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1
Lecture 2
Virus cultivation and detection
1. Cultivation of viruses
2. Detection of viruses in the host
3. Genetic analysis of viruses
1. Cultivation of viruses
Cell culture
Human fibroblasts Mouse fibroblasts Human epithelial cells
2
Cytopathic effects
A. Cell rounding by poliovirus
B. Syncythia by murine leukemia virus
Cytophatic effects of viral infection
Growth of viruses in embryonated eggs
3
Plaques formed by different animal viruses Dose-response curve of plaque assays
A. Single plaque by pseudorabies virus
In kidney cell culture
B. Different plaque morphology by
Influenza C virus strains
C. Poliovirus on human HeLa cells
Transformation assay
Chicken cells transformed by two strains of Rous sarcoma virus. Note the
loss of contact inhibition causes the cells to pile up.
A. One focus, B. three foci
Measurement of Virus particles and their
components
4
Hemagglutination assay
Orthomyxo, adeno, paramyxo
Serological methods
Direct and indirect methods for antigen detection
Herpes SV VP22
In the nucleus
5
Visualization of proteins by immunoprecipitation
Visualization of proteins by immunoblotting
Detection of viral antigen or antibodies
against viruses by ELISA
6
The use of green fluorescent protein
A. Imaging of neurons B. HIV and microtubule development
Pseudorabies virus (PRV)
Nucleic acid detection
Southern blot hybridization
7
DNA microarray technology
One-step growth curves
8
Classical genetics methods
Reassortment of influenza virus RNA segments
Engineering mutations into viral genomes
9
Genetic manipulation of RNA viruses
Mutagenesis by PCR: introducing
mutations into viral genomes
Genetic interference by DS RNA interference (RNAi)
Inhibition of poliovirus replication by siRNA
10
Viral vectors
DNA viral vectors
Adenovirus vectors
Adenovirus-associated vectors
11
Retroviral vectors
Homework for this week:
Read and understand Chapter 2 in the book. Put special
emphasis on:
-cythopatic effects of viral infection
-embryonated eggs
-detection of viruses
-plaque forming unit
-hemagglutination
-other immuno methods
-nucleic acid detection
-one-step versus two-step growth curves
-engineering mutations into viruses: genetic manipulation of
+ and –strand viruses
-virus vectors