MICR 420

Emerging and Re-EmergingInfectious Diseases

Lecture 8: Influenza VirusesDr. 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

What a virus? Not a living cell Acellular particle

No cell membrane Consist of nucleic acid and protein only

RNA or DNA, not both Protein coat : capsid Some are enveloped: membrane from host cell

Infects living host cells to replicate All forms of cells can be infected by a virus

Bacteria, archaea, eukaryotes Virus depends on host metabolism Viral genome subverts host cell’s machinery to

reproduce Estimate of 1032 viruses on earth

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

What is influenza? “Influenza (the flu) is a contagious respiratory

illness caused by influenza viruses. It can cause mild to severe illness, and at times can lead to death.” (http://www.cdc.gov/flu/about/disease/index.htm)

Figure 1. Gross and microscopic lesions from dog infected with highly pathogenic avian influenza (HPAI) H5N1. A) Severe congestion and edema in the lung. B) Lung histopathologic results showing severe pulmonary edema and hemorrhage with black-brown particles (hemosiderin) (magnification ×100). (cdc web site).

Normal LungInfluenza Lung

http://www.biologyofhumanaging.com/slides/lng120d1.jpg

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

Replication of Influenza Virus

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. Recombination by template switching

Between homologous segments of two different strains More common in avian isolates

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

How do we acquire viruses from pigs?

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 made it a potent inducer of pro-inflammatory

responses 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.

Changes in PB1 enhanced viral replication PB1-F2 - promotes apoptosis

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 (not produced by all strains)

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!

Cytokine storm

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

www.medicine.mcgill.ca/.../hemag_plate.jpg

Serological/Immunological Methods

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

Hemagglutination Inhibition

www.cdc.gov/.../16/2/images/09-1733-Ft.gif

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 by 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 Due to pneumonia that progresses to ARDS and multi-organ failure

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

Avian influenza

Direct bird to human transmission is also possible.

Pandemic disease cycle

Domestic birds

Natural avian influenza cycle

Water fowl Mammals (primarily swine)

Shore birds

Cycle of Avian Influenza Viruses in Animals & Humans

No human-human spread Avian HA proteins preferentially recognize and bind to

sialoligosaccharides terminated by N-acetylsialic acid linked to galactose by an α2,3 linkage. found on the respiratory epithelium of birds found on lower respiratory tract of humans along with α2,6

linkage only α2,6 linkage is found on the upper respiratory epithelium of

humans Pneumonia more common

Shift of drift could change this…….

Attachment of influenza viruses

Genes involved in H5N1 pathogenesis

Avian Influenza pathogenesis

Changes in polymerase allow for replication at lower temperatures of humans The avian viruses normally do not replicate well in the

upper respiratory tract of humans (33º C); normally replicate in intestinal tract of birds (41º C)

PB1-F2 causes apoptosis in macrophages which delays immune response; also enhances pro-inflammatory response

NS1 is a potent inducer of pro-inflammatory cytokines (TNF) through PDZ domain

HA binds to sialic acid residues with α2,3 linkage

Nations With Confirmed Cases H5N1 Avian

Influenza (February 2007)

Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO

6 May 2010

2003 2004 2005 2006 2007 2008 2009 2010 Total Country

cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths

Azerbaijan 0 0 0 0 0 0 8 5 0 0 0 0 0 0 0 0 8 5

Bangladesh 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0

Cambodia 0 0 0 0 4 4 2 2 1 1 1 0 1 0 1 1 10 8

China 1 1 0 0 8 5 13 8 5 3 4 4 7 4 0 0 38 25

Djibouti 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0

Egypt 0 0 0 0 0 0 18 10 25 9 8 4 39 4 19 7 109 34

Indonesia 0 0 0 0 20 13 55 45 42 37 24 20 21 19 3 2 165 136

Iraq 0 0 0 0 0 0 3 2 0 0 0 0 0 0 0 0 3 2

Lao People's Democratic Republic

0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 0 2 2

Myanmar 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0

Nigeria 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1

Pakistan 0 0 0 0 0 0 0 0 3 1 0 0 0 0 0 0 3 1

Thailand 0 0 17 12 5 2 3 3 0 0 0 0 0 0 0 0 25 17

Turkey 0 0 0 0 0 0 12 4 0 0 0 0 0 0 0 0 12 4

Viet Nam 3 3 29 20 61 19 0 0 8 5 6 5 5 5 7 2 119 59

Total 4 4 46 32 98 43 115 79 88 59 44 33 73 32 30 12 498 294

Total number of cases includes number of deaths. WHO reports only laboratory-confirmed cases. All dates refer to onset of illness. Indonesia numbers indicate cumulative total of sporadic cases and deaths which occurred during 2009.

Amantadine (Brand name Symmetrel: Treatment of influenza type A-2, but not type B). This drug cannot treat the cytokine storm associated with avian influenza

The pandemic H1N1 influenza virus

In 2009 a new H1N1 strain was passed from pigs to humans

The virus rapidly dispersed worldwide in a few months

Ultimately found to not be more virulent in humans than the seasonal strains

No genetic markers for severe disease detected

HA - has only 1 arginine at the HA cleavage site - no enhanced cleavability

PB1 - gene encodes a truncated, and most probably non-functional PB1-F2 protein

PB2 lacks mutation in 627 and 701 that contribute to better replication in mammalian cells

NS1 - has deletion of 11 carboxy-terminal encoding the PDZ domain

H1N1 Pandemic

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 Recombination by template switching

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, et al. 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 Arias, Carlos F, et al. 2009. Molecular anatomy of 2009 influenza virus A (H1N1),

Archives of Medical research 40:643-654. Thanh, Tran T., et al. (2008) Human H5N1 influenza: Current insight into

pathogenesis. The Int’l J. of Biochem. and Cell Biol 40:3671-2674. Kuiken, Thijs and Taubenberger, Jeffery K. (2008) Pathology of Human Influenza

revisited. Vaccine 26S:D59-D66. Basler, Christopher and Aguilar, Patricia V. (2008) Progress in identifying virulence

determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses. Antiviral Research 79:166-178.