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Virology > Lab 2 - Virus titration, CPEs and identification > Flashcards

Lab 2 - Virus titration, CPEs and identification Flashcards

1
Q

Reasons for virus titration

A
  • Serology: determines antibody content
  • Vaccine production: determines virion (antigen) content
  • Pathogenicity tests: for experimentally infection og susceptible animals
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2
Q

Methods for virus titration

A
  • Electronmicroscopic investigation-physical method
  • Determination of the infective titre in cell culture-biological method
  • In case of haemagglutinating viruses-haemagglutinating test
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3
Q

What is the electronmicroscopic investigation method for virus titration?

A

Counting the number of virus particles

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4
Q

How is determination of the infective titre in cell culture happening?

A

10-fold serial dilution of the virus suspension is inoculated into cell cultures, then detection of the appearance of CPE in the different dilutions

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5
Q

What is haemagglutinin?

A

Surface proteins, appearing on some viruses, and able to bind erythrocytes

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6
Q

What can virions with haemagglutinin do?

A

Agglutinate RBCs to form a lattice - haemagglutination

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7
Q

What is viral haemagglutination titre?

A

The end dilution of a virus suspension, where the haemagglutination still appears - contains 1 HA unit of viruses

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8
Q

What is cytopathic effects (CPEs)?

A

Alterations in the morphology of cells due to virus infection, therefore mainly observed in cell cultures

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9
Q

What is CPEs result of?

A
  • Direct damage of viruses on cells
  • Toxic effect of adsorption
  • Virus proteins inhibit cellular translation
  • Early proteins inhibit cellular nucleic acid transcription and replication
  • Viral proteins damage the cytoplasmic membrane permeability - osmotic changes
  • Cytosceleton depolymerisation, expression of fusion proteins
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10
Q

Main type of CPEs

A 
-Inclusion body formation: Intranuclear inclusion bodies
and intracytoplasmic inclusion bodies
-Cell rounding
-Syncytium formation
-Lumpy cell nucleus
-Cell vacuolisation
-Cytolysis
-Haemadsorption
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11
Q

What is inclusion bodies?

A

Virus depositions

Appear at the site of assembly of nucleocapsid

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12
Q

Where can inclusion bodies be observed?

A

In stained cells, with homogenous stainin, surrounded by a narrow light stripe

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13
Q

What is intranuclear inclusion bodies caused by?

A

DNA viruses replication in the nucleus, sometimes by RNA viruses

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14
Q

How can intranuclear inclusion bodies be observed when stained?

A

Can be basophyl, amphophyl or eosinophyl particles. In case of large number, caryomegalia and/or perichromasia can be observed

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15
Q

What is intracytoplasmic inclusion bodies caused by?

A

Usually by RNA viruses, sometimes by DNA viruses replicating in the cytoplasm

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16
Q

How can intracytoplasmic inclusion bodies be observed when stained?

A

Can be eosinophyl or rarely basophyl particles

17
Q

What is Pathognomic inclusion bodies?

A

Negri bodies and Guarnieri bodies

18
Q

What is the reason for cell rounding?

A

Cytosceleton depolymerisation and loss of electrolytes

19
Q

How can cell rounding be observed?

A
  • Unstained: double refraction, detachment, shrinkage

- Stained: cells is intensive stained

20
Q

What is syncytium formation caused by?

A

Exclusively by enveloped viruses. The fusion proteins of the virus are used prim. for penetration, but also cause fusion of neighbouring cells membranes

21
Q

What does syncytium formation make possible?

A

Intracellular spread of viruses, can hide from antibodies

22
Q

What is Lumpy cell nucleus caused by?

A

Chromatin conglomeration, rearrangement of changed refraction

23
Q

What does cell vacuolisation do?

A

Vacuoles are formed in the nucleus/cytoplasm of the cells

24
Q

Why does cytolysis occur?

A

Because of damage of the cell membrane or by lisosomal enzymes

25
Q

What is Haemadsorption?

A

Haemagglutinin incorporates into the cytoplasmic membrane of the infected cell -> able to adsorb erythrocytes on the surface of the monolayer

26
Q

In which case does Haemadsorption have diagnostic value?

A

Paramyxoviruses and ASFV

27
Q

What is plaque?

A

CPEs spread fro cells to neighbouring cells concentricall

28
Q

What is facilitation of plaque formation?

A

To supplement the maintenance medium with agar, carboxymethyl cellulose or metrizamide -> cause viruses to spread to neighboring cells

29
Q

What is plaque couting used for?

A

determine the quantity of virus particles or antibody content of the sample

30
Q

What should be supplemented to the cell culture for investigation of non-cytopathogenic viruses?

A

Immunperoxidase assay (IPA) or immunfluorescence (IF)

31
Q

Other test and assays that could be used for the investigation of non-cytopathic viruses

A
  • Artificial infection of suceptible test animals
  • Electronmicroscopic investigation
  • Immun-EM investigation
  • Complement-fixation test
  • Agargel-precipitation
  • Counter current immune electrophoresis
  • ELISA, RIA
  • Haemagglutination
  • Detection of viral nucleic acid by polymerase chain reaction
32
Q

Methods for classification into genus

A
  • Microscopic investigation
  • EM-investigation
  • Physio-chemical investigation of vegetative viruses
  • Investigation og group-specific antigens
  • Determination of the serotype
  • Determination of the subtype
33
Q

Type of microscopic investigation in virus identification?

A

CPE- tissue specificity, type of pocks, hemadsorption

34
Q

What is identified in EM investigation?

A

Dimension, size, shape of virion, its symmetry and surface

35
Q

Type of Physio-chemical investigation of vegetative viruses?

A
  • Enveloped/non-enveloped virus: chloroform treatment
  • DNA/RNA virus: halogenated deoxy-uridine
  • ssNA/dsNA: acridin-orange staining
36
Q

Type of Investigation og group-specific antigens?

A

AGP, IPA, IF, IEM, ccIEF, ELISA, RIA, HA

37
Q

Type of Determination of the serotype?

A

Virus neutralisation test, haemagglutination-inhibition test

38
Q

Type of Determination of the subtype?

A

Clinical signs, investigation of NA, investigation with monoclonal antibodies

Virology (6 decks)

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