Picornaviruses Flashcards
The family Picornaviridae currently consists of 147 species grouped into 63 genera.
Describe infection symptoms.
Picornaviruses cause a wide range of illness in humans (more so than other virus families).
Infection with various picornaviruses may be asymptomatic, mild illness such as the common cold, or more severe disease such as aseptic meningitis and encephalitis.
What type of picornavirus is poliovirus?
A member of the Enterovirus genus.
What are enteroviruses resistant to?
Enteroviruses are resistant to many common laboratory disinfectants that would inactivate many other types of viruses.
What are enteroviruses sensitive to?
Formaldehyde, which is used to denature the virus during vaccine production.
Enteroviruses are also sensitive to chlorine at levels of 0.3 to 0.5 ppm; however, the presence of organic matter may protect the virus from inactivation by the chlorine.
This finding might explain the transmission of poliovirus by ingestion of water from swimming pools and some sources of drinking water.
What characterizes paralytic poliomyelitis?
Poliovirus causes the disease paralytic poliomyelitis and is characterized by flaccid muscular paralysis due to the death of motor neurons.
It is estimated that less than 1% of poliovirus infections result in paralytic poliomyelitis.
When did the World Health Organization launch it’s global poliovirus eradication campaign?
What was the goal?
Did they succeed?
1988.
The goal of the campaign was the worldwide eradication of poliovirus by the end of the year 2001.
The campaign has had some successes but there is still much work to do to completely eradicate poliovirus as an infectious disease.
How are enteroviruses such as poliovirus transmitted?
Enteroviruses such as poliovirus are usually transmitted from one person to the next by the fecal-oral route.
Where does poliovirus replicate initially after ingestion?
What symptoms begin to appear?
After ingestion in contaminated food or water, the virus initially replicates in the oropharyngeal mucosafor 1–2 weeks.
At this time, the infected person may be asymptomatic or experience a mild illness (e.g., sore throat).
What happens after poliovirus replicates in the oropharyngeal mucosafor 1–2 weeks?
The virus then moves through the stomach and before infecting the epithelial cells of the lower intestinal tract.
In the intestinal epithelia, the virus undergoes extensive replication and may result in a transient viremia (i.e., virus in the bloodstream).
Once in the blood, the virus may target the Central Nervous System (CNS).
Poliovirus infects certain types of nerve cells and kills these cells after when the virus egress after replication.
The infected person excretes the virus in feces for several weeks
Describe poliovirus pathogenesis based on experimental findings?
The virus initially replicates in the oropharyngeal and intestinal mucosa, and eventually reaches the blood resulting in a viremia.
Invasion of virus into the central nervous system may occur either directly from the blood, or by retrograde axonal transport when virus enters the neuromuscular junction.
What is non-paralytic polio (aseptic meningitis)?
This form of polio is characterized with the same symptoms as abortive polio, but the patient experiences stiffness and pain in neck and back that lasts for 2–10 days.
The recovery is rapid and complete.
What responses may occur after infection with poliovirus?
- subclinical infection without symptoms
- mild illness (abortive polio)
- non-paralytic poliomyelitis (also called aseptic meningitis)
- paralytic poliomyelitis
What is abortive polio?
This is the most common form of polio, occurring in more than 95% of cases.
It is characterized as a febrile illness where the patient may experience non-specific symptoms such as fever, malaise, drowsiness, headache, nausea, vomiting and sore throat.
Complete recovery occurs in a few days.
It is only recognized and confirmed if virus is detected in the blood.
What is paralytic polio?
This form of polio is the major illness and occurs in about 1% of infections.
The paralysis may or may not follow the minor illness.
The predominating sign is flaccid paralysis resulting from lower motor neuron damage.
The amount of damage and destruction by poliovirus will vary from patient to patient.
The maximal recovery usually occurs within 6 months but sometimes takes longer.
Antibodies to the virus appear early in infection, and are usually present when paralysis appears.
What is post poliomyelitis muscle atrophy (PPMA)?
A “reappearance” of paralysis and muscle wasting has been observed in some patients decades after their illness and recovery from paralytic poliomyelitis.
It is not a consequence of a persistent infection or re-infection.
It is the result of the normal decline in the function of motor neurons that had compensated for the loss in function of motor neutrons killed by poliovirus.
Describe muscles commonly weakened by poliovirus infections.
Paralysis by poliovirus may involve any muscle of the body, but the legs muscles are the most commonly affected in survivors.
The paralysis is of the flaccid type (not stiff).
Some muscles may be weak or limp.
In time, the affected limb may not be able to straighten due to contractures (shortening) of certain muscles.
The muscles and bone of the affected limb become thinner than the unaffected limb, and does not grow as fast resulting in a shorter limb.
What were the approaches to control infection to poliovirus prior to vaccine development ?
The only approaches to control infection were passive immunization and non-specific public health measures (i.e., isolation of patients and closing schools, swimming pools etc.).
These measures were not successful in preventing epidemics of poliomyelitis.
What is a serotype?
A serotype is a variant of the virus that will elicit the production of a distinct population of antibodies.
These antibodies will bind to the serotype used as the antigen, but not to other serotypes of the virus.
Therefore, infection (and subsequent antibody production) with one serotype of poliovirus does not confer protection against the other serotypes.
How many poliovirus serotypes are there?
There are three poliovirus serotypes: 1, 2 and 3.
For poliovirus, where are the epitopes responsible for production of neutralizing antibodies located?
On the structural proteins VP1, VP2 and VP3.
For poliovirus, what are most of the neutralizing antibodies against?
Most of the neutralizing antibodies are against the epitopes on VP1.
Thus, immunization with poliovirus serotype 1 will elicit the production of antibodies that recognize VP1, VP2 and VP3 proteins of serotype 1.
These antibodies will not bind to VP1, VP2 and VP3 proteins of poliovirus serotype 2.
How many forms of a poliovirus vaccine exist?
There are two forms of poliovirus vaccine available:
IPV (Inactivated Poliovirus Vaccine, injected)
OPV (Oral Poliovirus Vaccine, ingested as a syrup).
How are the three serotypes of poliovirus used in the original IPV cultivated?
Using monkey kidney cells or human cell lines.
The viruses are concentrated, purified and inactivated with formaldehyde.
The vaccine is administered by injection and requires a “booster” (repeated) vaccination.
How does IPV work as a vaccine for poliovirus?
Induces the production of protective serum antibodies (IgG).
It does not confer localized immunity (i.e., there is no secretion of IgA antibody into the mucosal lining of the GI tract) and re-infection of the alimentary tract is possible.
The implication of this observation is that such a vaccinated person may become infected with poliovirus and shed the virus in their feces for several weeks. The vaccinated person would be protected from the virus because of the antibody in the bloodstream. However, if they were to ingest water contaminated with poliovirus, they could develop a gastrointestinal infection that produces virus particles that could be transmitted to a susceptible person.
Who developed the original poliovirus vaccine?
IPV was the original vaccine developed by Jonas Salk in the 1950’s.
What are advantages of IPV?
- confers protective antibody mediated immunity in the blood when given in sufficient doses,
- no active viruses excludes the potential for reversion to virulence by mutation and allows for its use in immunodeficient or immunosuppressed individuals.
What are disadvantages of IPV?
- requires boosters every 5 years to maintain immunity,
- does not induce antibody mediated immunity in the intestinal mucosa - vaccinated people might transmit wild poliovirus to susceptible people by the oral fecal route,
- expensive – requires sterile needles and syringes, require highly trained personnel to administer.
Who developed oral poliovirus vaccine?
OPV was developed by Albert Sabin and replaced the IPV in the early 1960’s.
How does OPV work as a vaccine for poliovirus?
OPV contains active attenuated strains of two poliovirus serotypes cultivated from monkey kidney cell culture.
In the OPV, the virus of the vaccine infects and replicates in the cells of the host.
It is an effective vaccine that confers both systemic (i.e., serum IgG) and localized (i.e., IgA in the mucosal lining of the GI tract) immunity.
What is attenuation?
Attenuation is a procedure that selects for the non-virulent strains of a pathogen that is still capable of inducing immunity.
What are two significant problems with OPV?
- there is a low rate of mutation where the virus reverts back to its virulent form and can cause paralytic poliomyelitis in the vaccinated recipient, and
- the virus is shed in the feces of the vaccinated recipient for several weeks and can be disseminated to unvaccinated contacts (ironically, this problem is exploited in vaccination programs).
Describe current protocols for polio vaccination.
Over the years, there have been changes in the vaccination protocols for poliovirus.
Instead of administering only OPV, the proposed changes recommend that IPV be used in the initial vaccination, followed by OPV as a booster.
After vaccination with IPV, the person would have serum antibodies against poliovirus.
OPV would be used to induce localized intestinal immunity - the serum antibodies would protect the person if the poliovirus reverted back to its virulent form.
What are advantages of OPV?
- confers antibody mediated immunity at the intestinal mucosa - mimics the natural infection,
- induces antibody production in recipient very quickly, does not require boosters, oral administration is more acceptable to recipient, easier to accomplish than injection,
- relatively cheap to produce, administer (does not require highly trained individuals to administer vaccine), inexpensive mass immunization without the need for sterile equipment (i.e., needles).
What are disadvantages of OPV?
- virus can mutate and (in rare instances) revert back to the form of neurovirulence that is sufficient to cause paralytic polio in vaccine recipient or susceptible contacts,
- vaccine progeny spreads to others in contact with recipient (i.e., household) - often the virus that is excreted has mutated and may not be safe as the original virus used in the vaccine,
- cannot be given to immunodeficient or immunosuppressed individuals or members of their household.
Describe the proportionality of the different types of poliovirus infection.
95% of infections are abortive Polio, characterized by mild cold-like illness for 72 hours.
1–2% of infections result in non-paralytic polio, characterized by Influenza-like symptoms lasting 1–2 weeks.
Less than 0.5% of infections result in paralytic poliomyelitis.
Compare the two vaccine’s are available against poliovirus.
Salk’s vaccine is an inactivated virus that requires a booster and is administered by injection.
Sabin’s vaccine is a live attenuated virus that is administered orally.
What does the picornavirus family consist of?
A large number of small RNA viruses and are amongst the simplest of vertebrate viruses.
Is poliovirus enveloped?
The virus particle is non-enveloped and is approximately 30 nm in diameter.
What does the poliovirus capsid consist of?
A densely packed icosahedral arrangement of 60 protomers.
What is each poliovirus protomer made up of?
Each of the protomer is made of one copy of VP0, one copy of VP1 and one copy of VP3.
How many protein molecules construct the poliovirus capsid?
Thus, the poliovirus capsid is constructed with 180 protein molecules to form a procapsid before its genome is packaged inside.
How is an infectious poliovirus particle generated?
In the later part of the replication cycle (after the genome has been packaged), the capsid undergoes a maturation process to generate a virus particle that is infectious (the immature virus particle is not infectious).
VP0 is cleaved to form VP2 and VP4; VP2 is localized to the exterior of the capsid, whereas VP4 is localized to the interior of the capsid.
What is VPO cleaved to form?
VP0 is cleaved to form VP2 and VP4;
VP2 is localized to the exterior of the capsid, whereas VP4 is localized to the interior of the capsid.
Describe the organization of the poliovirus genome?
Poliovirus genomes are organized as structural (capsid) genes followed by genes involved in the replication of their genomes.
The entire RNA strand is translated on a ribosome to generate a polyprotein.
The polyprotein is cleaved by two viral-encoded proteases to produce structural proteins and non-structural proteins.
What does the poliovirus genome consist of?
The genome consists of one single-stranded (+) sense uncapped RNA molecule.
A small, basic protein VPg (~ 23 amino acids long) is covalently attached to the 5’ end and a polyA tail at the 3’ end.
What is the 5’ UTR of poliovirus RNA important for?
And the 3’ UTR?
The RNA has a long untranslated region (UTR) at the 5’ end and a shorter untranslated region at the 3’ end.
The 5’ UTR is important in translation of the RNA and possibly the packaging of the genome into the capsids.
The 3’ UTR is important in the synthesis of the (–) strand for the replication of the genome.
Describe the restricted host range of polioviruses among lab animals.
Humans are the only known reservoir of poliovirus and close human contact appears to be the primary avenue of spread.
Mouse cells cannot be infected with poliovirus.
Transformed monkey cells can be infected with poliovirus.
What are the receptors for poliovirus?
Normal cellular proteins known as CD155.
This molecule is a member of the Ig gene superfamily (which includes Ig and other cell surface molecules such as CD4, CD8, MHC class I and II molecules).
What is CD155?
CD155 is a cell surface molecule that is expressed on monocytes, macrophages, thymocytes and central nervous system neurons.
CD155 appears to be an adhesion molecule, but is function in the immune system is unclear.
CD155 is sometimes referred to as the poliovirus receptor (PVR).
Describe attachment of poliovirus to the host cell and entry of its genome.
The poliovirus binds to the CD155 molecule of a susceptible cell.
The binding of the virus particle induces its endocytosis into the cell’s cytoplasm as well a conformational change in the VP1 protein such that it can insert into the lipid bilayer of the host cell. The conformational change results in the N-terminus on the VP1 protein and the VP4 protein being exposed on the virus surface.
The VP1 protein is inserted in the plasma membrane resulting in a transmembrane pore that allows the viral RNA to leave the capsid and enter into the cytoplasm of the host cell.
What is an internal ribosome entry site?
Eukaryotic ribosomes typically assemble on capped mRNA, but the poliovirus genome lacks a 5’ methylated cap.
Instead, the virus relies on an internal ribosome entry site (IRES).
This structure allows the ribosome to assemble on the 5’ end of the RNA to allow for translation
What happens after entry of the poliovirus genome into the host cell?
A cellular enzyme cleaves off the VPg molecule attached to the viral RNA.
The cell’s ribosome assembles onto the virus’ RNA using the IRES to initiate translation.
Describe the poliovirus IRES structure and its use in the assembly of the ribosome.
In the 5’ UTR, the RNA has asecondary structure that is formed when the RNA folds upon itself and forms H-bonds between complementary bases.
This structure is known as the internal ribosome entry site (IRES) and allows the ribosome to assemble on the 5’ end of the RNA to allow for translation.
Where is translation of the poliovirus RNA initiated?
Translation of the RNA is initiated at the internal start site 741 nucleotides from the 5’ end.
Describe the poliovirus genome.
The genome has one open reading frame and encodes for a single polyprotein 2100 to 2400 amino acids long.
Part of the polyprotein transcribed from poliovirus RNA becomes an active protease. What does it do?
Carries out several sequential cleavages of the polyproteis resulting in 20 different proteins.
Some of these proteins are structural proteins while other proteins are involved in the replication of the genome.
How soon does the poliovirus shut of cellular protein synthesis?
Initially, the viral RNA is at a disadvantage, but the virus quickly shuts off cellular protein synthesis (within 30 minutes).
Describe host translation shutoff in poliovirus.
Poliovirus protease 2A cleaves eIF4G host translation factor in such a way that the translation machinery is still functional for virus translation but insufficient for host mRNA translation.
Describe modification of VPg to VPg-U-U.
The RDRP (3D protein) and two 3CD proteins (also derived from the polyprotein) bind to two areas of the virus RNA (the CRE loop and the cloverleaf) and add the uracil nucleotides to the VPg protein so that it can be used as a primer.
Describe the replication and translation of the poliovirus genome.
The (+) strand is copied to synthesize the (–) strand.
The VPg-pUpU-OH-3’ serves as a primer by binding (complementary base pairing) to the 3’ end PolyA tail of the (+) strand.
The RDRP synthesizes the (–) strand by reading the base sequence of the (+) strand and adding the complementary base to the primer.
This produces a double stranded RNA intermediate.
The (–) strand of the double stranded RNA then serves as the template for the synthesis of new (+) strands of RNA (a new VPg protein would bind to the 3’ end of the (–) strand)
Some of the newly made (+) RNA strands are translated by the cellular ribosomes (the VPg would be cleaved off by the cellular protease prior to translation).
Some of the newly made (+) RNA strands are used for the genome of the progeny virus.
For poliovirus, describe the synthesis of capsid proteins and assembly of a procapsid.
The cleavage of the polyprotein resulted in the proteins (VP0, VP1 and VP3) required to build the viral capsid.
The capsid proteins VP0, VP1 and VP3 associate with each other to form the protomer, which then associates with other protomers to form a pentamer.
The pentamers (14S) assemble into the procapsid self-assembly followed by progeny genome packaging (see Figure 6.9A) or by capsid condensation around a progeny genome.
The final step of maturation involves the cleavage of VP0 to generate VP2 and VP4, a step which is induced by the packaged RNA.
Describe Genome Packaging and Virus Egress for poliovirus.
After the genome is inserted into the pro-capsid, the VP0 is cleaved to yield VP2 and VP4.
The VP2 is on the surface and the VP4 is on the inside of the capsid. This seals up the capsid and protects the RNA from degradation.
The RNA genome appears to catalyze this cleavage by acting as a ribozyme (i.e., an RNA molecule with enzymatic activity).
One of the proteins encoded in the polyprotein is a viroporin that serves to disrupt the plasma membrane. As a result, the cell membrane is more permeable and the poliovirus is released from the cytoplasm when the host cell lyses.
Define: picornavirus
Small RNA virus
Describe how IPV works.
- No gut immunity (IgA)
- IgG immunity in blood
Describe how OPV works.
- local immunity (IgA secretions in gut)
- might spread to bloodstream, could lead to IgG in blood (systemic immunity), or infection of motor neurons (leading to paralytic polio)
What are the two forms of Poliovirus?
Immature form (non-infectious): 3 different polypeptides VP0, VP1 and VP3.
- RNA genome* inserted into the capsid, cleaves the VP0 polypeptide into two - now called VP2 and VP4.
Mature form (infectious): 4 different polypeptides VP1, VP2, VP3, VP4.
*The RNA genome acts as a ribozyme an RNA molecule with enzyme activity.
Describe the structure of the Poliovirus genome.
The poliovirus genome is made of positive-sense ss RNA, about 7,500 nucleotides long.
Both ends are modified, the 5’ end by a covalently attached small, basic protein VPg (~23 amino acids), the 3’ end by polyadenylated tail - there is no 5’ methylated guanine cap.
The genome contains a single open reading frame which encodes a 247 kDa polyprotein.
Poliovirus RDRPs require a primer - a molecule that can provide a 3’ OH group to add the next nucleotide. How is this primer provided?
It encodes a gene for a primer - VPg.
This small protein is modified by adding to uracil nucleotides to it - thus providing the required 3’ OH group
(VPg - U - U 3’ OH)
Eukaryotic ribosomes recognize mRNAs with a 5’ cap and a poly-A tail. Poliovirus genome (positive-sense RNA) lacks a 5’ cap. How then does Poliovirus initiate translation?
Instead of using a 5’ cap, polio RNA uses an IRES structure – a secondary structure in the RNA which can interact with the small ribosome subunit and other cell proteins, resulting in the full assembly of the ribosome.
Describe cap-independent translation.
IRES recruits the cellular initiation factors to allow for ribosome assembly.
The host cell has RNases that degrade RNA molecules.
This is needed to recycle cellular mRNAs, tRNAs and rRNAs.
How does the polioviral genome escape degradation?
Replicate the genome in a compartment - protect the genome from the RNases.
Added bonus: RDRP replicates viral genome only - not host cell mRNA.
Describe genome entry for Poliovirus.
The binding of VP1 to the CD155 induces conformational changes in the capsid of the virus particle.
The N-terminal end of the VP1 protein is inserted into the cell membrane to form a pore.
VP4 which was inside the capsid prior to binding CD155 is now outside and may form part of this pore structure.
The RNA passes through the pore and into the cell’s cytoplasm.
What is VPg?
VPg is a small protein to which uridine is covalently attached.
It is part of the primer for RNA synthesis – both (+) and (–) strands.
A primer is a starting molecule to which nucleotides are added to build a nucleic acid.
What are 2 simple rules that apply to polymerases?
- polymerases read the template in a 3’ to 5’ direction,
- polymerase synthesize the complementary strand in a 5’ to 3’ direction.
Recall BIOL 112!
Once viral RNA replication is initiated, it ramps up quickly!
Describe how.
- Original (+) RNA template 1 copy
- (–) RNA template 1 copy
- (+) RNA copies X 50 each acts as a template to make more (–) RNA.
- New (–) RNA template 1 copy made from each (+) RNA copy.
- (+) RNA copies X 50 from each of the new (–) RNA template.
Use this image to describe the Poliovirus replication cycle in its entirety. (long answer!)
- (A, B) Picornavirus uses different receptors to enter the cell, some implicated in the signaling internalization (A), meanwhile others can act as carriers that transport the viral particle to meet the primary receptor (B).
- (C, D) This infection event can be impeded by the action of specific neutralizing antibodies that can destabilize the viral particle (C) or opsonize or stabilize the particle to impair receptor binding or conformational changes required for infection (D).
- (E) Once the virus enters the cell, the viral RNA delivery mechanism is triggered, and the viral genome (black wavy line) is released into the cytoplasm.
- (F) Upon removal of VPg (magenta oval), the genome starts the IRES-driven translation leading to the production of the viral polyprotein.
- (G) The proteolytic cascade produces all viral proteins, structural and nonstructural.
- (H) Some proteins act by hijacking the host cellular systems such as the nuclear pore, the cell translation machinery, and the apoptotic systems and initiate the remodeling of the internal cell membranes.
- (I) The structural proteins assemble into the capsid intermediates, the protomer and the pentamer, and also procapsids (L).
- (J) The formed replication complex assembled from non-structural proteins and modified internal membranes firing the picornaviral genome replication by the 3D polymerase via RNA complementary (red wavy lines) and using VPg as a primer.
- (K) The new progeny genomes including eventual mutations (yellow stars).
- (M) Mature virions assemble from pentamers that surround and package the new viral genomes.
- Viral particles escape from the cell by cell lysis or budding within membranes that can protect the viral progeny (P).
- (N) Some progeny virus with mutations in their capsids (yellow star) may escape from to the action of specific NAbs.
- (O) Empty capsids can act as molecular decoys for Abs to protect the infecting particles from neutralization
