Virus Isolation - Methods of Laboratory Diagnosis of Viral Diseases

Methods of Laboratory Diagnosis

Laboratory diagnosis of viral infections can be carried out by many methods. These methods include 

(a) demonstration of virus-induced cytopathic effects (CPEs) in the cells, 

(b) direct detection of viruses, 

(c) virus isolation and viral assays, 

(d) detection of viral proteins and other enzymes, 

(e) detection of viral genome, and 

(f) viral serology (Fig. 53-1).

Virus Isolation

Demonstration of virus in appropriate clinical specimens by culture establishes diagnosis of viral diseases. Isolation of virus is always considered as a gold standard for establishing viral etiology of a disease. Collection of appropriate clinical speci-mens depends on type of the viral disease. For example, cere-brospinal fluid (CSF) is the specimen of choice for diagnosis of viral infections of the central nervous system (CNS) caused by arboviruses, picornavirus, or rabies virus. Blood is the speci-men frequently examined for diagnosis of HIV and hepatitis B, C, and D infections and other blood-borne viral infections. The tests of other clinical specimens to be collected for diagnosis of other types of viral diseases are summarized in Table 53-2.

Timing of collection of specimen is important. The speci-mens collected early in the acute stage infection—before the shedding of virus is stopped—are most important. For exam-ple, enteroviruses are present in the CSF for only 2–3 days after the onset of CNS manifestations. Herpes simplex virus and varicella zoster virus are found in lesions only within first 5 days of onset of symptoms and respiratory viruses are present in respiratory secretions during only first 3–7 days of onset of symptoms.

      Immediate transport of the specimen to laboratory for pro-cessing also facilitates better isolation of virus from clinical specimens. The viruses are usually heat labile, and the clini-cal specimens may be infected secondarily by contamination with bacteria and fungi. Hence, clinical specimens for viruses are usually transported and stored on ice. They are transported in special transport media that contain antibiotics to inhibit bacterial and fungal contaminants and also contain proteins, such as serum albumin or gelatin. Some of the viruses, such as influenza virus, HSV, and VZV lose their infectious titer when clinical specimens are stored at room temperature or kept fro-zen at220°C. Most of the viruses can be cultivated in (a) exper-imental animals, (b) embryonated eggs, or (c) tissue culture.

◗ Animal inoculation

Mouse is most frequently used for isolation of viruses by animal inoculation. In addition, rabbits, hamsters, newborn or suckling rodents are also used. Experimental animals are rarely used for cultivation of viruses but play an essential role in study of pathogenesis of viral infections and that of viral oncogenesis.

Intracerebral, subcutaneous, intraperitoneal, or intranasal routes are various routes of inoculation. After inoculation, the animals are observed for signs of disease or death. The infected animals are then sacrificed and infected tissues are examined for the presence of viruses by various tests, and also for inclusion bodies in infected tissues. Furthermore, infant (suckling) mice are used for isolation of coxsackie virus and rabies virus.

◗ Embryonated eggs

Embryonated eggs were used initially for the growth of viruses. Embryonated chick egg was used first for cultivation of viruses by Goodpasture in 1931. The method further developed by Burnet was used for cultivation of viruses in different sites of the embryonated egg. Usually, 8–11 days’ old chick eggs are used for culture of viruses. The viruses are isolated in different sites of the egg, such as yolk sac, amniotic cavity, and allantoic cavity, and chorioallantoic membrane (CAM) (Fig. 53-2).

      Many of these viruses cause well-defined and characteris-tic foci, providing a method for identification, quantification, or assessing virus pathogenicity. The embryonated egg is also used for growing higher titre stocks of some viruses in research laboratories and for vaccine production.

Yolk sac: Yolk sac inoculation is used for cultivation of Japaneseencephalitis, Saint Louis encephalitis, and West Nile virus. It is also used for growth of chlamydia and rickettsia.

Amniotic cavity: Inoculation in the amniotic cavity is usedmainly for primary isolation of influenza virus.

Allantoic cavity: Inoculation in the allantoic cavity is used forserial passages and for obtaining large quantities of virus, such as influenza virus, yellow fever (17D strain), and rabies (Flury strain) viruses for preparation of vaccines. For production of rabies virus, duck eggs were used due to their bigger size than that of hen’s egg. This helped in production of large quanti-ties of rabies virus, which are used for preparation of the inacti-vated non-neural rabies vaccine.

Chorioallantoic membrane: Inoculation of some viruseson CAM produced visible lesions known as pocks. Each infec-tious virus particle produces one pock. The pox viruses, such as variola or vaccinia are identified by demonstration of typical pocks on the CAM inoculated with the pox virus. Nowadays, in a virology laboratory, chick embryo inocula-tion has been replaced by cell cultures for routine isolation of viruses.       

◗ Tissue culture

Cell culture is most widely used in diagnostic virology for culti-vation and assays of viruses. The tissue culture was first applied in diagnostic virology by Steinhardt and colleagues in 1913. They maintained the vaccinia virus by culture in tissues of rabbit cornea. Subsequently, Maitland (1928) used cut tissues in nutrient media for cultivation of vaccine viruses. Enders, Weller, and Robins (1949) were the first to culture poliovirus in tissue cultures of nonneural origin. Since then, most of the virus had been grown in tissue culture for diagnosis of viral dis-eases. Different types of tissue cultures are used to grow viruses. Tissue culture can be of three different types as follows:

Organ culture

This was used earlier for the isolation of some viruses, which appear to show affinity for certain tissue organs. For exam-ple, coronavirus, a respiratory pathogen, was isolated in the tracheal ring organ culture. In this method, small bits of the organs are maintained in vitro for days and weeks preserving their original morphology and function. Nowadays, organ cul-ture is not used.

Explant culture

In this method, components of minced tissue are grown as explants embedded in plasma clots. Earlier, adenoid tissue explant cultures were used for isolation of adenoviruses. This method is now seldom used in virology.

Cell culture

Cell culture is now routinely used for growing viruses. In this method, tissues are dissociated into component cells by treat-ment with proteolytic enzymes (trypsin or collagenase) followed by mechanical shaking. The cells are then washed, counted, and suspended in a growth medium containing essential amino acids and vitamins, salts, glucose, and a buffering system. This medium is supplemented by up to 5% of fetal calf serum and antibiotics. The cell suspension is dispensed in glass or plastic bottles, tables, or Petri dishes. On incubation, the cells adhere to the glass surfaces and divide to form a confluent monolayer sheet of cells covering the surface within a week (Fig. 53-3). The cell culture may be incubated either as a stationery culture or as a roller drum culture. The latter is useful for growth of some fastidious viruses due to better aeration by rolling of the culture bottle in special roller drums. The cell cultures are clas-sified into three different types based on their origin, chromo-somal characters, and number of generations for which they can be maintained, as follows (Table 53-3):

Primary cell culture: These are a culture of normal cellsobtained freshly from the original tissues that have been cultivated in vitro for the first time and that have not been subcultured. These cell cultures can be established from whole animal embryo or from selected tissues from adult, newborn, or embryos. These cells have the normal diploid chromosomal number and are capable of only limited growth (5–10 divisions) in culture. They cannot be maintained in serial culture, but can be subcultured to obtain large number of cells. Monkey kidney cell culture (Fig. 53-4, Color Photo 54), human embryonic kidney cell culture, and chick embryo cell culture are the common examples of primary cell culture. Primary monkey kidney cell cultures are highly useful for the primary isolation of myxovirus, paramyxovirus, many enteroviruses, and some adenoviruses.

Diploid cell strains: Diploid cell strains are of a single celltype that retains their original diploid chromosome number and karyotype. However, they have specific characteristics and compositions and are usually composed of one basic cell type. They are usually fibroblasts and can be cultured for maximum 50 serial passages before they undergo senescence (die off) or undergo a significant change in their characteristics. Diploid cells derived from human fibroblasts are useful for isolation of some fastidious viruses. They are also used for production of vaccines; for example, WI-38 human embryonic lung cell stem is used for the cultivation of fixed rabies virus, and human fetal diploid cells for isolation of adenovirus, picornaviruses, HSV, CMV, and VZV.

Continuous cell lines: Continuous or immortal cell lines arecells of a single type, which are derived from cancerous tissue and are capable of continuous serial cultivation indefinitely without senescing. The cells are usually derived from diploid cell lines or from malignant tissues and have altered and irregular number of chromosomes. Immortalization may occur spontaneously or can be induced by chemical mutagens, tumorigenic viruses, or oncogens. Hep-2, HeLa, and KB derived from human carcinoma cervix, human epithelioma of larynx, and human carcinoma of nasopharynx and other cell lines are excellent for recovery of a large number of viruses. These cell lines have been used extensively for the growth of a number of viruses. These cell lines are usually stored at 270°C for use when necessary or are maintained by serial subculture. The type of cell line used for virus culture depends on the sensitivity of the cells to a particular virus; for example, Hep-2 cell line is excellent for the recovery of respiratory syncytial viruses, adenoviruses, and HSV. Most of the viruses can be isolated by using one of these cell lines. Growth of viruses in cell cultures can be detected by the following methods:

·           Cytopathic effect :Many viruses can be detected and initiallyidentified by observation of the morphological changes in the cultured cells in which they replicate. The CPE pro-duced by different types of viruses are characteristic and help in the initial identification of virus isolates. Nuclear shrinking, vacuoles in the cytoplasm, syncytia forma-tion, rounding up, and detachment are the examples of  alteration of morphology of the cells. Most CPEs can be demonstrated in unfixed and unstained monolayer of cells under low power of microscope. For example, adenoviruses produce large granular changes resembling bunches of grapes, SV-14 produces well-defined cytoplasmic vacuola-tion, measles virus produces syncytium formation, herpes virus produces discrete focal degeneration, and enterovi-ruses cause crenation of cells and degeneration of the entire cell sheet.

·             Hemadsorption:Hemadsorption is the process of adsorp-tion of erythrocytes to the surfaces of infected cells which serves as an indirect measurement of viral protein synthe-sis. This property is made use of to detect infection with noncytocidal viruses as well as the early stage of cytocidal viruses. Viruses, such as influenza virus, parainfluenza virus, mumps virus, and togavirus, when infect cell lines code for the expression of red cell agglutinins, which are expressed on the infected cell membrane during infections. These hem-agglutinins bind some erythrocytes to the infected cell sur-face. Sometimes, viruses can be detected by agglutination of erythrocytes in the culture medium.

·             Heterologous interference:This property is used to detectviruses that do not produce classic CPEs in the cell lines. In this method, the growth of non-CPE-producing virus in cell culture can be tested by subsequent challenge with a virus known to produce CPEs. The growth of the first virus will inhibit infection by the cytopathic challenge virus by inter-ference. For example, rubella virus usually does not produce any CPE, but prevents the replication of picornaviruses, which is inoculated as a cytopathic challenge virus.

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·             Transformation:Oncogenic viruses that are associated withformation of tumors induce cell transformation and loss of contact inhibition in the infected cell lines. This leads to surface growth that appears in a piled-up fashion producing microtumors. Examples of such oncogenic viruses that pro-duce transformation in cell lines are some herpes viruses, ade-noviruses, hepadanoviruses, papovavirus, and retroviruses.

·             Light microscopy:Viral antigens in infected cell cultures aredemonstrated by staining virus-infected cells of tissue sec-tions with specific viral antibody conjugated with horse-radish peroxidase. This is followed by addition of hydrogen peroxide along with a benzidine derivative substance. In a positive reaction, a red insoluble precipitate is deposited on the cell line, which is demonstrated by examination under ordinary light microscope.

·             Immunofluorescence:Direct immunofluorescence usingspecific antibodies is frequently used to detect viral antigens in inoculated cell lines for identification of viruses.

·           Electron microscopy:The viruses can also be demonstrated ininfected cell lines by EM.