UNIVERSITY OF NAIROBI

ISOLATION OF VIRUSES

Prepared by

Kennedy O. Sigodo MLS

CONTENTS

Introduction

Purpose of virus cultivation

Virus cultivation systems

Tissue culture

Embryonated eggs

Laboratory animals

Viral quantification.

INTRODUCTION Viruses are obligate intracellular organisms Must be grown in living cells. They can't be grown in culture media or on agar plates alone,

they must have living cells to support their replication. The easiest viruses to grow are bacteriophages…..WHY? Animal viruses – difficult, due to the properties of the animal host

Under natural conditions, many viruses are relatively host-specific. Moreover, they may show a marked predilection for certain tissues of the host such as nervous tissue, epithelial tissue etc.

The majority can be adapted to foreign hosts by passage

VIRUS CULTIVATION Also known as viral propagation or growth.

Necessary to supply the virus with appropriate cells in

which it can replicate. Phages are supplied with bacterial cultures. Plant viruses may be supplied with specially cultivated plants

or with cultures of protoplasts (plant cells from which

the cell wall has been removed), Animal viruses may be supplied with whole organisms, such as mice,

eggs containing chick embryos, insect larvae or animal cells.

Viruses can be isolated from different specimens.

SPECIMENS USED TO ISOLATE VIRUSES

-Blood specimens

EDTA HeparinSerum

-Stool

-Throat swabs

-Naso-pharyngeal aspirates

-Stools, rectal swabs-Urine -Saliva-Cerebro-spinal fluid-Biopsy

Skin (filoviridae)Organs (fixation with formaldehyde 10%)

PURPOSE OF VIRUS CULTIVATION The primary purposes of viral cultivation are:1. To isolate and identify viruses in clinical specimens

2. To prepare viruses for vaccines

3. To do detailed research on viral structure, multiplication cycles, genetics, and effects on host cells.

VIRUS CULTIVATION SYSTEMS

Tissue culture system

Embryonated eggs system

Whole animal systems a) Natural host

b) Experimental animals

c) Transgenic animals

TISSUE CULTURE SYSTEM

HISTORY OF CELL CULTURE

Cultured cells could only survive for a few days. In 1951, cells taken from Henrietta Lacks (cervical cancer

patient) Cell line was found to be remarkably durable and prolific. George Gey was able to isolate one specific cell, multiply

it, and start a cell line. Named the sample HeLa. First human cells grown in the

lab that were immortal. The use of the antibiotics, chemically defined medium

and use of trypsin greatly enhanced the cell culture technique.

HeLa cells cont….

1.polio vaccine 2. Gardisil developed by studying

HeLa cells 3.MMR vaccine. 4. Understanding of TB, HIV, HPV-HeLa

cells have been used to understand how these diseases impact cells

5. Human chromosome number and mitosis-scientists used HeLa cells to determine the exact number of chromosomes in

TISSUE CULTURE SYSTEM cont….

Use isolated cell from animal that are cultured invitro.

It is the preferred type of growth medium for viruses.• Three discoveries greatly enhanced the usefulness of cell cultures for

virologists and scientists

1. The discovery and use of antibiotics made it possible to prevent bacterial and fungal contamination

2.The discovery of proteolytic enzymes (e.g. trypsin) can free animal cells from surrounding tissues without injuring freed cells

3.This technique has also become possible by the development of growth media for animal cells.

STEPS IN TISSUE CULTURE TECHNIQUE Cultivating animal viruses using tissue culture

technique involves following three main steps:

1. Monolayer preparation

2. Clonal cell line preparation

3. Infection with virus

The first two steps are summarised with the notes on cell culture in the next slides.

1.Monolayer and clonal cell line preparationCELL CULTURE

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favourable artificial environment.

The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been already established.

Can be classified under the following cell lines.

i) Primary culture

ii) Diploid cell lines

iii) Continuous cell lines

PRIMARY CULTURE Primary culture refers to the stage of the culture after the cells

are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence) (e.g. Primary monkey kidney, mice fibroblasts)

-Heterogeneous – many cell types

-Technical hassle-5 to 20 cell divisions-Normal chromosome

number-Contact inhibition-need constant source -Closest to animal

Making a primary cell line

DIPLOID CELL LINES After the first subculture, the primary culture becomes

known as a Diploid cell line or subclone. E.g(human fetal lung)

-Lines-up to 100 cell divisions-Homogeneous population of a single type. -Typically derived from tumors. Remain diploid-Further from animal-Technically less hassle

CONTINUOUS CELL LINES When a finite cell line undergoes transformation and acquires the ability

to divide indefinitely, it becomes a continuous cell line. Become immortal through a process called transformation. Can occur spontaneously or can be chemically or virally induced. -Immortal

-Most homogeneous-Genetically weird – furthest from animal-Hassle free-Suspension or monolayer -Aneuploid- abnormal in

chromosome morphology and number, Grow rapidly. (e.g.various types of cancer cells - HeLa cells, Hep 2 cells, or human amnion cells, continual monkey kidney cell line, dog kidney cell line, etc.).

Summary.

3.Infection with virus

The clonal cell lines suspended in suitable media are infected with any desired virus which replicates inside the multiplying cells. If the virus is virulent, they cause lysis of cells and virus particles are released in the surrounding medium.

These newly produced virus particles (virions) infect the adjacent cells. As a result localized areas of cellular destruction and lysis (called plaques) often are formed

CULTURE CONDITIONS Culture conditions vary widely for each cell type. The artificial media invariably consist of a substrate or medium

that supplies the essential nutrients (amino acids, carbohydrates, vitamins,minerals), growth factors, hormones, and gases (O2, CO2).

It also regulates the physicochemicalenvironment (pH, osmotic pressure, temperature).

Most cells are anchoragedependent and must be cultured while attached to a solid or semi-solid substrate(adherent or monolayer culture),

Others can be grown floating in the culture (suspension culture)

SUSPENSION ADHERENT

Differences between Adherent and Suspension CulturesADHERENT CULTURES SUSPENSION CULTURES

Most cells can be cultured this way Cells which are adapted to suspension cultures or non-adhesive cultures

Passaging required at certain intervals

Passaging is much easier, can dilute culture to stimulate growth

Allows easy visualisation of cells Harder to view cells

Cells dissociated enzymatically or mechanically

Not require enzymatic or mechanical dissociation

Surface area limits growth Cell concentration in medium limits growth Used for cytology, harvesting products Used for bulk protein production, batch continuously and also research harvesting and also research applications applications

CELL INCUBATOR

Viral detection:Detection of a growth of a virus is observed by the changes in

the cell culture monolayer - cytopathic effect (CPE).

CPE are Changes of morphology of cells e.g:

1. Lysis of the cells 2. Vacuolation,

3. Formation of syncytia 4. Presence of inclusion bodies

Uninfected Cell Culture Infected Cell Culture with CPE

Timecourse of polio infection. Note how cells round up and die

CPE cont..

Formation of multinucleated cellssyncytium = CPE

Syncytium formation induced by Murine leukemia virus

Cont… As some viruses do not cause CPE in cell lines,

they can be detected by other techniques.

Hemadsorption of erythrocytes onto cells

infected with viruses which do not form CPE and contain hemagglutinin can be used in myxovirus and paramyxovirus detection.

Influenza viruses can be released into the culture medium and then detected by hemagglutination.

DISADVANTAGES OF CELL CULTURES

Long period (up to 4 weeks) required for result.

Often very poor sensitivity, sensitivity depends on a large extent on the condition of the specimen.

Susceptible to bacterial contamination.

Susceptible to toxic substances which may be present in the specimen.

Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.

ADVANTAGES OF CELL CULTURES

EMBRYONATED EGGSINTRODUCTION

The Embryonated hen’s egg was first used for cultivation of viruses by Good Pasteur and Burnet (1931).

Cultivation of viruses in organized tissues like chick embryo necessitates a different type of approach..

For all practical purposes they all themselves behave as tissue cultures.

The process of cultivation of viruses in embryonated eggs depend on the type of egg which is used.

The egg used for cultivation must be sterile and the shell should be intact and healthy.

Cont…

Use embryonated chicken, duck or turkey for inoculation of viral suspension

Are used especially for the influenza viruses isolation.

7 - 10 days old embryonated eggs are used. The egg must be cleaned, the shell

decontaminated with a disinfectant and checked in ovoscope if it is alive

Ovoscope is the equipment used for candling.

SITES SUITABLE FOR CULTIVATION OF VIRUSES

Cont..

TISSUES VIRUSES CULTIVATED

Chorioallantoic membrane

Herpes,Small pox virus,Rous Sarcoma virus

Amniotic cavity Mumps virus ,Influenza

Yolk sac Yellow fever,Rabies

Embryo Influenza ,Mumps

PROCEDURE 1. CANDLING

2.DRILL THE HOLE

3.INJECT THE SUSPENSION WITH SYRINGE

4.HOLE SEALED WITH PARAFFIN WAX

5. EGGS INCUBATE AT 36C FOR 2-3 DAYS

HARVESTING OF EMBRYOS

DETECTION OF VIRAL GROWTH The signs of viral growth include:

i) Death of the embryo,

ii) Defects in embryonic development, and

iii) Localized areas of damage in the membranes, resulting in discrete, opaque spots called pocks (a variant of pox).

iv)The embryonic fluid and tissue can be prepared for examination with an electron microscope.

v) Some can also be detected by their ability to agglutinate red blood cells or by their reaction with an antibody of known specificity that will affix to its corresponding virus, if it is present.

POCK LESIONS ON CAM

HARVESTED EMBRYOS

ADVANTAGES

Isolation and cultivation of many avian and few mammalian viruses

Ideal receptacle for virus to grow

Sterile & wide range of tissues and fluids

Cost- much less

Maintenance-easier

Less labour

Readily available

DISADVANTAGES

WHOLE ANIMALS- using live animal eg.mice, rats, rabbits, guinea pigs, hamster, chickens, and monkey.

- the animal is exposed to the virus by injection of a viral preparation or specimen into the brain, blood, muscle, body cavity, skin, or footpads.

- use in example research to study the immune system’s response to viral infections.- HIV: immunodeficient mice grafted to produce human T cells and human gamma globulin.- Only system for studying pathogenesis & immune responses- Used if it’s the only method through which the virus can be isolated.

ANIMAL MODEL usually a purpose-bred animal

Mouse model

Advantages• in-breed strains reduce genetic variability

• genetics are well understood

• Introduce, mutate or inactivation specific genes thought to control the immune response.

Disadvantages• Sometimes not infected-therefore virus has to be adapted or use a closely related surrogate virus

• Does not always cause same disease state

• Mice are not humans

TRANSGENIC MOUSE MODEL

DETECTION OF VIRAL GROWTH

The signs of viral growth include

i)death of the animal

Ii) defects in animal development.

The infected animal tissue can be prepared for examination with an electron microscope

VIRAL QUANTIFICATION

Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration.

It is utilized in both (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable

The methods used include but not limited to:

i) Hemagglutination assay

ii) Plaque assay

iii)TCID₅₀

HEMAGGLUTINATION ASSAY

A direct method to titre virus.

Based on the ability of some viruses to agglutinate RBCs

Virus is tittered by making serial two fold dilutions of the virus and determining the highest dilution of virus that causes agglutination of RBCs.

PLAQUE ASSAY When cells grow as monolayers, they can be

used to quantify the number of viruses using plaque assay.

- The virus is serially diluted in a liquid medium.

- For each dilution a set amount is added to separate plate containing monolayer of tissue culture cells and the viruses in that solution are allowed to attach to the tissue culture cells.

- After attachment has been allowed to occur, a semi solid medium is added to restrict the movement of new viruses produced so that only adjacent cells will be infected.

CONT…Where virus has infected the tissue culture cells, the infected

cells will die causing the formation of a clear zone amongst the otherwise intact monolayer of cells

This clear zone is called a plaque and it theoretically represents an area where one virus has infected a single tissue culture cell, has multiplied and been released, and has gone on to infect adjacent cells.

The number of plaque forming units (pfu)/ml can be calculated based on the dilution of the original viral solution.

The term pfu/ml is used rather than the number of viruses/ml because it is possible that occasionally more than one virus infects a single cell.

Often the cells or plaques are stained to help in visualization of the plaques.

SERIAL DILUTION

…..

PLAQUE ASSAY RESULTS

CALCULATION OF PFU/Ml Plaques are

enumerated Plaque Counts are

averaged over wells

The average is then divided by the dilution times the volume

(43+40+38)/3(10-4 x 0.1)

= 3,730,000 pfu/ml

43 4 1 040 3 0 038 6 2 0Plaques formed per well

TCID₅₀ TCID50 is the measure of infectious virus titer.

This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells

This assay may be more common in clinical research applications where the lethal dose of virus must be determined or if the virus does not form plaques.

When used in the context of tissue culture, host cells are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death (i.e. infected cells) is manually observed and recorded for each virus dilution, and results are used to mathematically calculate a TCID50 result.

TCID₅₀ calculation

The outcome of a TCID50 determination can be used

to estimate a virus titre in pfu, or vice versa, using the formula

100

75

50

1 TCID₅₀ = 0.7 pfu

10−1 10−2 10−3 10−4 10−5

10−6

TCID₅₀

Other methods for TCID50 calculation Two methods commonly used to calculate TCID50 are:

i) Spearman-Karber

ii) Reed-Muench method

REFERENCES

www.invitrogen.com/cellculturebasics.

http://web.lfhk.cuni.cz/klinmikrob/en/laboratory_diagnosis.htm

http://www.microbelibrary.org/component/resource/laboratory-test/2875-cytopathic-effects-of-viruses-protocols

http://www.slideshare.net/kimareew/hela-cells

http://en.wikipedia.org/wiki/Virus_quantification

Flint, S.J.; Enquist, W.; Racaniello, V.R.; Skalka, A.M. (2009). "Virological Methods". Principles of Virology

John Carter; Venetia Saunders.(2007) Virology principles and applications

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