4. Microorganisms: viruses Flashcards
Define a virus
Virus - nucleoporin (protein + nucelic acid) complex - infects cells and uses their metabolism to replicate itself - smallest known infective agent
Why are viruses said to be metabolically inert?
Metabolically inert - can’t replicate on their own - must enter host cell to perform metabolic activity
What imaging tools can be used to view viruses?
- Electron microscopy
- X-ray
What is the average size of a virus?
10 - 400 nm
What are the common structural components of viruses?
- nucleocapsid
- envelope (not all have)
- viral genome: nucleic acids, segmented, DNA / RNA, ss / ds, linear / circular
Why is viral genome segmented?
Segmentation of viral genomes allows exchange of intact genes between related viruses when they coinfect the same cell
Explain why is the capsid needed and what are the possible structures of it
Capsid functions:
- protect genome
- aids in viral transfer into host
Capsid structure:
- protein coat - made up of capsomeres (capsid subunits) -> arrangements of capsomeres to compose a capsid: polyhedral, helical, complex
Why are some viruses naked and others enveloped?
Non-enveloped viruses - naked viruses - typically more virulent than enveloped viruses - because usually cause host cell lysis - non-enveloped also more stable, can have broader host range
- Enveloped: ex: poliovirus
- Naked: ex: SARS -CoV-2
Enveloped viruses better protected by envelopes under certain conditions - non-enveloped viruses have evolved more resistant protein capsids to environmental stressors - better suited to survive and persist in a variety of environments
covid naked or enveloped??
Explain what are the functions, sources and components of viral envelope
Envelope functions:
- protecting the RNA / DNA
- evading immune system recognition
- facilitating virus entry
Envelope sources - acquired when exiting:
- host plasma membrane
- host nuclear membrane
- host ER
Envelope components:
- phospholipids
- proteins
- glycoprotein spikes (+/- could have or not)
What are the possible variations of ssRNA in viruses?
ssRNA in viral genomes can be:
- positive (+) sense: coding - can be directly translated into protein
- negative (-) sense: will need to be copied into pos (+) to be translated into protein
What is the Baltimore classification of viruses?
Baltimore classification - classification of viruses into families based on thier structure, genomic and replication properties
(+)ssRNA -> (-)ssRNA -> +mRNA
(-)ssRNA -> +mRNA
What are the names of the six Baltimore viral classes?
I. Herpesvirus
II. Parvovirus
III. Reovirus
IV. Picornavirus
V. Influenza
VI. Retrovirus
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How are viruses cultured in labs?
Viruses are grown in tissue cultures - tissue cells - their hosts
Ex:
- human fibroblasts
- mouse 3T3 fibroblasts
- human HeLa epithelial cells
- fertilised chicken eggs
What technique is used for virus quantification?
- Plaque assay - if virus forms plaques
- Cytopathic effect - if virus doesn’t form plaques
What is the plaque assay used for?
Viral quantification if virus forms plaques:
plaque count * 1/(dilution factor) = plaque forming units (PFU) per unit volume of inoculate
What is cytopathic effect (CPE) used for?
Viral quantification if virus doesn’t form plaques:
viral invasion induces structural changes in host cells - by observing different dilutions of virus in known cell conc - determine where tissue culture infectious dose is 50% (TCID50) - quantity of virus needed to produce signs of infection in 50% of cultures
What is a one step growth curve?
One step growth curve - curve used to make determinations about the life cycle of a virus in a particular host
What is the general sequence of events in a viral life cycle?
- Adsorption (attachment)
- Entry
- Uncoating
- Replication and transcription
- Synthesis of viral components
- Assembly
- Release (and maturation)
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How do viruses recognise their hosts?
They recognise host cell surface components - each virus has specific receptors for their specific host surface components (proteins, glycoproteins, etc)
Explain how viral adsorption to host cell occurs
Adsorption:
1) Random collision of virus and host cell
2) Interactions of specific host cell surface components and viral receptors occurs (tropism, host range) - some viruses may use several host cell receptors
3) Not all cells recognised as potential host cells, can be successfully invaded
Most neutralising antibodies are specific for virion attachment points
receptors on viruses or on host cells - or no matter how you call it
What are the receptors on host cell for Influenza virus?
Influenza virus ligands bind to sialic acid - receptor on host cell surface
Viruses bind with ligands to host cell surface receptors
What are the natural species barriers in viral infections?
Natural species barriers - pathogens specific to species - infectious to one but not other - genetic resistance
Ex: avian and human influenza viruses
Explain viral entry into hosts cells
Entry mechanisms:
- endocytosis
- fusion of virus envelope with cell memebrane
- also injection of genetic material - ex bacteriophages
Explain how viral uncoating occurs in host cells
Uncoating - release of viral genome - cell lysosomes strip off virus protein coat - virion can no longer be detected - eclipse period
- uncoating at plasma membrane
- uncoating within endosomes
- uncoating at nuclear membrane
Explain HIV processes of attachment and entry into a host cell
Attachment:
- SU (gp120) protein attaches to CD4 expressed on host CD4+ T-lymphocyte (Th) - co-receptors required - chemokine receptors
Explain Influenza virus processes of entry and uncoating
- Attachment: hemagglutinin (HA) protein on the surface of the influenza virus (ligand) attaches to sialic acid receptors on the surface of host cells
- Internalization: virus is internalized into host cell by receptor-mediated endocytosis - virus is taken up into a vesicle called an endosome
- Fusion: endosome acidic environment causes conformational change in the HA protein - triggers fusion of the viral envelope with endosomal membrane - viral RNA + protein release into host cell cytoplasm
- Uncoating: viral RNA and associated proteins must be uncoated to initiate replication - first RNA transcribed into complementary RNA (cRNA) because (-)RNA can’t be read by host machienry - viral broight reverse transcriptase used - cRNA - a template to produce more viral RNA + proteins
Explain SARS Cov processes of attachment and entry into a host cell
- Spike protein binding to ACE2
- Internalization through endocytosis
- Fusion of the viral and endosome membranes
- Release of the viral genome into the host cell cytoplasm
- Replication and production of new virus particles
- Release from the host cell to infect new cells
Explain processes of poliovirus entry and uncoating in a host cell
- Poliovirus enters host cells through receptor-mediated endocytosis
2.Uncoating and releases viral genome into the host cell cytoplasm - Viral genome is translated, replicated, and assembled into new virus particles
- Virus particles released from the host cell to infect new cells
Explain adenovirus entry into a host cell
- Adenovirus enters host cells through receptor-mediated endocytosis - escapes endosome into the host cell cytoplasm
- Transported to the nucleus for replication and assembly of new virus particles
- Released from the host cell to infect new cells
Where does viral assembly take place in a host cell?
- cell nucleus
- cytoplasm
- plasma membrane
Assembly may occur together with viral release
Explain the process of viral release form host cells
Release:
- sudden rupture of host cell (non-enveloped viruses)
- gradual budding through cell membrane (enevloped viruses)
Release may occur together with viral assembly
What is special about Influenza virus RNA packing into virions?
2 theories to packing:
- random
- specific - recent data suggests specific
What is HIV maturation?
Viral maturation - transition process from an initial, non-infectious, assembly product to an infectious virion
Explain herpes virus assembly and release
- Herpesviruses enter host cells through receptor-mediated endocytosis
- Replicate their DNA genome in the host cell nucleus, assemble their capsids and envelopes
- Released by budding from the host cell surface
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Explain SARS Cov assembly and release
What is the possible variation in viral genome?
Viral genomes can be:
- dsDNA / ssDNA / dsRNA / ssRNA
- circular / linear
- non-segmented / segmented
What is the possible variation in all existing polymerases? What are their functions?
- DNA dependent DNA polymerase
- DNA dependent RNA polymerase
- RNA dependent RNA polymerase
- RNA dependent DNA polymerase
Why do all RNA viruses encode an RNA dependent RNA polymerase?
RNA viruses themselves must encode RNA dependent RNA pol (RdRP) because host cells only code for DNA pol - can’t provide RNA pol
How do the processes of mRNA generation differ in +ve / -ve strand viruses?
+ve strand (coding) RNA:
- genomic RNA can act as mRNA on cell entry
- translated to produce the pol
-ve strand (non-coding) RNA:
- need to carry pol in virion - to convert -ve strand into mRNA after entry
Explain how do DNA viruses produce proteins inside host cell
- enter the host cell, DNA molecule - transported to nucleus -> replication + transcription into mRNA
- mRNA into cytoplasm - translated into viral protein
- Viral assembly in host cytoplasm
Same info flow as the cell: DNA -> RNA -> protein
What are the different possible viral genomes?
- dsDNA
- ssDNA
- dsRNA
- ssRNA (+)
- ssRNA (-)
- ssRNA with DNA intermediate
Explain how do RNA viruses produce proteins inside host cell
- eukaryotic host cells don’t have RNA dep RNA pol - need to make themselves
-
(+)RNA - as mRNA - into cytoplasm to be translated + into nucleus for replication
(-)RNA + RNA dep RNA pol -> mRNA -> into cytoplasm -> protein
(-)RNA -> (+)RNA - template for transcription - Viral assembly in cytoplasm
Explain how do RNA viruses with DNA intermediate produce proteins inside host cells
Produce proteins inside host cells by reverse transcribing their RNA genome into a DNA intermediate -> integrated into the host cell genome -> serves as a template for transcription and translation of viral mRNA -> used to produce viral proteins
What are the problems that must be overcome by an RNA virus inside eukaryotic host cell?
- Eukaryotic cells don’t have RNA dep RNA pol -> need to make themselves to replicate genome
- Eukaryotic cells don’t encode for 1+ protein from one mRNA - transcriptiona and translation decoupled by the nucleus -> virus has to adapt to produce multiple proteins from single mRNA
What is the strategy employed by poliovirus to produce more than one protein from one RNA?
Poliovirus: polyprotein - single large protein - produced by poliovirus - cutting it to produce different smaller proteins => mRNA used once but many proteins produced
What is the strategy employed by coronavirus to produce more than one protein from one RNA?
Coronavirus: original genomic +ve RNA -> -ve RNA template produced -> Gene I for RNA dep RNA pol + multiple transcripts produced for each protein
What are the possible strategies used by viruses to produce multiple proteins froma a single RNA molecule?
- Produce a polyprotein - cut it (poliovirus)
- Produce multiple -ve RNA transcripts from original RNA for each protein (coronavirus)
- Have a segmented genome (influenza virus)
- Splice mRNA into different transcripts -> different proteins (HIV)
What is the strategy employed by influenza virus to produce more than one protein from one RNA?
Influenza: segmented genome (several molecules instead of one compose the genome) + re-assortment (re-assortment of genomic sequences between different viruses -> zoonotic emergence when non-human virus gets human infection sequence) + alternative splicing (host cell’s machinery used for splicing of single sequences)
What potential problem can occur by genome re-assortment in segmented genome viruses?
Segmented genome can produce new pathogenic strains by re-assortment of viral sequences -> ex: pigs great host for new strain emergence - can be infected by both avian and human viruses - avian virus acquires human infection sequence in re-assortment => new strain of human virus - human contact with pigs -> new virus spread in humans
What is the strategy employed by HIV to produce more than one protein from one RNA?
HIV: retroviruses splice their mRNA transcripts to produce different proteins form same RNA
What are the unique aspects of viruses that are targeted by anti-viral drugs?
Anti-viral drugs can target unique aspects of viral replication - host cells not affected:
- attachment -> drugs - attachment antagonists
- uncoating -> drugs - inhibiting viral uncoating
- DNA / RNA synthesis -> drugs - inhibit DNA / RNA synthesis
- viral maturation -> drugs inhibit viral maturation
What are the specific drug targets of HIV life cycle?
HIV life cycle targets:
- inhibit fusion / entry / uncoating
- reverse transcriptase action - inhibit HIV DNA production
- viral integrase action - inhibit HIV DNA integration in host genome
- viral protease action - inhibit viral maturation inside host cell
Highly active antiretroviral therapy (HAART) - use more than one anti-viral drug to reduce probability of virus acquiring drug resistant mutations for all used drugs
What are the factors affecting viral epidemiologies?
Factors affecting viral epidemiologies:
- mode of transmission
- age
- gender
- ethnic background / country of origin
- travel history
- occupation
- season
- underlying medical conditions (ex: immune sppression)
What are the types of viral transmission routes?
- Horizontal transmission: direct contact / respiratory / contaminated inanimate objects / faecal/oral / insect vector / zoonosis
- Vertical transmission: mother to fetus (ex: HIV)
What can be the results of a viral infection?
- primary infection
- disease (symptomatic / asymptomatic)
- secondary disease (+/-)
What are the main factors influencing the development and outcome of an infection?
Interaction between the host and pathogen affects development + outcome of infection (disease):
- primary host physical barriers
- host’s immunulogical ability to control + eliminate the pathogen
- pathogen’s ability for immune evasion
- pathogen’s virulence factors
- ability of the pathogen to spread in the host
What is the cycle of infection?
Cycle of infection (not viral life cycle):
1. Entry
2. Primary site of replication
3. Spread within the host
4. Secondary site of replication
5. Shedding
6. Transmission
What is the site of entry in the cycle of infection
Entry: physical barriers - mucous membranes / skin:
- respiratory tract
- oral - GI tract
- sexual
- ocular
- percutaneous (infected needles / wounds / animal bites / insect bites)
Explain replication process in the cycle of infection
Replication: replicating itself -> shedding
Explain what is the course of infection of mousepox
- invasion
- Replication in LN
- Spread in bloodstream - primary viremia
- Replication in other organs
- Further spread in bloodstream - secondary viremia
- Skin infection - focal infection -> ulceration
What are the three types of infection?
Categories fo types:
- acute
- chronic
- latent
Explain acute infections
Acute infection: rapid onset of disease - resolved /killed quickly by robust innate immune responses exerted by the host or, instead, may kill the host
Explain chronic infections
Chronic infection: continued presence of infectious virus after primary infection - may include chronic / recurrent disease - prolonged incubation period followed by progressive disease
Explain latent infections
Latent infection: lack of demonstrable infectious virus between episodes of recurrent disease
What are the possible effects of infection to the infected cells?
Infection effects on infected cells:
- abortive infection: restrictive factors block / limit viral replication
- cytopathic effect (cpe): morphological changes of cells
- translation switch off: host cell’s proteins not produced - only viral
- apoptosis
- transformation: immortalisation of cell -> tumours (cancer)
- immune response: PRRs - innate immune response, immune evasion, interferons
How do the cells change in cytopathic effect?
Morphological cell changes in cytopathic effect (cpe):
- cells round up -> detach from grown surface
- cell fusion -> multinucleate syncytia formed
- plaques - necrotic cells
- inclusion bodies formed (virus factories)
How do virus shut off host cell protein translation?
Ways how viruses shut off host cell protein synthesis:
- cleavage of ‘cap-binding complex’ (CBC) -> needed at translation initiation -> no translation of uncapped host -> viral RNA contain IRES -> translated
- host mRNA cap stealing -> viral endonucleases cut off cap - reduced host protein expression -> attach to own mRNA -> translated
IRES - sequence only on viral RNA - allows uncapped translation
Explain what is IRES
Internal ribosome entry site (IRES) - viral sequences that can recruit ribosomes - allow cap-independent translation - used by viruses to exploit host cell machinery
What are the possible mechanisms of apoptosis as an effect of viral infection?
Apoptosis mechanisms:
- intrinsic - mitochondrial pathway: cell injury
- extrinsic - death receptor pathway: receptor-ligand interaction
Explain what is the effect of HPV on host cells
Human papillomavirus (HPV) - disrupts the cell cycle: cancer-causing HPV genes mediate destruction of tumour surpressing proteins: E6 gene -> p53, E7 gene -> pRB => cell cycle uncontrolled - cell division increases - HPV replicates more efficiently
How does the HPV vaccine work?
HPV vaccine:
- recombinant virus-like particles based on HPV capsid protein
- quadravalent (4 strains) / nonavalent (9 strains) vaccines available
At which points of the viral lifecycle can PRRs recognise the virus?
At all stages of the life cycle:
- Entry
- Uncoating
- Replication, transcription
- Assembly, release
What are interferons?
Interferons (IFN) - small molecules - produced as a response to viral infection - major role in inhibiting infection - activate the adaptive immune system + induce neighbouring uninfected cells - anti-viral state
Explain what is a cytokine storm
Cytokine storm - severe immune reaction - body releases too many cytokines into the blood too quickly - overproduction of cytokines
What are the ways in which viruses try to limit the immune response?
Immune modulation strategies used by viruses:
- secrete modulators: cytokine / chemokine mimics and binders, mimics / antagonists of immune signalling
- stealth / latency: express few / no proteins - hard to detect
- Ag hypervariability: adapts and changes to avoid adaptive response
- block adaptive immunity receptors
- inhibit Complement System
- interfere with PRRs
- Block interferons / inflammatory cytokines
