Growing viruses from clinical samples.
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.
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
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
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
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.
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.
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.
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.
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
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.
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.
systems used for virus culture.
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
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.