CELL CULTURE AND DIAGNOSTIC VIROLOGY

• Since the discovery by Enders (1949) that polioviruses could be cultured 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

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

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.

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

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

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

• 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

Standard Virus Isolation from Samples

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

Microtiter Method of Virus Isolation from Samples

Sample 1 (4 dilutions)

Sample 2(4 dilutions)

Sample3

Sample4

6 cell lines 6 cell lines

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

A. Monolayer of uninfected Hep-2 cells (x20)B. Hep-2 cells infected with respiratory syncytial virus showing typical

syncytial cytopathic effect (x40)

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

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

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

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

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

Plaque Assay

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

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

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

Quantification of Viruses

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)

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)

Cytopathic Effect (CPE)

• 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

• https://www.youtube.com/watch?v=QlIRg7fnjyQ#t=177