Microbiology presentation

1. M. Talha Iftikhar
Roll no.15821
Topic: Virus Culturing and Prions
Submitted to: Prof Dr. Muhammad Ismail
Department of Bioinformatics and Biotechnology
Government College University, Faisalabad

2. Culturing of Viruses
Isolationof Viruses
Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a
living host cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and
grown, and then the growth medium can be harvested as a source of virus. Virions in the liquid
medium can be separated from the host cells by either centrifugation or filtration. Filters can
physically remove anything present in the solution that is larger than the virions; the viruses can
then be collected in the filtrate.
Membrane filters can be used to remove cells or viruses from a solution. (a) This scanning electron micrograph shows
rod-shaped bacterialcells captured on the surface of a membrane filter. Note differences in the comparative size of the
membrane pores and bacteria. Viruses will pass through this filter. (b) The size of the pores in the filter determines what
is captured on the surface of the filter (animal [red]and bacteria [blue]) and removed from liquid passing through.Note
the viruses (green) pass through the finer filter.

3. Cultivation of Viruses
By Bacteria Culture:
Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro
(outside a living organism in cells in an artificial environment, such as a test tube, cell culture
flask, or agar plate). Bacteriophages can be grown in the presence of a dense layer of bacteria
(also called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask
(see Figure 2). The agar concentration is decreased from the 1.5% usually used in culturing
bacteria. The soft 0.7% agar allows the bacteriophages to easily diffuse through the medium.
For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear
zone called a plaque is detected. As the phage kills the bacteria, many plaques are observed
among the cloudy bacterial lawn.
(a) Flasks like this may be used to culture human or animal cells for viral culturing. (b) These plates contain
bacteriophage T4 grown on an Escherichia coli lawn. Clear plaques are visible where host bacterial cells have been lysed.
Viral titers increase on the plates to the left.
In an Embryo:
Animal viruses require cells within a host animal or tissue-culture cells derived from an animal.
Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in
clinical specimens, 2) production of vaccines, and 3) basic research studies. In vivo host sources
can be a developing embryo in an embryonated bird’s egg (e.g., chicken) or a whole animal. For
example, most of the influenza vaccine manufactured for annual flu vaccination programs is
cultured in hens’ eggs.
The embryo or host animal serves as an incubator for viral replication. Location within the
embryo or host animal is important. Many viruses have a tissue tropism, and must therefore be
introduced into a specific site for growth. Within an embryo, target sites include the amniotic
cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may damage tissue

4. membranes, producing lesions called pox; disrupt embryonic development; or cause the death
of the embryo.
(a) The cells within chicken eggs are used to culture different types of viruses. (b) Viruses can be replicated in various
locations within the egg, including the chorioallantoic membrane,the amniotic cavity, and the yolk sac.
In Animal Cells:
For in vitro studies, various types of cells can be used to support the growth of viruses. A
primary cell culture is freshly prepared from animal organs or tissues. Cells are extracted from
tissues by mechanical scraping or mincing to release cells or by an enzymatic method using
trypsin or collagenase to break up tissue and release single cells into suspension.
Because of anchorage-dependence requirements, primary cell cultures require a liquid culture
medium in a Petri dish or tissue-culture flask so cells have a solid surface such as glass or plastic
for attachment and growth. Primary cultures usually have a limited life span. When cells in a
primary culture undergo mitosis and a sufficient density of cells is produced, cells come in
contact with other cells. When this cell-to-cell-contact occurs, mitosis is triggered to stop. This
is called contact inhibition and it prevents the density of the cells from becoming too high. To
prevent contact inhibition, cells from the primary cell culture must be transferred to another
vessel with fresh growth medium. This is called a secondary cell culture. Periodically, cell
density must be reduced by pouring off some cells and adding fresh medium to provide space
and nutrients to maintain cell growth. In contrast to primary cell cultures, continuous cell lines,
usually derived from transformed cells or tumors, are often able to be subcultured many times
or even grown indefinitely (in which case they are called immortal). Continuous cell lines may
not exhibit anchorage dependency (they will grow in suspension) and may have lost their
contact inhibition. As a result, continuous cell lines can grow in piles or lumps resembling small
tumor growths.

5. Cells for culture are prepared by separating them from their tissue matrix. (a) Primary cell cultures grow attached to the
surface of the culture container. Contact inhibition slows the growth of the cells once they become too dense and begin
touching each other. At this point, growth can only be sustained by making a secondary culture. (b) Continuous cell
cultures are not affected by contact inhibition. They continue to grow regardless of cell density.
An example of an immortal cell line is the HeLa cell line, which was originally cultivated from
tumor cells obtained from Henrietta Lacks, a patient who died of cervical cancer in 1951. HeLa
cells were the first continuous tissue-culture cell line and were used to establish tissue culture
as an important technology for research in cell biology, virology, and medicine. Prior to the
discovery of HeLa cells, scientists were not able to establish tissue cultures with any reliability
or stability. More than six decades later, this cell line is still alive and being used for medical
research. See “The Immortal Cell Line of Henrietta Lacks” below to read more about this
important cell line and the controversial means by which it was obtained.
Prions
What they are?
Prions are infectious agents composed entirely of a protein material that can fold in multiple,
structurally abstract ways, at least one of which is transmissible to other prion proteins, leading
to disease in a manner that is epidemiologically comparable to the spread of viral infection.

6. Prions composed of the prion protein (PrP) are believed to be the cause of transmissible
spongiform encephalopathies (TSEs) among other diseases.
Prions were initially identified as the causative agent in animal bovine spongiform
encephalopathy (BSE)—known popularly as "mad cow disease".
Human prion diseases include Creutzfeldt–Jakob disease (CJD) and its variant (vCJD),
Gerstmann–Sträussler–Scheinker syndrome, fatal familial insomnia, and kuru. All known prion
diseases in mammals affect the structure of the brain or other neural tissue.
No effective medical treatment is known. The illness is progressive and always fatal.
How they propagate:
Prions may propagate by transmitting their misfolded protein state. When a prion enters a
healthy organism, it induces existing, properly folded proteins to convert into the misfolded
prion form. In this way, the prion acts as a template to guide the misfolding of more proteins
into prion form.
In yeast, this refolding is assisted by chaperone proteins such as Hsp104. These refolded prions
can then go on to convert more proteins themselves, leading to a chain reaction resulting in
large amounts of the prion form.
Structure:
Basic Structure Normal prions contain about 200-250 amino acids twisted into three telephone
chord-like coils known as helices, with tails of more amino acids
Basic Structure The mutated, and infectious, form is built from the same amino acids but take a
different shape.100 times smaller than the smallest known virus.

8. All known prions induce the formation of an amyloid fold, in which the protein polymerises into
an aggregate consisting of tightly packed beta sheets. Amyloid aggregates are fibrils, growing at
their ends, and replicate when breakage causes two growing ends to become four growing
ends. The incubation period of prion diseases is determined by the exponential growth rate
associated with prion replication, which is a balance between the linear growth and the
breakage of aggregates The propagation of the prion depends on the presence of normally
folded protein in which the prion can induce misfolding; animals that do not express the normal
form of the prion protein can neither develop nor transmit the disease.
Prion aggregates are extremely stable and accumulate in infected tissue, causing tissue damage
and cell death. This structural stability means that prions are resistant to denaturation by
chemical and physical agents, making disposal and containment of these particles difficult.
Transmission:
Current research suggests that the primary method of infection in animals is through ingestion.
It is thought that prions may be deposited in the environment through the remains of dead
animals and via urine, saliva, and other body fluids. They may then linger in the soil by binding
to clay and other minerals.
Sterilization:
Infectious particles possessing nucleic acid are dependent upon it to direct their continued
replication. Prions, however, are infectious by their effect on normal versions of the protein.
Sterilizing prions, therefore, requires the denaturation of the protein to a state in which the
molecule is no longer able to induce the abnormal folding of normal proteins. In general, prions
are quite resistant to proteases, heat, ionizing radiation, and formaldehyde treatments
although their infectivity can be reduced by such treatments. Effective prion decontamination
relies upon protein hydrolysis or reduction or destruction of protein tertiary structure.
Examples include sodium hypochlorite, sodium hydroxide, and strongly acidic detergents such
as 134 °C (274 °F) for 18 minutes in a pressurized steam autoclave has been found to be
somewhat effective in deactivating the agent of disease. Ozone sterilization is currently being
studied as a potential method for prion denaturation and deactivation.