Case Study: H1N1/09 “Swine Flu” and Neuraminidase Inhibitors
What is “Swine Flu”?
Swine flu, also referred to as H1N1/09, is a term which categorizes a type of the virus
influenza A. The letters represent the two proteins that can be found on the surface of the
virus, hemagglutinin (H) and neuraminidase (N). The numbers indicate the different
strains. These proteins are found on the surfaces of all influenza A viruses.
In early 2009, there was some media attention devoted to an H1N1 virus outbreak, first
reported in Mexico City on March 18. It is now accepted that this virus most likely
originated in Asia and was brought to North America by infected individuals. Initially
dubbed the “swine flu”, H1N1/09 is a combination of four viruses, one each from human
and avian, and two from swine. Although mild in most cases and considerably less
virulent than H5N1 (“avian flu” – SARS), the World Health Organization (WHO)
declared H1N1/09 to be a pandemic on June 11, 2009. This was mainly due to the global
scale and rapid rate at which the virus spread.
The seasonal flu of 2009 preceded H1N1/09 in North America. In contrast, the Southern
Hemisphere experienced the seasonal flu at the same time as the arrival of H1N1/09. The
presence of two viruses affecting a population at the same time gives potential for
recombination and development of resistance.
The Influenza virus targets the respiratory tract. Affected individuals display symptoms
not unlike the common flu: coughing, sore throat, sneezing, muscle pain, headaches and
fever. This makes it difficult to track how many people have already been infected with
H1N1/09 as it is underreported and mistaken for other types of influenza A. There has
also been the tendency by some countries to underreport cases for fear of economic
repercussions, especially those countries that are heavily reliant on tourism.
Historically, there have been three major epidemics of the twentieth century. The first of
these, the Spanish flu, occurred from 1918-20. It was an Influenza A virus of the subtype
H1N1, and killed an estimated 50-100 million people. The Asian flu (1956-58) subtype
H2N2, claimed approximately 2 million lives, and the Hong Kong flu (1968-69) subtype
H3N2, killed about 1 million. This can be compared to the seasonal flu (Influenza
A/H3N2, A/H1N1 and B) which is responsible for approximately 250,000-500,000
deaths per year.
In most cases, treatment of viruses is symptomatic, meaning the illness has to run its
course. Vaccines that target the Influenza A virus have been developed, these include
Oseltamivir (Tamiflu), Zanamivir (Relenza), and Peramivir (experimental drug –
BioCryst Pharmaceuticals). These vaccines, generally administered to the young, elderly
and immuno-compromised, are not always effective. Viruses can recombine and mutate,
becoming resistant to vaccines. Such is the case with the seasonal H1N1 flu virus and the
Neuraminidase Structure and Function
Infected particles contain two major proteins on their surface (fig. 1). The first of these,
hemagglutinin, binds the virus to sialic acid residues that are found on the surface of the
host cell. Hemagglutinin has three shallow binding sites. The other protein is
neuraminidase, a glycoside hydrolase (viral surface antigen) that removes the sialic acid
from host cell glycoproteins, and enables the newly synthesized virus particles to be
released from the host cell to infect other cells. In humans, the neuraminidase homologs
(NEU 1, NEU 2, NEU 3, NEU 4) is called sialidase. Neuraminidase has a deep pocket
for an active site which makes it a great target protein for drug inhibition. A competitive
inhibitor will bind instead of sialic acid because of its similar chemical properties.
Neuraminidase inhibitors act on neuraminidase by binding to the active site. This
prevents the action of the neuraminidase to remove sialic acid, thus hindering the release
of virus particles into the mucus of the respiratory tract. This slows down the virus and
gives the immune system a chance to respond. Some viruses mutate (H1N1, seasonal flu)
and have an altered active site (pocket) increasing virus resistance.
Figure 1 a) virus and plasma membrane interaction b) neuraminidase structure, c)
Influenza and Sialic Acid-Based Neuraminidase Inhibitors
Effective sialic acid-based inhibitors have the following characteristics as shown in Table
1. Inhibitors should closely resemble the substrate as a successful structure based rational
Table 1: Functional groups for sialic acid-based inhibitors
1 Double bond in ring between C2 and C3
2 Carboxyl group at C2
3 Positively charged group at C4
4 N-acetamido group at C5
5 Glycerol side chain at C6
Oseltamivir, commonly known as Tamiflu, is the most common and widely used antiviral
medication for the treatment of Influenza A and B. Part of the popularity of this drug is
the ease of use – it is taken orally in tablet form. Oseltamivir (fig. 2) is a neuraminidase
competitive inhibitor that is synthesized in a multi-step process (fig. 3). This process is
limited by the availability of shikimic acid, which is extracted from star anise. A concern
with this drug is that there have been cases of resistance and therefore failed ability to
inhibit the virus. Part of the issue is because if the virus is not fully suppressed during
treatment, it can mutate and develop a resistance to the drug. This happened in some
cases of H5N1 avian influenza and seasonal influenza (H1N1). A combination of
oseltamivir with zanamivir (another Influenza A drug) could prove to be more effective
in the treatment and prevention of resistance, which is of concern if the H1N1/09 “Swine
Flu” acquires resistance.
Figure 2 – Oseltamivir structure
Figure 3 – Synthesis of Oseltamivir
Discovered in 1989 by scientists in Melbourne, Australia, Zanamivir (Relenza) is a
neuraminidase inhibitor (fig. 4) for the treatment and prevention of Influenza A and B.
The drug is taken by inhalation, and targets cells of the host that are infected with the
virus. It also prevents the virus from spreading. Zanamivir works by binding to the
active site of neuraminidase.
Figure 4 – Zanamivir structure
Taken intravenously or intramuscularly, this drug for the treatment of the influenza virus
is in experimental stages of development. Peramivir (fig. 4) is also a neuraminidase
inhibitor and prevents new viruses from coming out of infected cells.
Figure 5 – Peramivir structure
Neuraminidase and neuraminidase inhibitors continue to be investigated by the
researchers at AICCS. There are practical applications and significance to this area of
study, considering the societal implications of the Influenza virus. To hear more about
cell membrane glycoproteins and inhibitors, check the CMASTE podcast featuring Dr.
Christopher Cairo, principal investigator at the AICSS.
Case Study Questions
1. Identify and state the function of two proteins that are found on the surface of a
2. What are the symptoms that someone infected with the Influenza virus will
present with, and why is it difficult to determine whether an individual has
H1N1/09 (swine flu)?
3. What are the three major epidemics of the 20th century?
4. What is drug resistance, and how does it relate to H1N1/09 “swine flu”?
5. Compare the active sites for neuraminidase and hemagglutinin. Which makes a
greater target for drug inhibition?
6. Make a table to compare the three H1N1 vaccines that are found in the case study.
Include the name, function and mode of administration (ie. Pill form). Be sure to
include a title for your table.
7. What are the characteristics of an effective sialic acid-based neuraminidase
8. Draw the inhibitor Zanamivir, circle and label the functional groups.