key: cord-0252768-5yw4zwo4
authors: Doane, F. W.
title: Virus morphology as an aid for rapid diagnosis.
date: 1980
journal: Yale J Biol Med
DOI: nan
sha: a8c1928d0295a8f2f13a679f4536ceff03508bde
doc_id: 252768
cord_uid: 5yw4zwo4

Standard methods of virus diagnosis may take many days to complete. As antiviral drugs are being used with more effectiveness, it becomes more important to develop rapid diagnostic methods. It takes only a few minutes to prepare and examine a specimen for electron microscopy (EM), using the negative staining technique. Viruses in the specimen can readily be identified by their morphology. In order to be detected by EM there must be at least 10(7) virus particles per milliliter of sample. This concentration is frequently found in certain types of specimens. The sensitivity of EM is increased 100-fold if homologous antibody is used to aggregate the virus. Visualization of virus-antibody aggregates forms the basis for serotyping by immunoelectron microscopy (IEM).

The commonly practised laboratory methods to detect the presence of a virus in clinical material have changed little in the past 25 years. A specimen is inoculated into a host system (tissue culture, eggs, animals), and subsequent detection and identification of an isolated virus depends on indicators such as cytopathic effect, hemagglutinin, complement fixation, etc. Results obtained by these procedures usually take several days-if not weeks-to complete. Thus, although they continue to serve as the backbone of most virus laboratories, current procedures are rarely capable of providing a rapid diagnosis.

With the advent of specific antiviral drugs, especially for herpesviruses, there is now more pressure on the diagnostic virologist to provide results quickly. Direct microscopic examination of the clinical specimen for the virus itself is one obvious approach. For all practical purposes, viruses are too small to be seen by light microscopy, but they can easily be visualized, and morphologically identified, by electron microscopy (EM).

The detection and identification of vruses by EM offers many advantages. Most important-the method is fast; total time required for specimen preparation and EM examination is rarely more than 30 minutes. Requirement for an isolation system is removed, and loss of infectivity of virus in the specimen becomes unimportant. Thus viruses that are difficult or impossible to culture can be identified by EM. Family or group identification can be made immediately, entirely on the basis of virus morphology. And by immunoelectron microscopy-serotyping directly on the EM specimen grid-type-specific identification of a virus can often be accomplished.

The following brief review will examine some of the ways in which virus morphology can serve as an important aid for rapid virus diagnosis, some of the limitations of this approach, and current and future developments in electron microscopy relating to diagnostic virology. For detailed information on electron microscopy in diagnostic virology, the reader is referred to more comprehensive reviews [1, 2] .

DIRECT EM EXAMINATION OF CLINICAL SPECIMENS Direct EM examination of clinical specimens, using the negative staining technique, provides the simplest and most rapid method for virus detection. The technique consists of mixing a drop of specimen with a drop of heavy metal staining solution. The mixture is quickly air-dried on the support film covering the EM specimen "grid," and is ready for examination within 1-2 minutes. The metal provides an electron-dense background ("negative stain"), in sharp contrast to the more electron-transparent virus particles. The success of this method for virus detection depends almost entirely on the concentration of virus particles in the specimen. The quantity of material that can be examined on a specimen grid is so small that, in general, a positive detection requires at least 107 particles per milliliter in the original specimen [2] . Unfortunately many clinical specimens do not contain this quantity of virus; consequently, they are inappropriate for EM examination.

Undoubtedly the best type of clinical specimen for rapid virus diagnosis comes from vesicular eruptions associated with poxvirus or herpetic infections. The cells at the base of the lesions produce large quantities of virus, which can be found by EM in vesicle fluid or scrapings [3, 4] . The large brick-shaped poxvirus can readily be distinguished from the smaller icosahedral herpesvirus (Plate 1), and this difference in morphology, combined with the speed of the negative staining technique, has long made EM the method of choice in the differential diagnosis of smallpox and chicken pox [5] [6] [7] .

Another type of clinical specimen which often contains a high concentration of virus is the stool specimen. EM examination of stools is useful in the detection of rotaviruses and other agents associated with acute gastroenteritis [8] [9] [10] [11] [12] , enteroviruses [13] , adenoviruses [8, 13] , coronaviruses [14] , hepatitis A virus [15] , etc. Nasopharyngeal secretions may contain detectable quantities of parainfluenza virus, respiratory syncytial virus, coronavirus, and mycoplasma [16, 17] . Herpesviruses and mumps virus have been found by EM in cerebrospinal fluid [16, 18] , and CMV and BK virus in urine [19] [20] [21] .

Because of the growing number of promising reports concerning the effectiveness of antiviral chemotherapy at the early stages of herpes encephalitis, the virus laboratory is called on increasingly to perform virus studies on brain biopsies. If the sample has been correctly collected from an infected area, virus particles can usually be detected by EM, using the negative staining technique. A more dependable procedure, especially with low levels of virus, is to fix and embed the tissue by one of the newer rapid embedding techniques [22] and systematically examine sections in the EM. Sections are also useful in confirming a viral etiology in diseases such as subacute sclerosing panencephalitis (SSPE) and progressive multifocal leukoencephalopathy (PML) [2] . TISSUE CULTURE ISOLATION; EM IDENTIFICATION Although EM examination of the clinical specimen offers the fastest method of detecting a virus, many specimens received by a virus laboratory may contain insufficient virus for EM detection. For this reason, EM-equipped virus laboratories receiving large numbers of specimens may elect to perform direct examination only on selected specimens, such as biopsies, vesicle fluid or scrapings, and stools from acute gastroenteritis, and inoculate all other specimens into cell cultures. Those viral isolates that cannot be identified by cytopathic effect (CPE) alone can be identified on the basis of morphology by negative staining an aliquot of the infected culture (Plate 2). As shown in Table 1 , viral isolates can often be identified by EM 24-48 hours before they produce a CPE that is visible by light microscopy.

VIRUS SEROTYPING BY IMMUNOELECTRON MICROSCOPY Identification of a virus by morphology alone allows it to be classified with respect to family or group (e.g., picornavirus, adenovirus, herpesvirus, poxvirus, orthomyxovirus, paramyxovirus). This is usually all that is required by the clinician in order to dictate appropriate treatment of the patient. From an epidemiological point of view, however, it may be important to know the precise serotype of a virus, and this information cannot be obtained from virus morphology only. This problem is well exemplified in the case of the picornaviruses, where all members, including rhinoviruses and enteroviruses, have an identical morphology. Enteroviruses are commonly encountered in a diagnostic laboratory, and their serotyping is usually performed by means of a virus neutralization test in cell cultures. Results are obtained within l/2 to 2 weeks. The same results can be obtained within one hour if one uses immunoelectron microscopy (IEM), by which the virusantiserum mixtures are negatively stained and examined on an EM specimen grid. In the electron microscope a virus-antibody complex appears as an aggregate of virus held together by antibody molecules (Plate 3). Although still in the development stage, serotyping by IEM has been successfully applied not only to enteroviruses [23, 24] , but also to adenoviruses, papovaviruses, and myxoviruses [21, [25] [26] [27] [28] [29] [30] . I EM has also been used to increase the sensitivity of detection of virus in a sample. As little as 1035 TCID50 / ml of poliovirus can be detected by IEM-approximately 100 times less than that needed for EM detection in the absence of antibody [23] .

The ability of specific antibody to form visible aggregates of homologous virus has been utilized to detect unknown viruses that have remained elusive either by virtue of their indistinctive morphology, as in the case of rubella virus [31] , or because they have been difficult or impossible to culture, as in the case of viruses such as hepatitis A and B, and wart virus [15, [32] [33] [34] [35] . A clinical specimen is mixed with the patient's serum, on the theory that any virus present in the specimen may be aggregated by antibody in the serum. The mixture is then negatively stained and examined by EM.

Virology in all its forms relies heavily on cell cultures, which unfortunately may become contaminated with adventitious agents. One of the most notorious groups of contaminants comprise the simian viruses, which may occur in over 50 percent of "normal" primary monkey kidney cell cultures {36]. Established cell lines may be plagued with mycoplasma contamination, or with low-grade viral infections acquired by cross-contamination from infected cultures in the laboratory. Such contaminants are often extremely difficult to detect, as they may produce little or no cytopathic effect. They can be seen by electron microscopy, however, using methods such as negative staining and thin sectioning [37] . Virus pools passed in contaminated cultures can themselves become contaminated. A quick check by electron microscopy provides a means of monitoring pools for possible contaminants. CONCLUSION Although many virus diagnostic laboratories continue to rely heavily on the wellestablished isolation and identification procedures, increasing attention is being paid to the development of more rapid techniques, the majority of them involving antibody-labeling. The fluorescent antibody technique, when adequately controlled, can be used to detect viral antigens in clinical specimens and inoculated host systems. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) can now be applied with confidence for rapid quantitation of selected viral antigens and antibodies. Yet with all of these techniques the virologist must make a preliminary decision concerning the possible nature of the causative agent in order to select the appropriate reference antisera. A major advantage of using electron microscopy for rapid virus diagnosis is that one can actually see the virus and identify it by its morphology. Subsequent serotyping, if required, can then be carried out more precisely. Serotyping by IEM is extremely rapid, and offers promise for the future. Its value may increase through the use of more purified monospecific antisera.

The EM techniques discussed above have all made use of the standard transmission electron microscope (TEM). There are indications that the scanning electron microscope (SEM) may also eventually be of use to the diagnostic virologist. At present, its practical application, albeit a valuable one, is limited to screening cell cultures for mycoplasma contamination [38] .

Identification of Viruses by Immunoelectron Microscopy

Electron and Immunoelectron Microscopy

The use of the electron microscope in diagnosis of Variola, Vaccinia and Varicella

Smallpox diagnosis with special reference to EM

Reliability of the rapid EM diagnosis of smallpox

Electron microscopy in differential diagnosis of poxvirus infections

Electron microscopy in the rapid diagnosis of smallpox

Diagnostic electron microscopy of faeces. 1. The viral flora of the faeces as seen by EM

Diagnostic EM of faeces. I1. Acute gastroenteritis associated with reovirus-like particles

Clinical Observations and Diagnosis of Gastroenteritis

28 nm particles in faeces in infantile gastroenteritis

Comparison of the features of astroviruses and caliciviruses seen in samples of faeces by electron microscopy

Agar diffusion method for negative staining of microbial suspensions in salt solutions

Coronavirus particles in faeces from patients with gastroenteritis

Hepatitis A: detection by immune electron microscopy of a virus-like antigen associated with acute illness

Rapid laboratory diagnosis of paramyxovirus infections by electron microscopy

Diagnosis of viral respiratory infections by electron microscopy

Electron microscope studies of the vesicle and spinal fluids from a case of Herpes Zoster

Electron microscopy in the rapid diagnosis of cytomegalovirus: Ultrastructural observation and comparison of methods of diagnosis

Rapid diagnosis of cytomegalovirus infection in infants by electron microscopy

New human papovavirus (B.K.) isolated from urine after renal transplantation

Two-hour embedding procedure for intracellular detection of viruses by electron microscopy

Specific identification of enteroviruses by immuno-electron microscopy using a serum-in-agar diffusion method

Serotyping of coxsackieviruses by immune electron microscopy

Rapid adenovirus typing by immunoelectron microscopy

Serotyping of adenoviruses using immune electron microscopy

Visualization by immune electron microscopy of viruses associated with acute respiratory disease

Studies of the antigenic relationships of the new human papovaviruses by electron microscopy agglutination

Virions from progressive multifocal leukoencephalopathy: rapid serological identification by electron microscopy

Differentiation of Myxoviruses by Electronmicroscopy and Immunoelectronmicroscopy

Morphological characteristics of rubella virus

Particles associated with Australia antigen in the sera of patients with leukemia, Down's syndrome and hepatitis

Antibody to wart virus in human sera demonstrated by electron microscopy and precipitin tests

Detection and identification by immune electron microscopy of fastidious agents associated with respiratory illness, acute nonbacterial gastroenteritis, and hepatitis A

Immune electron microscopy as a method for the detection, identification, and characterization of agents not cultivable in an in vitro system. Manual of Clinical Immunology

Latent virus infections in primate tissues with special reference to simian viruses

Microscopic detection of adventitious viruses in cell cultures

A survey of viral cytopathology by scanning electron microscopy