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

1

Cytopathic effects

A. Cell rounding by poliovirus

B. Syncythia by murine leukemia virus

Cytophatic effects of viral infection

Growth of viruses in embryonated eggs

2

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

3

Hemagglutination assay

Orthomyxo, adeno, paramyxo

Serological methods

Direct and indirect methods for antigen detection

Herpes SV VP22 In the nucleus

4

Visualization of proteins by immunoprecipitation

Visualization of proteins by immunoblotting

Detection of viral antigen or antibodies against viruses by ELISA

5

The use of green fluorescent protein
A. Imaging of neurons B. HIV and microtubule development

Pseudorabies virus (PRV)

Nucleic acid detection

Southern blot hybridization

6

DNA microarray technology

One-step growth curves

7

Classical genetics methods

Reassortment of influenza virus RNA segments

Engineering mutations into viral genomes

8

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

9

Viral vectors

DNA viral vectors

Adenovirus-associated vectors

Adenovirus vectors

10

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

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