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Principle of molcular virology > Lecture 1 - Introduction > Flashcards

Lecture 1 - Introduction Flashcards

1
Q

Why are viruses described as pseudo-living?

A

They are non-living particles with some chemical characteristics similar to those of life

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2
Q

What is the baltimore system?

A

A system of specification of viruses, where viruses are placed into one of seven groups based on their nucleic acid structure, strandedness, sense and method of replication

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3
Q

Who is Rameses? (1100BC)

A

First recorded smallpox death

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4
Q

Why would we say that viruses are the most abundant mircobe on the human body?

A

Far more bacterial cells on human body than actual human cells, however viruses can infect both mammalian cells and bacterial cells, so viruses will be present on the bacteria on the human body (can also infect plant and insect cells)

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5
Q

How are viruses split into a life tree?

A

The microbial world is split into living organisms (including prokaryotes (algae, bacteria), eukaryotes (including fungi, helminths, protists (algae, protozoa)) and infectious, non-living agents (Viruses and prions/Viroids)

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6
Q

What is the definition of life?

A

Living things tend to be complex, highly organised, able to take in energy from the environment and transform it for growth and reproduction. Respond to stimuli in the environment and are reproducible, allowing evolution to function through mutation and natural selection.#
By definition, viruses are not alive - have no manner of transforming their own energy

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7
Q

What is the definition of a virus?

A

Ultramicroscopic microbes (20-400nm in diameter)
Metabolically inert infectious agents that replicate only within the cells of living hosts (bacteria, plants, animals, insects) and are therefore obligate parasites
Has a capsid
Does not have ribosomes
The ability to disintegrate and reconstitute itself

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8
Q

What is the basic structure of a virus?

A

Composed on an RNA/DNA core, protein coat (capsid) and in complex types, a surrounding envelope

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9
Q

What is Edward Jenner’s contribution to the study of viruses?

A

(1749-1823)
Father of vaccinations as the pioneer of the smallpox vaccination
Inserted pus from a cowpox pustule into a inscision on a boys arm, testing a theory based on folklore that milkmaids who suffered from cowpox never contracted smallpox. Having been innoculated with cowpox the boy was immune to smallpox

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10
Q

What is Louis Pasteur’s contribution to the study of viruses?

A

(1822-1895)
Left culture of bacteria accidentally outside of the fridge overnight, bacteria were injected into chickens and fell ill but did not die as expected. The experiment was repeated with cholera injections and it was found that the chickens were resistant to the disease. Led to the creation of vaccines in the lab using dead/inactive bacteria, as deactivated viruses/bacteria have the ability to stimulate the immune system

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11
Q

What was Robert Koch’s contribution to the study of viruses?

A

(1843 - 1910)
Developed an experimental method to test whether a particular microorganism is the cause of a disease. Took bacterium from a sheep that had died of anthrax, grew it and injected it into a mouse, which then also developed the disease. Repeated in over 20 generations to prove causative agent.

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12
Q

What are the basic function of viruses?

A
  1. Introduced into the population (e.g. influenza may begin in birds and enter the human population aka bird flu)
  2. Infectious - viruses spread cell-cell, host-host
  3. Escape the body’s defenses
  4. Pathogenic, capable of causing disease
  5. Major cause of death and attract publicity
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13
Q

How might a virus have developed traits to escape the immune system?

A
  1. May change rapidly to prevent becoming recognised by the host immune system. Involves a fitness/cost playoff
  2. Viruses may be ancient and have developed traits that allow the safe escape from the immune system
  3. May be highly infectious but not good at escaping the immune system and so are able to infect another host before the immune response kicks in
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14
Q

What is the purpose of Koch’s postulates?

A

Must be fulfilled to categorically prove whether a particular organism is the causative agent of disease

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15
Q

What are Koch’s postulates?

A
  1. Organism must be present in every case of the disease
  2. Organism must be isolated from a host containing the disease and grown in pure culture
  3. Samples of the organism must be taken from the pure culture and must cause the same disease when innoculated into a healthy susceptible animal in the lab
  4. Organism must be reisolated from the innoculated individual and identified as the same original organism that first originated from the diseased host
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16
Q

In what cases are Koch’s postulates not able to be fulfilled?

A

In some countries HIV is not stated as the cause of AIDS. HIV is human specific and infecting a human with the disease would not be ethical. However has been proven accidentally when those working with HIV in labs have been infected.

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17
Q

Viruses are a major cause of death worldwide - what is this dependent on?

A

A variety of factors including variations in the microbial agent, the host and the environment, and the interplay of these three.

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18
Q

How might a virus vary in ways which would affect its ability to cause death and disease?

A
  • genetic adaptation and change

- polymicrobial diseases

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19
Q

How might the environment affect the ability of viruses to cause death and disease?

A
  • climate and weather
  • changing ecosystems
  • economica development and land use
  • technology and industry
  • poverty and social inequality
  • lack of public health services
  • animal populations
  • war and famine
  • lack of political will
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20
Q

How might the host affect the ability of viruses to cause death and disease?

A
  • susceptibility to infection
  • demographics and behaviour
  • international trade and travel
  • intention to harm (bioterrorism)
  • occupational exposures
  • antibiotic use
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21
Q

List three non lethal viruses

A

Orthomyvovirus
Picoravirus
Herpesvirus

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22
Q

List two viruses that remain in the body for different times

A

Influenza, HIV

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23
Q

What is a Class I virus?

A

Double stranded DNA virus

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24
Q

Give an exmaple of a Class I virus

A
  • Herpes virus (Herpes simplex, chicken pos, Epstein-barr virus)
  • Papillmavirus (Warts, cervical cancer)
  • Pox virus (small pox)``
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25
Q

What is a class II virus?

A

Single stranded DNA virus

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26
Q

Give an example of a class II virus

A

Can infect:

  • Vertebrates (Amelloviridae, Circoviridae, and Porvoviridae)
  • Plants (Germiniviridae, Nanoviridae)
  • Prokaryotes (Microviridae)
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27
Q

What is a class III virus?

A

ds RNA virus

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28
Q

Give an example of a class III virus?

A

Rioviridae (reovirus, rotavirus)

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29
Q

What is a class IV virus?

A

ss RNA virus positive sense

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30
Q

Give an example of a class IV virus

A
  • Picornavirus (Polio, Rhinovirus, Hep A & C)

- Flaviviridae (Dengue, west nilevirus)

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31
Q

What is a class V virus?

A

ss RNA virus negative sense

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32
Q

What is a class VI virus?

A

ss RNA virus positive sense that replicates through a DNA intermediate

33
Q

Give an example of a class VI virus?

A

HIV, HTLV

34
Q

What is a class VII virus?

A

ds DNA virus that replicates through a ss RNA intermediate

35
Q

Give an example of a class VII virus?

A

Hep B

36
Q

What are the features of viral shapes

A
  1. Viruses have a capsid layer of protein that protects the genetic material, capsid form is highly repetitive
  2. Have extracellular and intracellular (virion) phases
  3. Vary in shape andsize
  4. typically small but not always the case (smallpox: 200nm, poliovirus 28nm - must be looked at under elctron microscope)
37
Q

What are the features of viral genomes?

A
  • DNA/RNA
  • ss or ds
  • genome smaller than host cell (5000 - 230,000 bp)
38
Q

What are the features of the virion structure?

A
  • vary in shape and chemical composition
  • some surrounded by a membrane (enveloped virus)
  • some are not (naked virus)
39
Q

What structures are capsids made out of?

A

Capsomers

40
Q

How is infection of target cells mediated by attatchment?

A

Proteins or spikes on the surface of the virus binds to host surface molecules (glycoproteins) for attachment to stimulate entry

41
Q

What are the two methods or viral entry into host cell?

A
  1. Membrane fusion

2. Endocytosis

42
Q

What is the process of membrane fusion for the entry of a virus into the host cell?

A
  1. Adsorption - spikes of virion attach to the specific host cell receptors
  2. Membrane fusion - envelope of virus fuses with the plasma membrane
  3. Nucleocapsid is released into the cytoplasm and the viral envelope remains part of the plasma membrane
  4. Nucleic acid separates from capsid coat protein
43
Q

What is the process of endocytosis for the entry of a virus into the host cell?

A
  1. Adsorption of virion to the host cell, initiation of endocytosis
  2. Plasma membrane surrounds the virion, forming a vesicle
  3. Envelope of the virion fuses with the plasma membrane of the host cell and nucleocapsid is released from vesicle
  4. Nucleocapsid is free within the hosts cytoplasm, nucleic acid separates from the capsid protein
44
Q

How must a naked virus enter the host cell?

A

By endocytosis not membrane fusion

45
Q

Define virion

A

The complete, infectious form of a virus outside a host cell with a core of RNA/DNA and a capsid

46
Q

How do class I viruses replicate?

A

dsDNA viruses, e.g. Herpes virus
DNA virus must be transported to the nuleus for replication
1. Viral DNA is transported to the host nucleus
2. Host RNA polymerase converts viral DNA to viral mRNA in the nucleus that is then transported to the cytoplasm
3. Viral RNA is translated to new viral proteins and transported back into the nucleus
4. Viral DNA replicated by host DNA polymerase and viral factors
5. Reassembly of viral genome and proteins, followed by release

47
Q

How is a class IV virus replicated?

A

positive strand ssRNA virus
Viral genome can stay in the cytoplasm
1. Viral RNA (=mRNA) translated to proteins by the cytoplasm ribosomes
2. Viral RNA genome replicated by viral synthase
3. Viral proteins form the capsid and encapsulate the replicated RNA genomes forming new vectors
4. New virions bud

48
Q

How does a class V virus replicate?

A

negative strand ssRNA virus
Influenza
1. taken up by endocytosis
2. RNA negative strand converts to mRNA using viral RNA polymerase
3. mRNA translated in cytoplasm to produce viral proteins

49
Q

How are class VI viruses replicated?

A

Retroviruses, HIV
ssRNA + sense replicating through a DNA intermediate
1. Entrance via bidning and fusion of viral envelope proteins to specific host cell receptors
2. Virion uncoating
3. Viral reverse transcriptase reverse transcribes one RNA strand into ssDNA and then into dsDNA, which enters nucleus
4. Viral DNA integrates into host DNA through viral integrase forming a provirus
5. Viral DNA is transcribed to viral mRNA and progeny RNA
6. Synthesis of HIV polyprotein
7. Processing of HIV polyproteins by viral protease and generation of mature functional proteins
8. Encapsidation of viral ssRNA
9. Budding
10. Release

50
Q

How are class VII viruses replicated?

A

Paretroviruses
Hep B virus
1. HBV fuses to the host cell membrane and core enters the cytoplasm
2. DS genome completed by the enzymes within the core
3. DNA enters nucleus
4. DNA is transcribed to genomic and mRNA
5. RNA enters the cytoplasm
6. Viral proteins are translated
7. Core assembles around positive sense RNA
8. positive sense RNA copied to negative sense DNA
9. negative sense DNA partially copied to positive sense DNA
10. core associates with HbS antigen in cell membrane
12. Virus with partial dsDNA leaves the cell

51
Q

What is the process of budding?

A
  1. Viral proteins which are to become spikes of virus attach to host plasma membrane
  2. Viral matrix protein coats inside of the plasma membrane
  3. Nucleocapsid becomes enclosed by viral envelope (composed of host plasma membrane)
  4. Budding
52
Q

What are four consequences of viral infections following attachment and penetration?

A
  1. Transformation into a tumour cell, which then divides and can transform normal cells to tumour cells
  2. Multiplication of cell leading to the death of the cell and release of the virus (lytic infection)
  3. Slow release of the virus without cell death (persistent infection)
  4. Virus present but not causing cell damage, later emerges in lytic infection (latent infection)
53
Q

Define an acute infection

A

Lytic
virus disapears after the disease ends
e.g. measles

54
Q

Define a latent infection

A

After initial infection, virus relplicates in non-infectious state. Virus can be activated to produce new disease symptoms e.g. chicken pox

55
Q

Define persistent infection

A

After inital infection with or without disease symptoms, infectious virus is released with no symptoms

56
Q

What is the size of the herpes virus?

A

Huge

100 proteins

57
Q

What is needed for - sense RNA to be used as mRNA?

A

Needs viral polymerase

58
Q

What makes a virus a virus?

A

Capsid
Lack of ribosomes
Capsid encoding organisms/Organisms that do not have their own ribosomes: Viruses of archaea, bacteria, eukaryota
Non-capsid encoding organisms/Have their own ribosomes: Bacteria, archaea, eukaryota

59
Q

What is the purpose of the capsid?

A

To protect genetic material

60
Q

How might a virus be defined in terms of characteristics of its lifecycle?

A

Viruses have the ability to dissassemble and reconstitute themselves
If during an infection experiment a particle was to lose its integrity but keep its genetic info intact, and later reassemble into progeny, it would be a virus

61
Q

What unspecific clinical signs would suggest a person is infected with a virus

A

High white blood cell count

Enlarged spleen

62
Q

What can be clinically looked at in a persons blood to test for a viral infection?

A

The viral genomes

An immune response against a specific virus

63
Q

What are five ways of detecting viral infection?

A

A. Growh of live virus in cultured cells in vitro (cell culture)
B. ELIZA (enzyme linked immunoassay) to measure antigen levels
C. Serology - flurorchome-tagged antisera used to detect viral antigens in cells from specimen
D. Electronmicroscopy
E. Directly measure RNA/DNA copies via qPCR

64
Q

How can viral infection be detecting by cell culture (plaque forming units)?

A
  1. Take cell type in the lab which is known to be infected by the virus
  2. Take patients blood and make serial dilutions
  3. Mix with the infection ready cells
  4. Grow in such a way that they form plaques
65
Q

What are the benefits of detecting viral infection using plaque forming units?

A
  1. Have an indictation of the viral load by the number of plaque forming units per dilution of sample
  2. Quantitative
  3. Tests for fully functional virus, shows it is infectious and can cause the formation of plaques
  4. Not over sensitive
66
Q

What are the disadvantages of detecting viral infection using plaque forming units?

A
  1. May not be able to find cells that can be infected in vitro
  2. Works on the notion that every cell that is infected proliferates and forms a plaque
  3. Slow (2 days to >3 weeks)
  4. Labour intensive
  5. Requires skill and experience to prep cellsm and read any cytopathic effect
67
Q

How can an ELIZA be used to detect viral infection?

A
  1. Take blood sample that may have an infection
  2. Column is coated with antibodies which recognise virus/antigen of the virus
  3. Put sample through column and wash three times, all that is stuck on the plate will be antibodies that have captured the virus/antigen of virus
  4. Add enzyme-conjugated antibody which if given the right substrate will have a colour change and wash three times
  5. Add substrate, the specific anti-target enzyme conjugate binds to the captured antigen, incubate at 37 degrees to view if there is a colour change
68
Q

What are the benefits of detecting viral infection using an ELIZA?

A
  1. Quantifiable
  2. Can make specific antibodies for a specific protein easier than can find cell line to respond to viral infection (PFU)
  3. Quick (2-3h)
  4. Can be read by machine
  5. Suitible for large numbers
  6. Commercial kits available
69
Q

What are the disadvantages of detecting viral infection using an ELIZA?

A
  1. Need to know the antigen you are looking for
  2. Measures presence of the viral antigen
  3. Need two types of specific antibody which may not be able to get
  4. No feedback on specimen quality
  5. Problems over false positives and boarderline results
  6. Sensitivity uncertain
70
Q

How can serology be used to detect viral infection?

A

Identify whether patient has antibodies against a particular viral antigen in the blood
Measures the immune response against the virus
Use a titration. Take patients blood and dilute ten fold. Slowly see fluoresence/colouration signall dropping through dilutions to a background level of signal.
Virus titre (Ab titre): highest dilution that gives a positive signal
Titre of 10000 (or 1 in 10,000) compared to a titre of 100 (or 1 in 100) has a higher immune response

71
Q

What are the disadvantages of detecting viral infection using serology?

A
  1. Measuring the effectiveness of the immune response not the level of infection e.g. in HIV immune system is destroyed so titre would be higher even though might have high levels of infection
  2. Requires specific antisera
  3. Requires skill and experience to read
  4. Requires darkened reading room and fluoresence microscope
72
Q

What are the advantages of detecting viral infection using serology?

A
  1. Specific
  2. Semi-Quantifiable
  3. Measures antibodies against infection
  4. Quick
  5. Provieds feeedback on speciment quality
  6. Good sensitivity for purpose
73
Q

How can electronmicroscopy be used to detect viral infection?

A

Take a biopsy and look at tissue under EM

Uses high resolution to visualise virus particles directly

74
Q

What are the disadvantages of detecting viral infection using electronmicroscopy?

A
  1. Have to be careful with the sample
  2. Cant do an on the spot check, requires high cost instuments and highly trained individuals
  3. Difficult to do
  4. Sensitivity difficult to define
75
Q

What are the advantages of detecting viral infection using electronmicroscopy?

A
  1. Direct evidence of the presence of the virus, absolute proof
  2. Very quick (15mins - 3hours)
  3. Specific
  4. Can detect mixed infections easily
  5. Catch all method suitable for unknowns
76
Q

How can qPCR be used to detect viral infection?

A

Directly measure DNA/RNA copies/genomes y an amplification to assessable levels

  1. Use standard curve and unknown patient samples
  2. The later the viral DNA is detected on the qPCR the less DNA present in the sample
77
Q

What are the benefits of using qPCR to detect viral infection?

A
  1. Highly sensitive
  2. Can be same day
  3. In multiplex form can detect mixed infections
  4. Can be automated for larger numbers
78
Q

What are the disadvantages of using qPCR to detect viral infection?

A
  1. May be over sensitive
  2. No indication of specimin quality
  3. Expensive
  4. Requires good technique to avoid cross contamination
  5. Any response, no matter how late, is a positive response. However those at the upper limit of detection may have to have further tests to confirm the presence/level of infection. e.g. for HIV testing
79
Q

What is the best method for detecting viral infection?

A

Depends on the virus, person. Better to do a combination of tests. E.g. for HIV:
HIV screening using ELISA, if reactive use a confirmatory ELISA, if not reactive report as negative, recommend repeat testing if in 3-6 week window
If confirmatory ELISA reactive, report as positive. If confirmatory ELISA non-reactive, follow up tests are needed, repeat ELISA, molecular testing (HIV PCR/viral load)

Principle of molcular virology (7 decks)

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