Lecture 7
Animal Virology
The problem with Polio: Virus cultivation
• 1949: Enders, Weller and Robbins grew
poliovirus in cultured (non-neuronal)
cells Major breakthrough
• Enabled discovery of new viruses and
large scale vaccine development
• Basic technology for molecular and cell
biology
• Enables growth of large amounts of
pure virus, making possible studies on
virus composition and structure
Cell culture
• Most common method used to propagate viruses.
• Cells can grow as monolayers or in suspension
cultures.
• Primary cell culture
– From animal tissues. Limited to 5-20 cell divisions
• Diploid cell lines
– Homogeneous population of a single type. Typically
derived from tumors. Can divide up to 100 times. Remain
diploid
• Continuous cell lines.
– Immortal cells derived from tumors or mutagenesis of
primary cells. Aneupolid. Don’t resemble progenitors.
Can be tumorigenic. Many types, e.g. HeLa (Henrietta
Lacks), HEp-2 (Hu. Epithelial), BHK (Baby Hamster
Kidney).
Different types of
cell culture used in
virology
A. Primary human foreskin fibroblasts
B. Established (immortalized)
mouse fibroblasts (NIH 3T3)
C. Continuous (transformed) line of human epithelial cells (HeLa)
Note differences in morphologies among the primary, established and
transformed cell lines. HeLa cells overgrow one another – loss of
contact inhibition.
The Immortal Life of Henrietta Lacks – HeLa Cells
• Her name was Henrietta Lacks, but scientists know
her as HeLa.
• She was a poor black tobacco farmer whose cells—
taken from her cervical cancer at Johns Hopkins
without her knowledge in 1951—became one of the
most important tools in medicine.
• Vital for developing the polio vaccine, cloning, gene
mapping, in vitro fertilization, and most recently
vaccines against human papilloma virus, which
causes cervical cancer, the disease to which she
succumbed.
• Henrietta’s cells have been bought and sold by the
billions, yet she remained virtually unknown, and
her family could not afford health insurance.
• The book addresses numerous issues regarding the
legacies of slavery, issues of race, class and
education, and medical ethics.
• A movie produced by Oprah Winfrey is currently in
production.
Cytopathology
of virus
infections
Cytopathology
A. Timecourse of polio infection. Note how cells round up and die.
Cytopathology
B. Syncytium formation induced by Murine leukemia virus.
Embryonated eggs
• Many viruses grow very well in embryonated
chicken eggs.
• Robust yield enables use for research and
vaccine production
Terms describing viral infection
of cells
Multiplicity of Infection (m.o.i.)
• Ratio of input virus to the number of target cells in
an infection.
• Usually used to describe the infection of a particular
cell type grown on a plate or in culture.
• Can be used to statistically evaluate the total number
of cells that will be infected and the number of
viruses that will infect each cell during an infection.
Lytic infection- Virus enters cell and usurps cellular
machinery to rapidly multiply and in the process kill
the cell (many flu and cold viruses).
Terms describing viral
infection of cells
PFU: plaque forming unit
• Lysis of cells (lytic
infection) creates holes in
the monolayer of cells.
• Called ‘plaques’
• PFU = the concentration of
Viral plaques. The red region is the
viruses that can form
solidified blood agar, while the white
plaques in a volume of a spots are the plaques. The viruses
spread through the agar, destroying
virus stock.
the blood cells within the agar and
1
4
12
13
5
15
16
25
26
3
7
6
14
23
24
2
19
8
17
18 20
21
28
29
27
9
10
11
22
30
31
32
generating these characteristic
spots.
Terms describing viral
infection of cells
CFU: colony forming unit
• Some viruses can ‘transform’
cells
• i.e. allow them to grow when
they otherwise would not
(lecture 26)
• Cells that grow form colonies
• CFU is a measure of viruses
per volume of stock that can
transform cells.
Terms describing infections of an
organism
Acute infection- Rapid onset of
disease symptoms result in
eradication of the virus or death of
the infected animal (cold and flu
viruses, ebola).
Terms describing infections of an
organism
Persistent infection
• Virus remains associated with the cell without
rapidly multiplying or killing the cell.
• Three types
– (1) virus genome persists within the cell but virus is not
released, ex. Some retroviruses.
– (2) Virus is released sporadically but remains in a so
called "latent" state most of the time (herpes simplex).
– (3) Virus is continually released without lysis of host
cell, ex. hepatitis B virus. Some bacteriophages
(lambda) have developed complex molecular systems to
that sense when the conditions warrant a shift.
Terms describing infections of an
organism
• Two classes of persistent infections
– Chronic Infection- prolonged infection in
which the organism is not immediately killed
and the virus is not eradicated (hepatitis,
HTLV, HIV). Always symptomatic.
– Latent infection – chronic infection that
can be periodically reactivated. e.g.
herpes simplex virus. Sporadically
symptomatic.
Terms describing virus
transmission
• Zoonosis- disease which is naturally transmitted
between vertebrate animals and man (rabies, hanta
virus).
• Often, but not always transferred by an insect
vector (arboviruses). Insect transfer can be:
– Mechanical transmission - virions transferred directly
with no propagation in insect.
– Propagative transmission - virus replicates in othropod
(insect vector) prior to transfer. Often the virus will
first infect the gut cells of the insect and then be
transferred the salivary glands to be transmitted during
feeding.
– Those viruses not transferred by insects may be by
direct contact (rabies for example), or contact with
bodily wastes or fluids (hantavirus for example).
Quantitation of virus activity in
whole animals
• Infectious dose50 (ID50)
– Dose required to infect 50% of the inoculated
animals. With most viruses several PFU are
required to infect an animal.
• Lethal dose50(LD50)
– The dose required to kill 50% of the inoculated
animals.
• Incubation period
– The time between the initial infection and the
onset of disease symptoms.
– Can range from a few days (cold viruses) to years
(HIV) at times (show HIV typical curve showing
viremia and onset of disease.
How do viruses enter?
Skin: Not common, however, can enter thru:
• small wounds in skin like insect bites (yellow
fever virus)
• large wounds (rabies from animal bites)
More generally, thru epithelial cells
How do viruses enter?
Entry thru respiratory tract epithelia
• Depth of entry may be important, Coxsackie virus cause a
nasal cold if only to nose but deeper inhalation of a
contaminate mist can cause a sever chest cold.
• Many viruses that enter through epithelium may cause may
cause more generalized disease (example, African swine
fever, smallpox, mumps, measles, rubella).
Entry through epithelium of alimentary tract
• mouth, esophagus, intestinal mucosa
• Some cause local disease
• ex. diarrhea by orbi and rotaviruses
• Others cause more general (Hepatitis A virus).
Entry thru reproductive tract: Venereal
• HSV-2, HIV, HTLV-1
Modes of transmission
• Horizontal transmission: from one
organism to another via direct or
indirect contact
– Transmission via secretions of
epithelial cells
e.g. Herpesviruses including Herpes 1 (cold
sores) and Epstein-Barr virus (EBV) which
can cause mononucleosis (kissing disease).
Modes of transmission
• Vertical transmission- transfer of virus
form parent to offspring.
– blood exchange through the placenta
(HIV, cytomegalovirus, rubella)
– congenitally (HSV),
– direct inheritance of germ line
integrated virus (mouse type B and C
retroviruses).
Modes of transmission
• Nosocomial transmission: occurs in a
clinical setting
• Iatrogenic transmission: occurs during a
medical procedure.