Virology... Virus Cultures
A Joint Venture of London Health Sciences Centre and St Josephs Health Care London

Growing viruses from clinical samples.

Introduction to culturing viruses.

In the early days of investigating the etiology of infectious diseases a series of criteria were put forward by Robert Koch, in which he laid out what needed to be demonstrated before an agent thought to cause a particular disease could be acceptably demonstrated to be the specific agent linked to that disease. These are known as Koch’s postulates and in a nutshell they required that a sample had to be obtained from person with the disease under investigation. The sample then had to be cultured for bacteria. Each type of organism isolated had to be shown to be in a pure culture e.g. consisting only of a single bacterial species not mixed with others. Then that pure culture of the bacteria needed to be introduced into another host and be able to create the same disease that was present in the original host.

These postulates were immensely useful in determining the causes of a number of infectious diseases: tuberculosis, cholera, dysentery and a host of others were soon identified, but the etiology of many other infectious diseases remained a mystery. It was recognized that in a number of common infections clinical samples could be filtered to remove all the bacteria yet the supposedly "sterile filtrates" was capable of passing the disease on to other host species. It was also recognized the treatment of these filtrates with heat, acid,formaldehyde or other chemicals often rendered them noninfectious. These infectious substances were originally called "non-filterable agents". Eventually as microscopy became better able to identify smaller structures and techniques to propagate these non-filterable agents in living systems such as suckling mice or fertile eggs, viruses as they became known as, began to be better defined. The big breakthrough in propagating viruses came about when techniques to maintain living cells in test tubes were developed and soon viruses were able to be "grown" from clinical samples, in the laboratory. It was a time-consuming and expensive process initially and this diagnostic tool was not routinely available at first but nowadays most virology labs provide virus culture as part of their diagnostic armamentarium.

Using tissue cultures for virus isolation.

The ability to take cells from tissues of humans, animals, plants etc. and keep them alive in test tubes or flasks is called tissue culture. In order to make the cells easy to work with it was first necessary to discover ways of making the cells stick to the surface of the glass or plastic and letting them form a continuous sheet of cells one layer thick. This is called a monolayer. These monolayers could be kept going by taking a few cells from an established cell culture and setting them up and a fresh flask or test tube where they could form a new monolayer. In this way various cell types could be propagated for many generations. It was soon recognized that some cells have a limited number of generations that could occur in vitro before the cells died. Some others appeared to be able to be propagated in perpetuity. Those cells that lasted only a few generations were calls primary cell lines, those with long-term propagation became known as continuous cell lines. Continuous (or immortalized) cell lines were often derived from malignant cells but it was later discovered that some virus infected cell lines (Raji cells, which are EB virus infected cells) also could become immortalized.

Viruses are usually quite host specific and in order for them to be grown from clinical samples they need to be inoculated into a cell line that permits them to grow. In order to give the viruses the best chance of growing the sample is usually sets up on several different cell lines of both primary and continuous type tissue cultures.

 

 


One of the most permissive cell lines for human viruses is derived from various types of monkey kidney cells. Initially Rhesus monkeys were used as they were plentiful but over the years they became in short supply. Some vaccine manufacturers kept colonies of Rhesus monkeys simply be to maintain their supply but now this type of cell line is generally unavailable due to a shortage of Rhesus monkeys. Other primate kidney cell lines have also been used, the most famous of which was from African green monkeys. The use of these cells encountered a setback when a number of German lab workers in the city of Marburg died when handling cultures from this species. It was discovered that they were infected with an Ebola-like virus, now called Marburg virus. Since then the trend has been away from using primate cell lines although at least one primate cell line is still usually inoculated in diagnostic virology tissue culture facilities.

The hallmark tthat a tissue culture monolayer is infected with a virus is that the virus causes a cytopathic effect (CPE). This usually consists of the virus infected cells being lysed and little "islands" of clearing occur in the monolayer. Once a virus was demonstrated to be present then additional methods to identify the type of virus could be used. These were usually immunologically based tests such as immuno electronmicroscopy, enzyme immuno assays, neutralization tests, or immuno fluorescent tests.

Virus culture is still important in diagnostic virology labs but it is cumbersome, expensive and requires a great deal of skill on the part of the virology technologists. Many of the traditional culture methods have been supplanted by "shell vial" cultures and some labs use shell vial culture techniques as the sole method for virus isolation. LLSG is one such laboratory.

The above is a very abbreviated and incomplete description of virus culture using tissue culture techniques but a full description is beyond the scope of this article. Manuals relating to diagnostic virology would need to be consulted for more complete descriptions.

Advantages of standard to tissue culture. Many different cell lines can be used. Incubation can continue until CPE occurs. If the virus doesn't adapt to the cells at first and no CPE occurs, a "blind passage" can be done. Blind passage requires cells from the original culture to be lysed and the supernatant then transferred to a new monolayer. For hard to grow viruses multiple passages can be done. Large quantities of virus can also be grown using tissue cultures.

Disadvantages. Tissue cultures are expensive, it requires expertise from the technologist reading the monolayers, it is time-consuming. It is also potentially dangerous to technologists working with animal cell lines

Shell vial cultures.

The shell vial technique is a variation on standard tissue culture in that it takes advantage of using a living cell system and enhances viral recovery by centrifuging the clinical sample onto the monolayer. In this technique a small bottle (vial) with a removable round glass cover slip is used to grow the cells as a monolayer on the cover slip.



Nowadays mixed cell types can be put in a single monolayer providing a variety of cell types for the virus to infect in a single vial. Once these monolayers are ready to be inoculated, the growth medium is removed from the vial and the clinical sample placed directly on the monolayer. The vial is then centrifuged, the clinical sample is removed and fresh growth medium is then added to the vial. Although the vials can be kept until CPE occurs the CPE can't be seen unless the cover slip is removed from the vial. Usually it is possible, using this technique, to identify the presence of a virus before CPE occurs. A description of how a clinical sample might be handled using the shell vial method could go something like this. Let's say that the sample is from someone suspected of having a respiratory virus infection. Three shell vials would be set up. After 48 hours of incubation, two of the vials would be used. The supernatants would be pipetted off and saved. The glass cover slips would be washed gently and then fixed. One of them would be stained with a single reagent containing influenza A and B.. Differentiation between A and B would be done by tagging the antibodies with different fluorescent dyes. The other cover slip would be stained for respiratory syncytial virus. The third vial is saved for staining at 72 hours for parainfluenza viruses. If the cover slip stains positive for say influenza A the report is sent out as influenza Avirus isolated. The supernatant from the original vial (which should contain live virus, can then be re-inoculated into a fresh vial which is then sent off for typing of the influenza A virus.

An important point to remember is that virus culture is often more sensitive than trying to detect viruses directly in clinical samples. This is particularly true in respiratory viruses where a direct test on the sample may give a negative result but a couple of days later a report from the culture may be positive. It is common practice in our institution to order direct testing for infection control purposes. The caution is that a positive direct result is useful, a negative direct result does not mean that the patient does not have active disease and must not be used as a reason for removing someone from isolation or for not taking precautions when a patient is to be admitted.

Advantages of shell vials.

They are fast, easy to do, no skill in looking for CPE necessary. It is also quite economical.

Disadvantages of shell vials.

Can not do "blind passages". Some viruses may require longer incubation to adapt to the particular cell lines. Once used, the monolayer can not be re-incubated or re-inoculated as the fixed cells and viruses are dead.

Other living systems used for virus culture.

Fertile eggs.

The fertile egg is a unique living system cells and affords a number of different sites where viruses might grow. These are nicely compartmentalized, the amnion, the chorion, the yolk sac and the embryo itself. Each of these sites can be inoculated with clinical material and each area supports the growth of specific virus types. While fertile eggs used to be a mainstay of virus propagation it has now been supplanted by other techniques (shell vials, tissue culture) and this method is rarely used in a diagnostic virology lab now. Some viruses grow well in eggs and can produce very large numbers of viruses so they are still sometimes used for obtaining large amounts of viral particles for vaccine manufacture. One of the viruses grown commercially in this way is the influenza virus. This is why people who are allergic to eggs are cautioned about receiving vaccines that use a source of virus that has been propagated in eggs.

Other live animal systems.

Viruses can grow in a number of different animal species. The most commonly used was the suckling mouse. It is mainly of historic interest but Coxsackie viruses grow well when injected intracerebrally in mice that are a few days old. While the technique of using a living animal to study viruses is still used in research labs it is not used for routine isolation of viruses from clinical specimens.

Reference. Clinical Virology Manual. Steven Specter, Richard L. Hodinka, Stephen A. Young. 3rd Edition. ASM Press. 2000.

 

Revised November 17, 2005 12:57 PM