- Current estimates of the case fatality rate for H5N1 infection are between 50-80%, but these rates may be skewed high because reporting criteria are too stringent and do not account for mild or asymptomatic cases. - Molecular studies show that H5N1 NS1 protein contributes to the virus's virulence by triggering apoptosis and hypercytokinemia, helping explain H5N1's associated rapid viral pneumonia and acute respiratory distress syndrome. - Limited human cell tropism of H5N1, restricted largely to cells in the lower respiratory tract, is thought to currently prevent efficient human-to-human transmission but adaptation remains possible through antigenic drift or shift.

1. 2012
Abstract:
Current World Health Organization (WHO) estimates give a case fatality rate of 50-80% for
people infected with H5N1. Molecular studies into the virulence displayed by H5N1 provide
some basis for an increased case fatality rate, however they cannot explain it completely.
Criteria for reporting H5N1 infections are currently too stringent to allow accurate and
representative reporting of true H5N1 case fatality rates. Molecular studies can, however,
explain the abnormal symptoms of primary viral pneumonia and Acute Respiratory Distress
Syndrome (ARDS) displayed by patients infected with H5N1. Limited human cell tropism is
currently thought to be responsible for preventing human-human H5N spread, preventing
further fatalities beyond the 603 witnessed as of May, 2012. Intense interest in producing an
adaptable or universal H5N1 vaccine has lead to an ever improving field of H5N1 vaccine
production.
Introduction:
A highly virulent pathogen, H5N1 is yet to display a high degree of pathogenicity in humans,
the few infections observed thus far often resulting in fatal acute respiratory distress
syndrome (ARDS) (Lee et al, 2009). Current epidemiology suggests an extremely high
mortality rate amongst humans, though estimates are skewed by monitoring methods. There
is sufficient molecular evidence to raise concerns on the potential for H5N1 to achieve a
human-human transmissible form and cause a pandemic. As a result there have been a
number of recent varied studies undertaken to determine the molecular basis of H5N1
virulence. Due to the current public concern surrounding H5N1 there have been a number of
efforts targeted at producing an H5N1 vaccine and prospects for an H5N1 vaccine produced
in time to immunise against a pandemic strain are constantly improving.
Reasons for the absence of a human H5N1 pandemic
Influenza pathogenicity (the ability to cause disease in a host) is dependent on the tropism of
the viral haemmagglutinnin for host cell surface sialic acid-galactose linkages (Siaα-2,3 or
2,6Gal) and is potentially limited by compatibility with essential host enzymatic systems (van
Riel et al, 2012). H5N1 haemagglutinin (HA) is specific for Siaα-2,3Gal cell surface
linkages which are present in some cells of the human respiratory tract but which are also
largely outnumbered by the predominant Siaα-2,6Gal linkages.
Cells binding MAA agglutinin (recognises Siaα-2,3Gal) appear red, those binding SNA agglutinin (recognises Siaα-2,6Gal) appear green,
cells were counterstained with blue DAPI (Shinya et al, 2006).
The distribution of Siaα-2,3Gal linkages on the surfaces of human respiratory cells was
confirmed with the aid of lectin histochemsitry, using fluorescently tagged tree –derived
agglutinins specific for Siaα-2,6Gal and Siaα-2,3Gal (Shinya et al, 2006). The data shown

2. 2012
above displays the relative paucity of cells displaying Siaα-2,3Gal linkages on their cell
surfaces in the lower respiratory tract relative to those which display Siaα-2,6Gal.
Considering Siaα-2,3Gal linkages are generally restricted to the lower respiratory tract, this
shows the overall lack of Siaα-2,3Gal in the human respiratory system. Viral histochemistry
has since confirmed that host cell range in the lower respiratory tract is predominantly limited
to type II pneumocytes and alveolar macrophages as well as Clara cells and non-ciliated
epithelial cells in the terminal bronchioles (van Riel et al, 2007), binding to the upper
respiratory tract cells occurring as well but only relatively rarely (Nicholls, 2007 and van Riel
et al, 2012).
Attachment of human and avian viruses to ciliated epithelial cells, goblet cells, and submucosal glands in the human URT, Upper respiratory
tract cells to which the virus bound display a red border which is indicative of virus-cell binding and thus virus-cell recognition. (van Riel
et al, 2010)
Virus histochemistry (use of labelled virus) has confirmed that tropism is associated with
sialic acid distribution and that tropism is associated with ability to spread (van Riel et al.,
2010). H5N1 has a different cell binding profile relative to seasonal (H1N1 and H3N2) and
pandemic (H1N1) forms of human influenza. As can be seen, H5N1 does not display a clear
binding to upper respiratory tract cells, with the exception of cells of the submucosal glands.
The human influenza strains tested are much more widespread amongst humans than H5N1
and so it is currently thought that tropism to upper respiratory tract epithelia may allow an
increased ability to undergo human-human spread (van Riel et al, 2010).
H5N1 human-human transmission may require compatible enzyme systems in human cells
allowing viral function and replication to occur (Webster et al,2006). This is not true in the
case of limited proteolysis of HA during the maturation of H5N1 (an extremely important
step in influenza replication) because the proteases responsible are virtually ubiquitous in our
cell membranes, more so than those responsible for seasonal and pandemic influenza HA
cleavage (Kido et al, 2012) suggesting that host proteases do not limit H5N1 as much as was
previously thought.
Thus, H5N1 has not caused more deaths in the human population probably because it is not
currently adapted to our upper respiratory tract, although adaptation is possible through
antigenic drift, as was seen in a reassorted H5N1 virus in ferrets (Kawaoka et al, 2012), or
antigenic shift through reassortment with human-adapted strains in intermediate reservoirs

3. 2012
such as pigs (Cong et al, 2010). Both processes are currently occurring in native H5N1
populations (Kawaoka et al 2012 and Ibid.) and so monitoring of the situation is a key factor
in our preparedness for a potential H5N1 pandemic.
General Molecular mechanisms of H5N1 virulence:
Rapidly progressive primary viral pneumonia is a major feature of most reported H5N1-
related fatalities, a relatively rare event in other forms of influenza (Abdel-Ghafar,2008).
Resulting acute respiratory distress syndrome (ARDS) is associated with damage to the
alveoli and is the main source of mortality amongst H5N1 patients (Peiris et al, 2009).
Molecular studies have sought to find the answers behind these abnormal symptoms, an
important and much-studied virulence factor being the H5N1 NS1 protein. H5N1 NS1 is
implicated in caspase-dependent apoptosis (Zhang et al, 2010) and hypercytokinemia (Lam et
al, 2011), allowing the symptoms of viral infection to extend far beyond a localised area in
the lungs (deJong et al, 2006). Systems-level comparisons between the H5N1 and the
seasonal form of H1N1 have found that H5N1 induces a similar pro-inflammatory cytokine
response to human-adapted influenza in vitro but that this response is quantitatively stronger
(Lee et al, 2009). Further study has also implicated NS1 in this response in vitro (Lam et al,
2011).
Transmission electron microscopy (TEM) on normal (left) and plasmid transfected cells (right) X5,000 magnification (Zhang et al, 2010).
NS1 is a non-structural protein which normally has the role of inhibiting the host
interferon(IFN)-mediated innate immune response by binding double-stranded RNA, thereby
preventing the activation of the downstream cytosolic viral-detection machinery (Peiris et al,
2009). The role of H5N1 NS1 in triggering apoptosis was discovered with the use of
plasmid transfection on carcinomic alveolar epithelial cells. Use of recombinant techniques
and generation of an Escherichia coli (E. coli) library lead to the production of plasmids
encoding H5NS1. The cells were transfected with these plasmids as a surrogate for viral
infection and incubated for 24 hours (Zhang et al, 2010). Shown above are two transmission
electron micrographs on non-transfected and H5N1 NS1-transfected cells. Note how the
transfected cells are characteristic of post-apoptotic cells with condensed chromatin
aggregated along the nuclear membrane (red arrow) and apoptotic bodies (orange arrow).

4. 2012
Enzyme activities of caspase-3 and caspase-9 in NS1-transfected alveolar epithelial cell line. Enzyme activities based on transformed y-axis
indicating levels of caspase activity based on absorbance according to Caspase detection kits(Zhang et al, 2010)
Western blotting showed that Caspases 3 and 9 (two downstream effectors of apoptosis in
mammalian cells) were present in transfected cells. Subsequent colorimetric assays for
activities indicated that both were activated during the infection of the alveolar epithelial cells
(Zhang et al, 2010). This study was done on an immortal cell line rather than primary ex vivo
alveolar epithelium in addition to only the NS1 gene in a modified form being substituted for
a native H5N1 particle with its associated myriad of virulence factors, limiting how
applicable these findings would be to the role of NS1 in a whole virus infection. However a
causative link between the presence of NS1 and caspase-dependent apoptosis has been
established (Zhang et al, 2010), providing a further clue into the molecular basis of H5N1
virulence as apoptosis on other forms of cell lines has also been observed and found to be an
essential mechanism for viral propagation (Wurzer et al, 2003) as well as being a contributing
factor to the viral pneumonia in fatal cases of H5N1 infection (Peiris, 2009).
Heatmap for averaged microarray gene expression profiles of H1N1 and H5N1 infected primary human macrophages. Results are averaged
across macrophages from three different individuals (Lee et al, 2009).
A systems-level approach showed that an infection of primary alveolar macrophages from 3
different individuals by a native H5N1 virus strain in vitro led to a much more pronounced
pro-inflammatory cytokine response than for a low-pathogenicity seasonal H1N1 subtype

5. 2012
inoculated at an equivalent multiplicity of infection (Lee et al, 2009). This study came in lieu
of a number of studies which collectively showed that H5N1 infection caused a hyper-
induction of the cytokine response by alveolar macrophages and alveolar epithelial cells (Lee
et al, 2009). Global gene expression of macrophages infected with either H5N1 or H1N1 was
recorded using GeneChip arrays. A heat map of the relative gene expression between two
sets of alveolar macrophages, those infected with H1N1 and those with H5N1, shows that
similar pathways were upregulated at similar time points but that H5N1 infection caused a
much more intense upregulation. Overall it was found that components of cytokine pathways
for IFN-β and TNF-α were hyper-expressed in vitro (Lee et al, 2009) indicating the H5N1
viral particle was responsible for the hypercytokinemia seen in H5N1 patients (deJong et al,
2006).
Proportion overall cells displaying signs of early apoptosis upon transfection with influenza NS1 mRNA. (CC= non transfected controls).
(Lam et al, 2011)
Transfection of a lung epithelial cell line with mRNA encoding viral NS1 confirmed that
H5N1 NS1 is involved in the induction of apoptosis and inducing the abnormal pro-
inflammatory cytokine response behind ARDS (Lam et al, 2011). The proportion of early
apoptotic cells sampled at irregular intervals was determined flow cytometrically with the aid
of a fluorescent dye specific for early apoptotic cells. Note how large the difference between
H5 and other influenza subtype NS1 is in terms of both biological and statistical significance,
especially at 6 hours. H5 subtype NS1 mRNA transfection resulted in a maximum proportion
of early apoptotic cells double any other influenza NS1 in addition to having a 95%
confidence interval which was not overlapping any of the other ones, indicating a statistically
as well as biologically significant difference (Lam et al, 2011). This increase in apoptosis
could explain why primary viral pneumonia with diffuse alveolar damage is so common in
H5N1 infections, especially considering that the virus is generally limited to the lower
respiratory tract.

6. 2012
Cytometric bead array was used to quantify the levels of relevant cytokines and chemokines.
As shown below, all influenza subtype NS1 mRNA transfections lead to levels of cytokine
both biologically and statistically significantly different from non-transfected cells, however
H5 subtype NS1 mRNA clearly caused a greater amount of expression of the pro-
inflammatory cytokines measured, indicating that it the hypercytokinemia displayed in
ARDS is at least partially due to the effect of H5N1 NS1(Lam et al, 2011).
Cytokines/chemokines induced by transfection of NS1 mRNA of different influenza subtypes. H5 subtype NS1 mRNA transfected cells are
shown in purple. Cytokine/chemokine levels are expressed as fold-changes compared to the non-transfected cell controls measured at the
same time points (Lam et al, 2011).
High case fatality rate
The current case fatality rate for reported H5N1 infections is between 50-80% (“WHO:
Cumulative number of H5N1 cases”, accessed 18.5.2012), extremely high compared to the
1918 H1N1-attributed case fatality rate of 2.5% (Marks et al, 1976). The difference between
case fatality rates is highly unlikely to be due to differences in virulence at the molecular
level alone. A good point has been made that the case fatality rate may be falsely high
because the WHO requires a very stringent number of conditions to be met before confirming
a case of H5N1 (Palesea,2012). A patient must report to a hospital with a laboratory
advanced enough to allow for serological or genetic testing with advanced symptoms of
influenza infection (“WHO: H5N1 Case definition”, accessed 14.05.12). These selection
criteria may provide a skewness towards patients who are severely ill (Peiris et al, 2009),
leaving little room for those who may have an asymptomatic H5N1 infection (if this is
possible) or people who cannot reach a hospital with the necessary facilities. Rural areas
where waterfowl and other reservoir species are abundant are also under-represented,
meaning that this stringent method is very likely giving a non-representative estimate of true
fatality rates amongst those infected with H5N1.

7. 2012
Cumulative number of confirmed human cases for avian influenza A(H5N1) reported to WHO, 2003-2012 (“WHO: Cumulative
number of H5N1 cases”, accessed 18.5.2012)
Presence of unreported H5N1 infection with a successful outcome is possible as shown by the
discovery of anti-H5N1 HA antibodies in a sample of the poultry workers in the Chinese
province of Jiangsu where previous H5N1 infections and a death attributed to H5N1 had
occurred (Huo et al, 2012). Amongst the sample taken there were no people with prior severe
respiratory illness and the poultry in the villages sampled were not vaccinated against H5-
subtype avian influenza. Hemagglutination inhibition assays on blood samples for anti-H5N1
HA antibodies showed that seropositivity was present in two of the three villages and that
that the true population seropositivity rate for these two villages concerned was significantly
higher than zero (as high as between 2.19% and 10.78% in Gaoyou (95% confidence)). If
we take the number of H5N1 seropositive individuals just from Gaoyou (n=7) and added it to
all reported cases in China at the time of the study (n=46 from the above table) then we can
see that the case fatality rate for China shifts immediately from 66.6% (100*[28/42]) down
roughly 10% to 57.1% (100*[28/49]) if we assume that the anti-H5N1 antibodies came from
H5N1 infection with a successful outcome. This crude calculation serves to show that
making case fatality rate calculations with such stringent selection criteria leads to the
generation of inaccurate and error-prone statistics. This study was limited by a number of
factors, not the least of which being the restricted geography of sampling and a small sample
size, but it showed that it is possible to have H5N1 infections which do not result in
extremely adverse effects and that go unreported (Huo et al, 2012). Therefore WHO
estimates of case fatality rate may be skewed and more research into the true rate of H5N1
infection worldwide must be done before any reliable estimate of the true worldwide case
fatality rate of H5N1 can be determined.
Prospects for H5N1 vaccine:
H5N1 is constantly and quickly evolving, meaning that genetic diversification may occur so
quickly that even geographically close isolates of H5N1 may differ sufficiently to prevent
immunological cross-recognition of these strains should a vaccine based on either virus be
produced (Balish et al, 2010). Thus a vaccine must be adaptable with an ability to be
produced in large quantitites in a small amount of time (Zhou et al, 2012). Widespread, rapid
deployment and mass production of such a vaccine also pose major challenges to the
production of an H5N1 vaccine (McKenna, 2007).
As H5N1 is constantly evolving, it does not make financial or practical sense to mass produce
a standard vaccine based on any particular strain of H5N1 as such a vaccine may be made
irrelevant in the space of a few years or may not be relevant to a pandemic strain. Biannnual
WHO reports on global monitoring of H5N1 clades and their antigenic and epidemiological
properties allows for constant adjustment, depending on the storage of many different

8. 2012
candidate viruses so that in the case of a pandemic it might be possible for the mass
production of any of these viruses, one of which would hopefully be close or identical to the
pandemic strain. As of February 2012 there were 20 known and available candidate viruses
for vaccines with 3 pending full development (“WHO: Antigenic and genetic properties of
zoonotic viruses”, 2012).
Production of a vaccine in large enough amounts to matter and whether this vaccine would
generate sufficient immunogenicity is another matter. Cell culture in bioreactors would allow
for a great increase in production capacity of an H5N1 vaccine without having to rely on the
low-yield and potentially at-risk embryonated hen egg techniques, a high-yield production
method based on the use of the continuous Vero cell line (kidney epithelium isolated from
Chlorocebus spp.) being recently developed (Zhou et al, 2012). The strength behind pairing
this production technique with an existing candidate virus and reverse genetics technology is
that this would allow high-level production of virtually tailor-made live attenuated viruses for
use in an inactivated or split virus vaccine based on relevant pandemic H5N1 antigens
(Watanabe, 2012).
H5N1 vaccines cause poor immunogenicity in patients and animal models inoculated and so
novel adjuvants increasing immunogenicity are sought after (McKenna, 2007). Multiple
different adjuvants are currently being trialled and developed. An alum-based adjuvant
showed a good immunogenic response in Japanese adults but induced febrile reactions in
minors (Nakayama, 2012) whereas a novel silver nanoparticle-based adjuvant has also been
developed (Jazayeri et al, 2012). The latter inducing pro-inflammatory cytokine release and
enhanced antibody and cell-mediated immune responses in chicks, however this adjuvant has
not yet been trialled in humans (Ibid., 2012).
There are several candidate H5N1vaccines which have been developed, however they are not
yet ready for widespread use (WHO: FAQs, accessed 18.5.12). Much work remains to be
done on an H5N1 vaccine. With constant monitoring and improved production capacities
coupled to reverse genetics, in addition to continuing development of improved novel
adjuvants, the outlook for an H5N1 vaccine and our ability to respond quickly to a pandemic
is looking better as time progresses.
Conclusion:
It is apparent that H5N1 could be the basis for a future pandemic, albeit at a true case fatality
rate likely to be less than 50-80%. This paper has only captured very few of the many
molecular studies on H5N1 virulence, focusing on the role of the multifunctional NS1. It
should be remembered that H5N1 virulence is a polygenic trait (Lam et al, 2011) not
dependent on NS1 function or cellular tropism alone. Epidemiology on H5N1 is currently
error-prone and should not be trusted as the sole measure on which predictions of an H5N1
pandemic should be based. However, the clearly increased molecular virulence has caused
enough concern and sparked enough fear and interest that the outlook for production of a
successful H5N1 vaccine in a short amount of time is constantly improving.
Word count (not counting abstract, figure legends and titles): 2,740 words.

9. 2012
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