Isolation And Identification

Isolation and identification is the gold standard technique for detection of many viral agents. The power of isolation is that a particular agent is not targeted and that, as the result of viral multiplication, even one infectious particle may be amplified millions of times, greatly facilitating the identification.

Specimens

The selection, transport, storage, and processing of the specimen are crucial for isolation attempts to be meaningful. The ideal specimen is taken from the site of the lesion or symptoms as early in the course of the illness as possible. The risk of fetal exposure or infection is determined by the status of the mother. Herpes I or II, enterovirus, rubella, and varicella-zoster virus (VZV) are some of the viruses that may be isolated and that are clinically relevant to the fetus or newborn. Other important agents such as hepatitis B virus, HIV, and parvovirus B-19 are either extremely difficult to culture or cannot be cultured.

In some cases, specimens taken from sites other than the site of the symptoms are useful. For example, enterovirus infections are frequently diagnosed from stool or respiratory specimens, which may then provide useful information on the etiology of systemic symptoms such as myocarditis or encephalitis.

It is advisable to inoculate culture systems immediately after obtaining the specimen or as soon as possible for isolation attempts to be successful. If the specimen will be inoculated within 48 hours of collection, it should be held at 4°C. However, when inoculation will be delayed for longer periods, stools and rectal swabs should be stored frozen at -20°C and respiratory and other specimens at -70°C. To select the optimal host system for culture, it is crucial that information on the symptoms of the patient, the date of onset, the date the specimen was taken, the disease suspected, and other relevant information accompany the specimen.

Specimens such as stools and cerebrospinal fluid (CSF) should be placed in a sterile container for holding and shipment. Swabs should be placed in a container with sterile viral transport media. The transport media may be purchased from a commercial supplier or may be prepared in-house. A transport media satisfactory for most purposes consists of a sterile buffered broth or balanced salt solution with approx 0.5% gelatin.

Specimen processing depends on the type of specimen. Specimens that are sterile, such as CSF, are inoculated directly into a suitable host system. Stools and rectal swabs are put into solution, and antibiotics are added. Penicillin and streptomycin in combination or gentamicin are satisfactory. Gentamicin is used by many laboratories because it has a broad spectrum of action and can be stored at 4°C. Fungizone (amphotericin B) is added to control fungal growth. Antibiotics are added directly to respiratory wash specimens. Tissue obtained at autopsy or biopsy tissue is ground and resuspended to 10 or 20% in an appropriate diluent and then inoculated.

Viral Culture Systems

Viruses are obligate intracellular parasites; therefore, cultivation requires an appropriate living host system. There is no one host system suitable for cultivation of all viral agents. In the historical development of diagnostic virology laboratory tests, the discovery and utilization of new host systems were critical events in advancing the discipline. The first host systems were laboratory or experimental animals. In the 1930s, embryonated eggs were first used to cultivate viruses. They supported the growth of nearly all viruses known at the time. However, the real breakthrough was the use of cells in culture. The report of growth of poliovirus in cultured, nonneural cells with the production of cytopathic effects (CPEs) in 1949 (1) marked the start of the age of cell culture for in vitro cultivation of viruses, which resulted in the discovery of a number of new viruses.

Suckling mice may still be used and are the only or best host for certain of the group A coxsackieviruses (2). Otherwise, they are not used for isolation. Embryonated eggs may have some limited use, but for the most part are of historical interest only. Cell cultures have replaced the other host systems and are the host of choice for growing most viruses. Because no single cell type is permissive for all of the viral agents that could be cultured, relevant clinical information is necessary to choose the appropriate cell types.

Cells grown in culture are the in vitro progeny of cells obtained from living tissue. Cultures are started from dissociated pieces of tissue placed in nutrient media in a sterile culture vessel. Cells may grow in suspension or adhere to the surface of the vessel. Adherent cultures form sheets of cells attached to a clear glass or plastic surface. Primary culture is the first passage of the cells outside the parent organism. When cells are removed from the surface of the growth vessel and transferred into two or more vessels, they are said to be passaged or subcultured. Once subcultured, cells are called cell lines. Cell lines may be finite, capable of a limited number of doublings, or infinite, capable of an unlimited number of doublings. Finite cell lines are diploid and exhibit contact inhibition. Examples are the human fetal diploid cells MR5 and WI38. Continuous cell lines, theoretically capable of infinite growth in culture (e.g., HeLa, Hep2, RK13, and A549), are less expensive and easier to maintain. Among the cells types most frequently used for viral isolation are primary monkey kidney cells, human fetal diploid cells, and a variety of continuous cell lines. Despite the ongoing effort to identify better host systems, permissive hosts for many important viral agents, such as those causing hepatitis and gastroenteritis, have not been found.

Viral Culture

Viral isolation attempts are initiated by inoculating specimen aliquots into tubes or bottles of cell cultures. Companion control and inoculated cells are incubated at 33°C for growth of respiratory viruses and at 36°C for optimal growth of other viruses. For some viruses, growth is enhanced by continuous slow rotation of the tubes. The cultures are examined microscopically at periodic intervals for CPE or hemadsorption (HAd). CPE is the degeneration of cells caused by the growth of virus. The type of CPE is characteristic for a given group of viruses, as for example herpes causes a ballooning of cells, enterovirus causes rounding up and lysis, and respiratory syncytial virus (RSV) causes syncytia formation. From the appearance of the CPE, the cells in which the virus grew, the time from inoculation until the CPE appeared, and the rate of progression it is possible to make an informed guess about which virus has been isolated. Although CPE is characteristic for particular virus groups, the CPE alone is not sufficient evidence to establish the identity of the virus. It is important to be aware that cell degeneration may occur for reasons other than the growth of a virus. Toxicity caused by the inoculum, culture medium, or aging of cells may be confused with CPE. Contaminants such as mycoplasma or parasites that can cause effects not easily differentiated from those caused by viruses are occasionally introduced with the specimen. Primary monkey cells frequently carry indigenous viruses that grow in the culture and can cause CPE or hemadsorb (3).

HAd is the attachment of red blood cells from various species to the cells in culture. It is useful as a marker for the growth of viruses that modify the cellular membrane by inserting viral-coded proteins, which attach to red blood cells. HAd does not identify which virus is present, but it is a good indicator for growth of members of the Myxo (influenza virus) or Parmyxovirus families. It is important to be aware that HAd in primary monkey kidney cells can be caused by an indigenous monkey virus.

Interference of viral CPE is used to monitor for the growth of rubella, a virus that does not cause CPE or HAd. At the California Department of Health, Viral and Rickettsial Disease Laboratory, when rubella is suspected, RK13 and BSC-1 cells are inoculated. Other cells that are susceptible include Vero and African green monkey kidney. Following several days of incubation, echovirus 11, a cytopathic enterovirus, is added to inoculated and control cultures. The failure of the enterovirus to cause CPEs in inoculated cultures is evidence that rubella has grown. Interference is not proof that rubella is present, but if interference does not occur, it proves that rubella has not grown. Rubella can then be identified using a specific labeled antibody.

Shell Vials

Another approach to make culture more rapid and to identify if a particular agent is present prior to the appearance of CPE or detectable HAd is the shell vial technique. The specimen is centrifuged onto the cell sheet, incubated for 24 or 48 hours, and then stained with specific antibodies. The technique is especially useful for detection of cytomegalovirus (CMV), which requires days or weeks to grow and may require subculture before viral growth can be recognized. Monoclonal antibody (MAb) to CMV early antigens, which are synthesized before CPE develops, allow for detection of viral infection before CPE is apparent (4). Shell vials are also used for herpes I and II, VZV, enteroviruses, and respiratory viruses. This method makes the identification of virus more rapid, but because it requires staining for specific agents, it cannot detect all the agents that might be present.

Virus Identification

Some laboratories identify the virus group based on CPE, the cells the virus grew in, and the fact that the CPE can be passed. However, as mentioned in the Viral Culture section, there are problems with the identification of viruses when relying on the presence and type of CPE. The identification is subjective, and even the most experienced virologist may make errors in the reading and interpretation of the cause and type of CPE. Confirmation of the identification for all isolates is highly recommended.

The identification is accomplished by a specific test for viral antigen or nucleic acid or by the morphology seen by electron microscopy (EM). Identification by nucleic acid and by EM is discussed in the section on direct detection. The most common method for viral identification is by specific antibodies from either polyclonal serum or MAbs. The discovery and utilization of MAbs have had a major impact on diagnostic virology. They can be selected to have a high sensitivity and specificity, and the identical

Fig. 1. Direct assay using labeled antibody. F, fluorescence; S, substrate; E, enzyme; Ag, antigen; FA, fluorescent assay; EIA, enzyme immunoassay; RIA, radioimmunoassay.

antibody is potentially available in unlimited supply. The reproducibility and unlimited supply have made it possible to develop highly standardized reagents and tests. The high specificity of MAbs can result in false-negatives because of antigenic variation, but this can be overcome using mixtures of MAbs.

Unlabeled Antibody Assays

Depending on the assay, the antibody may be unlabeled or labeled with a fluorescent molecule, a radioisotope, an enzyme, or biotin. In assays using unlabeled antibody, a specific antibody-antigen reaction is detected by a resultant biological effect. Examples of assays that use unlabeled antibodies are hemagglutination inhibition (HI), complement fixation, and neutralization. HI is the standard test for strain typing of influenza isolates but otherwise now has little use in virus identification. Complement fixation has been used for typing enteroviruses, adenoviruses, and others, but like the HI test is only infrequently used for virus identification. In the neutralization test, viral infectiv-ity is interfered with or blocked by the reaction of specific antibody with the virus. The failure of the virus to infect cells results in the cells remaining healthy without exhibiting CPE or cell death. Neutralization is highly specific and is the standard test for typing of entero- and adenoviruses. Tests identifying viruses by specific neutralization are still important but possibly are being replaced by more rapid tests.

Labeled Antibody Assays

Labeled antibodies are versatile and may be used in a variety of formats. An important distinction in the use of labeled antibodies is the difference between the direct and indirect formats. In the direct format (Fig. 1), the labeled antibody is specific for the target antigen. For instance, to identify a suspected herpes, virus cells suspected to be infected with the virus are fixed onto a glass slide and stained with labeled antibodies to herpes I and herpes II. At the same time, uninfected cells and herpes I and herpes II

Fig. 2. Indirect assay using labeled antibody. EIA, enzyme immunoassay; IFA, indirect fluorescence assay; RIA, radioimmunoassay.

infected control cells are also stained. If the controls react properly (i.e., no staining of uninfected cells and specific staining of herpes I and herpes II controls with the proper conjugates), then the test can be read.

In the indirect format, the labeled antibody is specific for immunoglobulin of the species in which the primary antibody was made (Fig. 2). This is sometimes known as the sandwich technique because the specific antibody is sandwiched between the fixed target antigen and the labeled antispecies antibody. Because the detecting antibody is antispecies immunoglobulin, a single conjugate can be used to detect a variety of primary antibodies specific for different viruses. The direct method is relatively more specific and less sensitive than the indirect method.

In the fluorescent antibody method, fluorescein isothiocyannate (FITC) or another fluorescent molecule is conjugated to antibody molecules specific for a given antigen without destroying the binding activity of the antibody. With FITC-labeled antibodies, specific staining for herpes will be seen as crisp, green fluorescence in the nucleus and cytoplasm of infected cells. In addition to high specificity and sensitivity, another important advantage of the fluorescent-labeled antibody technique is that the morphology of staining is read; that is, one is able to determine if the staining occurs only in the nucleus or cytoplasm or both. Particular staining patterns are typical of a given virus. Disadvantages are that reading requires a fluorescent microscope, and that when using the standard glycerol-based mountants, the slide preparations are not permanent. At the Viral and Rickettsial Disease Laboratory a polyvinyl alcohol, glycerol mountant that preserves full fluorescence for up to a year or more is used.

Enzyme-labeled antibodies are also versatile reagents used in a variety of assays. Similar to assays using fluorescent-labeled antibodies, enzyme immunoassays are highly sensitive and specific. They are used in one of two formats for antigen detection: in situ detection and solid-phase, quantitative assays. For the in situ detection of antigen, horseradish peroxidase is used in the immunoperoxidase assay. This assay is analogous to the immunofluorescent assay. Following the reaction of the peroxidase-labeled antibody with the test specimen and controls, the enzyme substrate is added so that the action of the enzyme on the substrate results in the production of a colored product. The product is insoluble, so it precipitates at the site of the of the enzyme substrate reaction. The colored precipitate can be viewed by light microscopy. Advantages of this assay over FA assay is that it does not require a fluorescent microscope and it provides a permanent record. Quantitative solid-phase assays are described in the section on direct detection.

Antibodies may also be conjugated with the low molecular weight molecule biotin. Because of its relatively small size, biotin is less likely to interfere with the antibody-to-antigen binding reaction than the larger enzyme molecules. Avidin or strepavidin both bind with a high affinity to biotin and can be labeled with enzyme. The direct reaction has two steps: (1) addition of biotin-labeled primary antibody, and (2) addition of avidin enzyme complex. The substrate is then added, and color development is monitored. In theory, these assays are extremely sensitive because one biotin molecule binds four avidin enzyme complexes.

Assays using radioisotope label are not in general use because of safety and disposal problems. The use of these and other assays such as latex agglutination and time-resolved fluoroimmunoassay for detection of viral antigen are discussed in an excellent chapter on the subject (5).

Interpretation

When a virus is isolated and identified, the significance of the isolate is based on the known association of the agent with the patient's syndrome and the site from which the virus was isolated. For instance, enteroviruses are known to be possible etiological agents of encephalitis. However, an isolate from stool is much less significant than one from a central nervous system specimen. Failure to isolate an agent in no way rules out the possible role of the agent in the syndrome

For many viruses, isolation and identification are the "gold standard" methods for detecting virus in a specimen. They offer the important advantages that the agent is greatly amplified, that a particular agent does not need to be targeted, and that successful culture provides a live replicating virus for further characterization. However, not all viruses grow in culture. Cell culture is time consuming and expensive and requires special expertise and training.

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