METHODS IN VIROLOGY

Recap Good or bad, virus is important Neither living nor non-living Basic virus structure Virus morphology Properties of naked and enveloped virus Chemical composition of virus Classification of virus

Growth & Quantification of Viruses • Viruses need to be grown in their host cell – animal, plant, bacteria ….. • Animal viruses – cell culture – cells from animals grown artificially in the laboratory – Often in monolayers on glass or plastic overlaid with suitable liquid media for the cells to grow Complex medium – amino acids, vitamins, salts, pH buffers, glucose, antibiotics, pH indicator ..

Growing Viruses Bacteriophages form plaques on a lawn of bacteria

Cultivation of Viruses First started in early 1900’s Successfully done in 1949 in case of poliovirus. 3 main methods: Embryonated chicken eggs Tissue Culture Animal model

Virus Cultivation (Cont) Embryonated Chicken Method Most economical and convenient method Fertile eggs of 5-14 days inoculated through shell aseptically. Opening sealed with paraffin wax Incubated at 36oC until the virus grow Different types of viruses are grown in different tissues of chick embryo Can be used for vaccine production

Embryonated Chicken Method

Virus Cultivation (Cont) Tissue Culture Method Most common method for propagation of virus Convenient Economical for maintenance Observable cytopathic effects Choice of cells for their susceptibility to particular viruses

Virus Cultivation (Cont) Preparation of tissue culture Dissociate tissue into single cell Mechanical disruption Proteolytic enzymes Cell suspensions are placed into plastic flask or plates Cells grow in monolayer in presence of chemically defined media, with vitamins, coenzymes, and amino acids.

Isolation of Cells .

Cell Culture Some cells will grow indefinitely – permanent cell lines; others will remain alive only for a short period – primary cell lines.

Tissue Culture Method- 3 types Primary culture-Derived from normal tissues of animal or human, have limited life span. E.g. primary human foreskin fibroblasts Diploid cell strain- Establish from developing embryo, possess diploid karyotype, can divide up to 100 times. E.g. WI-38, human embryonic lung Continuous cell line- Propagate for indefinite time, derived from tumor tissues or cells treating with tumor virus. E.g. HeLa

Cell Line Primary human fibroblast NIH3T3 HeLa

Cytopathic Effect

Purification of Virus Particles Concentration of Virus Particles by: -(NH4)2SO4 -PEG -Ultracentrifugation -Haemagglutination Virus particles can be separated by: -Differential centrifugation -Column chromatography

Measurement of Infectious Virus Done by determining the titer in a sample. Plaque assay Fluorescent Focus assay Infection center assay Transformation assay End point dilution assay

Plaque Assay Discovered by Renato Dulbecco, 1952. Cell monolayer is incubated by virus and covered with nutrient media and agar. Each infectious virus produce a zone of infected cells, known as plaque. Plaques can be distinguished from other cells as the cells in plaque are lysed. No. of plaques in a given dilution is proportional to the no. of infectious particles. Can be done only for cytopathic viruses

Plaque Assay

Plaque Assay (Cont.) One -hit- kinetics: when one infectious particle is sufficient to initiate infection. And the curve is linear. Two- hit- kinetics: two different types of virus particles must infect a single cell to ensure infection. The curve is parabolic. One-hit-kinetics Titer of virus, p.f.u./ml Two-hit kinetics Time in hr

Fluorescent Focus Assay Useful for measurement of non-cytopathic virus. An antibody to the virus is added A fluorescin conjugated secondary Ab is added A foci of infected cells fluoresce under UV microscope. Each focus is initiated by a single virus particle. Titer of virus can be expressed as FFU/ml.

Fluorescent Focus Assay

Infectious Centre Assay To determine the fraction of cells in a culture infected with a virus. Cells from the first monolayer separated before the progeny virus is produced. Cells are then seeded on another (indicator) susceptible cell line. No. of plaques on indicator cells measures the no. of cells infected in the original culture. Can be used to measure the persistently infected cells.

Transformation Assay Useful to measure the titer of oncogenic retroviruses. Oncogenic virus transform the cells. The cells lose contact inhibition and heap upon one another. The transformed cells form distinguishable small piles or foci. The count/infectivity is expressed as focus forming unit/ml.

End Point Dilution Assay Very old method, used for non-cytopathic viruses to determine the virulence. Serially diluted virus stock is inoculated in replicate test units. No. of test unit become infected is determined for each dilution. The end point of a dilution affection- 50% of the test unit is the ID50/ml. Virulence or pathogenecity is expressed as LD50/ml or PD50/ml.

Measurement of Virus Particles & Their Components Electron Microscopy Hemagglutination Measurements of viral enzymes Serological methods Neutralization Heamagglutination inhibition Complement fixation Immunoblotting EIA NA detection

Particle-to-PFU ratio Number of virus particles in sample/number of infectious particles ~1 for many bacteriophages High for many animal viruses A single particle can initiate infection High particle-to­ pfu ratio, not all viruses are successful- Damaged particles, mutations, Complexity of infectious cycle

A Viral One-Step Growth Curve Figure 13.10

One-Step Growth Curve Synchronous infection- key to one-­step growth cycle Multiplicity of infection (MOI)- Number of infectious particles added per cell- Not the number of infectious particles each cell receives Infection depends on the random collision of virions and cells The distribution of virus particles per cell- Poisson distribution

What’s the chemical formula for poliovirus??? Question? What’s the chemical formula for poliovirus???