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Isolation of viruses and Viral quantification

The document outlines the methods and purposes of virus cultivation, emphasizing the need for living cells to support viral replication. It describes various cultivation systems such as tissue culture, embryonated eggs, and whole animal systems, highlighting advantages and challenges of each. Additionally, it covers viral quantification techniques, including hemagglutination assay and plaque assay, as well as the historical context and technological advancements in cell culture methods.

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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 Heparin Serum -Stool -Throat swabs -Naso- -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 preparation CELL 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 Cultures ADHERENT 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 cells syncytium = 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 EGGS INTRODUCTION  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 0 40 3 0 0 38 6 2 0 Plaques 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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