TISSUE Culture and Virology Supervision of: DR.GHULAM MUHAMMAD Presented By: NOMAN HAFEEZ KHOSA Subject: Cell and tissue culture technology

Tissue Culture 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. A single cell culture can be used to cultivate a broad spectrum of viral agents. Viral culture also facilitates the production of high tittered 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 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 ECACC: European collection of cell culture ATCC: American Type culture collection

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

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

Micro titer Method of Virus Isolation from Samples Using this method six cell lines are seeded in suspension on 96 micro titter plates thereby improving the sensitivity of virus isolation Up to four specimens can be inoculated with each plate The cell lines selected for micro titer 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 6 cell lines 6 cell lines Sample 1 (4 dilutions) Sample 2 Sample 3 4 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 Monolayer of uninfected Hep-2 cells (x20) Hep-2 cells infected with respiratory syncytial virus showing typical syncytial cytopathic effect (x40)

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.

Cytopathic Effect (CPE)   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

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.

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

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Plaque assay- additional notes Viral Plaques of Herpes Simplex Virus Plaque-based assays are the standard method used to determine virus concentration in terms of infectious dose. Viral plaque assays determine the number of plaque forming units (pfu) in a virus sample, which is one measure of virus quantity. This assay is based on a microbiological method conducted in petri dishesor multi-well plates. Specifically, a confluent monolayer of host cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar orcarboxymethyl cellulose, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus infects a cell within the fixed cell monolayer. The virus infected cell will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated.  

Plaque assay- additional notes(2) The infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen visually or with an optical microscope. Plaque formation can take 3 – 14 days, depending on the virus being analyzed. Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL). The pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle.