Virology - oncogenesis and laboratory diagnostics

Question 1

Carcinoma origin

Answer 1

of epithelial cell origin

Question 2

Sarcoma origin

Answer 2

of mesenchymal cell origin

Question 3

Lymphoma origin

Answer 3

from leucocytes; solid tumors

Question 4

Leukemia origin

Answer 4

from leucocytes; circulating cells involved

Question 5

How can a viral infection transform a cell from normal to abnormal (potentially oncogenic) ? (3)

Answer 5

1. Cytopathic effects must be reduced or eliminated
   The infected cell does not die
2. Viral replication must be reduced or eliminated
   Transformed cells do not produce virus particles
3. The cell must continue to divide
   It becomes immortal

Question 6

What percentage of human cancers are caused by viruses?

Answer 6

15-20% of all human cancers are associated with infection with 7 viruses
◦ Epstein-Barr virus
◦ Hepatitis B virus
◦ Hepatitis C virus
◦ Human herpesvirus 8
◦ Human T-cell lymphotropic virus type 1
◦ Human papillomaviruses
◦ Merkel cell polyomavirus

Question 7

2 examples of oncogenic DNA animal viruses:

Answer 7

◦ Marek’s disease virus (Herpesviridae) this is an avian disease
◦ Papillomaviruses

Question 8

5 examples of oncogenic reverse transcribing animal viruses:

Answer 8

◦ Avian leukosis viruses
◦ Rous sarcoma virus

◦ Ovine pulmonary adenocarcinoma virus (Jaagsiekte virus)

◦ Feline leukemia virus (FeLV)
◦ Bovine leukemia virus

Question 9

What virus do all chickens carry?

Answer 9

Avian leucosis retrovirus (ALV)

◦ Sporadic leucosis (3%)
◦ 97% birds have transient viremia, no leucosis
◦ the birds achieve immunity if they live long enough

Question 10

RSV

Answer 10

Rous sarcoma virus

is an avian leucosis retrovirus but its recombinant

◦ Older birds develop tumors
◦ Connective tissue tumors or sarcomas (solid tumors)
◦ Viruses isolated from these solid tumors rapidly cause sarcomas, not leucosis

Question 11

3 methods of Oncogenesis by Retroviruses

Answer 11

rapid tumor formation by transducing retroviruses

intermediate kinetics of tumor formation by cis-activating retroviruses

slow kinetics for tumor formation by trans-activating retro viruses (blocks transcription termination)

Question 12

What are cis-acting elements?

Answer 12

Cis-regulatory elements, such as promoters, enhancers, and silencers, are regions of non-coding DNA, which regulate the transcription of nearby genes.

Question 13

Oncogenesis by DNA viruses (4)

Answer 13

◦ Oncogenes have no homologs or direct ancestors (c-onc genes) among cellular genes of the host

◦ Viral oncogenes present

◦ Bind and compromise tumor suppressor genes

◦ Alter cellular signal transduction signals

Question 14

Laboratory diagnosis of viral diseases methods (5)

Answer 14

Visualization (of viral particles or of cell damage caused by them)
Virus isolation

Detect viral nucleic acids (PCR, whole genome sequences)

Infectivity assays
Chemical/physical measures (this includes various serological assays)

Question 15

Light microscope in viral infection diagnosis

Answer 15

Can detect

◦ Large viruses (e.g. Mimiviruses)
◦ Changes in the cells such as

CPE (cytopathic effects)
Inclusion bodies (e.g. Negri bodies)

Question 16

What is a common biological method for virus cultivation?

Answer 16

Embryonated eggs infected with viruses

Question 17

What is a plaque assay?

Answer 17

A type of infectivity assay

The plaque assay is a well-established method for measuring virus concentration as it relates to infectious dose. The assay relies on determining the number of plaque-forming units (PFU) created in a monolayer of virus-infected cells.

Question 18

What is a Endpoint dilution assay?

Answer 18

A type of infectivity assay

The end-point dilution assay was used to measure virus titer before the development of the plaque assay, and is still used for viruses that do not form plaques. Serial dilutions of a virus stock are prepared and inoculated onto replicate cell cultures, often in multi-well formats.

Question 19

Why are not all virus particles infectious?

Answer 19

A single particle can initiate infection but not all viruses are successful in causing infection. There will be damaged particles, mutations and the complexity of
the infectious cycle will affect infectivity too.

To know viral infectivity, the ratio between number of physical viral particles and number of infectious particles in calculated.

A particle/PFU ratio of 1 means that basically every single viral particle is infectious and a particle/PFU ratio of 10,000 means that only one viral particle in 10,000 will cause infection.

Question 20

what is a viral plaque

Answer 20

A viral plaque is a visible structure formed after introducing a viral sample to a cell culture grown on some nutrient medium. The virus will replicate and spread, generating regions of cell destruction known as plaques.

Question 21

What is an HA assay?

Answer 21

Hemagglutination assay (HA) is a method for quantifying the relative concentration of viruses.

sialic acid receptors on the surface of red blood cells (RBCs) bind to the hemagglutinin glycoprotein found on the surface of influenza virus (and several other viruses) and create a network, or lattice structure, of interconnected RBCs and virus particles

The formation of the lattice depends on the concentrations of the virus and RBCs, and when the relative virus concentration is too low, the RBCs are not constrained by the lattice and settle to the bottom of the well.

Question 22

What is a HI assay?

Answer 22

The hemagglutination inhibition (HI) is a method for quantifying the relative concentration of viruses, bacteria, or antibodies.

HI is closely related to the HA assay, but incvolves anti-viral antibodies as “inhibitors” to interfere with the virus-RBC interaction.

Question 23

4 types of ELISA for viral infections

Answer 23

◦ Direct ELISA – Ab1 (primary antibody) chemically linked to enzyme (or fluorescent
molecule)

◦ Indirect ELISA – Ab1 + tagged Ab2 (secondary antibody)

◦ Sandwich ELISA – antigen is “sandwiched” between two antibodies

◦ Competitive ELISA – preincubation of antibody with antigen

Question 24

Basic steps of PCR (3)

Answer 24

◦ Denaturation (96°C)
◦ Annealing (55- 65°C)
◦ Extension (72°C)

Question 25

What is Ct in PCR?

Answer 25

Cycle threshold (Ct) is the number of PCR cycles required for the fluorescent signal to
cross the threshold for exceeding background levels.

Question 26

What is cell transformation

Answer 26

Cell transformation describes the changes associated with loss of normal homeostatic
control, particularly of cell division, which ultimately results in the development of a
neoplastic phenotype in cells.

Transformed cells continue to divide under circumstances in which the normal cells do not. Transformation leads to oncogenesis, the development of cancer.

Question 27

What type of viruses can PCR detect?

Answer 27

PCR can be used to identify and/or quantify viral DNA genomes in a sample. PCR is a very sensitive method and uses oligonucleotide primers designed to detect suspected virus.

Question 28

What is RT-PCR for?

Answer 28

RT-PCR – Reverse transcription PCR

◦ The enzyme reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from the small amount of viral RNA in the specimen. The cDNA can then be amplified by PCR.

Question 29

What is qPCR for?

Answer 29

quantitative PRC or real-time PCR

◦ Used to detect, characterize and quantify nucleic acids with fluorescent labelling of PCR
products.