Methods of Isolation, Culture, and Identification of Viruses

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isolation of a virus

Viral diseases are diagnosed by demonstrating virus in appropriate clinical specimens via culture. The isolation of a virus is always regarded as the gold standard for determining the viral etiology of a disease. The collection of appropriate clinical specimens is determined by the type of viral disease. Cerebrospinal fluid (CSF), for example, is the preferred specimen for diagnosing viral infections of the central nervous system (CNS) caused by arboviruses, picornaviruses, or rabies virus. Blood is the most commonly examined specimen for the diagnosis of HIV, hepatitis B, C, and D infections, as well as other blood-borne viral infections.

The timing of specimen collection is critical. The specimens collected early in the acute stage of infection before virus shedding stops are the most important. Enteroviruses, for example, are only present in the CSF for 2–3 days after the onset of CNS manifestations. Herpes simplex virus and varicella zoster virus are only found in lesions within the first 5 days of symptoms, and respiratory viruses are only found in respiratory secretions within the first 3–7 days of symptoms. The table below displays tests of other clinical specimens to be collected for diagnosis of other types of viral diseases.

SystemDirect examinationIsolationSerology
Respiratory systemNasopharyngeal aspirate (IF, EM)Throat swab, throat washingsPaired sera
SkinVesicular/pustular fluid (EM, ID), ulcer scrapings (EM), and crusts (EM, ID)Macular/papular scrapings, vesicular/pustular fluid, ulcer scrapings, crust, urinePaired sera
CNSBrain biopsy (IF, EM), CSF (EM, IF)Feces, blood (for arbovirus), CSF, and brain biopsyPaired sera
EyeConjunctival scrapings and smears (LM, IF)Conjunctival scrapings or swabsPaired sera
Gastro-intestinal tractStool (antigen detection, EM for rotavirus)Not culturedPaired sera (ELISA)
Congenital infectionsNilThroat swab, products of conceptionSingle sera (mother and baby)
LiverSerum and fecesBlood (for yellow fever)Serum
Terminology: IF, immunofluorescence; EM, electron microscope; ID, immunodiffusion; LM, light microscopy.

Immediate transport of the specimen to the laboratory for processing also allows for better virus isolation from clinical specimens. Viruses are typically heat labile, and clinical specimens may be infected secondarily by bacterial and fungal contamination. As a result, clinical samples for viruses are typically transported and stored on ice. They are transported in special transport media containing antibiotics to inhibit bacterial and fungal contaminants, as well as proteins such as serum albumin or gelatin. When clinical specimens are stored at room temperature or frozen at 20°C, some viruses, such as influenza virus, HSV, and VZV, lose their infectious titer. The majority of viruses can be grown in

  • Experimental animals
  • Embryonated eggs
  • Tissue culture.

Animal inoculation

The mouse is the most commonly used animal inoculation method for virus isolation. Rabbits, hamsters, and newborn or suckling rodents are also used. Although experimental animals are rarely used for virus cultivation, they play an important role in the study of viral pathogenesis and 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.

Intracerebral animal inoculation
Intracerebral animal inoculation

Embryonated eggs

Embryonated eggs were initially used to grow viruses. Goodpasture was the first to use embryonated chick eggs for virus cultivation in 1931. Burnet’s method was later used to cultivate viruses in various locations of the embryonated egg. Viruses are typically cultured in 8–11 day old chick eggs. The viruses are isolated from various parts of the egg, including the yolk sac, amniotic cavity, allantoic cavity, and chorioallantoic membrane (CAM)

EMBRYONATED CHICK EGG inoculation for isolation of a virus
EMBRYONATED CHICK EGG inoculation for isolation of a virus

Many of these viruses cause well-defined and distinct foci, which can be used to identify, quantify, or assess virus pathogenicity. The embryonated egg is also used in research laboratories to grow higher titre stocks of some viruses and for vaccine production.

(a) Yolk sac:

Japanese encephalitis, Saint Louis encephalitis, and West Nile virus are all grown using yolk sac inoculation. It is also used to promote chlamydia and rickettsia growth.

(b) Amniotic cavity:

Inoculation in the amniotic cavity is primarily used for primary influenza virus isolation.

(c)  Allantoic cavity:

Inoculation in the allantoic cavity is used for serial passages and to obtain large quantities of virus for vaccine preparation, such as influenza virus, yellow fever (17D strain), and rabies (Flury strain). Duck eggs were used for the production of the rabies virus due to their larger size than hen eggs. This aided in the production of large amounts of rabies virus, which are used in the preparation of the inactivated non-neural rabies vaccine.

(d) Chorioallantoic membrane:

When some viruses were injected into CAM, they caused visible lesions known as pocks. One pock is produced by each infectious virus particle. Pox viruses, such as variola or vaccinia, are identified by the presence of typical pocks on the CAM that has been inoculated with the pox virus. For routine virus isolation in a virology laboratory, chick embryo inoculation has been replaced by cell cultures.

Tissue culture

Cell culture is most widely used in diagnostic virology for cultivation and assays of viruses. The tissue culture was first applied in diagnostic virology by Steinhardt and colleagues in 1913. They kept the vaccinia virus alive by cultivating it in rabbit corneal tissues. Maitland (1928) then used cut tissues in nutrient media to cultivate vaccine viruses. Enders, Weller, and Robins (1949) were the first to culture poliovirus in non-neuronal tissue cultures. The majority of the virus has since been grown in tissue culture for the diagnosis of viral diseases. Viruses are grown in a variety of tissue cultures. Tissue culture can be classified into three types:

1.     Organ culture

This was previously used to isolate some viruses that appear to have an affinity for specific tissue organs. In the tracheal ring organ culture, for example, coronavirus, a respiratory pathogen, was isolated. In this method, small bits of the organs are maintained in vitro for days and weeks preserving their original morphology and function. Nowadays, organ culture is not used.

2.     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.

3.     Cell culture

Cell culture is now routinely used for growing viruses. In this method, tissues are dissociated into component cells by treatment with proteolytic enzymes (trypsin or collagenase) followed by mechanical shaking. After that, the cells are washed, counted, and suspended in a growth medium that contains essential amino acids and vitamins, salts, glucose, and a buffering system. This medium contains up to 5% fetal calf serum as well as antibiotics. Cell suspension is given out in glass or plastic bottles, tables, or Petri dishes. Within a week of being incubated, the cells adhere to the glass surfaces and divide to form a confluent monolayer sheet of cells covering the surface.

Prepatation of cell culture for isolation of a virus
Prepatation of cell culture for isolation of a virus

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 classified into three different types based on their origin, chromosomal characters, and number of generations for which they can be maintained, as follows

      I.            Primary cell culture:

The cell culture can be incubated as a stationary culture or a roller drum culture. Because of the improved aeration provided by rolling the culture bottle in special roller drums, the latter is beneficial for the growth of some fastidious viruses. Cell cultures are classified into three types based on their origin, chromosomal characteristics, and the number of generations that can be maintained, as follows. They cannot be grown in serial culture, but they can be subcultured to produce a large number of cells. Primary cell culture is most commonly seen in monkey kidney cell culture, human embryonic kidney cell culture, and chick embryo cell culture. Primary monkey kidney cell cultures are extremely useful for isolating myxovirus, paramyxovirus, many enteroviruses, and some adenoviruses.

   II.            Diploid cell strains:

Diploid cell strains are single cell types that retain their diploid chromosome number and karyotype. They do, however, have distinct characteristics and compositions, and are typically made up of a single basic cell type. They are typically fibroblasts that can be cultured for a maximum of 50 serial passages before senescence (die off) or a significant change in their characteristics occurs. Diploid cells derived from human fibroblasts can be used to isolate some difficult viruses. WI-38 human embryonic lung cell stem cells, for example, are used for the cultivation of fixed rabies virus, and human fetal diploid cells for the isolation of adenoviruses, picornaviruses, HSV, CMV, and VZV.

III.            Continuous cell lines:

Continuous or immortal cell lines are single-type cells derived from cancerous tissue that can be serially cultured indefinitely without senescence. The cells are usually derived from diploid cell lines or malignant tissues, and they have an abnormal and irregular number of chromosomes. Immortality can happen naturally or be induced by chemical mutagens, tumorigenic viruses, or oncogens. Hep-2, HeLa, and KB cell lines derived from human carcinoma cervix, human epithelioma of larynx, and human carcinoma of nasopharynx, as well as other cell lines, are excellent for virus recovery. These cell lines have been used extensively for the growth of a number of viruses. These cell lines have been extensively used in the growth of a variety of viruses. These cell lines are typically stored at 70°C for future use or are maintained through serial subculture. The type of cell line used for virus culture is determined by the cells’ sensitivity to a specific virus; for example, the Hep-2 cell line is excellent for recovering respiratory syncytial viruses, adenoviruses, and HSV. The majority of viruses can be isolated using one of these cell lines. The following methods can be used to detect virus growth in cell cultures:

(a) Cytopathic effect:

Many viruses can be detected and identified by observing the morphological changes that occur in the cultured cells where they replicate. The CPE produced by various types of viruses is distinct and aids in the initial identification of virus isolates. Nuclear shrinkage, cytoplasmic vacuoles, syncytia formation, rounding up, and detachment are examples of cell morphology changes. The majority of CPEs can be seen in an unfixed and unstained monolayer of cells under a low-power microscope. Adenoviruses, for example, cause large granular changes that resemble grape bunches, SV-14 causes well-defined cytoplasmic vacuolation, measles virus causes syncytium formation, herpes virus causes discrete focal degeneration, and enteroviruses cause cell crenation and degeneration of the entire cell sheet.

(b) Hemadsorption:

Hemadsorption is the process of erythrocyte adsorption to the surfaces of infected cells that serves as an indirect measure of viral protein synthesis. This property is used to detect noncytocidal virus infection as well as the early stages of cytocidal virus infection. Viruses that infect cell lines, such as influenza virus, parainfluenza virus, mumps virus, and togavirus, code for the expression of red cell agglutinins, which are expressed on the infected cell membrane during infections. Some erythrocytes are bound to the infected cell surface by these hemagglutinins. Viruses can sometimes be detected by agglutination of erythrocytes in culture medium.

(c)  Heterologous interference:

This property is used to detect viruses in cell lines that do not produce classic CPEs. The growth of non-CPE-producing viruses in cell culture can be tested using this method by challenging them with a virus known to produce CPEs. The first virus’s growth will interfere with infection by the cytopathic challenge virus. Rubella virus, for example, does not usually produce CPE but does prevent the replication of picornaviruses, which are inoculated as a cytopathic challenge virus.

(d) Transformation:

Oncogenic viruses that cause tumor formation cause cell transformation and loss of contact inhibition in infected cell lines. This results in surface growth that appears piled-up, resulting in microtumors. Some herpes viruses, adenoviruses, hepadanoviruses, papovaviruses, and retroviruses are examples of oncogenic viruses that cause transformation in cell lines.

(e)  Light microscopy:

By staining virus-infected cells of tissue sections with a specific viral antibody conjugated with horseradish peroxidase, viral antigens in infected cell cultures are demonstrated. Following this, hydrogen peroxide and a benzidine derivative substance are added. A red insoluble precipitate is deposited on the cell line in a positive reaction, as evidenced by examination under an ordinary light microscope.

(f)   Immunofluorescence:

For virus identification, direct immunofluorescence with specific antibodies is frequently used to detect viral antigens in inoculated cell lines.

(g) Electron microscopy:

EM can also be used to show the viruses in infected cell lines.

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