Animal Virology
Lecture 6. BSCI437
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).
Evidence of viral growth in cultured cells -- Cytopathology (see Table 2.1)
Viral infection can change the appearance of cells – from no effect all the way to death.
Cytopathic effects can be seen under the microscope.
Include morphological alterations (changes in shape) and inclusion bodies (big blobs
inside of cells).
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:
Persistent infection (also used in describing infection of organism)- virus remains
associated with the cell without rapidly multiplying or killing the cell. This can occur
when the viral genetic material integrates into the host genome (retroviruses) but
integration need not occur for persistence (Herpesvirus). Three types are known to occur
(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.
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. The number 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
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infection.
PFU:
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CFU:
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plaque forming unit
Lysis of cells (lytic infection) creates holes in the monolayer of cells.
Called ‘plaques’
PFU = the concentration of viruses that can form plaques in a volume of a virus
stock.
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:
Lytic infection- Virus enters cell and usurps cellular machinery to rapidly multiply and in
the process kill the cell (many flu and cold viruses).
Acute infection- Rapid onset of disease symptoms result in eradication of the virus or
death of the infected animal (cold and flu viruses, ebola).
Chronic Infection- prolonged infection in which the organism is not immediately killed
and the virus is not eradicated (hepatitis, HTLV, HIV).
Terms describing virus transmission:
Horizontal transmission- transmission of virus or other pathogen to host at any age after
birth.
Vertical Transmission- passage of a viral genome from one host generation to the next,
either as an integrated provirus (HIV) or in close association with the host gametes
(Herpes viruses).
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
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contact with bodily wastes or fluids (hantavirus for example).
Quantitation of virus activity in animals:
Infectious dose50 (ID50)-The 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- (show Fig. 12.1)- The time between the initial infection and the onset
of disease symptoms. This 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?
-Generally not through the skin
-small wounds in skin like insect bites (yellow fever virus)
-large wounds (rabies from animal bites)
-epithelial cells of respiratory tract, 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).
-epithelium of alimentary tract (mouth, esophagus, intestinal mucosa). Some cause local
disease (ex. diarrhea by orbi and rotaviruses) while others cause more general (Hepatitis
A virus).
-oral secretions-Herpesviruses including Herpes 1 (cold sores) and Epstein-Barr virus
(EBV) which can cause mononucleosis (kissing disease).
-venereal-HSV-2, HIV, HTLV-1
-vertical transmission- transfer of virus form parent to offspring. May occur by blood
exchange through the placenta (HIV, cytomegalovirus, rubella) or congenitally (HSV),
also by direct inheritance of germ line integrated virus (mouse type B and C retroviruses).
Note that endogenous retroviruses have been found in many mammals and retroviral
sequences have also been found in humans. These endogenous genomes may be
defective and unable to complete replication producing only some of the viral proteins or
in some cases complete virions can be produced although no virus has ever been
cultivated from humans. Data indicates that these viruses have evolved along with the
species they infect. The viruses share more sequence homology when compared from
related species than from less closely related. For examples endogenous viruses
recovered from apes are closer to those of monkey
than those of cats.
Stages of viral infection:
1. Primary infection- Infection of cells in the general location where virus enters, This is
common for example with cold viruses and Diarrhea causing viruses. Many viruses
including HIV initially infect cells in the infected area.
2. Viremia- Virus enters the blood system and can be detected in the blood stream. Not
all viruses do this. Some remain only localized.
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3. Secondary infection- Infection of other organ of cell types by the virus. Many viruses
have high affinity for specific organs due to the presence of receptors or specific cell
metabolic functions. Examples are infection of salivary glands by mumps virus, brain
tissue by encephalitis virus, and liver tissue by hepatitis virus.
Incubation period- The time between the initial infection and the onset of disease
symptoms. This can range from a few days (cold viruses) to years (HIV) at times (show
HIV typical curve showing viremia and onset of disease).
Virulence-The ability of an infectious agent to cause disease.
-The term is relative in the sense that it depends on the particular host that is
infected and the state of that host as well as the site of infection. Rabies causes
neurological symptoms in 30% of infected individuals bitten on the hand by a rabid
animal while 70% of those bitten on the head show symptoms. In all cases some people
show no disease but have antibodies to the virus. Lassa fever which is caused by a
arenavirus can cause severe hemorrhagic fever in infected individual coming to Africa
while most natives get no disease. They develop antibodies to the virus with no
symptoms. This illustrates the selection of a more resistant host in the environment
where the virus has been present for a long time. Some viruses infect animals but are
virulent only when the host immune response is suppressed. Examples are certain herpes
and hepatitis B virus that become virulent when human hosts are treated with
immunosuppressant drugs after organ transplant. It has been hypothesized that
cytomegalovirus (also HHV-6?), which is a herpes virus is the causative agent of
Kaposi's Sarcoma (A cancer effecting the epithelial cells lining blood vessels prominent
in AIDS patience). EBV shows a spectrum of disease some individuals get no disease or
mononucleosis while in others the virus can lead to B cell lymphomas and
nasopharyngeal cancer.
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