1. Cell Culture and Diagnostic Virology
part 3

2. Introduction
Since the discovery by Enders (1949) that polioviruses could be cultured in tissue, cell culture has become a very useful and convenient method for isolating viruses in vitro
Viral isolation in cell culture still remains the "gold standard" for many cultivable viruses
A single cell culture can be used to cultivate a broad spectrum of viral agents
Viral culture also facilitates the production of high titered virus which can be used in:
Antibody testing
Viral characterization
or molecular analysis

3. Monolayer Cell Cultures
Most diagnostic virology laboratories use monolayer cell cultures to propagate viruses
The main advantage of using monolayer cultures is the ease with which the infected cultures can be monitored microscopically
Many viruses present themselves in cell culture by producing degenerative changes in the cells, the so-called cytopathic effect (CPE)
The CPE is often characteristic of a specific virus and this allows the experienced observer to make a presumptive diagnosis based on the type of CPE present on the monolayer

4. Transport Medium
Virus viability is crucial for successful isolation in cell culture
Enveloped viruses are particularly labile
Viruses such as respiratory syncytial virus, influenza and the herpes viruses, may lose infectivity if they are not adequately protected
Rapid transportation to the laboratory under the proper conditions can greatly enhance effective isolation
Viruses should be transported to the laboratory in the appropriate transport medium (viral transport medium) which can be bought commercially or made up in Lab.

5. Isolation of Viruses in Cell Culture
The ability to culture viruses successfully in the laboratory depends on a number of important factors which include:
The sensitivity of the cells used
The viability of the virus
The type of specimens sent to the laboratory
The stage of the patients illness when the specimen is taken
and the way they are processed
The culture conditions
Even when all these considerations are taken into account, not all viruses can be cultured
There are certain viruses that are very difficult to grow or require very specialized culture conditions

6. Isolation of Viruses in Cell Culture
However, most of the more common human pathogenic viruses can be cultured relatively easily provided the proper conditions are satisfied
A wide variety of virus-sensitive cell lines are available either commercially or through one of the national or international cell bank collections such as ATCC & ECACC
ECACC: European collection of cell culture
ATCC: American Type culture collection

7. Standard Virus Isolation from Samples
Seed cell suspensions into culture vessels using freshly made medium
For viral isolation it is usual to prepare at least three different cell types for inoculation to increase the chances of isolation
While some cell lines have a broad range of viral susceptibility, no single cell line is sensitive to every virus
Herpes Simplex
Vero Hep-2, human diploid (HEK and HEL),human amnion
CMV
human diploid fibroblasts
Adenovirus
Hep2, HEK,
Poliovirus
MK, BGM, LLC-MK2, human diploid, Vero, Hep-2,Rhadomyosarcoma
Influenza A
MK, LLC-MK2, MDCK
Influenza B
Parainfluenza
MK, LLC-MK2
Mumps
MK, LLC-MK2, HEK, Vero
Rhinovirus
human diploid (HEK, HEL)
Measles
MK, HEK
Rubella
Vero, RK13

8. Standard Virus Isolation from Samples
Vessels are incubated and allowed to reach 90% confluent
0.2 ml of freshly prepared specimen is inoculated into each vessel in duplicate
Incubation at 37°C
Some viruses (influenza, parainfluenza) need to be cultured at lower temperatures
Examine the cultures daily for CPE

9. Microtiter Method of Virus Isolation from Samples
Using this method six cell lines are seeded in suspension on 96 microtiter plates thereby improving the sensitivity of virus isolation
Up to four specimens can be inoculated with each plate
The cell lines selected for microtiter plate work should represent a broad range of viral susceptibility
The plates are monitored daily for CPE using an inverted microscope

10. Microtiter Method of Virus Isolation from Samples
6 cell lines
6 cell lines
Sample 1
(4 dilutions)
Sample 2
Sample 3
Sample 4
6 cell lines

11. Identification of Virus Isolates
Development of characteristic CPE in cell culture is often useful in making a presumptive identification of the viral isolate
This identification would also be based on the specimen source and the cell type in which the virus has grown
However, final identification of the viral isolate needs to be confirmed
Monolayer of uninfected Hep-2 cells (x20)
Hep-2 cells infected with respiratory syncytial virus showing typical syncytial cytopathic effect (x40)

13. The upper left panel shows uninfected cells, and the other panels show the cells at the indicated times after infection. As the virus replicates, infected cells round up and detach from the cell culture plate. These visible changes are called cytopathic effects.

14. Tubule Lining Cells

15. Cytopathic Effect (CPE)
hela cells infected with rhinovirus.mp4
This video shows the cytopathic effect of human rhinovirus infection on HeLa cells
Uninfected cells are adherent, they normally grow flat and stuck down firmly on the tissue culture flask
After infection with rhinovirus, the cells change shape, becoming round and more refractile (brighter) under phase contrast microscopy
Some infected cells detach from the tissue culture flask and float in the medium

16. Identification of Virus Isolates
A more definitive viral diagnosis is carried out by further testing of the viral isolate
This can be achieved by performing:
Infectivity assays
A viral neutralization assay
The application of immunoassay techniques such as:
IF staining of infected cells
ELISA
Western blotting
Molecular techniques
nucleic acid hybridization
The application of these techniques are particularly useful for detecting specific viral replication in cultures in the absence of a CPE

17. Identification of Virus Isolates
Not all viruses will produce a CPE and some viruses are slow to grow
Immunoassay techniques can also allow early detection of viral replication prior to the formation of a CPE and allow more rapid viral diagnosis
The availability of specific and sensitive monoclonal antibodies directed against viral antigen has greatly enhanced the use of these techniques in viral diagnosis

18. Infectivity Assays: Virus Neutralization
Neutralization assays are based on the principle that viral antibody will bind specifically to the virus with a resultant neutralization of viral infectivity
Virus neutralization tests may utilize any assay that measures viral infectivity
These in vitro assays may include inhibition of plaque formation or cytopathic effect (CPE)
A confirmed identification of the viral isolate is made when development of CPE has been effectively inhibited by the specific antiserum

19. Infectivity Assays: Virus Neutralization
Viral neutralization may be performed:
for virus identification,
to determine the antigenic relationship of different viral isolates,
and the Identification and quantification of viral antibody
Known reference antiserum is necessary for virus identification
and known infectious virus is necessary for identification and quantification of viral antibody

20. Quantification of Viruses
Involves counting the number of viruses in a specific volume to determine the virus concentration
Many approaches are available to determine the concentration of viruses in a given tissue
Infectivity assays,
molecular assays
and direct counting of virus particles using electron microscopy
These enable the virologist to calculate the number of infectious viral particles per unit volume

21. Quantification of Viruses
It is utilized in many procedures including:
Research
Diagnosis of early viral infection
Monitor a patient's response to anti-virus therapy
as well as in production situations where the quantity of virus at various steps is an important variable
For example, the production of viral vaccines
recombinant proteins using viral vectors

22. Quantification of Viruses
The most commonly used methods to quantify viruses can be subdivided into three broader categories:
Techniques measuring viral infectivity
Viral plaque assay
TCID50 
Those that examine viral nucleic acid and protein
qPCR (real time PCR)
Western blotting
Immunoassyas
ELISA
Those that rely on direct counting of physical viral particles
viral flow cytometry
and transmission electron microscopy

23. Quantification of Viruses

24. Plaque Assay
Based on the ability of infectious virus particles to form small areas of cell lysis or foci of infection on the cell monolayer
This is achieved by first adsorbing the virus onto a confluent cell monolayer and then overlaying the monolayer with agar
The overlay medium restricts the spread of secondary infection so that only areas of the cell monolayer adjacent to the initially infected cells will become infected and form plaques or small areas of CPE
These plaques can then be counted and the viral titer calculated
Plaque assays can be carried out in 24-well cell cluster plates or cell culture plates

25. Plaque Assay

26. ..\practical virology\virology\lab.10\Lab. 10.2plaque assay.mp4

27. TClD50 (Tissue Culture Infectious Dose)
This is a quantal assay which determines the dilution of virus required to infect or cause CPE in 50% of inoculated cell cultures
The assay can be carried out in culture tubes or 96-well microtiter plates
Different viral dilutions are prepared and inoculated on cell culture
The virus replicates and the progeny virus that is released into the supernatant fluid is free to infect any other cell
The cytopathological damage is allowed to develop usually over a period of days (depending on the given virus and the cells)

28. Flow Cytometry
There are a few commercially available flow cytometers that can be used for virus quantification
A virus counter quantifies the number of intact virus particles in a sample using fluorescence to detect proteins and nucleic acids
Samples are stained with two dyes and analyzed as they flow through a laser beam
one specific for proteins
and one specific for nucleic acids
The quantity of particles producing simultaneous events on each of the two distinct fluorescence channels is determined, along with the measured sample flow rate, to calculate a concentration of virus particles (vp/mL)

29. cell culture video.mp4

30. The END