Orthomyxoviruses Flashcards
What are orthomyxoviruses?
Viruses with a segmented, (–) strand RNA genome.
They are responsible for the highly contagious, acute respiratory illness known as influenza, or simply, the “flu.”
How many types of influenza virus are there?
There are four types of influenza virus; A, B, C and D.
Which influenza type causes the seasonal epidemics of disease?
Influenza A and B viruses cause most of the seasonal epidemics of disease.
Which influenza virus can cause flu pandemics?
Influenza A is the only influenza virus known to cause flu pandemics.
What does influenza C cause in humans?
Influenza C causes a mild illness in humans.
What does Influenza D cause in humans?
Influenza D infects cattle and is not thought to infect humans.
How are influenza viruses typically transmitted?
Influenza viruses are typically transmitted from one person to the next by the respiratory route.
Describe the Influenza A virus particle.
The virus particle is highly pleomorphic (i.e., many shapes), most commonly spherical or ovoid in shape, and approximately 80–120 nm in diameter.
The virus has a lipid bilayer envelope with viral glycoproteins embedded in the membrane.
The glycoproteins are often called spike proteins because they protrude from the envelope
What are the two types of glycoproteins in the Influenza A virus’ envelope?
Hemagglutinin (H) and neuraminidase (N).
What is hemagglutinin?
A trimer of identical subunits.
Hemagglutinin is the anti-receptor that attaches to the host cell.
The virus attaches to the sialic acid found on host cell glycoprotein to begin the replication cycle.
Originally named because of the ability of the virus to agglutinate red blood cells.
What is neuraminidase?
Neuraminidase is a tetrameter of identical subunits that the virus uses to detach from the host cell by digesting the sialic acid after the replication cycle is completed.
Neuraminidase is important for release from the host cell in egress.
How many glycoproteins are in the lipid envelope of the influenza A virus, and what is the proportionality between H and N glycoproteins?
There are about 500 of these glycoproteins in the lipid envelope of the influenza A virus.
There are approximately 5 H glycoproteins to every N glycoprotein.
In addition to hemagglutinin and neuraminidinase, what are two other important envelope proteins for the influenza A virus?
The M2 and M1 proteins.
What is M2?
M2 is an ion channel that allows protons (H+) to enter into the interior of the virus particle.
The acidification process is important in the releasing of the virus’ genome from the M1 protein at the early stages of the replication cycle.
What do the M1 envelope proteins do?
The M1 proteins lines the inside of the lipid envelope and serves to allow the nucleocapsid, the envelope and the glycoproteins to assemble correctly during the late stages of the replication cycle.
What makes up the Influenza A genome?
10 genes distributed on 8 pieces of (–) sense RNA molecules.
Each segment is coatec with nucleoprotein and has its own RNA polymerase complex associated with it (PA, PB1, and PB2).
Six of the segments code for only one protein, and each of the other two segments code for two proteins.
Each segment is replicated and transcribed independently
Note that the segments 1, 2 and 3 are approximately the same size, segments 4, 5 and 6 are approximately the same size, and segments 7 and 8 are approximately the same size.
Which segments are approximately the same size in the Influenza A genome?
Segments 1, 2 and 3 are approximately the same size, segments 4, 5 and 6 are approximately the same size, and segments 7 and 8 are approximately the same size.
What is a RiboNucleoProtein?
The segments of the Influenza A genomes are called RiboNucleoProteins (RNPs) and consist of the (–) RNA, the nucleoprotein (NP) and an RNA-dependent RNA polymerase complex (PA, PB1, PB2).
One copy of NP binds to approximately 20 nucleotides in the RNA strand, thus there are multiple copies of this structural protein
How are Influenza A viruses classified?
By subtypes based on the properties of their H and N surface proteins.
There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1 through H18 and N1 through N11, respectively).
While there are potentially 198 different influenza A subtype combinations, only 131 subtypes have been detected in nature.
Which subtypes of influenza A viruses routinely circulate in people?
A(H1N1) and A(H3N2).
How many different H and N subtypes are there?
There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes
(H1 through H18 and N1 through N11, respectively).
How is the nomenclature to describe isolates of influenza virus strains of human origin expressed?
In this order:
- virus type,
- geographic site where it was first isolated,
- strain number,
- year of isolation,
- virus subtype (for type A viruses only)
For example, seasonal influenza A (H3N2), A/Perth/16/2019
For influenza strains of non-human origin, how is the nomenclature to describe the virus expressed?
In this order:
- virus type,
- species of host,
- geographic site where it was first isolated,
- strain number,
- year of isolation,
- virus subtype (for type A viruses only)
For example, avian influenza A (H1N1), A/duck/Alberta/35/76.
Discuss possible resevoirs of Influenza A viruses.
All known influenza A subtypes exist in the wild bird reservoir and are associated with interspecies transfer and the emergence of new influenza strains with pandemic potential.
The link between humans and pigs is significant in the development and transmission of new flu virus strains.
What will Influenza A infect?
Humans, animals and birds.
Pigs and birds are thought to be particularly important reservoirs for influenza A.
What is the host cell receptor for influenza virus?
The N-acetylneuraminic acid (also called sialic acid) that residues on mucoproteins.
Where is sialic acid found?
Sialic acid is very common on cell surfaces and in mucus, common of cells in the respiratory tract
Where is the natural environment for the influenza virus for mammals, and why?
Upper respiratory tract.
This is because the virus is dependent on a protease in respiratory secretions to activate the H protein so that the virus can release its genome into the cell.
How does the influenza virus infect birds?
Influenza viruses typically infect the gastrointestinal tract of domestic and wild birds.
The influenza RNA dependent RNA polymerase is a complex of two proteins named PB1 and PB2.
True or false?
False.
The influenza genome contains eight segments of negative sense RNA.
True or false?
True.
The influenza virus is an enveloped virus.
True or false?
True.
Influenza (and all orthomyxoviruses) have (+) RNA genomes.
True or false?
False.
The influenza virus particle contains RNA-dependent RNA polymerase.
True or false?
True
The matrix protein sticks out the furthest from the envelope bilayer.
True or false?
False.
A genome segments is called an RiboNucleoProteins (RNPs) and consists of the (–) RNA, the nucleoprotein (NP) and an RNA-dependent RNA polymerase complex.
True or false?
True.
An influenza genome can have missing segments if one of the other segments is duplicated.
True or false?
False.
Some of the influenza RNA genome segments are positive sense, whereas other segments are negative sense.
True or false?
False.
There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes.
True or false?
True.
The current subtypes of influenza A viruses that routinely circulate in people include A(H5N1) and A(H3N2).
True or false?
False.
A virus with the influenza nomenclature “A/Perth/16/2019” would have been the 16th strain isolated.
True or false?
True
The host cell receptor for influenza virus is the N-acetylneuraminic acid (also called sialic acid).
True or false?
True.
Sialic acid is found on many proteins in the human body.
True or false?
True.
In mammals, influenza A preferentially infects cells in the respiratory tract.
True or false?
True.
Pigs can be a reservoir of influenza A viruses for humans.
True or false?
True.
Where do influenza viruses replicate their genomes?
Influenza viruses are (–) sense RNA viruses that replicate their genomes in the host cell nucleus.
Assembly of the virus particle starts with what?
What completes it?
Assembly of the RNPs in the nucleus, and is completed in the cytoplasm near the plasma membrane of the cell.
How fast is the replication cycle of the influenza virus?
The replication cycle of influenza viruses, from the time of entry to the production of new virus, is very quick.
The release of the first influenza viruses from infected cells occurs after only 6 hours.
What is the affinity of the virus particle with sialic acid on mucoproteins of the epithelial cells?
Probably of low affinity, but there is high avidity because of multiple low affinity interactions.
How does the influenza virus enter the epithelial cell?
By receptor-mediated endocytosis
What happens prior to the endocytosis of the Influenza virus particle?
The virus’ hemagglutinin proteins (H, H0) are modified by cleavage by the protease “tryptase Clara.”
The Clara cells of the bronchiolar epithelium secrete this protease into the mucus layer of the respiratory tract.
The cleavage of H0into H1and H2is necessary for the full infectivity of the influenza virus.
What is needed for release of the fusion peptide?
This H protein has been cleaved by tryptase Clara to yield H1(HA1 in the diagram) and H2 (HA2 in the diagram).
The N-terminal region of the H2peptide contains the fusion peptide needed to allow the viral envelope to fuse with the endosome membrane.
What is the ‘fusion peptide’ and why is it important?
The N-terminal portion of H2 has an area called the “fusion peptide” which is critical for the subsequent fusion events of the endosome membrane and the virus’ envelope.
After the H protein has been cleaved by tryptase Clara, what happens to the resultant proteins?
H1 binds to the SA on the mucoprotein and the H2 remains integrated in the bilayer of the envelope.
What happens after endocytosis of an Influenza A particle into an epithelial cell?
The endosome is acidified, resulting in conformational change in the H protein that allows the fusion peptide to be inserted into the endosome membrane.
The fusion of the two lipid bilayer structures of the endosome membrane and virus envelope is required for the release of the virus’ genome into the cytoplasm of the cell
In addition to the endosome acidification, how else is the virus particle acidified?
Why is this necessary?
The interior of the virus particle is also acidified by the movement of protons through the M2 channel in the envelope.
This acidification is necessary for the release of the RNPs from the M1 protein lining the inside of the virus envelope.
The released RNPs are transported into the nucleus.
What happens once the Influenza A genome has been released into the cytoplasm?
The intact RNPs enter the nucleus through the nuclear pore.
NPs have a special targeting sequence that gets the genome into the nucleus.
NPs also protect the genome from any RNA nucleases.
The transport of RNP into the nucleus does not seem to involve the cell’s cytoskeleton structures.
Where does the synthesis of influenza virus mRNA?
The synthesis of virus mRNA occurs in the nucleus of the cell.
How is the influenza virus transcribed?
The (–) strand of RNA is used as a template by the viral RNA polymerase complex to synthesize viral mRNA.
Each of the 8 RNA segments of the influenza genome is transcribed independently.
RNA splicing of the mRNA of two segments yields two mRNA products (for each segment)
Where does the influenza RNA polymerase complex anchor for transcription?
The influenza RNA polymerase complex anchors the 5’ end of the genomic (–) strand RNA.
What is ‘cap-stealing’?
The RNA polymerase binds to a host cell mRNA molecule near the 5’ end and uses its endonuclease to cleave the 5’ cap with about 10 – 13 nucleotides from the mRNA (the rest of the host cell mRNA is released).
This process is called “cap-stealing ”
The influenza RNA polymerase complex requires a primer for the synthesis of viral mRNA.
True or false?
True
The influenza virus polymerase complex binds to the cells mRNA can cleaves the RNA 5’ Cap to use as a primer for the synthesis of its mRNA.
Why is the viral mRNA often called ‘incomplete’?
The entire sequence of the genomic (–) RNA is not copied into mRNA.
For this reason, the mRNA is often called the “incomplete” (+) strand of RNA in the virus’ replication cycle.
The mRNA is incomplete because of the “panhandle” structure that is formed when the complex anchors to the 5’ end of the viral (–) strand RNA.
This results in the last 15–22 nucleotides of the genomic (–) RNA not being copied into the mRNA.
What does the influenza RNA polymerase use as a primer for the synthesis of viral mRNA?
Uses a fragment of host cell mRNA as a primer for the synthesis of the viral mRNA.
How does the viral RNA polymerase complex complete the synthesis of the (+) strand of RNA?
By using the (–) strand as the template and adding the complementary nucleotides to the cap/host cell derived nucleotides
Describe the synthesis of Influenza mRNAs.
RNA dependent RNA polymerase complex initiates transcription by binding to the leader sequence in 3’ of the genomic negative strand RNA.
The RdRp transcribes a 5’ triphosphate-leader RNA, then stop and restart on a transcription initiation signal.
The RNA initiated on this signal is capped.
At the end of viral genes is a transcription stop signal on which the RdRp will produce a polyadenylation signal by stuttering on a U stretch before releasing the mRNA.
How does the RDRP add a polyA tail to viral mRNA?
Near the 5’ end of the (–) RNA template, there is a polyadenylation sequence that is read multiple times by the RNA polymerase (i.e.,the polymerase stutters at this sequence), resulting in the addition of a poly(A) tail.
Describe polyadenylation of the influenza mRNA.
The stop signal present at the end of each gene comprises a stretch of U on which the viral polymerase acquires a stuttering behaviour: after each Adenine (A) inserted, it moves back one nucleotide along with the mRNA.
It resumes transcription adding a new A, then again moves back.
In this way a U on the genomic template is copied hundreds of times at the end of viral mRNA thereby producing a polyA tail.
Eventually the polymerase will release the polyAdenylated mRNA and stop transcription.
What are two distinct advantages with the cap-stealing strategy to synthesize viral mRNA?
- the mRNA that is produced has many features of a cellular mRNA (5’ methylated cap, poly(A) tail) that enable it to bind to and be translated by the cell’s ribosomes,
- the host cell mRNA is degraded and competition for the ribosomes has been eliminated.
Where are the viral mRNAs translated?
The viral mRNAs are transported out of the nucleus to the cytoplasm for translation by the ribosomes.
The viral mRNAs that specify viral envelope proteins are translated by the ribosomes bound to the ER whereas all other mRNAs are translated by cytoplasmic ribosomes.
What is the function of newly synthesized PA, PB1, PB2, and NP proteins?
Newly synthesized PA, PB1, PB2 and NP proteins are transported back to the nucleus where they catalyze the synthesis of the genomes for the progeny virus.
Initially they catalyze the synthesis of full-size (+) strand RNAs (anti-genomic RNA, used as a template for making genomic (–) RNA), then the synthesis of (–) strand RNAs (genomic RNA, used as the nucleic acid for progeny viruses).
Both strands are synthesized in the forms of nucleocapsids
What happens after the viral genome has been transcribed and the mRNA has been translated?
Several proteins return to the nucleus to initiate the replication of the genome and the assembly of the virus particle.
Describe the replication of the viral Influenza A genome.
This round of RNA synthesis is for the purpose of creating a (+) strand of RNA (i.e., antigenome) that will be used as a template for the synthesis of (–) strand RNA for the virus progeny.
This process would use the RNA polymerase complex to copy the (–) strand into a (+) strand, but the process of replicating the genome does not require a primer and does not involve cap-stealing.
Each of the eight segments of the genome is replicated independently.
The switch to the production of full-sized (+) RNA may be regulated by the amount of “free” NP protein molecules present in the cell.
The genome/antigenome ratio is about 10.
Describe replication of the viral genome again!
>:)
The RNA polymerase complex binds to the leader sequence on the encapsidated (–) RNA genome, and starts replication.
This process is unprimed.
The (+) RNA (antigenome) is also coated with NP during replication.
The RNA polymerase complex ignores all transcription signals when in replication mode.
The (+) RNA is then replicated under the same process, the RNA polymerase complex binding first to the trailer sequence.
What results in the cessation of viral mRNA synthesis and induces the export of the progeny nucleocapsids into the cytoplasm?
Some of the newly made (–) strand RNA can be used as a template for the synthesis of additional viral mRNA.
The binding of M1 to the newly synthesized (–) strand RNA.
What drives the formation of the ‘bud’ at the plasma membrane?
The H and N proteins (synthesized by ER ribosomes) are transported to the cell surface and become incorporated into the plasma membrane of the cell.
It is the H and N proteins that drives the formation of the “bud” at the plasma membrane.
The matrix protein M1 associates with the cytoplasmic tails of the H and N proteins.
In turn, the M1 protein recruits the virion RNPs and the second matrix protein M2 to this site of virus assembly.
Briefly describe the budding of the Influenza virus particle at the plasma membrane.
The virus particle assembles at the plasma membrane.
The virus envelope proteins attract other proteins which in turn attract the virus RNPs.
Describe the two models for the packagin of the segmented genome of influenza.
The random model implies that the virus acquires a complete genome purely through chance, as there is no mechanism to distinguish between the different genome segments.
The specific packaging model suggests a mechanism ensures that only one copy of each different segment is specifically selected during viral assembly.
There is evidence to support both models, so the aspects of genome packaging are still unclear.
What is the random model?
The random model suggests that during packaging, more than eight RNPs are packaged to ensure that some virions contain a complete genome (i.e.,at least one copy of each segment). in a segment non-specific manner such that a reasonable proportion of virions contain at least one copy of each segment.
What is the specific model?
The specific model proposes that unique segment-specific packaging signals operate to form a defined array of eight RNPs containing one copy of each segment.
Describe influenza virus egress.
The new virus then buds from the plasma membrane of infected cells.
Host cell membrane proteins appear to be excluded from virion particles.
Once the particle has budded from the plasma membrane, the M2 protein promotes membrane fission resulting in the detachment or release of the virus bud.
The neuraminidase (N) removes the sialic acid residues so that the virus particle can be freed from the cell.
The removal of the sialic acid ensures that the virus particle won’t re-infect the same cell.
What removes the sialic acid residues so that the influenza virus particle can be freed from the cell?
What does this ensure?
The neuraminidase (N) removes the sialic acid residues so that the virus particle can be freed from the cell.
The removal of the sialic acid ensures that the virus particle won’t re-infect the same cell.
Some influenza viral mRNAs need to be spliced.
True or false?
True.
(these mRNAs initially contain the genetic information of two proteins).
What must happen for the influenza virus to be infective?
The host cell receptor for Influenza is sialic acid (SA) found on mucoproteins,.
H binds to SA, but in order for the virus to be infective, H must be cleaved by tryptase Clara into H1 and H2.
What will reveal the fusion peptide?
The virus enters the host cell by receptor-mediated endocytosis and fuses with an endosome.
The low pH of the endosome causes the H2 to change conformation to reveal the fusion peptide.
How are the RNPs released into the host cell?
The envelope fuses with the endosomal membrane.
The RNPs are released into the host cell cytoplasm because the interior of the virus particle was acidified as well.
Antigenomic and genomic RNA are made by primer-dependent RNA synthesis.
True or false?
False!
Antigenomic and genomic RNA are made by primer-independent RNA synthesis.
What causes membrane fusion in virus budding?
M2 causes membrane fission and N cleaves the sialic acid to release the new virus particle.
What has been the worst pandemic in recorded history?
Throughout recorded history, influenza has caused several pandemics, the worst being in 1918 – 1919 in which 50 – 100 million people died.
Influenza is commonly called the flu; it’s the same stomach flu that causes diarrhea and vomiting.
True or false?
False.
In humans, influenza is a viral infection that attacks the respiratory system: the nose, throat and lungs.
Influenza is commonly called the flu, but it’s not the same as stomach “flu” viruses that cause diarrhoea and vomiting.
Describe transmission of influenza viruses.
Influenza viruses are usually transmitted from one person to another by the aerosol route (i.e., from droplets formed during coughing or sneezing) but it can also be spread by direct contact.
Which enzyme is the Influenza virus dependent on and why?
The Influenza virus is dependent on an enzyme (tryptase Clara) found in the upper respiratory tract for cleavage of the envelope hemagglutinin protein (H); a step that is necessary for the release of the virus’ genome into the host cell cytoplasm.
As this enzyme is not found elsewhere in the body, Influenza virus infections are usually limited to the respiratory tract.
Where does influenza virus replicate?
In the epithelial cells of both the upper and lower respiratory tract, and causes an acute respiratory illness that lasts about 3 – 7 days.
What is responsible for many of the symptoms associated with upper respiratory infections such as fever, chills, muscle aches, headaches and fatigue?
The virus replication and the immune response to infection result in the destruction of the cells lining the respiratory tract.
The epithelial damage and the cytokines produced during an immune response to the virus are responsible for many of the symptoms associated with upper respiratory infections.
As the viral infection subsides, the epithelium slowly regenerates.
True or false?
True.
Most healthy individuals recover fully after a few days, but complications and death are common in certain high-risk groups. Name the groups.
- Very young children,
- elderly persons,
- persons with underlying chronic health problems such as cardiac and pulmonary diseases,
- individuals with immunosuppressive disorders.
What are common complications of influenza infection?
What is the most severe?
Sinus infections and bronchitis are common.
Pneumonia is among the more severe complications of influenza infection, an event most frequently observed in children or adults.
The pneumonia can be caused directly by the influenza virus (especially if it infects the alveolar epithelia), or it can be the result of a secondary infection caused by bacteria of the microbiota.
How long does an infected person transmit influenza?
Influenza replicates throughout the respiratory tract (the replication cycle is about 6 hours) and is shed from the infected person in high amounts about 48 hours after infection.
An infected person can transmit influenza virus before they are symptomatic and for about 5 – 7 days after infection.
What do new influenza strains change?
Every few years a major epidemic occurs where many people become ill with influenza.
This is because there is a strain of influenza that is unfamiliar in the human population and few (if any) people have previous immunity to it.
These new influenza strains have changed the genes encoding the hemagglutinin (H) and/or neuraminidase (N) glycoproteins.
How do H and N glycoproteins change?
H and N glycoproteins can change in two ways: antigenic drift or antigenic shift
What is antigenic drift?
These are minor changes in the H and/or N glycoproteins due to the accumulation of changes in the amino acid sequence and result in minor antigenic differences.
These changes are mutations caused by the viral RNA dependent RNA polymerase.
These changes result in H and N that are immunologically similar to the previous strain.
Therefore, the existing antibodies might still be (but it is also possible they may not be) effective at neutralizing the virus.
What is antigenic shift?
This results in a new virus strain with novel H and/or N proteins that are immunologically distinct
(i.e., the existing antibodies will not be able to neutralize the virus).
These changes arise from the genetic re-assortment of previously circulating human and animal Influenza viruses.
The H and N are encoded on different RNA segments of the Influenza genome.
If a host cell is infected with two different influenza viruses (simultaneously), there can be a high frequency of re-assortment producing different combinations of H and N variants.
Describe prevalence of antigenic shift and antigenic drift in Influenza A viruses.
Influenza A viruses continually undergo antigenic drift.
This process accounts for most of the changes that occur in viruses from one “flu season” to the next.
In contrast, antigenic shift occurs only occasionally and accounts for major pandemics.
However, after a new pandemic influenza strain has evolved, it may undergo antigenic drift and evolve into a seasonal influenza strain.
Describe the drift of influenza virus.
After a re-assortment process has occurred, if the virus became established in humans, the virus would begin to drift like any other seasonal influenza virus.
During drift, small antigenic changes in the H protein generated by mutation are selected to increase immune evasion of the virus particle.
Why are pigs susceptible to both human and avian influenza viruses?
Avian viruses bind preferentially to α2-3-linked sialic acids moieties whereas influenza viruses adapted to humans have a binding preference for cell receptors that contain α2-6-linked sialic-acid moieties.
The respiratory epithelial cells of pigs have receptors that express both α2-3- and α2-6-linked sialic-acid moieties, thus pigs are susceptible to both human and avian influenza viruses.
Dual infections can occur and re-assortment of gene segments can give rise to new strains.
The pigs serve as a “mixing bowl” for the generation of new strains of influenza viruses; these strains can then be transmitted back to humans
What is the primary resevoir of Influenza A viruses?
Aquatic birds of the world, but viruses have adapted to alternate hosts such as humans.
How do human and avian influenza virsues differ?
Human and avian viruses have preferences for different conformations of the cellular receptor, sialic acid.
Because of this, viruses adapted to humans replicate in and transmit well to other humans, but poorly to aquatic birds.
Conversely, viruses adapted to aquatic birds replicate in and transmit well to other aquatic birds, but poorly to humans.
What is the mode of action of neuraminidinase inhibitor drugs?
N inhibitors prevent viruses from being released from infected cells, thus halting virus replication.
What is a ‘plug drug’?
Zanamivir and Oseltamivir are sialic acid analogues and are referred to as “plug drugs” because they block the active site of the N protein.
Without active N, the virus is unable to cleave sialic acid and escape from the cell in order to infect a new host cell.
These drugs are effective for both Influenza A and B infections.
What is Oseltamivir?
Different Influenza anti-viral medications may have different targets.
For example, NA inhibitors include Oseltamivir (Tamiflu), Zanamivir (Relenza), and GS 4104.
What is Amantadine?
Other anti-viral drugs block the release of the influenza virus genome into the cytoplasm by affecting the M2 ion channel.
Amantadine (Symmetrel) and Rimantadine (Flumadine) are examples of ion channel inhibitors.
How can influenza virus be cultivated for vaccine production?
In embryonated chicken eggs
New vaccines are produced by genetic re-assortment to produce a strain with the desired H antigen.
Describe the manufacture of Influenza A virus vaccine.
- The circulating strain of influenza is co-infected with a vaccine strain into eggs.
- A virus that has the H and N derived from the circulating strain and the remaining genetic elements from the vaccine strain is selected from the pool of re-assortments.
- The new virus strain is amplified in eggs and collected from the allantoic fluid.
- The virus is inactivated, treated for with detergents to solubilize the envelope and subjected to partial purification to enrich for the H and N components for the final injectable vaccine.
What do the vaccines we currently use for influenza include?
- whole virus (WV) vaccine which contains intact but inactivated virus,
- subvirion (SV) vaccine contains the isolated envelope portion of the virus,
- surface antigen (SA) vaccines which contain the isolated H and N glycoproteins.
How do we prepare an adequate amount of vaccine stocks for the upcoming winter season?
A decision has to be made as to which type of H antigen to include in the vaccine strain.
This decision is usually made in August and is based on data collected by epidemiological surveillance programs.
Describe primary and secondary exposure to influenza virus.
- The first exposure to an antigen (in this case, influenza virus) activates B cells that have receptors (i.e., B cell receptors) that recognize the antigen.
- These activated B cells proliferate and differentiate into memory B cells and antibody secreting plasma cells.
- The primary response is slow to develop, but upon exposure to the same antigen, the memory B cells respond more quickly and vigorously to the antigen (the secondary response).
- With influenza infections, the B cells that recognize the H or N antigens will become activated and differentiate into memory B cells.
- Upon re-infection with the same Influenza strain, the secondary response generates lots of anti-H and anti-N antibodies.
- The anti-H antibodies particularly important because they bind to the H protein and neutralize the virus before it can bind to the host cell.
- These antibodies can be generated either by natural exposure to the influenza virus or by vaccination.
Influenza results in destruction of respiratory epithelial cells and can lead to viral and/or bacterial pneumonia.
True or false?
True
Antigenic drift occurs all the time and is the main reason for changes in seasonal flu. It results in small differences resulting in a different subtype.
True or false?
False
Antigenic drift occurs all the time and is the main reason for changes in seasonal flu.
It results in small differences but the subtype remains the same.
When does antigenic shift occur?
Antigenic shift, however, occurs when two different strains enter the same cell and re-assortment of RNA segments occurs, which may result in the generation of new sub types.
Antigenic shift happens less frequently than antigenic drift but may result in pandemics.
Because of antigenic drift, new influenza vaccines are generated each year. Vaccine can be inactivated whole virus (WV), disrupted virus vaccines that contain the envelope structure (SV) or vaccines that contain the purified envelope proteins H and N.
True or false?
True
Bacterial pneumonia is the most frequent complication of influenza infection.
True or false?
True
With seasonal influenza, people between the age of 19 and 34 have the highest rates of influenza-associated hospitalizations.
True or false?
False
The current subtypes of influenza A viruses that routinely circulate in people include A(H5N1) and A(H3N2).
True or false?
False.
Re-assortment of the genes in influenza virus is facilitated by the fact that the genome is segmented.
True or false?
True
New strains of influenza continually arise because the virus mutates and recombines the gene encoding the protein that makes it resistant to antibodies.
True or false?
True.
Influenza virus may also cause “stomach flu”, associated with nausea, vomiting and diarrhea.
True or false?
False.
The common cold and influenza are caused by the same virus.
True or false?
False.
The flu is typically spread through coughs and/or sneezes.
True or false?
True
The very young and very old are usually those most susceptible to major complications from influenza infection.
True or false?
True
Influenza viruses can cause a latent infection.
True or false?
False.
Inhalation is the most common route of viral infection.
True or false?
True
Influenza is a vaccine preventable diseases of travelers.
True or false?
True
Antigenic drift happens when influenza virus acquires a different gene segment from another strain of influenza virus.
True or false?
False.
Antigenic shift does not happen frequently; when it does, there is a potential for a pandemic
True or false?
True
Antigenic drift could potentially happen every time the virus’ genome is replicated.
True or false?
True.
Describe the host range of Influenza A.
A has a broad host range (humans, other mammals, birds).
Where does the Influenza virus derive its envelope?
Envelope derived from the plasma membrane of the cell it previously infected.
What are three viral proteins embedded in the influenza A envelope?
Why are each of them important?
Hemagglutinin (H) - attachment to host cell.
Neuraminidase (N) – release from host cell.
Matrix protein – allows H+ to enter – important for uncoating.
Where do most new influenza strains emerge?
The solid line between humans and pigs is also significant - most new influenza strains emerge from pigs.
Genetic and biologic observations suggest that pigs may serve as ‘mixing vessels’ for the generation of human-avian influenza A virus re-assortants with pandemic potential.
An H7N2 virus has an H7 protein and an N2 protein.
An H5N1 virus has an H5 protein and an N1 protein.
True or false?
True
What are two modifications to H that are needed for the influenza virus to get its genome into the cytoplasm?
- Tryptase clara cleaves H to reveal the fusion peptide (external). Likely happens before binding to SA, but not required for binding to SA.
- pH dependent change in the endosome – allows H to unfold so the fusion peptide inserts into the endosome membrane - the two lipid layers fuse together.
The assembly process for Influenza is quite complicated.
What are the key events?
- The mRNAs for the H and N proteins are translated on the rER; the H and N proteins are inserted into cell’s plasma membrane.
- The new genomic (–) RNA is coated with NP protein, and the RDRP (RNA polymerase complex) associates with it to form the RNPs.
- The M protein moves into the nucleus and associates with the RNPs.
- The RNPs are directed out of the nucleus and assemble with other proteins at the cell membrane.
What must each virus particle have in order to have a complete genome?
Each virus particle must have 1 of each of the 8 (–) RNA segments in order to have a complete genome.
No duplications, no missing ones!
Where does assembly of the new influenza viral particles take place?
The assembly of the new viral particles takes place at the plasma membrane of the host cell.
The RNPs and M1 proteins are directed towards the plasma membrane of the cell where the envelope proteins that had been inserted (H, N and M2).
What promotes bending of the membrane and final budding of the influenza viruses?
The accumulation of the viral subunits promotes bending of the membrane and final budding of the viruses.
What promotes membrane fission so that the virus particle can detach from the host cell?
M2 (the ion channel)
What controls the release of the virus particle?
The release of the virus particles is controlled by the glycoprotein neuraminidase (A) that cleaves the sialic acid( SA) residues both from the particles and from the host cell.
What does the removal of sialic acid from the cell’s plasma membrane ensure?
Ensures that the newly released virus particle does not re-infect the cell.
What does the removal of the sialic acid from the virus’ envelope prevent?
The self- aggregation of the virus particles.
The process of viral mRNA synthesis results from the destruction of the host cell mRNA.
True or false?
True
Why does the host cell eventually die by programmed cell death?
(apoptosis, necroptosis, and pyroptosis - the latter two are associated with inflammation)
Recall that the process of viral mRNA synthesis results from the destruction of the host cell mRNA.
As a result, the host cell hasn’t made protein for itself for some time.
Protein synthesis is a requirement for life.
Give an overview of the Influenza A replication cycle.
- Attachment.
- Entry of virus particle into cell, release of the viral genome into cell’s cytoplasm, and movement from cytoplasm to nucleus
- Gene Expression and Genome Replication:
- (+) RNA made from (–) RNA for mRNA (Translation)
- (+) RNA made for more (–) RNA (Genome replication).
- Assembly of progeny virions.
- Egress - release from the cell.
The viral genome is (–) sense RNA - it cannot be translated by ribosomes. How does the virus solve this problem?
The virus synthesizes a (+) RNA that can function as mRNA with the RNA polymerase complex that it has packaged with the genome.
How does the virus synthesize its mRNA?
It uses its package polymerase proteins to transcribe the (–) RNA to make mRNA.
Because it is (–) sense, it cannot make these proteins after it enters the cell – it must have them beforehand.
What are the two types of (+) RNA that influenza virus synthesizes during its replication cycle?
What is similar about their production?
Incomplete
- also known as mRNA
- needed for protein synthesis
- it is made early in the replication cycle
- it is made by stealing the 5’ caps from the cellular mRNA
Complete
- also known simply as (+) RNA
- needed as a template to replicate the genome
- it is made later in the cycle and is not used as mRNA
- it is thought that the alternate conformation of the RNA polymerase complex does not require a primer
Both are made using the (–) as the template and the viral RNA dependent RNA polymerase (RDRP) complex.
What is the default conformation of the RDRP complex?
mRNA synthesis
What are the three important sites in the RDRP complex?
Site that binds the 5’ end of the (–) RNA template and does not release it.
Site that acts as an endonuclease.
Site that reads the (–) RNA template and catalyzes the synthesis of the mRNA.
How many segments of the Influenza A virus genome undergo splicing before export to the cytoplasm?
Two of the 8 segments
Why is the mRNA for the H and N proteins translated on a ribosome associated with the endoplasmic reticulum?
This ensures that the protein product is inserted into the plasma membrane of the cell.
These mRNA would have a target sequence that guides them to the ribosomes associated with the ER.
How does the RDRP convert between its two conformations?
When the concentration of NP protein is sufficiently high enough, the RDRP complex dissociates, then reassembles into the alternate conformation for replication of the viral genome.
What is a trivalent vaccine?
Trivalent influenza vaccines contain the 3 strains of Influenza (2 Influenza A and 1 Influenza B) most likely to be circulating during influenza season.
Where do the most significant mutations occur in the influenza virus?
In the H protein
What is the consequence of antigenic shift?
- Everyone is potentially susceptible to infection with the new strain because most people don’t have immunity to the new strain.
- The disease caused by the new strain is likely to be serious.
- Younger people (like MICB 202 students) may be even more susceptible to infection than older people (like your instructor)
What are most vaccines against influenza designed to generate?
How is this done?
To generate IgG type antibodies (a trick is to release the antigen slowly from the injection site over time).
How does the influenza vaccine work?
The concept is that the person will make antibodies that neutralise the virus.
These antibodies might cause steric hindrance so the virus cannot bind, they may stabilise the structure so that the genome is not released, or the envelope does not fuse in the endosomal membrane.
Describe egg based vaccines.
- high production, relatively low cost
- production time is about 6 – 8 months
- (~1.5 billion doses/year – about 85% of the global supply)
- potential adaptation of virus to avian structures
- shortages of embryonated eggs
Describe cell based vaccines.
- flexible and scalable
- less likely to see virus adaptation
- seems to yield better quality of vaccine for senior citizens
- would be more costly and take longer to produce ~1.5 billion doses/year
Describe recombinant DNA vaccines.
- hemaggutinin and/or neuraminidase gene is cloned into an expression vector
- flexible and scalable
- would be more costly and take longer to produce ~1.5 billion doses/year
Use this image to describe the Influenza A replication cycle.
(Long answer)
- The binding of influenza virus to the host cell (1) is mediated by the spike protein hemagglutinin (HA).
- The internalization of the virus occurs in endosomes (2) where, upon acidification (3), HA undergoes a conformational change which leads to the fusion between the viral envelope and the endosomal membrane (4).
- Thus the viral genome enters the nucleus (5) where it is both replicated and duplicated (6).
- Despite of HA, NA and M2, whose synthesis occurs in the ER, the other viral components are synthesized in the cytosol, where also RNPs are formed (7).
- The viral subunits accumulate at the budding site for the subsequent assembly and release of the new viral progeny (9)
