Virology L12-17 Flashcards
What are the general components in a virus?
DNA/RNA genome (ds/ss, linear/circular)
Surface protein
Capsid protein
+/- envelope (lipid bilayer)
What are the characteristics of viruses?
Obligate intracellular parasites
Have specific hosts
Simplest life form
What are virions?
Purified virus particle
What do virions contain?
Numerous copies of one or more different proteins
One or more nucleic acid molecules (genome)
What is the viral genome enclosed in?
A protein shell known as a capsid made up of many identical capsomeres
What is the nucleocapsid?
The genome enclosed by the capsid
What are the 5 basic structural forms of viruses?
Naked icosahedral
Naked helical (no human viruses)
Enveloped icosahedral
Enveloped helical
Complex
What type of viruses can be used to treat cancer?
Oncolytic
What do many viruses cause?
Disease
Death
Economic burden
What is the general cycle of infection of a virus?
Entry
Primary site replication
Spread within host
Shedding
Transmission
What are the 3 modes of virus transmission?
Horizontal transmission
Vertical transmission
Zoonosis
How does respiratory transmission of a virus occur?
Droplets can be inhaled, infect conjunctiva, all onto surface and be transferred
What are examples of viruses spread by faecal-oral route?
Rotaviruses
Hepatitis A, leads to jaundice
Poliovirus, can lead to irreversible muscle paralysis
Which viruses are spread by sexual transmission?
Human immunodeficiency virus (HIV) leading to AIDS
Hepatitis B, leads to liver cirrhosis and liver cancer
Human papilloma virus (HPV), type 6&11 show genital warts and types 19&18 cervical and penile cancer
Herpes simplex virus (HSV), type 2&1 painful blisters, neonatal transmission can lead to fatal infection of brain and liver
Which viruses can be transmitted in urine?
Cytomegalovirus and poliovirus
What is mechanical transmission of viruses?
Blood-borne viruses, transfer blood from one another
e.g. in drug abuse
What is vertical transmission of viruses?
Mother to child transmission
Primary infection of mother while pregnant or reactivation of infection
What are examples of vertical transmission of viruses?
Transplacental: rubella, congenital defects
During birth: HSV type 1&2
After birth: HIV-1 in breast milk and Hepatitis B in saliva
What is zoonosis?
Animal to man transmission
Animal virus replicates in is reservoir and animal transmits virus is the vector
What are the 6 steps of virus multiplication?
Attachment, penetration, uncaring, biosynthesis, assembly and release
What is biosynthesis?
Genome replication, mRNA synthesis and translation
What is required for biosynthesis?
Host ribosomes, enzymes and precursors
What is the process of attachment of the virus to the host?
(e.g. HIV) Diffusion, low affinity receptor (heparin sulphate) , primary receptor (CD4) and coreceptors (CCR5/CXCR4)
When are the coreceptors CCR5 and CXCR4 used in HIV infection?
CCR5: macrophage and some CD4+T cells early infection
CXCR4: most CD4+T cells late infection
What are the different types of internalisation methods?
Fusion from without (genome and proteins only enter cytoplasm)
Receptor mediated endocytosis (enters as vesicle to fuse then be released)
How are RNA virus genomes replicated?
They have to be converted from RNA to DNA using reverse transcriptase
What is the function of the proteins encoded by the virus genome?
Replication of the genome
Package the genome into virus particles
Alter the metabolism of the infected cell
Which proteins are packaged into virus particles?
Structural proteins
How are post-translation modifications used to benefit viral proteins?
A single large viral poly protein has to be post-translationally cleaved to release individual viral proteins
How can viruses be released from the host?
By cell lysis and budding (plasma membrane into lumen of ER)
How can viruses spread from cell to cell?
Via pores between cells and by inducing fusion of they membranes
What is the process of localised and generalised viral infections?
Infection
Primary site
Localised replication (shedding)
Lymph node and blood (primary viremia)
Secondary sites (liver, bone marrow spleen, blood vessel endothelium)
Blood (secondary viremia) (shedding HepB)
Target organs (lungs, skin, brain)
What are the different types of bacteriophage?
ssDNA - microviridae, inoviridae
dsDNA - Myoviridae, Siphoviridae, Podoviridae, Corticoviridae, Fuselloviridae, Tectiviridae, Plasmaviridae, Lipothrixviridae, Rudiviridae
ssRNA - Leviviridae
dsRNA - Cystoviridae
What are the features of the M13 filamentous bacteriophage?
Circular ssDNA
Cloning and DNA-sequencing
Released without lysing host cell
‘Budding’ makes own channel
What happens in the lytic cycle of a bacteriophage?
Phage injects DNA into goat
Host genome is fragmented and phage proteins are made
Phage components (head, collar and tail) are made
Phages are assembled and released
What happens in the lysogenic cycle of the bacteriophage?
Phage inserts DNA
Phage DNA taken up into host plasmid DNA
Both DNA gets photocopied
2 Plasmids made
Cell replicated
Why are bacteriophages important?
Can turn bacteria virulent
Alternative to antibiotics
Tool in lab (Recombinant DNA tech)
How can HIV be transmitted?
Sexual: homo & heterosexual
Mechanical: Needles/tatto/piercing, surgery/blood transfusion
Vertical: prenatal, perinatal and post-natal
How can information about HIV help reduce cases?
Education: protected sex and needle hygiene
Drugs: triple therapy
How did HIV originate?
SIV- Simian immunodeficiency virus
What happens in the cellular response to HIV-1?
CD4 T cells activated which activates:
CD8 cytotoxic T cells
B cells (and antibodies)
CD4 T cells produce NFκB which allows transcription of the virus
What are the opportunistic infections associated with HIV-1?
Pneumocytis so.
TB
Malaria
Candida sp. (thrush)
Toxoplasma sp.
HPV
Which viruses are reactivated HIV?
Varicella zoster virus (Shingles)
Epstein Barr Virus (B-cell lymphomas)
Cytomegalovirus (Lymphomas)
HSV-8 (Kaposi’s sarcoma)
What are the challenges of using vaccination to prevent HIV?
Mutation rate
Mucosal infection (subcutaneous vaccine)
No good animal model
What are examples of anti-HIV therapies?
Triple therapy - highly active anti-retroviral therapy (HAART)
Nucleoside analogues - competitive inhibitor of RT
Peptide analogues - competitive inhibitor of HIV protease
Which viruses are morphologically similar to coronavirus?
Mouse hepatitis virus & infectious bronchitis virus
What are the components of coronaviruses have?
Genome: single strand positive-sense RNA virus
Enveloped vision
Envelope proteins
Internal nucleocapsid
What are the key proteins in the coronavirus?
Spike protein - cellular receptor
M protein - vision assembly
N protein - binds and protects the RNA genome
E protein - Efficient viral release from cell
How are coronaviruses replicated?
Receptor binding
Endocytosis
Translation of genome
Production of viral polymerase complex
Replication of genome, transcription/translation of nested mRNAs
Assembly and release
How is attachment and entry mediated in coronavirus?
Spike protein binds to ACE2 on cell surface
TMPRSS2 cellular protease cleaves spike protein
Viral and cellular membranes fuse
What are severe respiratory diseases have emerged?
Severe acute respiratory syndrome (SARS)
Middle East respiratory syndrome (MERS)
Corona virus disease 2019 (Covid-19)
What components of worldwide collaboration helped end the SARS epidemic?
Improvement in surveillance, virus identification
Drug development
Where did MERS originate?
Jeddah, Saudi Arabia
What are the common symptoms associated with COVID19?
Headache
Runny nose
Sore throat
Skin rashes
What are the risk factors for severe illness and mortality?
Age
Cardiovascular disease
Diabetes
Chronic lung diseases
Hypertension
BAME ethnicity
Chronic kidney disease
Obesity
Smoking
Cancer
What are the complications of COVID19?
Silent hypoxia
Acute respiratory distress syndrome (ARDS)
Sepsis
Secondary infections
What are the components of lateral flow tests?
Quick
Sensitivity, 50% reliable
Multiple tests, confidence in negative result
What therapeutics can be used for SARS-CoV-2?
Nirmatre/ritonavir - viral protease inhibitors
Baricitinib - immunosuppresive
Which vaccines are approved?
Moderna
Pfizer
AstraZeneca
Janssen
What immune response do the vaccines elicit?
Activation in lymph node
Role of dendritic cells
TLR activated by foreign mRNA
Cytokine profile
Spike protein production
T/B cell activation
What are the useful properties of viruses that we can exploit?
Natural gene-delivery vehicle
Trigger an immune response
Cytopathic effect
What are the main useful properties of viruses?
Easy to modify/manipulate
Target delivery to specific cells
Powerful gene expression
Carry foreign protein/genes
Remove the components that make a virus harmful
What does it mean when a virus is attenuated?
Human viruses have lost the properties to replicated efficiently in human cells (do not cause disease)
Which parts of virus gene expression do we want to exploit?
Human cytomegalovirus (HCMV) promotors/enhancers
Simian virus 40 (SV40)
Human immunodeficiency virus (HIV) long terminal repeats (LTR)
How can you use viruses to express genes?
Isolate ORF of interest
Clone into expression vector
Isolate/purify plasmid DNA
Transfect target cells & detect gene expression
What is the process of transfection?
It is the process of getting DNA in a cell
Using: calcium phosphate, liposomes
Why are viruses used as vectors for gene expression?
More efficient delivery
Uses own virus entry mechanism
Infects hard to transfect cell lines
What are the advantages and disadvantages of using adenoviral vectors for gene expression?
Advantages:
Small genome
Infect many cell types, high gene expression
Disadvantages:
Limited to ORFs<8kb
Immunogenic
Transient expression (on/off)
How can adenoviruses be used as gene vectors?
Amplify virus vectors
High yields
Hit nearly all cells
Modify promotor for cell type specific gene expression
What is needed to use adenoviruses as gene vectors?
E1 gene region (essential for replication)
Viral proteins
‘Helper’ or ‘Packaging’ cell-line
What are the only components from lentiviral vectors that are used in gene expression?
Only use LTRs (contain promoters and polo signals)
What are advantages and disadvantages of lentiviruses?
Advantages: Small genome
Infect many cell types
Infect/express in non-dividing cells
Stable gene expression over time
Disadvantages: Limited to ORFs <10kb
Safety concerned if transferee inserts near proto-oncogene
Constitutive expression
What are the generalities with vaccination?
Induces primary immune response (memory B & T cells)
Following exposure leads to rapid immune response
What happens when first vaccinated?
1st response weak, slow
Generates memory B & T cells
Adaptive immune response
System learns from each exposure
What are advantages and disadvantages of attenuated vaccines?
Advantages: Mimic wild type infection
Strong humoral and cellular responses
Induces innate immunity
Fewer doses required
Disadvantages: Reversion can occur
Cannot be used in immunosuppressed individuals
Required careful handling and storage
Why can’t a live vaccine always be made?
Virus cannot be cultured
Virus cannot be attenuated to safe level
What is the principle with subunit vaccines?
Vaccinate with immunogenic viral proteins rather than whole virus
How are recombinant viruses made?
Gene ligated from pathogenic virus to live virus using a vector producing live recombinant viral vaccine
What is gene therapy?
Replacement of deflective gene function by introducing a normal gene
What are the different approaches to gene therapy?
Ex vivo: cells removed from body, gene introduced and returned
In vivo: replacement gene introduced directly into the body via gene vector/virus
What is CAR-T gene therapy?
Chimeric T-cell receptors
Genetically modified T cells targeting cancer cells
Isolate T cells from cancer patient
Modified with lentiviral vector
Modified T cells re-introduced into patient
T cells now target cancer
Why are viral vectors used for gene therapy?
Target specific cells
Protects nucleic acid
Delivers genes into cytoplasm/nucleus
Gene expression may be transient or permanent
What is a disadvantage when using viral vectors for gene therapy?
Prior exposure may mean immunity attacking the virus
What is oncolytic virotherapy?
Use of a lytic virus to destroy targets
What is the difference between oncolytic and oncogenic viruses?
Oncolytic: destroy tumour cells
Oncogenic: promote development of tumour cells
What are the pros and cons of oncolytic virotherapy?
Pros: Combination with other cancer treatments (synergistic interactions)
Engineering and arming (efficacy)
Excellent safety profile
Dual mode of action (direct oncolysis and immune-mediated anticancer effect)
Alternative killing mechanisms
Cons: Resistant cancer cells
Antiviral immune responses
Limited replication and spreading
What are the advantages and disadvantages of phage therapy?
Advantages: specific to bacteria
Evolve and adapt to resistance
Self amplifying until bacteria are dead
Disadvantage: Lysis of gram-ive bacteria releases endotoxins
Theoretical potential to transfer DNA between bacteria
May need cocktail
Individualised
What are lysins/ endolysins?
Enzymes produced by some bacteriophages to cleave host cell wall during final stage of lytic cycle
How can viruses be counted and detected in labs?
Cell based assays:
Plaque assay
Tissue culture infection dose assay
Protein based assays:
Hemagglutination assay
Electron microscopy
Immunofluorescence
Enzyme linked immunosorbent assay (ELISA)
Why is detection and quantity of viruses important in medicine?
For diagnosis: what it is, genotyping, determination of resistance
Prognosis: viral load, genotype determines treatment options
Why is detection and quantity of viruses important in research?
Measure the amount of virus in stock solution
infect cells in tissue culture with known number of particles (multiplicity of infection m.o.i)
drug discovery tells the effectiveness of new drug
What are the steps to grow viruses in cells?
Step 1: grow cells in tissue culture
Step 2: infect cells with virus
Step 3: Incubate infected cells and observe for viral infection
Step 4: Harvest cells
What is CPE?
CytoPathic Effect
What is multiplicity of infection (m.o.i)?
The number of infectious viral particles used to infect 1 cell
(not all cells infected)
What does it mean if there is an m.o.i of 1?
There is a high probability of many uninfected cells
Why is m.o.i important?
May want all cells infected
Synchronise infections
Some viruses act differently
Viability of stocks
Studying cellular response to infection
What is the protocol of a plaque assay?
Perform serial dilution
Add cells to appropriate volume
Leave to absorb
Remove inoculum
Add fresh media containing agar
Overlay medium to keep moist
Wait
Fix and stain
What do the number of plaques show in a plaque assay?
Allow the calculation of the concentration of viable virus particles in the undiluted stock solution
What values are needed to find the concentration of viable virus particles in undiluted stock?
Dilution factor
Amount of diluted stock added to plate
Number of plaques produced
What are the limitation of plaque assay?
Virus must cause visible cytopathic effect, time required can be significant, sterility must be maintained
What happens in focus forming assay (FFU)?
Variation of plaque assay
After 24 hrs can fix immunostain for virus with fluorescence antibody
What is an end-point dilution assay?
A relatively simple assay
Sequential dilution of virus stock in micro-titre format
Allows multiple replicants of each dilution
Count plaques under microscope
What is tissue culture infectious dose 50?
TCID50 represents virus concentration that kills 50% of cells in culture
Infectious titre
End point dilution assay
What is the method of TCID50?
Grow cells in 96 well plate
Serial dilution across place
Leave to infect and kill cells
Score wells as infected (+/-)
What does TCID50 mean?
Each TCID50 unit can infect 1 out of 2 cells
Where is TCID50 used?
Assay for drug development
Allows non-plaguing virus to be quantified
What are the disadvantages of TCID50?
Time consuming
Medium changed regularly
Prone to drying out
Sterility due to length of incubation
How can electron microscopes be used to quantify viruses?
Best to identify virus particles
Physically count particles
Compare pfu
Get a measure of particle to infectivity ratio
What are the features of haemagglutination protein assays?
Measurement of viable and no-viable virus particles
Generic effect
Ability of virus to cross link RBCs
Relative quantification
To get absolute quantification, compare results to standard virus suspension containing known number virus particles per ml
What happens in a haemagglutination assay?
Many enveloped viruses agglutinate in RBCs
Glycoprotein on surface of virus binds to glycoprotein on surface of RBC
How do immunofluorescence assays work?
Add virus (specific antibody) and wash cells to remove unbound atibody
Add FITC (fluorescein isothiocyanate) conjugated anti-rabbit IgG antibody, wash any unbound, examine cells using UV microscope
When are immunofluorescence assays used?
To stain virus antigens on cell surface or in sections of virus-infected host cells
How are antigens detected in ELISA?
Monitoring HRP (horseradish peroxidase)
OPD - amber detects HRP
TMB - blue detects HRP, yellow when acid added
ABTS - green detects HRP
What is the process of ELISA testing?
Antigen added
Anti-virus antibody added
Interaction between antibodies
Substrate added
Interaction between antibody and substrate
Incubation
Plate is read
Where is ELISA used?
In clinics: p24 antigen assay- HIV
Related to viral titres using standards
