MICR 454L Emerging and Re-Emerging Infectious Diseases Lecture 10: Influenza Viruses (Reading: Capturing a Killer Flu Virus) Dr. Nancy McQueen & Dr. Edith Porter
Jan 14, 2016
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.