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Inhibition of Influenza H7 Hemaglutin-Mediated Entry

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on 19 November 2013

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Transcript of Inhibition of Influenza H7 Hemaglutin-Mediated Entry

The importance of NA spike
Binding
•It mediates the release from host cells by cleaving sialic acid residues to which newly formed virions are attached.
•It prevents newly formed virus particles from clumping, enhancing dispersal.
•It penetrates of virus particles through sialic acid-rich mucin bathing epithelial linings of the respiratory tract.

Influenza: The Virus
Type A is most common
30,000 – 50,000 deaths/year in USA
Segmented genome (8 helical nucleocapsids)
Each codes for different proteins
Envelope has 2 different types of peplomers
PEPLOMERS (protein spikes) are antigenic
H = hemagglutinin - attachment
Sixteen different H ( H1, H2, H3, etc)
N = neuraminidase - release from host cell
Nine different N (N1, N2, etc)
Different antigenic types of H & N from genetic changes

Influenza: A virus with high mutation potency


Influenza viruses are among the best viruses that mutate. There are new strains that appear on a yearly basis.
This is mainly due :
RNA genome which is error prone
Segmented (8 segments) genome which tends to recombine and reassort during viral replication to give a new virus with a new H and N combination
Inhibition of Influenza H7 Hemaglutin-Mediated Entry
Mandy Cho, Elena Galindo, Jacques Niamien, Shaurya Prakash
Influenza: the Virus
Single stranded RNA virus.
3 types of influenza viruses: A, B, and C
Influenza A causes epidemics and pandemics and has an animal reservoir especially in birds and swine.
Influenza B only cause epidemics and are not found in animals
Influenza C do not cause epidemics and give rise to minor respiratory illness.

Mims Medical Microbiology 5th edition
Binds via HA to sialic acid in alpha 2,3 or alpha 2,6 linkage with galactose on the host cell surface.
HA1 is the receptor binding subunit and HA2 is the subunit that mediates membrane fusion .
During the entry process, HA undergoes a series of binding and pH induced conformational changes that result to binding of the virus to the plasma membrane , entry of the virus into the endosome, exposure of the HA fusion peptide fusion, fusion of the viral and endosomal membrane and finally release of the viral RNA into the cytoplasm. (Antanajisevic et al, 2013)
Sialic acid in 2,6 linkages are characteristic of human cells
Sialic acid in 2,3 linkages are characteristic of avian cells.
Mims medical microbiology 5th edition
Antigenic drift
There are two types of changes...
Small point mutations affecting the H and N constantly. The new subtype is slightly different than the parent and is usually responsible for the new epidemics or cases occurring yearly.
Antigenic shift
Is less common and only occurs with influenza A. This is a sudden and major change in the H and/or N antigens creating a new strain. Ex: H3N2 to H1N1. This is due to major recombination occurring when 2 strains of viruses infect the same cell. Usually the best mixing vessel for such an event is a SWINE cell. Usually cause pandemics.

The role of pigs
The human and swine viruses evolved from a common origin. Virus goes from bird to pig to human. Swine cells have receptors for both avian and human influenza viruses. The pig is the “mixing” bowl for reassortment of avian and human viruses.
http: //paulkiser.files.wordpress.com/2012/03/pig-smiles.jpg
Influenza infection
http ://i1-news.softpedia-static.com/images/news2/How-the-Flu-Virus-Became-Resistant-to-Drugs-2.jpg
•Influenza infections are reported worldwide
•Transmission is by contaminated droplets and HANDS.
•The initial symptoms are due to direct damage and inflammatory responses in the respiratory epithelial cells.

•Within 1-3 days the cytokines released cause symptoms of chills, fever, malaise, muscle aches, runny nose and cough.

•Most mortality cases occur due to bacterial superinfections causing pneumonia by S.aureus, H. influenzae, Streptococcus pneumoniae.

•Most diagnosis is clinical or by rapid agglutination tests of throat swabs.

Influenza in the past...
Current treatment options
Tamiflu and Relenza which inhibit viral release from cells by acting on the neuraminidase (N) glycoprotein and thus decreases cellular infection by viruses.

They prevent new virions from being released from the surface of the infected cell therefore controlling the infection.
Drug resistance
Resistance is increasing in circulating influenza strains.
2008-2009 H1N1 strain exhibited 100% resistance against tamiflu.
One of the cause is probably the overuse of tamiflu by confusing flu symptoms with regular cold symptoms therefore increasing the resistance of the virus over time. (high mutation potency of flu virus)
http: //vaccinenewsdaily.com/wp-content/uploads/2013/01/tamiflu.jpg
http: //www.gsk.tw/pic/Products/medicines/RELENZA.jpg
Influenza in the past...
1918-1919:
H1N1 Spanish flu
In 1918, the Spanish influenza pandemic resulted in the death of about 20 million people worldwide. It was the most severe and famous influenza pandemic to date.

The pandemic was a result of the H1N1 influenza strain, which was 25 times more deadly than any previous strain, with a 5% mortality rate.

This strain of influenza resulted in high fevers, painful headaches, lassitude, and even hemorrhaging. Victims would turn purple and drown from the fluids in their lungs.

http ://dartmed.dartmouth.edu/winter06/html/cold_comfort.php
Influenza in the past ...
1957-1958:
H2N2 Asian flu
Influenza in the past ...
1968-1969:
H3N2 Hong Kong flu
Influenza now
Introducing H7N9
Influenza in the past ...
2008-2009: H1N1 Swine Flu
First reported in March of this year.

Avian influenza A: people infect after exposure to birds or environments
contaminated with virus. 77% of first 82 patients had some animal exposure.

As of now, 134 cases reported and 45 deaths in China alone.

There is currently no vaccine for this strain.

As temperatures begin to drop, what will happen with the strain?
Is there the potential for another pandemic?

Subject of the followng study.

htt p://abcnews.go.com/blogs/health/2013/04/08/h7n9-bird-flu-update-21-infected-6-dead/
Influenza:
htt p://www.cdc.gov/flu/pdf/avianflu/h7n9-reassortment-diagram.pdf
http://www.cdc.gov/flu/avianflu/h7n9-images.htm
H7N9
http://www.who.int/influenza/human_animal_interface/influenza_h7n9/10u_ReportWebH7N9Number.pdf
WaterLOGSY NMR indicates TBHQ binds to H7 HA. It also shows that it binds specifically to the loop since the antibody F49 which also binds to the loop is able to prevent theTBHQ from binding.
Then Saturation Transfer Difference (STD) NMR to find out how the binding might take place. This is based on the amount of interaction between different hydrogen in TBHQ. The hydrogen found to interact the most are indicated in the diagram above. Since the tert-butyl hydrogens did not interact as much it could indicate that they are not involved in binding and would therefore be good areas to alter to increase affinity.

Theoretical image of how TBHQ binds inside of an H14 HA protein. The H14 is still part of group 2 HA’s so its binding is pretty similar to how the binding in H7 would be. The t-butyl group in the back is not involved much in this interaction.


Source: Structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion.
How TBHQ Binds
WaterLOGSY NMR Results
STD NMR find which hydrogens interact the most
pH analysis of TBHQ
Virus life cycle
The life cycle shows how the virus binds to the cell, undergoes endocytosis, and after the M9 proton channel is activated, creates an acidic environment inside the endosome. After this fusion occurs, the RNPs are released to replicate and release new virus.
HA conformational rearrangement
At fusion pH, extensive structural reorganization occurs that involves extrusion of the “fusion peptide” from the interior of the neutral-pH structure, presumably toward the target endosomal membrane with which the virus membrane is to fuse.

HA fuses
membrane together
Another image of how the hemagglutinin fuses the membranes together. The red is the fusion peptide. The yellow portion zips/twirls together bringing the cell membranes closer to each other. If TBHQ were present, the fusion peptides (red) would never be released and the membranes would never fuse together.
TBHQ Changes Conformation Change pH
This shows that the H7 HA bound to TBHQ decreases the pH needed by H7 HA to undergo a conformational change. The midpoint pH changes from 4.9 to 4.6. This is enough to prevent conformational change and prevent release of the binding peptide.
TBHQ stabilizes X31 (a group 2 HA) since the temperature need to cause change increases by 3 degrees Celsius.

TBHQ also stabilizes other group 2 HA
Discussion Questions
Can the results of this study be applied to other types of flu? Other infectious viruses in general?
What are some of the ethical issues that could arise from the use of a food additive such as TBHQ?
Do you think influenza viruses will become resistant to TBHQ in the future?
What are some other targets for antiviral drugs, besides HA, NA, and M2 inhibitors?
What would be the best way to distribute to use TBHQ to prevent H7H9 and other avian flu?
Can influenza
be eradicated ?
Do you think that the new H7N9 outbreak has the potential to become a pandemic? Why or why not?
http://ww w.virginmedia.com/science-nature/amazing-bodies/outbreak-history-of-epidemics.php?ssid=5
The Asian flu pandemic lasted between 1957 and 1958. First identified in China in late 1957, the Asian Flu caused 70,000 deaths in the United States and nealy 2 million deaths worldwide.
Beginning in 1968, Hong Kong Flu, a strain of the H3N2 influenza virus, was first identified in Hong Kong. Between 1968 and 1969 this strain killed approximately 1 million people worldwide. The H3N2 influenza A virus is still in existence.
http://www.health.com/health/gallery/0,,20307381_4,00.ht ml
Phylogenetic tree of influenza A HAs. The 2 groups are colored cyan (group 1) and green (group 2), each of which can be further subdivided into 3 clades (H8, H9, and H12; H1, H2, H5, and H6; H11, H13, and H16) and 2 clades (H3, H4, and H14; H7, H10, and H15). The tree was drawn by using the program TreeIllustrator.
Pandemic: worldwide outbreak
TBH Structure:
https://www.google.com/search?q=TBHQ&oq=TBHQ&aqs=chrome..69i57j0l5.5781j0j7&sourceid=chrome&espv=210&es_sm=91&ie=UTF-8
Summary of conclusions

TBHQ Inhibits the entry of H3 and H7 HA
TBHQ binds to the stem loops of H3 and H7 HA
TBHQ stabilizes the neutral pH conformation of H3 and H7 HA
What can we do with TBHQ?
Targeting HA is a novel approach that has not been attempted before. It is unlike other antiviral treatments currently in use.

The authors of the study propose that TBHQ could be added to animal feeds in order to prevent infection in animals and the spread of H7N9 in humans.

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