MICR 454L

Emerging and Re-EmergingInfectious Diseases

Lecture 10: Influenza Viruses(Reading: Capturing a Killer Flu Virus)

Dr. Nancy McQueen & Dr. Edith Porter

Overview RNA viruses Influenza viruses

Brief history Nomenclature Morphology and nature of the genome Viral replication cycle Genetic variability Pathogenesis and clinical symptoms Diagnosis Treatment Prevention Threat

Transcription and Replication of RNA Viruses

RNA viruses

Why are so many of the newly emerging infectious diseases caused by RNA viruses? All must bring in their own RNA dependent RNA

polymerases (replicases) Are error-prone - error rate of 10-3- 10-5

Have no proof reading function Many quasi-species found in viral infections

Nitric oxide (NO) production by host accelerates

viral mutations

Rapid evolution

Brief history and epidemiology Influenza appears to have afflicted humans since

ancient times. Hippocrates in 412 BC Numerous epidemics in the middle ages

Epidemics of influenza Every winter > 20,000 deaths/year Elderly or immunocompromised individuals.

Pandemics Irregular 10-50 year intervals. The Spanish flu (1918-1920) - killed 20-40 million people

worldwide The Asian flu (1956-1957) - 60,000 deaths in North America.

Spread of the Asian Influenza pandemic in 1957

Nomenclature

Family Orthomyxoviridae Myxo = mucus - virons bind to sialic acid residues in

mucoproteins

Genus: Influenza Virus• Three groups which share a common structure and mode of

replication, but differ serologically based on M and NP antigens• Type A infect humans and animals; epidemics and pandemics• Type B infects humans only; epidemics• Type C infects humans and pigs; mild disease

Classification of Human Influenza Viruses

Type A or B Geographic source Isolate number Year of isolation Four HA: H0, H1, H2, H3 Two NA: N1, N2

More on the significance of HA and NA will be discussed later on

World Health OrganizationInfluenza Nomenclature

Influenza type

Hemagglutinin subtype

Geographic source

A/Panama/2007/99 (H3N2)

Year of isolation

Isolate number

Neuraminidase subtype

Morphology and nature of the genome

SS (-) RNA genome Segmented genome (8 segments encode

11 proteins) Virions may be spherical or filamentous

Influenza Virus

(RNA + 3 polymerase proteins - PA, PB1, and PB2)

(M2)

(HA)

(NA)

Matrix protein (M1)

medicineworld.org/images/blogs/9-2006/influenza-virus-82101.jpg

(NP)

Replication of Influenza Virus

www.northwestern.edu/.../ pinto2/pinto_1big.jpg

Why do we continue to have Influenza Virus Epidemics?

Genetic variability Influenza virus keeps changing its structure via two different

mechanisms: Antigenic drift - changes in the antigenic determinants of the HA

and NA that accumulate with time. (result in variants of the SAME NA or HA type) Viral RNA polymerase

Error prone No proofreading

Provides a selective advantage Antigenic shift - major changes due to a re-assortment of genes

that occurs when two different influenza viruses infect the same host.

Rapid evolution

Antigenic Shift

Two different viruses infect the same pig and through re-assortment of the gene

segments, a new virus is generated

Human influenza virus Avian influenza virus

New human influenza virus

Genetic variability The 1957 Asian influenza pandemic- antigenic shift

(new HA, NA, and PB1) The 1968 Hong Kong pandemic - antigenic shift

(new HA and PB1) 1918 Spanish influenza pandemic was not due to

shift- new studies indicate that it arose from an avian virus by drift. Enhanced cleavability of HA due to NA changes! Changes in NS1 (and maybe NP and/or M) A single mutation in HA resulted in a virus that had gained

the ability to bind to sialic acid residues present in the human respiratory tract.

Pathogenesis and clinical symptoms

Aerosol transmission 3 day incubation (influenza A) Virus initially infects epithelial cells in the upper

respiratory tract Loss of the ciliated epithelium

Direct effect of virus multiplication and release Due to toxic oxygen radical formation (host cell

response) Due to apoptosis

dsRNA and NA may trigger host cell responses that contribute to apoptosis

PB1-F2 sensitizes cells to apoptosis

Pathogenesis and clinical symptoms

With loss of ciliated epithelium: Loss in the ability of the respiratory tract to clear

viruses or bacteria by mucociliary flow Secondary bacterial infections

Virus replication induces interferons and other cytokines (IL-6, IL-8, TNF-) leading to local and systemic inflammatory responses.

This results in the symptoms that define the “flu” syndrome:

Death

Pathogenesis and clinical symptoms

Fever Headache Chills Malaise Muscle aches As the fever declines

runny nose coughing

Selected virulence characteristics

HA for attachment Inhibition of host mRNA translation (establishing control of the

host) Cap snatching Viral mRNAs compete more effectively for initiation factors. Inactivation of the cap binding reaction by removing the required

phosphate from eIF-4E, reducing available initiation factors NS1 interferes with host cell mRNA splicing, polyadenylation,

and transport to the cytoplasm

Inactivation of eIF-4

Summary of virulence characteristics

Evasion of host defenses NS1 binds to dsRNA to inhibit activation of IFN

Damage Induction of apoptosis - dsRNA, NA, and Pb1-F2 all play a

role NO and O2-

NO enhances development of more quasi-species Induction of cytokines

Strain Dependent Differences in Pathogenesis Strain differences may result in differences in the severity of

the disease for both human and avian viruses. Aquatic birds are the natural reservoir for avian influenza A

viruses Is usually asymptomatic in feral birds Highly pathogenic strains may cause serious systemic infections

in domestic poultry Due to the presence of a polybasic cleavage site in HA (Cleavage of HA at a basic residue by host cell proteases is

required for viral infectivity)

• For human viruses, systemic spread has not been documented. This may be due to:

Strain Dependent Differences in Pathogenesis

Lack, in other organs, of proteases that capable of cleaving the HA

Interferon activity In humans variations in pathogenicity may be due to

Differences in the effectiveness of NS1 to antagonize IFN / production

Differences in NA that allow binding of host proteases that assist in HA cleavage activation or activation of apoptosis

Those most likely to succumb to the disease are usually the elderly and the very young. Why? The 1918 strain was an exception to this rule - it caused more

severe symptoms in those who were the most immunocompetent!

Due to an overdeveloped immune response (“cytokine storm”) of the host against the virus!

Diagnosis

Nasopharyngeal swabs, washes, or aspirates taken early in the course of the disease are the best specimens The virus can be grown in the amniotic or

allantoic cavity of embryonated chicken eggs, or in tissue culture cells with trypsin added to cleave HA.

Diagnosis

• May assay directly for the virus (direct assay)

• May assay for antibodies, produced in the host, against the virus (indirect assay)• Hemagglutination assay-a direct method to identify the

presence of the virus and to get a rough titer of the virus. • Is based on the ability of influenza viruses to agglutinate

RBCs.

• Virus is titered by making serial two-fold dilutions of the virus and determining the highest dilution of virus that causes agglutination of the RBCs.

Hemagglutination assay

Hemagglutination assay

Serological/Immunological Methods

Hemagglutination-Inhibition Assay – an indirect test for antibody against specific influenza virus types -

Serological/Immunological Methods

Immunofluorescence Enzyme immunoassay (EIA) Optical immunoassay

Treatment Amantidine and rimantidine – targets the M2 protein,

blocking the ion channel it forms and preventing uncoating of the virus. Only effective against Group A influenza viruses

Treatment Zanamivar (Relenza) and Oseltamivar

(Tamiflu) – target the neuraminidase, inhibiting its activity and, therefore, inhibiting release of the virus. Effective against both Groups A and B

Prevention

Vaccination – need a new vaccine every year because of shift and drift of the virus Whole inactivated virus - flu shot Live, attenuated cold adapted virus (LAIV or FluMist)

Made by combining the HA and NA genes of the targeted virus strain with the six other gene segments from mutant viruses known to have restricted growth at 370C

Nasal-spray inoculation The reassortment viruses cannot replicate in the lung at core

body temperature, but grow well in the cooler nasal mucosa where they stimulate an excellent immune response.

Attenuated Vaccine Virus To New Virus Type

Virulent Wild Type Influenza

Virus

PB2

HA

PA

PB1

NA

NP

M

NS

Attenuated Influenza

Vaccine Virus

PB2

HA

PA

PB1

NA

NP

M

NS

PB2

HA

PA

PB1

NA

NP

M

NS

Vaccination

In development: Subunit vaccines

Poxvirus recombinants expressing single viral proteins Oligopeptides corresponding to the antigenic

components of the HA protein DNA-based vaccines

Target epitopes that are highly conserved in all influenza A viruses

WINTER - 2007Should we be afraid of the avian (bird) flu?

Starting in 1997, a highly virulent avian form of influenza (H5N1) spread through the commercial poultry farms in Hong Kong.

It has now been found in many sites in Southeast Asia and a number of humans have been infected, with several resulting deaths Due to apoptosis of alveolar epithelial cells and leukocytes? Due to enhanced proinflammatory cytokine response (especially TNF)?

Fortunately, these strains have not yet shown signs of spreading efficiently among humans……….

Avian Influenza The avian viruses do not replicate well in the upper respiratory

tract of humans (33º C) where the body temperature is cooler than the intestinal tract of birds (41º C) where the avian influenza virus normally replicates (may be due to polymerase proteins).

The avian influenza virus HA proteins preferentially recognize

and bind to sialoligosaccharides terminated by N-acetylsialic

acid linked to galactose by an α2,3 linkage. This linkage

is found on the respiratory epithelium of birds while an

α2,6 linkage is found on the respiratory epithelium of

humans. However, drift or shift could change this……..

Nations With Confirmed Cases H5N1 Avian

Influenza (February 2007)

Threats Every year in the United States, on average:

5% to 20% of the population gets the flu; more than 200,000 people are hospitalized from flu

complications about 36,000 people die from flu.

Research suggests that currently circulating strains of H5N1 viruses are becoming more capable of causing disease (pathogenic) in animals than were earlier H5N1 viruses.

Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka Y.Human infection with highly pathogenic H5N1 influenza virus. Lancet. 2008 Apr 26;371(9622):1464-75.

Blendon RJ, Koonin LM, Benson JM, Cetron MS, Pollard WE, Mitchell EW, Weldon KJ, Herrmann MJ.Public response to community mitigation measures for pandemic influenza. Emerg Infect Dis. 2008 May;14(5):778-86.

Song D, Kang B, Lee C, Jung K, Ha G, Kang D, Park S, Park B, Oh J.Transmission of Avian Influenza Virus (H3N2) to Dogs. Emerg Infect Dis. 2008 May;14(5):741-6.

Blumenshine P, Reingold A, Egerter S, Mockenhaupt R, Braveman P, Marks J.Pandemic influenza planning in the United States from a health disparities perspective. Emerg Infect Dis. 2008 May;14(5):709-15.

Take Home Message Influenza virus epidemics and pandemics have occurred

regularly since ancient times. Influenza virus is an enveloped virus that has a SS, - RNA

genome of eight segments Influenza virus epidemics and pandemics continue to occur

because of the genetic variability of the virus NA and HA due to Drift- genetic mutations Shift - genetic reassortment

Influenza infections are characterized by fever, headache, chills, malaise and muscle aches. Secondary bacterial infections are common

Diagnosis is usually by immunological means Treatment may target the HA or NA New vaccines are needed every year

Live, attenuated cold adapted virus made by reassortment (FluMist)

Resources The Microbial Challenge, by Krasner; ASM Press; Washington DC; 2002. Brock Biology of Microorganisms, by Madigan and Martinko, Pearson

Prentice Hall, Upper Saddle River, NJ, 11th ed, 2006. Microbiology: An Introduction, by Tortora, Funke and Case; Pearson

Prentice Hall; 9th ed, 2007. Fundamentals of Molecular Virology, by Nicholas Acheson; Wiley and Sons;

2007 Suzanne L. Epstein, Terrence M. Tumpey, Julia A. Misplon, Chia-Yun Lo,

Lynn A. Cooper, Kanta Subbarao, Mary Renshaw, Suryaprakash Sambhara, and Jacqueline M. Katz 2002. DNA Vaccine Expressing Conserved Influenza Virus Proteins Protective Against H5N1 Challenge Infection in Mice, Emerging Infectious Diseases

http://www.brown.edu/Courses/Bio_160/Projects1999/flu/vaccines.html http://www.hhmi.org/biointeractive/museum/exhibit99/4_6b.html http://www.pandemicflu.gov/ http://www.who.int/csr/disease/avian_influenza/en/ http://www.cdc.gov/flu/avian/outbreaks/current.htm

Viral replication cycle• Attachment

• The ligand on influenza virus is the hemagglutinin (HA) glycoprotein spike protein which is composed of three monomers to make a trimer. • Each monomer is composed of the 2 peptide subunits, HA1 and HA2,

• HA is synthesized as a precursor, HA0

• During transport of HA to the cell surface, HA0 is cleaved into HA1 and HA2 that remain associated with each other through disulfide bonding• HA1 has a globular head that contains a conserved region that binds to the N-

acetyl neuraminic acid (sialic acid) cellular receptor

• HA2 spans the envelope and contains a region at its amino terminus called the fusion peptide that is released upon cleavage of HA0 into HA1 and HA2• The fusion peptide functions to mediate fusion of the envelope of the virus

with a host cell membrane during viral entry.

• The fusion peptides of the three monomers are buried in the structure of the trimer and not available to mediate fusion until there is a pH dependent conformational change in the protein.

The Glycoprotein Spike HA Protein of Influenza Virus

HA trimers

HA1 HA2

Cleavage site

Fusion peptide

Conformational change in HA at low pH

Penetration and Uncoating The virus HA binds to its sialic acid containing receptor The virus enters the host cell through receptor mediated

endocytosis (penetration)• When the pH in the endosome decreases, there is a conformational

change in the HA of influenza which exposes the fusion peptides that were previously hidden within the trimer.

• The peptides mediate fusion of the viral envelope with the endosomal envelope.

• During acidification of the endosome, the M2 protein, which functions as an ion channel, allows H+ to penetrate the interior of the virion.

• The low pH within the virion weakens the interaction of the matrix protein, M1, with the nucleocapsids (RNP), facilitating their release into the cytoplasm upon membrane fusion (uncoating).

• The nucleocapsid (RNP) is transported into the nucleus via nuclear localization signals on the nucleoprotein and polymerase proteins

Fusion after receptor mediated endocytosis

pH dependent fusion

Biosynthesis

Unlike most other RNA-containing viruses, influenza viruses replicate in the nucleus: The nucleocapsids are first transported to the nucleus Viral mRNAs are sent to the cytoplasm for translation Viral polymerase and nucleocapsid proteins are

subsequently sent back to the nucleus to direct genome replication and form nucleocapsids

Nucleocapsids are transported back to the cytoplasm for assembly

Why does influenza replicate in the nucleus?

Biosynthesis

• In addition to viral enzyme activities, for transcription influenza A virus requires host cell enzymatic activities that are found in the nucleus• M1 and NS2 proteins mediate movement in and out of the

nucleus.

• The virus requires host cell RNA polymerase II which is responsible for making host cell mRNAs – WHY?

• The viral transcription complex for mRNA synthesis is composed of PB1, PB2 and PA.

• PB2 recognizes the 5’ caps of host cell mRNAs and in conjunction with the viral RNA and PB1, cleaves off the 5’ cap plus 10-13 bases to use as a primer for + strand mRNA synthesis on the negative strand genomic template (cap snatching).

Viral transcription• PB1 initiates the transcription and, with

PA, extends the primer. • Poly A tails are added at the 3’ ends.

Genome replication• PB1 and PA are involved in replication of the

genome. • Details of replication are unknown, but multiple

copies of newly synthesized NP proteins and PA are required. The presence of NP allows for de novo synthesis of RNA (i.e., no primer is needed).

Assembly

How does the virus with its segmented genome ensure that virions contain a copy of each segment? For influenza virus the ratio of virus particles to actual infectious

units is comparable to the ratio predicted for random packaging. However recent evidence suggests that during budding, viral

proteins recognize and interact with specific RNA sequences in each of the eight nucleocapsids.

They then incorporate them, one by one, into bundles that are packaged into virions during budding

Release

How do influenza viruses exit their host cells? Their envelopes are derived from the host cell plasma

membrane that has been modified by the insertion of viral HA, NA, and M2 proteins

Maturation and release via the process of budding (exocytosis) involves 4 steps Synthesis and insertion of viral glycoproteins in host cell plasma

membranes Assembly of the viral nucleocapsid The nucleocapsid and the modified membrane are brought

together (the C terminal domain of the envelope protein interacts via the matrix (M1) protein with the nucleocapsids)

Exocytosis or budding which may or may not kill the host cell

Budding

Influenza virus budding

Neuraminidase function during budding

• The neuraminidase is believed to function in preventing the virus from sticking to the host cell sialic acid residues or to other viruses containing sialic acid residues during exit of the virus from the host cell.