In addition, avian
influenza viruses, such as the H5 and H7 subtypes, sporadically cause
highly lethal infections in both animals and humans,[2-4] and animal
or human-animal influenza reassortant viruses occasionally cause global
epidemic or pandemic influenza.[5]
Vaccination is considered the most effective strategy for controlling
influenza in humans.[6] However, current influenza vaccines have
several limitations, including their limited efficacy due to antigenic
mismatches between the vaccine and circulating virus strains.[7] For
this reason, antiviral drugs are important for controlling influenza.
Representative classes of anti-influenza drugs include adamantane-based
matrix protein 2 (M2) ion channel blockers (e.g., amantadine and
rimantadine) and neuraminidase (NA) inhibitors (e.g., oseltamivir and
zanamivir).[8] However, the emergence of antiviral drug resistance
is a constant concern owing to the high mutation rates of influenza
viruses.[9] Since the first report of amantadine-resistant influenza
A viruses (IAVs) during the 1980 epidemic,[10] the prevalence of
these viruses among circulating IAVs (especially, H1N1 and H3N2
subtypes) has increased rapidly to nearly 100% of the cases.[11] In
response, the CDC has stopped recommending the use of adamantane in the
United States.[12] Increasing application of NA inhibitors
(especially oseltamivir) brings into focus the risk of developing
resistance to this class of anti-influenza drugs. Although the
prevalence of NA inhibitor-resistant influenza viruses is generally low
(oseltamivir <3.5%) or rare (zanamivir <1%),
[13-16] the problem of reduced susceptibility and resistance of
influenza viruses to NA inhibitors has been increasing recently.
Therefore, there is an utmost need to develop better or novel
anti-influenza drugs.
In this study, we first aimed to identify anti-influenza viral agents by
screening compound libraries. Aprotinin, a serine protease inhibitor,
presented as a candidate. Previous reports have suggested that aprotinin
has anti-influenza viral activity.[17-19] However, most reports
cover only a narrow range of IAV strains (especially seasonal IAV
strains) and strains of influenza B virus (IBV) that may no longer be
circulating. Therefore, we investigated the anti-influenza viral effects
of aprotinin on various subtypes of IAV, including i) human seasonal
IAVs, ii) avian influenza viruses with zoonotic potential (H5N2, H9N2,
and H6N5), iii) oseltamivir-resistant IAV, and iv) a currently
circulating strain of IBV in vitro . We also used a mouse model to
verify the anti-influenza activity of aprotinin. Our findings contribute
further evidence to the potential of aprotinin as a broad-spectrum
anti-influenza agent.