Influenza

Elysha Hussein Sarah Hall Ayesha Sattar

Tuesday, February 25, 2003

Structure of Virion
HA - hemagglutinin NA - neuraminidase helical nucleocapsid (RNA plus NP protein)
100 n m

lipid bilayer membrane polymerase complex

M1 protein Influenza virions are SMALL. The average eukaryotic cell diameter is 10,000 nm (10 microns), which is 100 times bigger than the influenza virion diameter.
http://www.med.sc.edu:85/pptvir2002/INFLUENZA-2002.ppt

Influenza Subtypes
Types A & B
3 IMPs
HA NA M2

Type C
1 IMP
HEF Serves functions of both HA and NA

8 Segments of RNA
Responsible for epidemics & pandemics

7 Segments of RNA Causes only mild infections

Influenza strains are subtyped A, B, or C based on the relatedness of the matrix (M1) and nucleoprotein (NP) antigens All 3 subtypes can infect human, subtype A can also infect other mammals and birds Within each subtype, there are many variant strains

Subtype Viral Structure/Carriers

Type A
Humans Swine Birds Horses Seals

Type B
Humans

Type C

Humans Swine

http://www-ermm.cbcu.cam.ac.uk/01002460a.pdf

Integral Membrane Proteins (IMP)

Hemagglutinin Trimeric Protein 500 copies per virion

Neuraminidase Tetrameric Protein 100 copies per virion

Matrix 2 (M2)
Tetrameric Protein 10 copies per virion
http://www.biotech.ubc.ca/db/TEACH/BANK/PPT/flu2.ppt

Fusion Schematic
1) HA binds a cell GP at a Sialic Acid Binding Site

http://ubik.microbiol.washington.edu/microm-pabio445/MM_445_lec3_2002_files/MM_445_lec3_2002.ppt

Fusion Schematic
1) HA binds a cell GP at a Sialic Acid Binding Site

Low pH

2) Clathrin-Coated pit endocytoses virion

http://ubik.microbiol.washington.edu/microm-pabio445/MM_445_lec3_2002_files/MM_445_lec3_2002.ppt

Fusion Schematic
1) HA binds a cell GP at a Sialic Acid Binding Site
3) Conformational Change: Hydrophobic binding of HA to vesicle membrane

Low pH

2) Clathrin-Coated pit endocytoses virion

http://ubik.microbiol.washington.edu/microm-pabio445/MM_445_lec3_2002_files/MM_445_lec3_2002.ppt

Fusion Schematic
1) HA binds a cell GP at a Sialic Acid Binding Site
3) Conformational Change: Hydrophobic binding of HA to vesicle membrane

Low pH

2) Clathrin-Coated pit endocytoses virion

4) RNPs are released into cytoplasm for replication and transcription (vRNA and mRNA)

http://ubik.microbiol.washington.edu/microm-pabio445/MM_445_lec3_2002_files/MM_445_lec3_2002.ppt

Hemagglutinin (HA)
IMP: homotrimer of non-covalently linked monomers
There are 15 variants of HA currently identified

Precursor (HA0) is synthesized in the RER & Golgi, then transported to the cell membrane Activated when cleaved into 2 chains (HA1 & HA2) that join by disulfide bond HA1 is critical for initial fusion event
Uses Sialic-acid-containing receptors on host cell glycoproteins. This receptor binding event is followed by endocytosis.

HA2 is critical for fusion of virion w/ endosomal membrane

Decrease in pH in endosome enables HA to undergo a confomational change that enables HA to fuse with the endosomal membrane
http://www.ccbb.pitt.edu/PDFFiles/150.pdf

HA Cleavage
Specific cleavage site is a basic sequence of AAs. The site is conserved for specific species. Cleaving enzyme can determine pathogenicity of virus. If the enzyme is ubiquitous in cells, then those cells can make virulent influenza. Humans: Argenine is present at cleavage site
Cleaving enzyme is a tryptase called Clara Only produced in Clara cells, which are only found in upper respiratory tract
Influenza infection is confined to this region of the body

Neuraminidase
IMP: heterotrimer
There are 9 variants currently identified & sequenced

Catalyzes cleavage of ketosidic linkage between sialic acid and adjacent D-galactose or D-galactosamine
HA binds sialic receptors, NA releases virus or progeny virus from receptor

Roles in viral entry/exit:

Help virion navigate mucusal lining of respitory tract Release progeny virion from surface of host cell

Newest Class of drugs: Neuraminidase Inhibitors

Matrix 2
IMP: Homotetrameric Single pass transmembrane protein Roles in last 2 steps of entry process
Facilitates membrane fusion in endosome
Low pH in endosome activates M2 to open ion channel. Hydrogens enter virus and activate HA to undergo conformational change that results in membrane fusion with endosome

As a consequence, RNPs are released into cytoplasm

http://www.northwestern.edu/neurobiology/faculty/pinto2/pinto_flu.pdf

Ribonucleoprotein Complexes (RNPs)

After virion fuses with the endosome membrane, RNPs are shuttled to nucleus Each (-) ssRNA segment associates with 3 polymerases and a nucleoprotein to form Ribonucleoprotein Complexes (RNPs) Replication: vRNAcRNAvRNA Transcription: vRNAmRNA(viral proteins) The RNA polymerase is unable to proofread during transcription
This enables the virus to alter surface antigens and accounts for its ability to evade the immune system

Nomenclature
3 Subtypes, coupled with variance of the antigenicity of surface proteins (HA & NA) and the long history of influenza epidemics necessitate a nomenclature system to catalogue each strain.

A/Moscow/21/99/H3N2
Subtype NP & MI Geographic Origin

Strain Number
Year of Isolation HA & NA Sub-strain

Genetic Reassortment (HA & NA)

Antigenic Drift
Minor changes in the antigenic character Mutation rate highest for type A, lowest for type C Most meaningful mutations occur in HA1 protein When 2 virions infect a cell, there are 256 possible combinations of RNA for offspring.

http://www.biotech.ubc.ca/db/TEACH/BANK/PPT/flu2.ppt

Antigenic Shift
Phylogenic evolution that accounts for emergence of new strains of virus Immunologically distinct, novel H/N combinations Genetic reassortment between circulating human and animal strains is responsible for shifts Segmented genome facilitates reassortment Only been observed in type A, since it infects many species

Antigenic Shift: 1997 Hong Kong

H5N1 virus, harbored in chickens, infected humans via direct contact, only 6 casualties What made H5N1 strain so virulent? Post-mortem examination revealed high levels of cytokines and TNF-.
Indicates an innate, but not specific, immune response Hong Kong researchers suggest that this strain of the virus exacerbates the cytokine response, possibly causing toxic-shock symptoms or death

Antigenic Shift: 1997 Hong Kong

Webster et al: Use reverse genetics to identify the gene responsible for increased virulence and immune system evasion
Remove nonstructural (NS) gene from H5N1 Insert this gene into benign strain Assess virulence of this new strain, compare to control

Conclusion: NS1 is critical for limiting antiviral effects of cytokines.

Downregulates expression of genes involved in the pathway which signals the release of cytokines Single point mutation is responsible for making NS1 a better downregulator

Where does influenza act in the body?

The influenza virus is a upper respiratory tract infection caused by one of the influenza virus pathogens (Type A, B, or C). Although it is called a respiratory disease, it affects the whole body, making you feel sick all over.

http://www.nlm.nih.gov/medlineplus/ency/imagepages/17237.htm

Transmission from person-to-person by:

Tiny droplets that come from a persons mouth and nose when they cough and sneeze. Touching objects contaminated with particles from an infected persons nose and throat.

http://www.lungusa.org/diseases/c&f02/influenza.html#what

Symptoms
Symptoms begin 1-4 days after infection. You can spread the flu before your symptoms start and 3-4 days after your symptoms appear. The following symptoms of the flu can vary depending on the type of virus, a persons age and overall health:
Sudden onset of chills and fever (101 103 degrees F) Sore throat, dry cough Fatigue, malaise Terrible muscle aches, headaches Diarrhea Dizziness

Is it a cold or the flu?

Symptoms
Fever:

Cold
Rare

Flu

Characteristic,high (102 104 F),lasts 3 4

days Headache: Rare Prominent General Aches: Pains Slight Usual Often severe Fatigue: Quite mild Can last up to 2 3 weeks Extreme Exhaustion: Never Early and prominen Stuffy Nose: Common Sometimes Sneezing: Usual Sometimes Sore Throat: Common Sometimes Chest Discomfort: Mild to moderate Common:can becom hacking cough severe

Complications Superinfection
A bacterial superinfection can develop when the influenza virus infects the lungs. The result?
The bacteria that live in the nose and throat can descend to the lungs and cause bacterial pneumonia.

Who is most at risk?

People over 50, infants, those with suppressed immune function or chronic diseases.

Other complications include bronchitis, sinusitis and ear infections.

http://www.ecureme.com/atlas/version2001/atlas.asp

Complications in children:
Studies show a link between the development of Reyes syndrome and the use of aspirin for relieving fevers caused by the influenza virus. The disease involves the CNS and the liver and children exhibit symptoms of drowsiness, persistent vomiting and change in personality.

Influenza outbreaks:
Outbreaks are associated with cold weather and therefore occur mostly in the winter months.
A reason for this: the contrast of the cold outdoor air and the heated indoor air can cause the drying of the respiratory tract tissues and render individuals more susceptible to contracting the flu.

Outbreaks are likely to occur among individuals living together in settings such as nursing homes or among people who gather together indoors during the winter months.

Diagnosis:
Individuals with symptoms of influenza should see their doctor for a thorough physical exam. Rapid influenza tests, viral cultures, and serum samples can be used to confirm infection by the influenza virus since the symptoms of the flu are similar to the symptoms caused by other infections.

Rapid influenza tests:

These tests are 70% accurate for determining if the patient has been infected with the influenza virus and 90% accurate for determining the type of influenza pathogen. Examples of rapid influenza tests: Directigen Flu A, Directigen Flu A + B, Flu OIA, Quick Vue, and Zstat flu. Rapid influenza tests provide results in 24 hours and can be performed in the physicians office.

Viral Cultures:
Samples to be tested by viral cultures need to
be collected from the first four days of infection. The viral culture can be performed from nasopharyngeal or throat swabs, nasal wash, or nasal aspirates. The results are made available within 3 to 10 days.

Serum samples:
Blood samples can be tested for the presence of influenza antibody to diagnose recent infection. Two samples should be collected: one sample within the first week of illness and a second sample 2-4 weeks later. If antibody levels increase from the first to the second sample, influenza infection likely occurred

How do you prevent infection?

The only proven method for preventing influenza is a yearly vaccination approximately 2 weeks before the flu season begins. Since the influenza virus is subject to genetic mutations with the HA and NA proteins, new vaccines that consist of different influenza strains need to be developed each year. Every year, the vaccine is trivalent, meaning that it provides resistance to three strains of influenza viruses. The vaccine consists of 2 influenza A virus pathogens and 1 influenza B pathogen.

Surveillance
The global surveillance network determines which strains of the influenza virus will make-up the vaccine. The networks is made up of 200 WHO laboratories in 79 countries and 4 WHO Influenza Collaboratory Centers coordinate the work of the labs. During the course of the year, influenza viruses from patients are sent to these centers. The centers, in conjunction with the FDA Vaccines and Related Biological Products Advisory Committee, make recommendations as to the IV strains they expect to circulating in the next year.

Surveillance Contd:
After both parties agree, the vaccine is manufactured from inactivated viruses.

More on vaccination:
Each years vaccine takes about six months to produce, package and distribute. The influenza vaccine is currently produced in embryonated chicken eggs. Future possibilities: a new growth medium could speed up vaccine production.

I already have the fluNow what?

Increase liquid intake like water, juice, and soups. Get plenty of rest for the 7 to 10 days during which the symptoms may persist. Take anti-fever drugs to relieve the fever. Anti-viral drugs have recently been designed to treat the flu. If patients begin taking these drugs within 48 hours after their symptoms begin, the drugs may reduce the length of the illness by about 1 to 2 days.

Anti-viral drugs: General background

All anti-viral drugs inhibit viral replication but they act in different ways to achieve this. Drugs that are effective against influenza A viruses: amantadine and rimantadine. Drugs that are effective against influenza A viruses and influenza B viruses: zanamivir and oseltamivir.
Amantadine Type of Influenza virus infection indicated for use Administration Ages approved for treatment of flu Ages approved for prevention of flu Influenza A Rimantadine Influenza A Zanamivir Influenza A Influenza B Oseltamivir Influenza A Influenza B

oral 1 year 1 year

oral 14 year 1 year

oral inhalation 7 years not approved

oral 18 years not approved

http://wdhfs.state.wy.us/epiid/fluvac.htm

Zanamivir and Oseltamivir

These drugs are neuraminidase inhibitors.
They prevent the NA proteins on the surface of the IV from removing sialic acid from sialic acidcontaining receptors. Viral budding and downstream replication of IV are inhibited when sialic acid remains on the virion membrane and host cell. The emerging IVs stick to the cell plasma membrane or other viruses since the sialic acid is still on the surface of the cell and the virion.

Neuraminidase inhibition

http://www.tamiflu.com/hcp/neuramin/neura_index.asp

Amantadine and Rimantadine

These drugs inhibit influenza virus A replication. They block they ion channel M2 protein which inhibits the delivery of IV RNPs from the endosomes to the cytosol. However, the gene that codes for M2 can mutate and confer resistance from these drugs.

http://www.tulane.edu/~dmsander/WWW/335/Orthomyxoviruses.html

Future Directions for protection:

Neirynck et al. suggest a universal vaccine for all influenza A viruses. HA and NA proteins are variant between the influenza A viruses, but the extracellular domain of the M2 protein is highly conserved. Neirynck et al. propose a vaccine based on the M2 protein would protect infection by influenza A viruses.

Historically Speaking
Influenza can be traced as far back as 400 BC In Hippocrates Of the Epidemics, he describes a cough outbreak that occurred in 412 BC in modern-day Turkey at the turn of the autumn season In Hippocrates Of the Epidemics, he describes a cough outbreak that occurred in 412 BC in modern-day Turkey at the turn of the autumn season

412 BC Outbreak
Actual disease that affected the camp is still under debate but is theoretically influenza High communicable rate and autumn season onset are notable characteristics of influenza Death and funerals were a daily spectacle Miasma rising from bodies was fatal to the sick and the sick were fatal to the healthy Hostile ranks were forced to withdraw from the camp

18th Century Outbreak

Between 1781-1782, an influenza epidemic infected 2/3 of Romes population and of Britains population Disease spread to North America, West Indies, and South America Spread of pandemic culminated in New England, New York, and Nova Scotia in 1789 1781 marked the beginning of the 10-40 year cycle of influenza epidemics and pandemics

19th Century Outbreaks

Asia 1829
Spread to Indonesia by January 1831

Russia 1830
Spread throughout Russian and westward between 1830 and 1831 By November 1831, the influenza outbreak reached America

Epidemics prevalent until 1851

19th Century Outbreaks

After a forty year dormant cycle, Russian Flu pandemic occurred between 1889 and 1890
Mostly deadly pandemic to that date (1889) Began in Central Asia during summer of 1889 and spread to Russia, China, North America, parts of Africa, and major Pacific Rim countries 500,000 750,000 mortalities worldwide

Influenza had been regarded as a joke, but the medical profession finally started to realize its severity

Influenza in the spotlight

1900 JAMA article recognized influenza as a serious health threat
Variable forms of influenza suggested Catarrhal type affects the respiratory or gastro-intestinal regions Neurotic type affects the cerebral, neuralgic, and the cardiac regions Blending of these types produces typhoid

20th Century Outbreaks

1918 Spanish Flu 1957 Asian Flu 1968 Hong Kong flu 1976 Swine Flu scare 1977 Russian Flu scare 1997 Avian Flu scare

1918 Spanish Flu

Most lethal and infectious pandemic ever Flu first appeared in Kansas in March of 1918
Within one week of the first reported case, the flu had spread to every state in the US Those who fell ill in the morning were dead by nightfall Those who survived symptoms of the flu often died of complications (such as pneumonia) caused by bacteria

By April, virus spread to Europe, China, Japan, Africa, and South America
Characterized as the First Wave high communicability, low lethality Despite low lethality, 800,000 worldwide had died by the summer

1918 Spanish Flu

In late August, a second more virulent form emerged
Characterized as the Main Wave

Virus killed over 100,000 people per week in some US cities Spread throughout Europe, the Alaskan wilderness, and remote islands of the Pacific By October 1919, flu strain vanished
At least 20,000,000 dead worldwide within 18 months 850,000 Americans

1918 Spanish Flu

Mortality was greater than the 4-year Black Death Bubonic Plague Mortality rate was 2.5%, other epidemics had been 0.1% Unusually, most deaths associated with young, healthy adults Researchers isolated a wide selection of bacteria virus for influenza unknown
Years later, H1NI strain found responsible for infection However, bacteria responsible for the severe secondary complications of pneumonia causing death

1957 Asian Flu

Began in China and spread through Pacific H2N2 Strain responsible Mortality rate of 0.25% Virus quickly identified Vaccine production began in May 1957 Virus entered US and spread through school children Deaths occurred between Sept 1957-March 1958
Highest rate of death in elderly 70,000 Americans dead

1968 Hong Kong Flu

First detected in Hong Kong in early 1968 H3N2 Strain responsible Wildly spread to US by December Mildest pandemic in 20th Century
Immunity may have developed from Asian Flu School children were home for the holidays Improved medical care and antibiotics for secondary infections were available

1976 Swine Flu Scare

Novel virus identified in Fort Dix labelled Killer Flu Thought to be related to 1918 Spanish Flu Mass vaccination campaign in US Virus never moved outside Fort Dix area
If it had spread, it would have been much less deadly than the Spanish Flu

1977 Russian Flu Scare

Started in northern China Influenza A/H1N1 responsible Epidemic disease in young children and young adults worldwide Persons born before 1957 had developed an immunity because of 1957Asian Flu
Not considered a true pandemic because illness occurred primarily in children Virus was included in 1978-1979 vaccine

1997 Avian Flu Scare

Isolated in Hong Kong A/H5N1 flu responsible Few hundred were infected 18 Hospitalized, 6 dead Flu did not spread from person to person
Cause for concern because virus moved directly from chickens to people Pigs were NOT the intermediate host Chickens (1.5 million) were slaughtered No further spread afterwards

1999 Avian Flu scare

Isolated in Hong Kong Influenza A/H9N2 responsible 2 children infected Pandemic was not started but incident is a cause for ongoing concern
Continued presence in birds Ability to infect humans without intermediate host Influenza virus able to change and become more transmissible among people

Weaponization & Bioterrorism

High mutation rate
Antigenic shifts Antigenic drifts Both changes produce new influenza virus variants and strains Strains which humans have no immunity against are likely to be causative agents of pandemics

Communicability

If Influenza Strikes Again

Influenzas destructive capacity resides in the pace and unpredictability of its virus evolution
Can easily subvert the bodys immune response and outstrip societys efforts at containment

Scenario of greatest concern for medical, public health, and political leaders
Lead to a catastrophic epidemic severely taxing societys ability to care for the sick and dying

 How can we prepare?

Build capacity for care for mass casualties
Physicians from all resources and space must be on hand Limited space sends the sick back home to further spread the virus Decentralized delivery of aid (i.e home care)

Respect social mores relating to burial practices

Proper treatment of the dead during an infectious disease emergency would require expeditious handling of corpses to prevent public health threats while avoiding dehumanizing mortuary practices

Focus on developing a pneumonia vaccine, to prevent secondary, often fatal, infections which are facilitated by influenza infection.

 How can we prepare?

Characterize outbreak accurately and promptly
Systematic reporting system would allow public health officials to keep the public informed For example www.cdc.gov gives a weekly influenza summary Latest reports are all available online

 How can we prepare?

Earn public confidence in emergency measures
Neither support nor resistance to public health recommendations by the community should be taken for granted Successful plan for managing an epidemic would be conveying consistent and meaningful messages, serving audiences with diverse beliefs and languages, and acknowledging citizen concerns and grievances

Guard against discrimination and allocate resources fairly

Need to explain the disease to prevent prejudice that reinforces existing social schisms and inequalities Fairly allocate resources

References
1. 2. 3. 4. 5. 6. 7. 8. Burnett, Chiu, and Garcea. Structural Biology of Viruses. Oxford: Oxford University Press, 1997. Mahy, Brian WJ. A Dictionary of Virology. 2nd Ed. San Diego: Academic Press, 1997. Fields, Barnard N. et al. Fields Virology vol 1. 3rd Ed. Philadelphia: Lippincott-Raven, 1996. http://www.med.sc.edu:85/pptvir2002/INFLUENZA-2002.ppt Structure and Genome Organization of Influenza Viruses. Expert Reviews in Molecular Medicine. Available: http://wwwermm.cbcu.cam.ac.uk/01002460a.pdf. Cambridge University Press, 2001. Antler, Christine, Boyler, Erin. Who Knew? The Flu and You! From: Biotechnology Laboratory, University of British Columbia. Available Online: http://www.biotech.ubc.ca/db/TEACH/BANK/PPT/flu2.ppt. No date. Isin, Basak, et. al. Functional Motions of Influenza Virus Hemagglutinin: A Structure-Based Analytical Approach. Biophysical Journal. Feb 2002: vol. 82, 569-581. Lagunoff, Michael. Viral Replication. Lecture notes from April 9, 2002 for Microbiology/Pathology 445. University of Washington. Available Online: http://ubik.microbiol.washington.edu/micrompabio445/MM_445_lec3_2002_files/MM_445_lec3_2002.ppt Pinto, Lawrence. The M2 Ion Channel Protein of Influenza Virus A. Detailed Research Summary from Northwestern University. Available Online: http://www.northwestern.edu/neurobiology/faculty/pinto2/pinto_flu.pdf. 8. Feliciano D, et. al. Five-year Experience with PTFE Grafts in Vascular Wounds. American Scientist 2003, 92: 122129. Pandemics and Pandemic Scares in the 20th Century from CDC: Pandemic Influenza [Online] Schoch-Spana M. Implications of Pandemic Influenza for Bioterrorism Response. Clinical Infectious Diseases 2000; 31:1409-13 Puskoor, Rohit et al. Invfluenza Virus Book Chapter. Not yet published.

9. 10. 11. 12. 13.