Cell Culture and Diagnostic Virology

1. Cell Culture and Diagnostic Virology

2. 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. 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 Monolayer Cell Cultures

4. 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. Transport Medium

5. 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

6. 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 Isolation of Viruses in Cell Culture

7. 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 Standard Virus Isolation from Samples

8. 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

9. 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

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

11. 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 Identification of Virus Isolates 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. 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 Cytopathic Effect (CPE)

16. 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

17. 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 Identification of Virus Isolates

18. 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

19. 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 Infectivity Assays: Virus Neutralization

20. 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 Plaque Assay • 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

21. Plaque Assay

23. 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

24. 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

25. 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

26. Quantification of Viruses

27. 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 microtiterplates 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) TClD50(Tissue Culture Infectious Dose)

28. 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) Flow Cytometry