In this article we will discuss about the isolation, assay and cultivation of viruses.
Isolation of Viruses:
Viruses are isolated from infected host cells containing mature virions. The cells are mechanically disrupted and the cell contents are released in a suitable buffer solution. The suspension containing the virions and cell ingredients is then subjected to centrifugation for several times at different speeds to fractionate the virions from other cell components. Such procedure is known as differential centrifugation.
A more refined technique is the density gradient centrifugation which is usually applied for getting a more purified sample of viruses. Before subjecting the sample of virus to density gradient centrifugation, it is initially purified by differential centrifugation. A density gradient is prepared in a centrifuge tube. For example, a sucrose gradient contains a linearly increasing concentration of sucrose from top to bottom of the centrifuge tube.
The partially purified virus sample is poured on the top and the tube is subjected to high-speed centrifugation for several hours in an ultracentrifuge. The centrifugal force drives the viral particles towards the bottom until they settle at a density gradient of sucrose which equals that of the virions, forming a concentrated zone or band.
The suspension of virions can then be removed from this band with the help of a pipette. The bacteriophages causing lytic infections can be isolated by a more or less similar method by differential centrifugation of the lysate to eliminate cell debris and the non-lysed intact cells of the host bacteria.
Assay of Viruses:
Viral assay means determination of number of viral particles per unit volume of a sample. Several methods are available for this purpose. The total number of viral particles in a sample including both viable and non-viable virions can be counted directly by means of electron microscopy.
For this purpose, a known volume of a purified sample of the virus is mixed with a known volume of a suspension of minute polystyrene latex beads of known concentration. The mixture is sprayed in droplets on a supporting membrane, dried and shadowed.
From the ratio of the number of beads and that of viral particles the number of virions per unit volume can be calculated, when the preparation is examined under an electron microscope. For example, if in a preparation, a droplet reveals presence of 200 viral particles and 20 latex beads, the concentration of virions / ml would be 200/20 multiplied by the number of beads per ml in the suspension.
If the bead concentration in the original suspension is 5 x 1010 per ml. The viral count would be 200/20 x 5 x 1010/ml = 5 x 1011/ml. The concentration of viruses in a sample is also known as its titre. Among the other methods for assay of viral titres in a sample, the plaque method is a very important one.
It makes use of the infectivity of the viruses and their capacity to destroy the infected cells. The plaque method was first developed for detection and counting of bacteriophages and later it was extended to the study of animal viruses. The plaque method is widely used for its simplicity, accuracy and reproducibility.
For assay of bacteriophages, a sample is diluted many-fold (~10-20 to 10-25) and mixed with a drop of a dense liquid culture of the host bacterium and a few milliliters of molten soft agar medium at 44°C. The mixture is poured on the surface of an already solidified hard nutrient agar in a plate and uniformly spread to allow the phages and bacteria to be evenly distributed.
Overnight incubation of the plates reveals the presence of a number of clear areas, known as plaques, on a lawn of continuous growth of the host bacterium. Plaques are formed due to infection and destruction of the host cells producing the clear areas. The number of plaques is proportional to the concentration of the virus.
Each plaque is produced by a plaque-forming unit (PFU). Thus, if an aliquot of 0.1 ml of a 10-20 dilution of the phage sample is plated and produce an average of 40 plaques per plate, the titre is 40/0.1 x 1020 PFU/ml. It should be noted that the number of PFUs cannot be taken as equal to the number of phages, but the two are proportional to each other.
The plaque method with necessary modification has also been used for assay of animal viruses. In place of bacteria, a suspension of cultured animal cells is used as host. The bacteriological nutrient medium is replaced by appropriate nutrient medium for animal cell culture.
As in the case of bacteriophage assay, the viral sample is serially diluted and aliquots of appropriate dilutions are spread on monolayers of host culture cells growing on a solid support e.g. in a petridish. The virions are allowed to get attached to the host cells for an hour or so, and then the monolayer is covered with a soft agar or some other gelling medium to prevent free horizontal diffusion of the viral particles.
The infectious progeny particles released by lysis of the infected cells remain more or less localized to produce foci or plaques. The plaques can be counted to determine the infectivity titre of the virus sample. Development of visible plaques may require from 1 to 2 days or even several weeks depending on the virus. For facilitating detection and counting of plaques, it may be necessary to stain the cell layer with a dye like neutral red which stains only the living cells, or a stain like trypan blue which stains only the dead cells. The accuracy of plaque assay depends on the number of plaques counted.
If too many plaques develop on a plate, some of these tend to fuse. As a result, the count becomes lower than what it should be. Many viruses produce sharp plaques, while others produce plaques with a diffuse margin.
Again some viruses produce large plaques and others small ones. Depending on the nature of the virus to be assayed, the dilution is to be accordingly determined to get reliable results. In case of bacteriophages, the number of plaques in plates is proportional to the concentration of the virus i.e. the relationship between plaque number and the viral concentration is linear.
Another method of assay of animal viruses which was used previously and now practically abandoned and replaced by the plaque assay is the pock assay. In this method the appropriately diluted viral sample is inoculated into the epithelial layer of the chrioallantoic membrane (CAM) of a chick embryo in an embryonated chicken egg. Characteristic infection lesions, called pocks appear after an incubation period of 36 to 72 hr. The pocks are opaque areas — usually white — and can be located on the transparent CAM.
The plaque method may also be suitably modified for enumerating plant viruses. For this purpose, a known volume of a properly diluted sample of the plant virus is applied on the leaf surface of a susceptible host plant after the leaf has been mechanically injured by mildly rubbing the surface with an abrasive like carborandum.
After incubation for several days necrotic lesions appear on the leaf. From the number of lesions per unit area, the dilution factor of the applied viral sample and the inoculum volume, the concentration (titre) of the virus can be calculated.
Cultivation of Viruses:
As obligate parasites, viruses cannot be grown in inanimate artificial media like bacteria or fungi. They can multiply only in living cells.
(i) Embryonated Egg:
One of the earliest — but still a common method of cultivating animal viruses — is the use of fertilized chicken eggs or embryonated eggs containing a young (6-12 days old) embryo. Different animal viruses can multiply in different parts of such eggs.
The different parts of an embryonated chicken egg are shown in Fig. 6.33:
Of the different parts of an embryonated egg, the allantoic cavity and the chorioallantoic membrane (CAM) are used for cultivating most animal viruses, though the yolk sac and the embryo are also used for certain viruses. The CAM is inoculated for growing pox viruses. The lesions (pocks) are visible grayish white spots. Mumps virus grows preferentially in the allantoic cavity. Influenza virus and rabies virus also grow in the allantoic cavity.
For inoculation, the egg surface is sterilized with a surface sterilizing agent. A hole is drilled through the shell and the shell membrane and the viral suspension is introduced into the proper part through the needle of a hypodermic syringe. The hole is then sealed with paraffin and the inoculated egg is incubated for 5 to 12 days.
(ii) Animal Cell Cultures:
The development of in vitro methods of culturing animal cells has greatly improved propagation of animal viruses. Cultured animal cells have provided quantitative techniques for studying animal viruses comparable to those applied for studying bacterial viruses. Cultures of different origin can be used for cultivating specific viruses, because just as all viruses cannot infect and multiply in all host organisms, so also all viruses cannot be propagated in all cell cultures.
Cultured animal cells differ in many ways from cultures of microorganisms. One of the most important differences is that cell cultures produced from normal tissues do not generally survive on repeated transfer. After some time they do not further divide and die. Such cell cultures derived from host tissues are called primary cultures. Primary cultures derived from embryonic tissues are able to continue growth for a longer time than cell cultures originating from adult tissues.
For establishing a primary culture a small piece of tissue of the animal is treated with trypsin to separate the cells. Trypsin is removed by centrifugation and the cells are suspended. The suspension is placed in a glass or plastic container together with a liquid medium, like Eagle’s medium.
On incubation, the cells attach to the surface of the container and divide mitotically to produce daughter cells which spread as a single-layer thick continuous confluent growth covering the surface of the container (monolayer).
Primary cell cultures in monolayers can be inoculated with animal viruses resulting in the formation of plaques. From the plaques, the viruses can be collected and purified. Primary cultures can be prepared from various tissues of different animals. The commonly used primary cultures are the Rhesus monkey kidney cell culture, chick embryo fibrolast cell culture, human amnion cell culture etc.
One of the limitations of the primary cell cultures is that they are relatively short lived and cannot be indefinitely maintained in subcultures, like those of microorganisms. The individual cells lose the capacity to divide after several cell generations. Most human cell cultures lose dividing capacity after about 50 duplications.
Sometimes, it so happens that a clone derived from a normal cell of a primary culture acquires the ability to grow indefinitely. Such a clone having an unlimited dividing capacity gives rise to a cell line. A cell line derived from a primary culture has a greater longevity than the mother primary culture, but is not truly immortal.
During repeated transfer of a cell line, a clone of transformed cells may originate spontaneously. These cells are cancer cells and are able to produce cancer when introduced into the proper animal. Such transformed cells constitute the permanent cell lines. Permanent cell lines can be generated directly from cultures of malignant tissues of hosts, or they can be produced infecting normal cell cultures with oncogenic viruses.
The cells of such permanent cell lines differ in morphology, cell orientation and chromosomal make up from the cells of primary cultures and of the cell lines derived from them. Different permanent cell lines have been generated from different tissues and different sources e.g. human cervix, liver, amnion, monkey kidney, hamster kidney, mouse connective tissue etc. one of the best known permanent human cell line is the HeLa cells derived from the cervix cancer of an Afro-American woman named Henrietta Lacks.
Cultivation of bacteriophages presents no difficulty, as they multiply in growing host bacteria either in broth or on solidified medium. When an inoculum containing a lytic phage is added to an actively growing liquid bacterial culture, the turbidity falls and the culture becomes almost clear due to lysis of the cells. By removing the cell debris by centrifugation, a rich crop of the bacteriophage can be obtained.
Similarly, when a bacterial culture growing in a petridish is inoculated with a phage suspension, the bacteria may be completely lysed, except a few phage resistant colonies. If the number of bacteria far exceeds the number of phages in the inoculum, then individual plaques are formed.
When a temperate bacteriophage Is inoculated into a liquid culture of the appropriate host bacterium, the turbidity decreases transiently and then increases. In agar cultures, the temperate phages produce turbid plaques. Turbid plaques result because a small proportion of the host bacteria undergo the lytic cycle, while the rest enters into lysogenic relationship.
(iv) Plant Viruses:
Plant viruses are generally propagated by direct inoculation of susceptible host plants. Viral inoculum is introduced into the leaf cells by causing artificial mechanical injury, such as rubbing with an abrasive. The viral particles enter through the disrupted cell walls into the cytoplasm of leaf cells where they multiply.
Some viruses, like TMV, may multiply in such great numbers that the progeny TMV particles may account for as much as 10% of the dry weight of the infected tobacco leaves. By disruption of the plant cells and by differential centrifugation TMV can be isolated in mass.
In more recent times, some success has been obtained in cultivating plant viruses in plant protoplast cultures. For example, protoplasts prepared from tobacco leaf mesophyll cells can be infected with TMV. Some plant viruses, like the Rhabdovirus causing necrotic yellow disease of lettuce, can multiply both in the plant host as well as in the insect vector. Such viruses can be grown in the vector cell culture. Lettuce necrotic yellow virus has been successfully grown in monolayers of cell cultures derived from leaf hopper giving an yield of about 10,000 virions per cell.