Conventional Cell Culture. Viruses are strict intracellular parasites, requiring a living cell for multiplication and reproduction. To detect virus using living cells, suitable host cells, cell culture media, and techniques in cell culture maintenance are necessary. Host cells, referred to as cell cultures (referred to by some as tissue cultures), originate as a few cells and grow into a monolayer (single confluent layer) on the sides of glass or plastic test tubes. The cells are kept moist and supplied with nutrients by keeping them continuously immersed in a cell culture medium (Figure1). Cell cultures are routinely incubated in a roller drum that holds cell culture test tubes tilted 5 to 7 degrees while they slowly revolve (0.5 to 1 rpm) at 35° to 37°C. Cell culture tubes can be incubated in a stationary rack rather than a roller drum. Rapidly growing viruses, such as HSV, appear to be detected equivalently by the two methods. Comparative studies are not available for most viruses.

Fig1. Cell culture tubes incubating on their sides in a stationary rack. Tubes are oriented with the same glass surface facing downward, because an emblem printed on one side of the glass near the neck is used for correct positioning in the rack.

Metabolism of growing cells in a closed tube results in the production of carbon dioxide and acidification of the growth liquid. To counteract the pH decrease, a bicarbonate buffering system is used in the culture medium to keep the cells at physiologic pH (7.2). Phenol red, a pH indicator that is red at physiologic pH, yellow at acidic pH, and purple at alkaline pH, is added to monitor adverse pH changes. Once inoculated with specimen, cell cultures are incubated for 1 to 4 weeks, depending on the viruses suspected. Periodically the cells are inspected microscopically with an inverted light microscope for the presence of virus, indicated by areas of dead or dying cells, called cytopathic effect. The degree of CPE is graded from 1+ to 4+; 1+ involves 25% of the cell monolayer; 2+ involves 50%; 3+ involves 75%; and 4+ involves 100% of the cell monolayer.

Virus-induced CPE also presents two other important considerations: the rate at which CPE progresses and whether the type of cell culture in which the virus grows may be used for presumptive identification. An example of rate can be seen with HSV, in which CPE progresses rapidly to involve the entire cell monolayer. In contrast, two other herpes viruses, VZV and CMV, grow slowly, mainly in human diploid fibroblast cells (HDFs), and CPE progresses over a number of days or weeks. The fact that the cell culture type may serve as an indicator of the presumptive identification can be seen with poliovirus and echovirus. Poliovirus and echovirus produce similar CPE in primary rhesus monkey kidney (RMK) cells, but echovirus does not induce CPE in continuous cell lines, whereas poliovirus does. A trained virologist can determine whether CPE is due to viral growth or is nonspecific because of toxicity of specimens, contamination with bacteria or fungi, or simply old cells. Inoculation into fresh cells should amplify viral effects and dilute toxic effects.

Two kinds of media, growth medium and maintenance medium, are used for cell culture. Both are pre pared with Eagle’s minimum essential medium (EMEM) in Hanks’ or Earle’s balanced salt solution (HBSS or EBSS, respectively) and include antimicrobials to prevent bacterial contamination. HBSS has a better buffering capacity with carbon dioxide (CO2), whereas EBSS has a better buffering capacity in ambient air. Typical added antimicrobials include vancomycin (10 µg/mL), gentamicin (20 µg/mL), and amphotericin (2.5 µg/mL). Growth medium is a serum-rich nutrient medium (10% fetal, newborn, or agammaglobulinemic calf serum) designed to support rapid cell growth. This medium is used to initiate the growth of cells in a tube when cell cultures are prepared in-house or to feed tubes of purchased cell cultures that have incomplete cell monolayers. “Feeding” refers to the removal of old medium, followed by the addition of fresh culture medium.

Maintenance medium is similar to growth medium but contains less serum (0% to 2%) and is used to keep cells in a steady state of metabolism. Fetal, newborn, or agammaglobulinemic calf serum is used to avoid inhibitors, such as specific antibody, and because it is free of mycoplasmas present in the serum of older animals.

Several kinds of cell cultures are routinely used for isolation of viruses. A cell culture becomes a cell line once it has been passed, or subcultured, in vitro. Cell lines are classified as primary, diploid (semicontinuous), or continuous. Primary cell lines have been passed only once or twice since harvesting (e.g., PMK cells). Further passage of primary cells results in a decreased receptivity to viral infection. Diploid cell lines remain virus sensitive through 20 to 50 passages. HDF cells, such as lung fibro blasts, are a commonly used diploid cell line. Continuous cell lines, such as human epidermoid carcinoma (HEp-2) cells, can be passed and remain sensitive to virus infections indefinitely. Unfortunately, most viruses do not grow well in continuous cell lines. Most clinically significant viruses can be recovered using one cell culture type from each group. A combination frequently used by clinical laboratories is RMK cells, MRC-5 lung fibroblast cells, and HEp-2 cells or A-549 cells (Table 1).

Table1.  Isolation and Identification of Common Clinically Encountered Viruses

Inoculated cell cultures should be incubated immediately at 35°C. After allowing virus to adsorb to the cell monolayer for 12 to 24 hours, the remaining inoculum and culture medium commonly are removed and replaced with fresh maintenance medium. This avoids most inoculum-induced cell culture toxicity and improves virus recovery. Incubation should be continued for 5 to 28 days, depending on the suspected agent (see Table 1). Maintenance medium should be changed peri odically (usually once or twice weekly) to provide fresh nutrients to the cells.

Blind passage refers to passing cells and fluid to a second cell culture tube. Blind passage is used to detect viruses that may not produce CPE in the initial culture tube but produce CPE when the “beefed-up” inoculum is passed to a second tube. Cell cultures that show non specific or ambiguous CPE are also passed to additional cell culture tubes. Toxicity, which causes ambiguous CPE, is diluted during passage and should not appear in the second cell culture tube. In both instances, passage is performed by scraping the monolayer off the sides of the tube with a pipette or disrupting the monolayer by vortexing with sterile glass beads added to the culture tube, followed by inoculation of 0.25 mL of the resulting sus pension into new cell cultures. Blind passage is less frequently used today, because the added time and expense do not justify detection of a few additional isolates after extended incubation in two cell culture tubes.

Shell Vial Cell Culture. The shell vial cell culture is a rapid modification of conventional cell culture. Virus is detected more quickly using the shell vial technique, because the infected cell monolayer is stained for viral antigens produced soon after infection, before the development of CPE. Viruses that normally take days to weeks to produce CPE can be detected within 1 to 2 days by detecting early produced viral antigens. A shell vial culture tube, a 15 × 45 mm 1-dram vial, is prepared by adding a round coverslip to the bottom of the tube, covering this with growth medium, and adding appropriate cells (Figure 2). During incubation, a cell monolayer forms on top of the coverslip. Shell vials should be used 5 to 9 days after cells have been inoculated. Shell vials can be purchased with the monolayer already formed. Specimens are inoculated onto the shell vial cell mono layer by low-speed centrifugation. This enhances viral infectivity for reasons that are not well understood. Coverslips are stained using virus-specific immunofluorescent conjugates. The presence and visualization of characteristic fluorescing inclusions are used to confirm the presence of an infecting virus (Figure 3).

Fig2. Shell vial cell culture tubes and stained coverslips. At the bottom of each shell vial tube under the culture medium is a round coverslip with a cell monolayer on the top surface. After incubation, the coverslip is removed, stained, and placed on a microscope slide for fluorescence viewing. Note that two stained coverslips are on the glass slide.

Fig3. Typical fluorescing nuclei of human diploid fibroblast cells infected with cytomegalovirus as seen in the shell vial assay. (Courtesy Bostick CC: Laboratory detection of CMV, 1992, Micro biology Tech Sample No MB-3.)

The shell vial culture technique can be used to detect most viruses that grow in conventional cell culture. It is best used for viruses requiring relatively long incubation before producing CPE, such as CMV and VZV. The advantage of the shell vial procedure is its speed; most viruses are detected within 24 hours. The disadvantage is that only a single type of virus can be detected per shell vial. For example, a specimen that might contain influenza A or B or adenovirus would need to be inoculated to three separate shell vials so that each vial could be stained with a separate virus-specific conjugate. Other strategies pool antibody for detection of many viruses with a single vial. Additional vials from positive specimens are then stained with individual conjugates to identify the specific virus present. The shell vial procedure with mixed cell types used to detect seven different respiratory viruses is outlined in Box 1.

Box1.  Overview of Respiratory Virus Detection by R-Mix  Shell Vials

Identification of Viruses Detected in Cell Culture. Viruses are most often detected in cell culture by the recognition of CPE. Virus-infected cells change their usual morpho logy and eventually lyse or detach from the glass surface while dying. Viruses have distinct CPEs, just as colonies of bacteria on agar plates have unique morphologies (Figure 4). CPE may be quantitated as indicated in Table 2. Preliminary identification of a virus frequently can be made based on the cell line that supports viral replication, how quickly the virus produced CPE, and a description of the CPE (see Table 1). Experienced virologists can presumptively identify most viruses isolated in clinical laboratories based on these criteria. When con firmation or definitive identification is required, additional testing can be performed. Fluorescent-labeled antisera, available for most viruses, are used for confirmation. In addition, acid lability is used to differentiate enteroviruses from rhinoviruses, and neutralization is used to identify viruses with many serotypes for which fluorescent-labeled antisera are not available. Some viruses that produce little or no CPE (e.g., influenza, parainfluenza, and mumps viruses) can be detected by hemadsorption, because infected cells contain viral hemadsorbing glycoproteins in their outer membranes. The addition of guinea pig red blood cells (RBCs) to the cell culture tube, followed by a wash to remove nonadsorbed RBCs, results in a ring of RBCs around infected cells (see Figure 4, G). Cell cultures demonstrating hemadsorption can be stained with fluorescent-labeled antisera to identify the specific hemadsorbing virus present.

Fig4. Cell culture morphology and viral cytopathic effects (CPE). A, Normal human diploid lung fibroblast cells (HDF). B, Normal HEp-2 cells. C, Normal primary monkey kidney cells (PMK). D, HEp-2 cells infected with adenovirus. E, HDF cells infected with cytomega lovirus. F, HDF cells infected with herpes simplex virus. G, PMK cells infected with hemadsorbing virus, such as influenza, parainfluenza, or mumps, plus guinea pig erythrocytes. H, HEp-2 cells infected with respiratory syncytial virus.  Continued

Fig4. cont’d I, HDF cells infected with rhinovirus. J, PMK cells infected with echovirus. K, HDF cells infected with varicella-zoster virus. (From US Department of Health, Education, and Welfare, Public Health Service, Centers for Disease Control, Atlanta, Ga.)

Table2. Quantitation of Cell Culture Cytopathic Effects

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مواضيع ذات صلة

Pathogenesis of SARS-CoV-2 and COVID-19

Viral Serology :General Principles

Virus detection methods: Molecular Detection Using Nucleic Acid Probes and Polymerase Chain Reaction Assays

Virus detection methods: Immunodiagnosis (Antigen Detection)

Arenavirus diseases

Bunyavirus diseases

Bunyavirus Encephalitis viruses

Dengue Virus

Clinical Features of Yellow fever

Virus detection methods: Electron Microscopy

Virus detection methods: Cytology and Histology

Specimen Transport and Storage in viral infections