Download MICR 454L Lec10 2008Influenza - Cal State LA

MICR 454L
Emerging and Re-Emerging
Infectious Diseases
Lecture 10: Influenza Viruses
(Reading: Capturing a Killer Flu Virus)
Dr. Nancy McQueen & Dr. Edith Porter
Overview
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RNA viruses
Influenza viruses
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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
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Why are so many of the newly emerging
infectious diseases caused by RNA viruses?
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All must bring in their own RNA dependent RNA
polymerases (replicases)
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Are error-prone - error rate of 10-3- 10-5
Have no proof reading function
Many quasi-species found in viral infections
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Nitric oxide (NO) production by host accelerates
viral mutations
Rapid
evolution
Brief history and epidemiology
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Influenza appears to have afflicted humans since
ancient times.
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Epidemics of influenza
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Hippocrates in 412 BC
Numerous epidemics in the middle ages
Every winter
> 20,000 deaths/year
Elderly or immunocompromised individuals.
Pandemics
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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
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Family Orthomyxoviridae
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Myxo = mucus - virons bind to sialic acid residues in
mucoproteins
Genus: Influenza Virus
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Three groups which share a common structure and mode of
replication, but differ serologically based on M and NP antigens
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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
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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 Organization
Influenza Nomenclature
Hemagglutinin subtype
Influenza
type
Year of isolation
A/Panama/2007/99 (H3N2)
Geographic source
Isolate
number
Neuraminidase subtype
Morphology and nature of the
genome
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SS (-) RNA genome
Segmented genome (8 segments encode
11 proteins)
Virions may be spherical or filamentous
Influenza Virus
(M2)
Matrix protein
(M1)
(HA)
(RNA + 3 polymerase
proteins - PA, PB1, and
PB2)
(NP)
(NA)
medicineworld.org/images/blogs/9-2006/influenza-virus-82101.jpg
Replication of Influenza Virus
www.northwestern.edu/.../ pinto2/pinto_1big.jpg
Why do we continue to have
Influenza Virus Epidemics?
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Genetic variability
Influenza virus keeps changing its structure via two different
mechanisms:
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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)
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Viral RNA polymerase
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Error prone
No proofreading
Rapid evolution
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.
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
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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.
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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
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Aerosol transmission
3 day incubation (influenza A)
Virus initially infects epithelial cells in the upper
respiratory tract
Loss of the ciliated epithelium
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Direct effect of virus multiplication and release
Due to toxic oxygen radical formation (host cell
response)
Due to apoptosis
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dsRNA and NA may trigger host cell responses that contribute
to apoptosis
PB1-F2 sensitizes cells to apoptosis
Pathogenesis and clinical
symptoms
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With loss of ciliated epithelium:
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Loss in the ability of the respiratory tract to clear
viruses or bacteria by mucociliary flow
Death
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:
Pathogenesis and clinical
symptoms
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Fever
Headache
Chills
Malaise
Muscle aches
As the fever declines
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runny nose
coughing
Selected virulence
characteristics
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HA for attachment
Inhibition of host mRNA translation (establishing control of the
host)
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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
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Evasion of host defenses
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NS1 binds to dsRNA to inhibit activation of IFN
Damage
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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
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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
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Is usually asymptomatic in feral birds
Highly pathogenic strains may cause serious systemic infections
in domestic poultry
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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
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In humans variations in pathogenicity may be due to
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Lack, in other organs, of proteases that capable of cleaving
the HA
Interferon activity
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?
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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
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Nasopharyngeal swabs, washes, or aspirates
taken early in the course of the disease are
the best specimens
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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
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May assay directly for the virus (direct assay)
May assay for antibodies, produced in the host,
against the virus (indirect assay)
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Hemagglutination assay-a direct method to identify
the presence of the virus and to get a rough titer of the
virus.
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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
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Hemagglutination-Inhibition Assay – an indirect
test for antibody against specific influenza virus
types -
Serological/Immunological
Methods
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Immunofluorescence
Enzyme immunoassay (EIA)
Optical immunoassay
Treatment
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Amantidine and rimantidine – targets the M2 protein,
blocking the ion channel it forms and preventing
uncoating of the virus.
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Only effective against Group A influenza viruses
Treatment
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Zanamivar (Relenza) and Oseltamivar
(Tamiflu) – target the neuraminidase,
inhibiting its activity and, therefore, inhibiting
release of the virus.
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Effective against both Groups A and B
Prevention
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Vaccination – need a new vaccine every year because of shift
and drift of the virus
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Whole inactivated virus - flu shot
Live, attenuated cold adapted virus (LAIV or FluMist)
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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.
PB2
PB2
PB1
PB1
PA
PA
HA
HA
NA
NA
NP
PB2
NP
M
PB1
M
NS
PA
NS
HA
Attenuated
Influenza
Vaccine Virus
NA
NP
M
NS
Attenuated
Vaccine Virus To
New Virus Type
Virulent Wild
Type Influenza
Virus
Vaccination
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In development:
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Subunit vaccines
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Poxvirus recombinants expressing single viral proteins
Oligopeptides corresponding to the antigenic
components of the HA protein
DNA-based vaccines
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Target epitopes that are highly conserved in all
influenza A viruses
WINTER - 2007
Should we be afraid of the avian (bird) flu?
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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
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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
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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
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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
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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
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Live, attenuated cold adapted virus made by reassortment (FluMist)
Resources
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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
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Attachment
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The ligand on influenza virus is the hemagglutinin (HA) glycoprotein
spike protein which is composed of three monomers to make a trimer.
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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
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HA1 has a globular head that contains a conserved region that binds to the Nacetyl 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
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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
Cleavage site
HA1
HA2
Fusion peptide
HA trimers
Conformational change in HA
at low pH
Penetration and Uncoating
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The virus HA binds to its sialic acid containing receptor
The virus enters the host cell through receptor mediated
endocytosis (penetration)
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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
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Unlike most other RNA-containing viruses, influenza
viruses replicate in the nucleus:
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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?
•
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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
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How does the virus with its segmented genome ensure that
virions contain a copy of each segment?
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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
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How do influenza viruses exit their host cells?
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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
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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.