Virology Chapter 5-6: Picornaviruses (Poliovirus) Flashcards
How are enteroviruses (such as poliovirus) usually transmitted?
fecal-oral route
Pathogenesis of Poliovirus – Route
- oropharyngeal mucosa
- replicated for 1-2 weeks
- asymptomatic, or mild illness (ie. sore throat) - stomach
- epithelial cells of lower intestinal tract
- extensive replication
- may result in transient viremia - [if it enters blood] may target CNS
- infect certain types of nerve cells
- kill these cells when egressing after replication - excreted in feces
- several weeks
What is transient viremia?
virus in bloodstream
What are the 4 responses that may occur after infection with poliovirus?
- subclinical infection without symptoms
- abortive polio (mild illness)
- non-paralytic poliomyelitis (aseptic meningitis)
- paralytic poliomyelitis
What is abortive polio?
- symptoms
- recovery period
- diagnosis
most common form of polio
symptoms:
- febrile illness
- non-specific symptoms – malaise, drowsiness, headache, nausea, vomiting, sore throate
recovery period:
- few days
diagnosis
- only recognized and confirmed if virus is detected in blood
What is non-paralytic polio (aseptic meningitis)?
- symptoms
- recovery
symptoms:
- same as abortive polio
- stiffness and pain in neck and back (2-10 days)
recovery:
- rapid
- complete
What is paralytic polio?
- symptoms
- recovery
major illness (< 1% of infections)
- antibodies to the virus appear early in infection, and are usually present when paralysis appears
symptoms:
- flaccid paralysis – results from LMN damage
- amount of damage varies
recovery:
- usually max. within 6 months, but can take longer
What is post poliomyelitis muscle atrophy (PPMA)?
‘reappearance’ of paralysis and muscle wasting observed decades after illness and recovery from paralytic poliomyelitis
What causes post poliomyelitis muscle atrophy (PPMA)?
result of the normal decline in function of motor neurons that had compensated for the loss in function of motor neutrons killed by poliovirus
not a consequence of a persistent infection or re-infection
How many poliovirus serotypes are there?
1, 2, 3
What is a serotype?
variant of the virus that will elicit production of a distinct population of antibodies
antibodies bind to the serotype used as the antigen, but not to other serotypes of the virus
infection (and subsequent antibody production) with one serotype of poliovirus does not confer protection against other serotypes
Where are the epitopes responsible for production of neutralizing antibodies?
on structural proteins VP1, VP2, and VP3
What are most of the neutralizing antibodies against?
epitopes on VP1
immunization with poliovirus serotype 1 will elicit 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
What are the two forms of poliovirus vaccine available?
- Salk’s IPV (inactivated poliovirus vaccine) –injected
- Sabin’s OPV (oral poliovirus vaccine) – ingested
How is the IPV vaccine made?
- the 3 serotypes of poliovirus are cultivated using monkey kidney cells or human cell lines
- viruses are concentrated, purified, and inactivated with formaldehyde
What does the IPV induce? What does it not?
production of protective serum antibodies (IgG)
does not confer localized immunity – no secretion of IgA antibody into mucosal lining of GI tract
Is reinfection after IPV possible?
reinfection of the alimentary tract is possible
What are the advantages of IPV? (2)
- 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 individual
What are the disadvantages of IPV? (3)
- requires boosters
- does not induce antibody mediated immunity in intestinal mucosa – vaccinated people might transmit wild poliovirus to susceptible people by oral-fecal route
- expensive – requires sterile needles and syringes, require highly trained personnel to administer
What does OPV contain?
active attenuated strains of two poliovirus serotypes cultivated from monkey kidney cell culture
Where do viruses of the OPV vaccine infect?
infects and replicates in cells of the host
What does OPV do?
confers both systemic (IgG) and localized (IgA in mucosal lining of GI tract) immunity
What are two significant problems with OPV?
- low rate of mutation, where virus reverts back to its virulent form and can cause paralytic poliomyelitis in vaccinated recipient
- virus is shed in feces of vaccinated recipient for several weeks, and can be disseminated to unvaccinated contacts
What is the recommended vaccination protocol?
IPV for initial vaccination
- would have serum antibodies against poliovirus
OPV for booster
- induces localized intestinal immunity
- serum antibodies would protect the person if the poliovirus reverted back to its virulent form
What are the advantages of OPV? (5)
- confers antibody mediated immunity at intestinal mucosa – mimics 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 (ie. needles)
What are the disadvantages of OPV? (3)
- virus can mutate and (in rare instances) revert back to 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 (ie. 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
Who can poliovirus infect?
humans only
unless artificially introduced into cells of other animals
Can enterovirus survive low pH conditions?
yes
For children who are traveling in endemic areas, would it be better to get IPV or OPV?
OPV – attenuated
attenuated virus vaccine induces stronger, longer lasting immunity that will protect them better
once they have cleared the virus from the vaccine, they will make IgA antibodies in their gut secretions
child could no longer be a carrier of the virus and cause infections in susceptible people
What is the structure of poliovirus?
- non-enveloped
- icosahedral capsid
What is the icosahedral capsid made of?
60 protomers – each made of one copy of VP0, VP1, and VP3
= 180 protein molecules total
What is the goal of the capsid maturation process?
to generate a virus particle that is infectious
immature virus particle is non-infectious
How does the capsid mature?
VP0 is cleaved to form VP2 (localized outside of capsid) and VP4 (localized inside)
What is packaged inside the capsid?
- genome
- Na+ or K+ ions
Why are Na+ or K+ ions packed inside the capsid?
to counteract negative charges of phosphate groups of RNA
What is the genome structure of poliovirus?
one single-stranded (+) sense uncapped mRNA
What is on the 5’ and 3’ ends of the poliovirus RNA?
5’ – Vpg protein
3’ – polyA tail
What are the UTRs of the poliovirus RNA?
5’ UTR
- long
- important for translation of RNA
- possibly important for genome packaging into capsid
3’ UTR
- short
- important in synthesis of (-) strand for genome replication
Is the genomic RNA of poliovirus infectious?
yes – if RNA is introduced (artificially) into host cell, virus progeny will be produced
What type of protein does poliovirus RNA generate?
polyprotein – cleaved by two viral-encoded proteases to produce structural and non-structural proteins
What are the receptors for poliovirus?
CD155 (poliovirus receptor – PVR) – normal cellular protein on cell surface of monocytes, macrophages, thymocytes, and CNS neurons
Attachment of Virus Particle and Entry of Virus Genome – Process
- Poliovirus binds to CD155 molecule of susceptible cell
- Induces endocytosis into cell’s cytoplasm, and conformation change in VP1 protein
- VP4 is exposed on virus surface - VP1 protein inserts into lipid bilayer of host cell and forms pore
- Viral RNA leaves capsid and enters into cytoplasm of host cell
Translation of RNA – Process
- Cellular enzyme cleaves off VPg molecule of RNA
- Cell’s ribosome assembles onto virus’ RNA using IRES on 5’ end to initiate translation
- One polyprotein is synthesized, and folds into secondary and tertiary structures as it is being synthesized
- Part of polyprotein becomes an active protease that carries out several sequential cleavages of polyproteins – structural or non-structural proteins produced
Translation of RNA
In poliovirus, why does the cell’s ribosome assemble onto IRES of RNA (normally capped mRNA)?
poliovirus genome lacks 5’ methylated cap
Translation of RNA
When and where is the internal ribosome entry site (IRES) formed?
in 5’ UTR, IRES secondary structure is formed when RNA folds upon itself and forms H-bonds between complementary bases
Translation of RNA
How many open reading frames does the poliovirus genome have?
one – encodes a single polyprotein
Translation of RNA
Initially, the viral RNA is at a disadvantage, but the virus quickly shuts off cellular protein synthesis (within 30 minutes). How does this occur?
one of the products of the polyprotein is a protease that cleaves one of the translation factor eIF4G needed for translation of cellular mRNA
translation machinery is still functional for virus translation, but insufficient for host mRNA translation
Replication and Transcription of Genome
What are two important products of the polyprotein (translation) needed immediately to replicate the genome and synthesize more viral mRNA?
- VPg
- RNA-dependent RNA polymerase (RDRP)
Replication and Transcription of Genome
What does the replication complex consist of?
- RDRP
- VPg
- genomic (+) strand of RNA
Replication and Transcription of Genome –Process
(+) strand is copied to synthesize (-) strand
- Modified VPg serves as primer by binding to 3’ polyA tail
- RDRP synthesizes (-) strand by reading base sequence of (+) strand, and adding complementary base to primer
- Produces double-stranded RNA intermediate
(-) strand of double-stranded RNA serves as template for synthesis of new (+) strand of RNA
- New modified VPg protein binds to 3’ end of (-) strand
- Newly made (+) strands of RNA are either:
- translated by cellular ribosomes
- used for genome of progeny virus (replication)
Replication and Transcription of Genome
Why does replication occur in a replication compartment?
possibly to avoid detection of double-stranded RNA intermediate by host cell defenses
Replication and Transcription of Genome
How are replication compartments formed?
replication complexes associate with membrane structure derived from ER
Replication and Transcription of Genome
What does VPg do?
serves as primer for RDRP for virus RNA synthesis
Replication and Transcription of Genome
How is VPg modified to act as primer?
VPg ––> VPg-U-U
RDRP and other proteins derived from the polyprotein bind to two areas of the virus RNA (CRE loop and cloverleaf), and add uracil nucleotides to 3’OH end
Synthesis of Capsid Proteins and Assembly of Procapsid
What are the capsid proteins required, and where do they come from?
VP0, VP1, and VP3
result of cleavage of polyprotein
Synthesis of Capsid Proteins and Assembly of Procapsid – Process
- Capsid proteins (VP0, VP1, and VP3) associate with each other to form protomer
- Protomer associates with other protomers to form pentamer
- Pentamers (14S) assemble into procapsid self-assembly
- Progeny genome packaging OR capsid condensation around progeny genome
- Packaging induces maturation: cleavage of VP0 to VP2 and VP4
Genome Packaging, Maturation, and Virus Egress – Process
- Viral genome is inserted into procapsid
- Maturation: VP0 is cleaved to VP2 and VP4 by ribozyme (RNA molecule with enzymatic activity)
- seals up capid
- protects RNA from degradation - Viroporin (encoded in polyprotein) disrupts plasma membrane by assembling into pores in the membrane
- Cell membrane becomes more permeable
- Host cell lyses, and poliovirus is released from cell cytoplasm
Genome Packaging and Virus Egress
How is it ensured that (+) strand of RNA is packaged into capsid?
(+) strand of RNA contains short nucleotide sequence called packaging site (PAC) that interacts with capsid proteins
Poliovirus Replication Cycle – Overview
Attachment and Entry → Translation → Replication and Transcription → Assembly → Egress
Where are host cell receptors for poliovirus (CD155) found?
on epithelial cells
What is the anti-receptor for poliovirus?
indentation in VP1 structural protein on capsid
Does capsid assembly occur spontaneously?
yes – once a critical level of virus protein is reached
Why is it vital that (+) strand is packaged into procapsids, and not (-) strand
no RDRP is packaged into the capsid
Would poliovirus be able to use the host cell RNA polymerase to replicate and transcribe its genome?
no – host cell RNA polymerase uses DNA as template for transcription
Why does the poliovirus utilize a polyprotein strategy when translating its mRNA? Why aren’t the individual viral genes translated separately into the individual proteins?
its genome is composed of single strand of (+) RNA that encodes for all its proteins
- eukaryotic protein translation
system is unable to translate individual genes of poliovirus genome due to polycistronic nature of viral RNA - eukaryotic ribosome is not able to re-assemble in middle of mRNA
- therefore, poliovirus creates large polyprotein that has autocatalytic abilities – cleaves itself into smaller units
