InfluenzavirusesHistory

Classification/Nomenclature

Virion structure

Genome structure/organization

Life-cycle

Pathogenesis

Clinical Disease

Immunity

Prevention (vaccines/antivirals)

History• Influenza-like disease described as early as 412 B.C. by

Hippocrates

• Major epidemics and pandemics of influenza have occurred throughout history

• Major cause of morbidity and mortality worldwide each year

• Many virologists think another influenza pandemic that could kill millions of humans is inevitable (H1N1 swine flu provided a trial run for pandemic preparedness)

Classification of Influenza Viruses

• Orthomyxoviridae family

• 3 Types of Influenza: A, B and C

• All 3 can infect and cause similar symptoms in humans

• Infection with one type does not confer immunity to another type of influenza

Virus Nomenclature• Influenza type (genus)• Species isolated form (unless human)• Place of Isolation• Number of the isolate• Year of isolation• H and N subtypes (16 H and 9 N subtypes)

Example: the 220th isolate of an H5N1 subtype virus isolated from chickens in Hong Kong is designated:

A/chicken/Hong Kong/220/97(H5N1)

Virion Structure (complex architecture)

-Envelope contains the H and N glycoproteins and M2 protein

-Matrix proteins M1 lies just below the envelope

-Ribonucleoprotein complex (RNPs) make up the core of the virus

- RNPs consist of the viral RNAs complexed with the polymerase proteins (PB1, PB2 and PA) and the nucleoprotein (NP)

Viral Genome Structure and Organization

8 negative strand RNA segments

Virion contains the RNA-dependent RNA polymerase (RdRp) upon infection

Genome first transcribed into mRNA

Functions of Influenzavirus proteins

Influenzavirus Life Cycle

1. Attachment to sialic acid2. Endocytic entry3. Uncoating and release of

vRNPs into cytoplasm4. Translocation of vRNPs into

nucleus5. Generation of mRNA and

positive (+) strands and more – strand RNA genomes

6. Production of viral proteins7. Genomes packaged into pre-

assembled capsids8. Budding and release of

progeny virions

-Binding to sialic acid linked to galactose on cell surface

-Mediated by Hemagglutinin (HA)

-Human viruses bind 2,6 Galactose (present in respiratory tract)

-Avian viruses bind 2,3 Galactose (present in bird intestines)

-Binding specificity influences pathogenesis in these two species

- Current work suggests that a single amino acid change within the HA of Spanish flu (1918 pandemic) resulted in the ability of this avian flu virus to now bind 2,6 Galactose

Influenzavirus Attachment

Influenzavirus Entry

• Virions enter by endocytosis

• Inside of the endosome, the virion is exposed to a low pH (from 7 to 5).

• The low pH causes HA to undergo a conformational change resulting in fusion of viral envelope with endosomal membrane

• Viral RNAs (vRNPs) released into cytoplasm and transported into nucleus

Influenza A Uncoating Step

• M2 ion channel in the viral envelope allows H+ ions to penetrate the virion

• Weakens the viral M1 matrix protein from the vRNPs

• vRNPs released into the cytoplasm

• Amantidine (sold as Symmetrel) and rimantidine (sold as Flumadine) block the M2 ion channel function, interfering with uncoating.

Nuclear import of Ribonucleoproteins and Viral mRNA Synthesis

• NP functions to translocate vRNPs into the nucleus (contains nuclear localization signals NLS)

• After the vRNPs enter the nucleus, mRNA synthesis begins

• Cap-snatching process

– Viral PB2 protein binds to the cap structure along with a short string of nucleotides of host mRNA in the nucleus of the cell.

Figure 12-10aAdapted from N. H. Acheson. Fundamentals of Molecular Virology. John Wiley & Sons, Inc., 2006.

Replication/Transcription of Viral RNA

Figure 12.10b: Differences between influenza virion (–) genome RNA, viral (+) mRNA containing the snatched cellular mRNA cap structure, and (+) viral complementary RNA or template RNA that is a complete copy of the genome RNA segment.

Adapted from E. G. Strauss and J. H. Strauss. Viruses and Human Diseases. Academic Press, 2001.

Virion Maturation and Assembly• Capped mRNAs are translated by ribosomes in the cytoplasm

• HA, N, and M2 are folded and glycosylated and transported to the trans- Golgi network and cell surface where virus assembly takes place

• The HA protein must be cleaved by cellular proteases inside the trans-golgi in order for the new virions to be infectious

• One copy of each genome segment is packaged into the virion

Release of Influenza Virions• Virions are released by

budding

• The viral NA protein cleaves the sialic acid on host cells to prevent clumping of viral particles at the host surface.

• Zamanivir (sold as Relenza) and oseltamivir phosphate (sold as Tamiflu) inhibit the function of NA.

Pathogenesis• Droplet transmission

• Virus enters respiratory tract

• Attaches to ciliated columnar epithelial cells lining the sinuses and airways

• Primary site of infection—tracheobronchial tree, involving nasopharynx

• As virus replicates, cilia are destroyed– Cleaning system in the lungs does not

work as well– More mucus stays in the airway, clogging

them, causing coughing– Virus replication peaks at 48 hours and

then declines– Virus shed for up to a week

• Destruction of cilia contribute to secondary bacterial pneumonia infections, sinusitis, otitis

Figure 12.3a: The cilia are attached to columnar epithelial cells

Influenza has a short INCUBATION PERIOD within the host

Time from infection to symptoms is approximately 1 to 2 days

Infected individuals act as ACUTE CARRIERS of the disease

Infected individuals readily TRANSMIT the virus by coming into contact with un-infected individuals or by expelling the virus into the environment in the form of FOMITES (for example by sneeezing and coughing)

Clinical Features Uncomplicated Infection

– Onset of symptoms:• Headache• Aching in the limbs and back• Fever (100-103oF)• Malaise• Dry cough• Tickling throat• Sore throat• Myalgia• Chest X-ray is normal

– Usually resolves itself after 7 days

Complicated Infection

– Age-dependent

– Young children—croup, secondary bacterial pneumonia, middle ear infections

– Elderly—life threatening secondary bacterial pneumonia, pre-existing conditions like congestive heart disease exacerbated

– Immune compromised individuals—at risk of death during an influenza epidemic

Immunity

• Infected individuals develop antibodies against the outer proteins of Influenza virus—neuraminidase (NA) and hemagglutinin (HA)

• Antibodies against HA neutralize the virus

• Antibodies against NA do not. but can reduce the release of virus from infected cells

Laboratory Diagnosis• Office-based rapid tests—ELISA assays

– Some can distinguish between Influenza A and B – Fast results—1- to 20 minutes– Some false-positive or negative results

• Other Tests– Cell Culture—inoculate MDCK cells, most

accurate test

• Serology– Analyze convalescent serum and analyze for an

increase in antibody titer

• “Flu chips” or microarrays

Courtesy and © Becton, Dickinson and Company

Figure 12.CS01: An influenza test.

Vaccines

• Most effective way to prevent influenza

• Flu vaccination time in the U.S. – October and November

• Vaccine grown in eggs

• Inactivated trivalent vaccine

Trivalent Vaccine Composition

• Recommendations made based on antigenic analyses of recently isolated influenza viruses, epidemiologic data and post-vaccination serologic studies in humans

• Vaccine is a cocktail of 3 virus strains– 2 strains of Influenza A– 1 Influenza B strain

Effectiveness of the Vaccine

• Age of the vaccine recipient

• Immunocompetence of the recipient

• Degree of similarity between the viruses in the vaccine and those in circulation

Target Groups for Vaccination• Persons aged 50 or older

• Individuals in chronic care facilities– Including nursing homes

• Individuals with pre-existing chronic problems– Asthma, other pulmonary or cardiovascular problems, immunosuppression

• Pregnant women

• Healthcare workers

• Travelers

Antivirals for Influenza Treatment• First drugs: M2 inhibitors (prevent uncoating step)

– Amantidine (sold as Symmetrel)– Rimantidine (sold as Flumadine)

• New class of antivirals: NA inhibitors (prevents neuraminidase from cleaving sialic acid during budding)

– Causes viruses to clump at the cell surface, reducing viral spread

– Oseltamivir (sold as Tamiflu, pill form)– Zanamivir (sold as Relenza, must be inhaled)

• Treatment must begin within 36 hours of onset of symptoms

• Used prophylactically in chronic care facilities

Steps of the Viral Life Cycle Targeted by Influenza Antivirals

Figure 12-21

The 1918 Influenza Pandemic

• Also called the Spanish flu• Killed 675,000 Americans• National average death rate was 4.39 out of every 1000

people • Death rates peaked in October of 1918• Unique epidemic: healthy adults ages 20-40 died of the

flu, in addition to children and elderly (who are usually high-risk)

• It decreased the life expectancy in the United States by 11 years.

1918 Flu Related Deaths

Figure 12-1a Adapted from PBS, “Influenza 1918,” American Experience, http://www.pbs.org/wgbh/amex/influenza/maps.

Influenza Mortality Curves: “U-shaped” vs. “W-shaped”

Adapted from J. K. Taubenberger, ASM News 65 (1999): 473-478.

Figure 12.1b: A comparison of the age distribution of influenza deaths in 1918 and 1911–1917.

U.S. Life Expectancy in 1918

Figure 12-1cAdapted from J. K. Taubenberger, ASM News 65 (1999): 473-478.

Influenza Statistics

• 1957-1987—there were 20 influenza epidemics recorded in the U.S.

• 36,000-50,000 people have died as a direct or indirect consequence of an influenza infection

Epidemiology

• Epidemics become unmanageable at alarming speeds because:– Short incubation period (1-4 days)– One droplet can contain 100,000 to 1,000,000 virus

particles.– Symptomatic people do not stay home = spread.– Lack of herd immunity

• Absenteeism from schools is the best indicator of the scale of an epidemic.

Time Course of Influenza A Infection

Figure 12-4Adapted from P. R. Murray, et al. Medical Microbiology, Third Edition. Mosby Elsevier Health Science, 1998.

• If a person has been infected in the past couple of years by a closely related strain of influenza H subtype:– Their antibodies may intercept and neutralize the

virus, protecting the lungs.

Cytokine Storm

• Also referred to as systemic inflammatory response syndrome (SIRS).

• SIRS may explain the devastating nature of the 1918 strain of influenza.

• The immune system overreacts toward the pathogen.• Cytokines signal macrophages to travel to the site of

infection, causing damage to the body and organ failure.

• H5N1 Avian Influenza virus also causes SIRS and is at least 50% lethal in humans.

Cytokine Storm

Adapted from M. T. Osterholm, N. Engl. J. Med. 352 (2005): 1839-1842.

Figure 12.5: Model for the mechanism of the cytokine storm evoked by highly virulent influenza viruses.

12.9 Genetic Variation

• Mutations are common during viral replication.

• Viral RNA dependent RNA polymerase lacks proofreading and correction ability.

• Two processes, antigenic drift and antigenic shift mediate genetic variation that causes new strains of influenza to appear.

Antigenic Drift

Figure 12.13a: Antigenic drift is the gradual accumulation of new epitopes on the H protein (and, to a lesser degree, the N protein).

Adapted from Annenberg Media. “Emerging Infectious Diseases.” Rediscovering Biology: Molecular to Global Perspectives. Annenberg Media, 1997. http://www.learner.org/channel/courses/biology/textbook/infect.

• Responsible for seasonal influenza strains

Antigenic Shift

Figure 12.13b: Antigenic shift occurs when the influenza A virus acquires a new H or N gene.

• Responsible for Pandemic Strains

Influenza Reassortments

Figure 12-14

Adapted from B. N. Fields, et al. Fields Virology, Fourth Edition. Lippincott Williams & Wilkins, 2001.

12.10 Pandemics in History

Is China the Incubator for Flu Viruses?• Close association of humans with animals and birds.• Influenza A is constantly circulating in birds, pigs and

horses.

Figure 12.15a: Farmers migrating in China, bringing their birds and animals with them.

© Bjorn Svensson/Alamy Images

Figure 12.15b: It is not uncommon for farmers in southeast Asia to live in close quarters with their birds and animals.

Why is Influenza More Prevalent During the Winter?

• Aerosol spread of influenza virus is dependent upon humidity and temperature.

• The virus was best transmitted at low humidity (20%) and colder temperatures (5oC or 41oF).

• Supporting evidence that weather conditions play a role in influenza virus transmission.

• Lowen, A. C. et al. 2007. "Influenza Virus Transmission is Dependent on Relative Humidity and Temperature." PloS Pathogens 3(10):1470-1476.

The 1918 Spanish Flu Statistics• Killed more people in 25 weeks than AIDS killed in 25 years.• Kill more people in a year than the plagues of the Middle

Ages killed in a century.• Estimated deaths—20 to 50 million, including 675,000

Americans.• In NYC, 21,000 children were orphaned by influenza.• 7 times as many people died of the 1918 Spanish Flu than in

WWI.

• True estimates will never be known.

It’s Effects were Unique• It killed 20-40 year old adults.• It killed quickly (2-3 days).• Those infected suffered from hemorrhagic symptoms.

Courtesy of the National Museum of Health and Medicine, Armed Forces Institute of Pathology, Washington, D.C., NCP 1603

Figure 12.16: Many young servicemen died in U.S. Army hospitals during the 1918 Spanish influenza pandemic.

Why was the 1918 Spanish Flu So Deadly?• Johan Hultin exhumed bodies of

flu victims buried in frozen ground at Brevig Mission, Alaska.

• Removed frozen lung tissues and provided them to Jeffrey Taubenberger (Armed Forces Institute of Pathology in Washington D. C.) who sequenced the viral genome of the 1918 strain.

• 1918 was an avian—not swine strain.

Figure 12-17: Johan Hultin Courtesy of Johan Hultin

1918 Influenza Strain was Re-Created in the Laboratory, 2005

• Terrence Tumpey, CDC researcher.• Reverse genetics.• Work done in an enhanced BSL-3 laboratory.

Figure 12.18: CDC researcher Dr. Terrence Tumpey working with the reconstructed 1918 pandemic influenza virus in a Biosafety Level 3 laboratory.

Courtesy of James Gathany/CDC

2007—Yoshihiro Kawaoka University of Wisconsin-Madison

• Infected macaques with the reconstructed 1918 Influenza virus.

• Carried out experiments in a BSL-4 laboratory• Macaques died of SIRS (cytokine storm)

1957 Asian Flu

• 70,000 Americans died.• Asian flu first identified in northern China

(February, 1957) and spread to the United States by June, 1957.

• Vaccine was made and available by August, 1957.

1968 Hong Kong Flu

• First detected in Hong Kong during early months of 1968.

• Reached the United States by September, 1968.• Deaths in the United States peaked in

December, 1968-January, 1969 (mostly elderly).• Mildest pandemic in the century, killing only

33,800 people in the U.S.

12.11 The Pandemic Scares

1976 Swine Flu Scare: A/New Jersey/76/H1N1

• February, 1976 Cadets at Fort Dix, NJ came down with the flu, one private died.

• CDC investigated the outbreak.• 4 out of 19 throat washings tested positive for H1N1

influenza A (at the time believed to be a “swine” flu closely related to the 1918 Spanish flu virus).

• 150 million doses of vaccine prepared in the U.S. • 46 million doses were administered within a few months.

1976 Swine Flu Vaccine, cont.

• Vaccine was fast-tracked• Congress passed a liability protect bill to protect

manufacturers of the vaccine• Early problems of the vaccine:

– Guillain-Barré Syndrome (532 people within 10 weeks)– 32 deaths

• Vaccine campaign suspended in the late fall of 1976• U. S. government paid more than $90 million on claims

cases.

1977 Russian Flu Scare: A/USSR/77/H1N1

• May, 1977 Influenza A H1N1 isolated in northern China.

• Primarily children and younger adults became ill.

• This strain was similar to the 1957 strain– Individuals born before 1957 would have no cross-

reacting/partial immunity toward the virus.

1997 Avian Flu Scare: H5N1

• May, 1997, 3-year-old boy in Hong Kong died.• Influenza A isolated from the boy but the H

subtype could not be identified.– Later confirmed to be H5 – H5 not known to infect humans before (subtype

isolated from birds only)• The “jump” of an avian strain directly to humans

had never happened before.• Same H5N1 strain was killing chickens.

1997 Hong Kong H5N1 Scare• Veterinary authorities slaughtered chickens to prevent the

spread of H5N1 to chickens and people.

Figure 12-19 © Vincent Yu/AP Photos

Lessons Learned from the 1997 H5N1 Hong Kong Flu

• Only 18 human cases in 1997• 33% fatality rate.

– 1918 flu was about 4% fatal in the U.S.• It is possible that a single insertion (mutation) could enable the

virus to spread much more efficiently to the brain and heart.• The most important control measures are

– rapid culling of infected or exposed bird and proper disposal of carcasses

– quarantining and rigorous disinfection of farms

Avian Strains Continue to Plague Eastern AsiaHuman cases of H5N1 January 2003-March 2007

Figure 12-20a Adapted from World Health Organization, 2007.

H5N1 Strains in Poultry and Wild BirdsJanuary 2003-March 2007

Figure 12-20b Adapted from World Health Organization, 2007.

12.14 International Influenza Surveillance

• 1946 WHO established influenza surveillance program

• U.S. sentinel physicians send flu statistics to the CDC– Patient visits for influenza-like illness– Age groups– Morbidity and mortality stats

Will There Be Another Killer Flu?Are We Preparing?

• Virologists say it is inevitable

• Many pandemic planning teams at the international, national, state and local levels

http://www.pandemicflu.gov/plan/states/stateplans.html

http://www.who.int/csr/disease/influenza/pandemic/en/

Will the Current Arsenal of Influenza Antivirals Work Against New Pandemic Strains of Influenza?

• Tumpey 2002 study says yes.• Viral resistance has been shown.• We won’t know until it happens.

12.15 Lessons Learned from the SARS Outbreak of 2003

• Within 6 weeks of its discovery—– SARS CoV infected thousands of people in 16

countries around the world.• Spread quickly by air travel.• Identification of pandemic strains and rapid

response to contain outbreaks are paramount.

Virus File 12-1 Retrospective Studies

• Study that looks backward in time.• University of Wisconsin Oshkosh, 1918 Flu

study

http://www.uwosh.edu/departments/biology/shors/textbook/UWOflustudy.html

Virus File 12-2 Preparing a Pandemic Influenza Vaccine

Reverse Genetics or a Universal Vaccine?

• Researchers working on a universal vaccine based on the use of the influenza A, M2 protein

Figure 12.VF01a: Reverse genetics technology.

Adapted from National Institute of Allergy and Infectious Diseases, National Institutes of Health, How the Flu Virus Changes, http://www3.niaid.nih.gov/healthscience/healthtopics/Flu/Research/ongoingResearch/FluVirusChanges.

Robert Webster, Influenza Expert Uses Reverse Genetics to Create Pandemic Avian Flu Vaccines

• Avian influenza strains cannot be grown in embryonated eggs.

• Avian strains kill the eggs.• Reverse genetics allows experts to

grow avian strains in cell cultures.• Developed by Webster so that

rapid avian flu vaccines can be made.

Figure VF 12-1bCourtesy of Seth Dixon, St. Jude Children's Research Hospital

NS1 Prevents Nuclear Export of Cellular Pre-mRNAs, Facilitating Cap-Snatching

Figure 12-12 Adapted from B. N. Fields, et al. Fields Virology, Fourth Edition. Lippincott Williams & Wilkins, 2001.

HA protein• Must be cleaved by cellular proteases inside of the

trans Golgi network for the virus to be infectious.

Adapted from N. H. Acheson. Fundamentals of Molecular Virology. John Wiley & Sons, Inc., 2006.

Figure 12.8c

Viral mRNAs are not Cannibalized for Their 5’ Caps

• The viral PB2 polymerase proteins selectively “snatch” caps.

• Binds to a specific sequence that is complementary to nucleotides 1-12 of the 3’ ends of each vRNA segment.

5’ AGCAAAAAGCAGG 3’

Low pH-dependent Fusion of Influenza A

Figure 12.8a: Influenza virus attachment and entry.

Adapted from B. N. Fields, et al. Fields Virology, Fourth Edition. 2 vols. Lippincott Williams & Wilkins, 2001.

Figure 12.8b: Conformational changes in influenza H protein trigger membrane fusion.

Adapted from B. P. Bergstrom, “Protein Sorting: Organelle Biogenesis and Protein Secretion,” BIOL336: Cell Physiology, http://www.muskingum.edu/~brianb/CellPhys/Lect11.

Fusion and Uncoating of Influenza A

Figure 12-9 Adapted from Fields, B. N., et al. Fields Virology, Fourth Edition. Lippincott Williams & Wilkins, 2001

Reye’s Syndrome

• Rare condition• Risk of developing Reye’s Syndrome (R.S.)

increases with aspirin use• R.S. affects all organs of the body– Most harmful to brain and liver– Causes pressure in the brain and massive

accumulation of fat in the liver and other organs

Influenza A RNA segment ORFs

Figure 12-7

Adapted from S. J. Flint, et al. Principles of Virology: Molecular Biology, Pathogenesis, and Control of Animal Viruses, Second Edition. ASM Press, 2003.

Influenza Exploits Host Nuclear-Splicing Machinery to Splice Viral mRNA Segments 7 and 8

Figure 12-11

Translational Control Mechanisms• Influenza A suppresses the interferon system in host cells.