Virology - oncogenesis and laboratory diagnostics Flashcards
Carcinoma origin
of epithelial cell origin
Sarcoma origin
of mesenchymal cell origin
Lymphoma origin
from leucocytes; solid tumors
Leukemia origin
from leucocytes; circulating cells involved
How can a viral infection transform a cell from normal to abnormal (potentially oncogenic) ? (3)
- Cytopathic effects must be reduced or eliminated
The infected cell does not die - Viral replication must be reduced or eliminated
Transformed cells do not produce virus particles - The cell must continue to divide
It becomes immortal
What percentage of human cancers are caused by viruses?
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
2 examples of oncogenic DNA animal viruses:
◦ Marek’s disease virus (Herpesviridae) this is an avian disease
◦ Papillomaviruses
5 examples of oncogenic reverse transcribing animal viruses:
◦ Avian leukosis viruses
◦ Rous sarcoma virus
◦ Ovine pulmonary adenocarcinoma virus (Jaagsiekte virus)
◦ Feline leukemia virus (FeLV)
◦ Bovine leukemia virus
What virus do all chickens carry?
Avian leucosis retrovirus (ALV)
◦ Sporadic leucosis (3%)
◦ 97% birds have transient viremia, no leucosis
◦ the birds achieve immunity if they live long enough
RSV
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
3 methods of Oncogenesis by Retroviruses
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)
What are cis-acting elements?
Cis-regulatory elements, such as promoters, enhancers, and silencers, are regions of non-coding DNA, which regulate the transcription of nearby genes.
Oncogenesis by DNA viruses (4)
◦ 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
Laboratory diagnosis of viral diseases methods (5)
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)
Light microscope in viral infection diagnosis
Can detect
◦ Large viruses (e.g. Mimiviruses)
◦ Changes in the cells such as
CPE (cytopathic effects)
Inclusion bodies (e.g. Negri bodies)
What is a common biological method for virus cultivation?
Embryonated eggs infected with viruses
What is a plaque assay?
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.
What is a Endpoint dilution assay?
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.
Why are not all virus particles infectious?
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.
what is a viral plaque
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.
What is an HA assay?
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.
What is a HI assay?
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.
4 types of ELISA for viral infections
◦ 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
Basic steps of PCR (3)
◦ Denaturation (96°C)
◦ Annealing (55- 65°C)
◦ Extension (72°C)
