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Microbiology & Immunology > 30-31 - Viruses As Research Tools and Assay of Animal Viruses > Flashcards

30-31 - Viruses As Research Tools and Assay of Animal Viruses Flashcards

1
Q

Name some of the useful properties of viruses which we can exploit…

A
  • Trigger immune response
  • Natural gene delivery vehicles
  • Cytopathic (cell killing) effects
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2
Q

An early example of viruses used in medicine is…

A

Viral vaccines

(Edward Jenner using cowpox to innoculated against smallpox)

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3
Q

Types of vaccines in general use (4)…

A
  • Live heterologous (cowpox - smallpox)
  • Live attenuated (weakened, MMR)
  • Killed whole virus (Salk vaccine for polio)
  • Subunit (purified from virus (Influenza A) or recombinant proteins (HBV, HPV))
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4
Q

Describe host range attenuation (4)…

A
  1. Virus isolated and cultured on human cells
  2. Incubated on cells from a new host (e.g. monkey) 🙊
  3. Spontaneous mutation allows viral growth on monkey cells
  4. This virus can be used as a vaccine as it can’t grow on human cells
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5
Q

The disadvantage of killed and subunit vaccines is that they…

A

Only induce a humoral (antibody) response, when cell mediated response is often critical to an anti-viral immune response

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6
Q

The advantage of live vaccines is that they elicit…

A

Both humoral (antibody) and cell-mediated immune response

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7
Q

Give two reasons why it may not be possible to make a live vaccine for a particular virus…

A
  • It can’t be grown in culture
  • It cant be attenuated to a safe level
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8
Q

A potential solution to the problems associated with live vaccines and killed/subunit vaccines is…

A

Live recombinant viral vaccines

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9
Q

To create a live recombinant viral vaccine, the immunogenic gene from the pathogenic virus is…

A

Ligated into the genome of an existing viral vector

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10
Q

Give examples of applications of viral vectors in research (In vitro and In vivo)

A
  • In vitro
    • Investigation of protein function
      • Express a protein not normally expressed
      • siRNA delivery to block gene expression
  • In vivo
    • To create transgenic animals 🐁
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11
Q

List 5 commonly used viral vectors…

A
  1. Bacteriophage
  2. Baculovirus
  3. Adenovirus**
  4. Pox virus (Vaccinia)*
  5. Retroviruses*

*Mammalian host cells

**Specific human host cells

Bold = safety issues

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12
Q

Other than viral vaccines, give 3 further examples of viruses used as medical tools…

A
  • Gene therapy (delivery of functional gene)
  • Oncolytic therapy (targeting cancer cells)
  • Phage therapy (targeting bacterial cells in infection)
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13
Q

Distinguish betwen ex vivo and in vivo approaches to gene therapy…

A
  • ex vivo (outside body)
    • cells removed from body-> gene introduced -> cells returned
  • in vivo (in body)
    • gene is introduced directly into body via virus or other gene vector
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14
Q

An example of ex vivo gene therapy is…

A

CAR-T (chimeric antigen receptor T cell) therapy

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15
Q

Phage therapy relies on enzymes produced by some bacteriophages, known as…

A

Lysins (or endolysin) which cleaves host cell wall

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16
Q

Oncolytic virotherapy is the use of a…

A

Lytic virus to destroy cancer cells

17
Q

Outline the pros and cons of virotherapy…

A
  • Pros
    • Can be combined w/ other cancer treatments
    • Excellent safety profile
    • Dual action (direct oncolysis and immune-mediated anticancer effect)
    • Alternative cell killing mechanisms - overcome resistance
  • Cons
    • Resistant cancer cells
    • Limited replication and spreading (because tumour cells must be effectively destroyed)
    • Antiviral immune response
18
Q

Oncorine

A

First oncolytic virus approved for clinical use. Genetically modified adenovirus H101 for nasopharyngeal carcinoma

19
Q

How can we detect and count viruses in the lab (2)…

A
  • Cell based assays
  • Protein based assays

(other techniques exists, but not covered in this module)

20
Q

4 Steps to growing viruses in the lab…

A
  1. Grow cells in tissue culture
  2. Infect the cells with virus
  3. Incubate and observe for effects of viral infection
  4. Harvest cells/ medium to measure viable virus particles per ml
21
Q

MOI (multiplicity of infection) ( = or ≠ )particles per cell

A

MOI (multiplicity of infection) ≠ particles per cell

22
Q

The two main types of cell-based assay are…

A
  1. Plaque assay
  2. End-Point Dilution Assay (EPDA)
23
Q

Plaque assays detect infectious virus particles. Each plaque observed represents…

A

1 viable infectious particle (PFU)

24
Q

Limitations of plaque assays…

A
  • The virus used must cause a visual cytopathic effect
  • requires 7+ days
  • requires maintainence of sterility
25
Q

Example of a variation of the plaque assay…

A

Focus Forming Assay (FFU)

  • No agar overlay
  • Immunostain (fluroscent Ig) fixing after 24hrs
  • DNA stain counterstain counts cells
  • Cells and infected cells counted via fluorescent microscopy
26
Q

End-point dilution assay (EPDA) is the second type of cell-based assay. It involves…

A

Sequential dilution of virus stock in a microtitre plate format.

(8 x 12 =96 wells allows multiple copies at each dilution level)

27
Q

Give an example of an EPDA…

A

Tissue Culture Infectious Dose 50 (TCID50)

  • Used in drug development (not clinically)
  • Can identify non-plaquing viruses
  • TCID50 = virus concentration capable of killing 50% of cells in culture
28
Q

List the limitations of Tissue Culture Infectious Dose 50 (TCID50) end-point dilution assays…

A
  • Time consuming
  • Labour intesive (medium must be regularly changed)
  • Prone to drying out
  • Sterility must be maintained
29
Q

List the 3 protein or biochemical-based assays…

A
  1. Electron microscopy
  2. Haemagglutination
  3. Immunofluorescence
30
Q

Electron microscopy (either TEM or SEM) allows for identification of virus particles, but not…

A

Infectivity

31
Q

Haemaglglutination measures ______ & ________ virus particles via a simple biochemical reaction.

A

Haemaglglutination measures viable & non-viable virus particles via a simple biochemical reaction.

32
Q

Haemogglutination relies on the ability of viruses to ______________ , which results in a visual difference between wells (see image).

A

Haemogglutination relies on the ability of viruses to cross-link RBCs , which results in a visual difference between wells (see image).

33
Q

Haemagglutination is ( specific / non-specific )

A

Haemagglutination is non-specific

34
Q

Immunofluorescence is used to stain _______ on cell surface or sections of infected cells

A

Immunofluorescence is used to stain viral antigens on cell surface or sections of infected cells

35
Q

Outline the 3 steps of immunofluoresence…

A
  1. add virus-specific antibody*
  2. add FITC (fluorescent) secondary antibody*
  3. identify fluorescent loci with UV microscope

*and wash to remove unbound antibodies

36
Q

ELISA stands for…

A

Enzyme Linked ImmunoSorbent Assay (and is a form of immunofluorescence)

Microbiology & Immunology (17 decks)

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