Intro Virology and Replication Flashcards
(34 cards)
1
Q
What is a Virus?
A
- Obligate intracellular parasite- needs host cell to survive (in order to replicate & complete its life cycle)
- Lacks organelles- no mitochondria/ energy source etc
- Extremely small - ‘filterable agents’- Need electron microscope to visualise
2
Q
Virion
(an entire virus particle)
A
- In its most simple form comprises of:
- Nucleic acid- RNA/DNA (confers infectivitity)
- A capsid (confers specificity)- structure & symmetry of this is important.
- Additional features may include:
- An envelope- Host derived lipid bilayer contains virus encoded glycoproteins
-Viral enzymes- incorporated into the envelope/ within the capsid
3
Q
DNA Viruses
A
- All monopartite (all viral genes on a single molecule)
- All ds (except parvoviridae and circoviridae)
4
Q
RNA Viruses
A
- Mostly single stranded & can be segmented
- Segmentation- allows virus to ↑ its diversity very rapidly (reassortment)
- Need an RNA polymerase to copy their RNA genome (no equivalent enzyme in the host)
- RNA dependent RNA polymerase
- RNA polymerases are error prone- No proof reading capability, as a consequence of this:
- RNA viruses are more variable:
- Within a species of virus are more subtypes/serotypes
- Often zoonotic (jump from animals to humans and cause disease)
- Can evolve rapidly if needed
- If a virus jumps from one species to another, RNA viruses can more readily adapt
5
Q
Capsids
(Structure)
A
Viral capsids: enclose the nucleic acid & have 3 forms of symmetry
1. Icosahedral-e.g.parvoviridae
- 12 vertices, 20 facets, 5:3:2 fold symmetry
- Composed of capsomers:
- *-Penton Capsomer**- 12 present, one at each vertex,has 5 capsid proteins
- *-Hexon Capsomer**- Composed of 6 capsid proteins
2. Helical Capsid-e.g.paramyxoviridae & rhabdoviridae
- Structural unit is one capsid protein- arranged as a helix
- All animal viruses with helical symmetry are enveloped
3. Complex Capsid-e.g.poxviruses
- Some of the large viruses have structures that are more complex
- >100 proteins -encode more proteins than other viruses
- Neither helical or icosahedral structure
6
Q
Enveloped VIrus
A
- Few viruses with icosahedral capsid
- All viruses with helical capsid
- Complex capsid
7
Q
Naked Virus
A
- Icosahedral Capsid
8
Q
Viral Envelope
A
Many acquire an envelope as they bud through the host cell membranes
- Host membrane = lipid bilayer- ‘coating ‘is effectively inert
- BUT would not permit recognition of receptor molecules on the host cell
- SO viruses modify the envelope by synthesis of viral encoded proteins which are associated with the envelope
9
Q
Biological Properties of Enveloped Viruses
A
- More pleomorphic (not a regular shape)
- More fragile than viruses with just a capsid
- More easily destroyed by: detergents, disinfectant & outside environment
10
Q
Naked Capsids
A
- Components: Protein
- Properties: Environmentally stable to:
- Temperature, pH, Porteases, Detergents, Drying, Released by cell lysis
- Consequences:
- Can be spread easily
- Can dry out and retain infectivity
- Can survive adverse conditions in the gut
- Resisitant to detergents
- Lyses cell to release; usually cause acute infections
11
Q
Enveloped Viruses
A
- Components: lipids, porteins, glyoproteins
- Prorperties: evironmentally labi
- destroyed by: acid, detergents, drying, heat, released by budding
- Consequences:
- Not easily spread (large droplets, secretions, transplants/transfusions)
- Must stay wet
- Cannot survive in the GIT
- Easilty destroyed by detergent
- Does not need to kill the cell to spread; can cause persistent infections
12
Q
Baltimore Classification System
A
classified by the mechanism of generating positive strand mRNAs
- Seven fundamentally different groups
13
Q
Hierarchical VIrus Classification System
A
classified according to characteristics:
- Presence or absence of viral envelope
- Capsid symmetry
- Size and shape
- Genome composition, polarity and structure
- Virus Taxonomy:
- Order (-virales)
- Example: family (-viridae)- Flaviridae, genus (- virus)- Pestivirus, species – Bovine viral diarrhoea virus 1
- Divided into genera containing species that further subdivide into serotypes and then subtypes
14
Q
Antigenicity
A
- Viruses can be differentiated on basis of antigenic sites on their surface = “serotypes”
- Viruses can be divided into “serotypes” and further sub grouped into subtypes
- Classified on the basis of their reactivity with antibody (serological reactivity)
15
Q
What viral proteins determine “serotype”
A
- Generally proteins on the virus surface that are involved in Virus entry & Antibody reactions
- In different serotypes, these proteins tend to vary in their precise amino acid composition–>immune system recognizes these proteins as slightly different
16
Q
Virulence
A
- Viruses can be divided into pathogenic (virulent) and subclinical (avirulent- low pathogenic)
- isolates e.g. avian influenza virus and feline infectious peritonitis virus have both
17
Q
Nucleotide Sequencing
A
- Viruses can be divided into genotypes depending on the nucleotide sequence of their genes
- e.g. BVDV1a ‐m, BVDV2a‐c
18
Q
Capsid Proteins
(structural proteins)
A
- Structural components of the virus capsid (icosahedral, helical and complex)
- Protect the viral nucleic acid
19
Q
Capsid Proteins of Naked Viruses
A
- Deliver the viral nucleic acid to the cell
- Receptors to attach to host cell
- Protein domains that fuse with the host membrane to allow entry
- Assembly‐capsid proteins self assembles into capsids during virus replication
- NB ‐contain sites that will induce an antibody response
20
Q
Envelope Proteins
A
- Structural components of the virus, often glycosylated
- Embedded in a lipid bilayer, derived from host membrane (virus envelope)
- Contain receptors that allows the virus to attach and then enter the host cell
- Two step process
- Attachment/binding
- Fusion
- Targets of the host immune response
- Antibodies will recognise these surface exposed viral proteins (antigenic determinants)
- Antibodies often protective (neutralising)
- Virion Associated Enzymes: the virion contains an enzyme (RNA-dependent-DNA polymerase or reverse transcriptase) which uses this RNA as a template for the synthesis of double stranded DNA which integrates into the cell DNA
21
Q
Non-structural proteins
A
- Are not structural components of the virus particle
- Often enzymes involved in viral replication Proteases; helicases; RdRpol(RNA dep RNA polymerase)
- Made in the virus infected cell following infection: Often proteins involved in transcription, replication & protein cleavage
22
Q
Viral Attachment
A
- Viral surface proteins bind to receptor on cell surface
- Receptor is cellular protein that happens to fit viral protein
- Virus-receptor interaction determines specificity of viruses for cells and tissues (no receptor–> no entry)
23
Q
Viral Entry
A
Two possible routes:
- Endocytosis: virus is released from endosome by pH change or fusion of viral envelope with endosomal membrane
- Some enveloped viruses fuse directly with the plasma Membrane
24
Q
VIral Uncoating
A
Release of viral nucleic acid from viral capsid
- Process is variable: In some viruses, nucleic acids is still in a nucleoprotein complex,
- In other viruses the capsid is only partially disintegrated
25
Viral Gene Transcription
* Viruses use different strategies to transcribe mRNA depending on their genome structure:
- RNA or DNA
- Single stranded or double stranded
- (ssRNA) Plus or minus sense RNA
**_DNA viruses_**
- mRNA transcription in the nucleus (few exceptions) _using cellular RNA polymerase_
- Transport of mRNA to ribosomes in cytoplasm for translation
**_RNA viruses_**
- Cells do not possess enzyme for transcription of RNA from RNA template
- RNA Viruses need to use their own enzymes to make messenger RNA (RdRp)
- **Most RNA viruses don’t need to enter the nucleus & so remain in the cytoplasm for replication**
26
Translation of viral mRNA into proteins
***Synthesis of viral proteins at ribosomes in the cytoplasm***
* **Early proteins (non‐structural)**:
- Regulate viral transcription and replication
- Shut down host cell processes
* **Late proteins (structural):**
- E.g. capsid and surface proteins
27
Assembly
* ***Packaging of new genomes with viral proteins to form new particles***
28
Viral Release
***Three different mechanisms of release:***
* **Budding from plasma membrane**
- Enveloped viruses need to acquire envelope by budding
- Occurs at plasma membrane or at internal membrane (nucleus/ E.R.)
* **Exocytosis**
* **Lysis** of cells due to toxic effect- Naked viruses are released by this method- can lead to some acute effects
29
VIrus Culture Overview
* Study/research
* Vaccine production- rinderpest eradicated by vaccine & small pox
* Virus as a tool –viral vectors/ ‘protein factories’
* Viruses require living cells for replication:
- **Experimental animals**: natural host/ lab animal. Expensive & has ethical issues so only really used to culture viruses that don’t work well in other systems/ attenuation of viruses
- **Fertilised hen ́s eggs**: Use embryonated egg (~10 days), sterile inoculation through hole in shell, egg provides a nutrient rich environment for growth of viruses- e.g. influenza virus
- **Cell culture**: Use appropriate cell cultures, derived from tissue samples (kidney, liver, skin.) Advantage: easy to handle, easy to scale up
30
Cell Cultures
**_Preparation of cells for culture_**
* Take **tissue sample** -STERILE --\> Cut tissue into very small pieces --\> Treat with **enzymes** (trypsin) to break up tissue further and to obtain individual cells--\> Put cells into **culture flasks** with cell culture medium (salt solution/ glucose/ growth factors etc)
**_Cell culture maintenance_**
Cells eventually cover whole growth area of flask--\> Treatment with trypsin detaches cells from plastic--\> Cells are transferred to new flasks = passaging
* **Primary cell cultures**- *Cells freshly prepared from tissue sample*
- Can survive for about 10‐15 passages before differentiation prevents further cell division
* **Continuous cell lines**- *Immortalised cells that continue to grow*
* Often derived from tumours (HeLa cells)
* Most widely used for virus culture
* Disadvantage: loss of receptors
31
Isolation of Viruses from clinical cases
* Sample: nasal swab, faeces, tissue from post mortem case
* Keep on ice or freeze for long‐term storage
* Suspend sample in cell culture medium with antibiotics
* Inoculate cell cultures and wait for changes
32
Cytopathic Effects
| (CPE)
***Visible changes to cells following virus infection***
* Cells round up and detach from the flask --\> **holes appear in cell layer**
* **Syncytia formation**: cells start to fuse creating very large cells with several nuclei, eventually cell death
* **Inclusion bodies** (masses of viral proteins within the cell)- within nucleus/ cytoplasm
33
Non-cytopathic Viruses
***No visible signs of virus replication but viral particles are produced***
* Can result in undetected contamination of cell lines e.g. IBR vaccine (modified live virus) with BVD virus
34
Infective Assays
* To **quantify number of infectious virus particles**-made in cell culture/ egg/ sample from infected animal
* **10 fold dilutions** of sample/virus stock are prepared
* Inoculate cell cultures (several replicates of each dilution)
* TCID50 (50% Tissue Culture Infective Dose) –endpoint dilution assay
* **Plaque Assays**: _Count plaques_ –calculate pfu/ml = virus titre
- Plaque forming units (pfu): using an agar overlay over infected plates, each virus forms a plaque (of lysed cells)
