MODULE 1-3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
OUTLINE
I.
II.
III.
IV.
V.
Introduction
Learning outcomes
Introduction to Virology
Diagnostic Virology
Prevention of Diseases with the use of Vaccines
INTRODUCRTION / OVERVIEW
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Viruses
o Nucleic acids which can either be DNA or RNA.
No mechanism to reproduce themselves, so they have
to get inside the host cell and dictate the host cell to
produce its own kind.
Three kinds of viruses:
o animal virus
o plant virus
o bacterial virus (bacteriophage (named as such bc
it affects the bacteria))
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NON-ENVELOPED
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INTRODUCTION TO VIROLOGY
VIRUSES
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filterable agent
•
obligate intracellular parasite
o cannot reproduce outside the cell.
o Thus, multiples inside the cell
Dmitri Ivanowski
o One who discovered viruses through his study of
mosaic disease in tobacco.
Contain single type of nucleic acid, either DNA or RNA
Contain a protein coat
o sometimes enclosed by an envelope of lipids,
proteins, and carbohydrates that surround the
nucleic acid
o
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can pass through filters
Viruses contain only those
components necessary to
invade and then utilize
host cell machinery for
their replication.
o
LEARNING OUTCOMES
At the end of the module, the student must be able to:
1. Describe the morphology and physiology of viruses
2. Discuss the diseases caused by viruses.
3. Discuss the methods of collection and examination of
specimen to isolate and identify the viruses.
4. Discuss the importance of controlling the growth and
spread of viruses.
5. Establish quality assurance in Virology.
BASIC STRUCTURE
Icosahedral
o solid, many geometric sides
Helical forms
o 20 triangular faces with 12 apexes
Complex
o Variable in size
A virus can be enveloped or non-enveloped
That’s why they don’t
survive outside the cell
and for their small size,
they heavily rely on the
host cell to multiply.
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They consist of two
primary components:
o Capsid
▪
an
external
protein coat
o DNA or RNA
▪
a core nucleic acid
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In the case of coronavirus, what kind of nucleic acid is
inside the virus? RNA.
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Nucleocapsid
o Nuclide acid core + capsid
Viral capsid
o Made up of Capsomers
▪
An accumulation of small, identical building
blocks of protein
o The configuration and arrangement of these
capsomers defines the overall shape of the
nucleocapsid which is either helical or
icosahedral.
o An icosahedron is a regular polyhedron with 20
equilateral triangular faces and 12 vertices.
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VIRUS SIZE
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Viruses range
in size from
~20nm
to
300nm.
This means
that
the
majority
of
viruses can
only be seen
through the
electron
microscope.
DOROTEO, MEGARA C. | 3CMT-4
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MODULE 1 - 3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
ENVELOPED
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Many viruses possess an
additional
covering
external to the capsid
known as the envelope.
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Inserted
into
the
envelope are viral encoded
proteins known as "spikes"
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Can take on a variety of
shapes due to the more fluid
nature of the envelope.
VIRAL STRUCTURE AND NOMENCLATURE
NUCLEIC ACID
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Either DNA or RNA
Perpetuation of species
In plants: viroid
Configurations of nucleic acid
o Single stranded
o Double stranded
o Single stranded circular
CAPSID
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Protein polypeptide of similar composition
Not for identification
Basis for identification
Each repetitions → capsomere
CAPSOMERE
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Potent Ag
Defense factor to produce immunity
Used for identification
ENVELOPE
BACTERIOPHAGE
• Also known simply as a
phage
• a virus that attacks and
infects bacteria.
• The infection may or may
not lead to the death of
the bacterium, depending
on
the
phage
and
sometimes on conditions.
• Each bacteriophage is
specific to one form of
bacteria.
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Not virus coded, not part of the genome
Part of the P.M. of the host
Retained by the presence of exocytosis
Functions together with capsomere
o Adsorption
o Identification
VIRAL INVASION
BASIS OF CLASSIFICATION OF VIRUS
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nature of the nucleic acid
structure of the nucleic acid
genome characteristics
presence or absence of cell divided envelope
symmetry of the nucleic acid
# of capsomere/diameter of the helix
MINOR CRITERIA
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Susceptibility to ether
Symptomatology
Host range
RECOGNITION
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PENETRATION
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VIRUS TAXONOMY
Taxonomic Characteristics
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For example, the family
Picornaviridae, the cause
of
such
diseases
as
Hepatitis A and polio, are
single
stranded
RNA
viruses with icosahedral
capsid symmetry.
These viruses do not have
an envelope and infect
only animal cells
DOROTEO, MEGARA C. | 3CMT-4
Host cell receptors must match the virus recognition
sites for the virus to subsequently absorb into the host
cell.
Most virus receptors is surface glycoproteins.
The distribution of these receptors plays a crucial role in
the tissue and host cell specificity of animal viruses.
Viral entry into host cells through one of the following
methods:
o Endocytosis
▪
active cellular process by which nutrients
and other molecules are brought into a cell
o Fusion
▪
viral envelope fuses with the plasma
membrane and releases the capsid into the
host cell's cytoplasm.
o Nucleic acids translocation.
ENDOCYTOSIS
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During endocytosis, the
entire virus is engulfed by
the cell and enclosed in a
vacuole or vesicle.
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MODULE 1 - 3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
FUSION
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With direct fusion, the
virus fuses with the cell
membrane of the host cell.
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NUCLEIC ACID TRANSLOCATION
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With some non-enveloped viruses,
the capsid adheres to the cell
membrane and the nucleic acid is
translocated into the cell.
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The mRNA transcript moves
into the cytoplasm of the host
cell and is translated on host
cell ribosomes.
The complex viruses such as
the adeno, herpes and
poxvirus must direct the
synthesis of their own DNA
dependent DNA polymerase in
order to replicate their DNA.
Smaller DNA viruses can use
host cell DNA dependent DNA
polymerase
The DNA dependent DNA polymerase migrates to the
host cell nucleus.
The virus DNA can now be replicated.
➢
A short segment of the
double stranded DNA is opened
(replication bubble).
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A DNA dependent DNA
polymerase
(HOST
POLYMERASE OR VIRUS CODED
POLYMERASE) then replicates
the DNA by synthesizing and
adding complementary nucleotides
to the parent strand.
VIRAL REPLICATION
RNA VIRUS REPLICATION
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DNA VIRUS REPLICATION
DOUBLE STRANDED DNA
➢ The double-stranded DNA
enters
the
host
cell
nucleus.
➢ Most viruses use the host
cell DNA dependent RNA
polymerase to make mRNA.
➢ What steps are involved in
this process?
o The viral DNA enters
the nucleus.
➢
Most viruses use the host
cell
DNA
dependent
RNA
polymerase to form a mRNA
transcript.
DOROTEO, MEGARA C. | 3CMT-4
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RNA viruses enter the host cell already in an RNA form
and the virus cycle occurs entirely in the cytoplasm.
RNA viruses bring with them one of the following genetic
messages:
o a positive sense genome
o a negative sense genome
o a dsRNA
o ssRNA which is converted to DNA (latent
retroviruses)
RNA VIRUS REPLICATION
POSITIVE SENSE ssRNA VIRUSES
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Examples
of
positive
sense
ssRNA viruses:
o Polio
o Hepatitis A
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With these viruses,
the RNA can be
read directly by the
host cell ribosomes.
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In other words, the
virus RNA is the
messenger.
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MODULE 1 - 3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
RNA VIRUS REPLICATION
REPLICATION AND EXPRESSION
A. Viral genome and
reverse transcriptase enter
cell.
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VIRION RELEASE
Mechanisms of virion release
differ between naked and
enveloped viruses.
Non-enveloped and complex
viruses that reach maturity in
the cell cytoplasm or nucleus
are released by lysis of the
host cell.
• Enveloped viruses are
released by budding or
exocytosis
from
the
membrane
of
the
cytoplasm,
nucleus,
endoplasmic reticulum, or
vesicles.
• The
nucleocapsid
attaches itself to the inside
of the membrane and is
pinched
off
with
its
envelope.
B. Single-stranded DNA
copy is synthesized by
reverse transcriptase
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C. RNA degraded; second
DNA strand synthesized
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D.
DNA
circulates
(unintegrated provirus) or
integrates into host cell
genome
(integrated
provirus)
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The area of the
cellular
membrane
where budding is to
occur first acquires a
cluster
of
viral
glycoprotein spikes
which
displaces
host cell proteins.
The
lipid
and
carbohydrate
components of the
envelope are those of
the host cell.
MODE OF TRANSMISSION OF A VIRUS
Respiratory
Fecal Oral
Close Contact
Arthropod/animal bite
EFFECTS OF VIRAL INFECTION
CYTOPATHIC EFFECT (CPE)
DIRECT
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lysis of motor neurons can cause loss of function of
the corresponding muscle
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Example: polio virus
CYTOPATHIC EFFECT (CPE)
INDIRECT
1. influenza v. damages the respiratory epithelium, and
ciliary activity is severely damaged.
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Result = accumulation of bacteria that normally
would be eliminated by ciliary action.
▪
Staph, strep, Hemophilus adhere to
respiratory tissue and cause disease.
2. AIDS virus affects immune cells, so patients die not
because of AIDS but because of secondary infections.
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Affects T helper cells, especially the CD4 and it lowers in
number, there will be and 2ndary infection.
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MODULE 1 - 3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
INCLUSION BODY FORMATION
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Replication of virus in nucleus or cytoplasm results in
the formation of viral and cellular products.
These accumulations which maybe nucleic acids,
proteins can be stained and referred to as inclusions
bodies.
Ex. Rabies virus - negri bodies
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CELL FUSION (SYNCYTIA FORMATION)
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Enveloped viruses (herpes v. paramyxovirus, and AIDS
v.) release specific proteins that become incorporated
into the cytoplasmic membrane of the infected cell.
These proteins act like magnets on the infected cell and
attract uninfected cells on their surface.
Repetition of the process results in aggregation of
several infected cells.
These aggregated cells eventually fuse, producing a
giant multinucleated cell or syncytium.
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Human Papilloma virus
o known to cause warts involving hands and other
parts of the body.
o may cause uterine cancer
Oncogenic RNA virus
o Belong to group called retrovirus
Human T lymphotropic virus (HTLV)
o causes of certain leukemias and lymphomas in
humans.
o One HTLV is the cause of AIDS, a disease that
predisposes its patients to cancer
Hepatitis B virus
o 300M people in Asia are carriers of Hep. B virus.
o A carrier is 100x greater in acquiring liver cancer
than that of a noncarrier.
Hepatitis C virus
o Major cause of chronic hepatitis
o potential cause of liver cancer
DIAGNOSTIC VIROLOGY
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3.
4.
Detection of viral molecules
o a. proteins
o b. nucleic acids
Microscopy
Culture
Serology
DIRECT DETECTION
ANTIGEN
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Syncytial cell
make the Ab.
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CHANGES IN SURFACE ANTIGENS
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Enveloped as well as nonenveloped viruses insert
novel antigen into the cell membrane of the infected
cells.
o These novel antigens make the cell a target for
immunological destruction by virus-specific
antibody
INTERFERON PRODUCTION
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Some cells infected by virus can produce a protein
called interferon.
Interferon has the capacity to prevent in infection of
healthy cells.
VIRUSES AND CANCER
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Oncogenic viruses are found within several groups of
DNA-containing viruses
o Adenovirus
o Herpesvirus
o Poxvirus
o Papovavirus
Herpesvirus
o Epstein-Barr virus is the only herpes virus that is
associated with certain neoplasm
o EBV has affinity for lymphocytes.
o It was isolated from patients afflicted with Burkitt's
lymphoma and nasopharyngeal ca.
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Some viral infections can be
detected
by
looking
directly for virus in tissue.
This
process
involves
making a specific antibody
against the virus in
question.
o Looking for the AG, so you
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The antibody is then
conjugated to a tag such as
a fluorescent dye (FA).
If
the
antibody
(Ab)
recognizes the antigen (Ag)
of the virus, they combine and can be seen directly in
the tissue/sample.
Examples:
o HSV in brain
o CMV in urine
o RSV in the respiratory tract
DIRECT DETECTION
NUCLEIC ACID PROBES
A. Nucleic acid (NA)
probes are made by
taking specific nucleic
acid fragments from a
virus..
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MODULE 1 - 3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
B. …and producing lots of these
in a cloning vector, for example
plasmid in a bacterium.
C. The DNA is denatured to
separate into its two strands.
OVERVIEW OF VIRAL DISEASES
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many diseases fail to produce any symptoms
one virus can produce a wide spectrum of diseases
different viruses may cause the same diseases
disease is the outcome of the virus host interaction
viral tropism
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in vitro growth or maintenance of cells, tissues, or organ
you can only culture them in living cells
2 methodological approaches
o primary culture
o cell line culture
Cell lines
o Either came from cancer cells that have been
propagated for many years and these are used for
some of the culture media or kidney tumor cells. It
can’t grow on artificial cells
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CELL CULTURE
PRIMARY CULTURE
D. The NA probes are then labelled
with either a radioactive probe or a
dye.
The probe is added to a specimen
and if a complementary sequence is
present, they will combine.
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start w organ of animal
1st in vitro culture
sensitive to original isolation of virus
has the same karyotype as the parent tissue
derived from tissues like monkey kidney cells
(MKC) and human embryonic kidney cells (HEK) and
die out after few generations
CELL LINE CULTURE
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E. The complex is detected by
radioactivity or color.
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subculture of primary culture
transformed cells that can be maintained for indefinite
generations.
Ex. Hela cell line- isolated from cervical carcinoma of a
woman named Hela, who died in 1951.
Culture serially
o diploid cell line
o haploid cell line
Table No. 1 Cell Line Culture
Characteristics
Diploid
Karyotype
>75% normal
Morphology
DIRECT DETECTION
Growth
ANTIBODY
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As with any other infecting organism, the presence of
circulating antibody in the host can be useful in detecting
infection.
Detection methods include:
o Ab detection by complement fixation
o Haemmaglutination
o Radioimmunoassay
o enzyme enhanced assays
o neutralization
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Contact Inhibition
Passage ability
Plating Efficiency
pH+ HCO- tolerance
Examples
Fibroblast like
Monolayer
1. Stationary
suspension
2. Roller
Yes
Limited
<10%
Narrow range
WI-38 Embryo
lung
HEK
Haploid
<75% normal
EC like
Fibroblast like
Monolayer
- suspension
No
>70 passages
>10%
Broad range
HELA-2
Hep 2
FL, Vero
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MODULE 1 - 3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
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CULTURE
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Remember,
viruses
are
intracellular parasites and
require specific host cell lines
to replicate.
Therefore,
propagation
requires tissue culture.
Tissue is removed from a
eucaryotic organism, and the
cells are grown in the laboratory.
COMPONENTS OF CELL CULTURE MEDIUM
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• When a specimen containing a
virus is added to the appropriate cell
line, cytological changes occur that
are characteristic for a particular
virus.
o These changes are referred to as
the Cytopathic Effect or CPE.
• Speciation of the isolate is
completed by testing the organisms
for specific antigens, for example
Herpes simplex Type l or Herpes
simplex Type 2.
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Most viral laboratories will use a combination of cell
lines to increase the range of viral isolation.
Remember that some viruses can reproduce only in
certain hosts or even only in certain organs of that
host.
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Vaccines
o whole vaccines
o split vaccines
o subunit
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Prevention
o natural active
o artificial active
❖ What kind of immunity that we get from the vaccines that
we receive? Artificial active
o Artificial – injected to you
o Active – the body is actively participating
ACTIVE (LIVE ATTENUATED)
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Primary cell Lines
o HEK
o MK
o Rabbit kidneys
o GPK
o Chick embryo
Passage Diploid
o HDF
o WI 38
o Mrc
Passage cell line human cancer cells
o HATH
o Hep 2
o A 549
o HeLa
o Rp
Passage cell lines derived from kidney cells
o BGMK
o Vero
o RK-1 13
o BHK-21
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The virus is alive, but the potency is reduced.
oral polio vaccine (trivalent)
measles
o Schwarz
o Morbilvax
o Rimevax
o Ed-monstan Zagrab
o Moratan
Mumps
o Mumpsvax
o Orovax
o Pariorix
Rubella
o Rubeaten
o Rudivax
o Ervevax
o Gunevax
MMR
o MMRII
o Pluserix
o Trimovax
Yellow fever
o YFV
Chicken pox
o Varitrix
VZ
Influenza
INACTIVATED RECOMBINANT
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balance salt solution
AA (arg, his, lys, met, tyr, leu, val, phe, cys, iso, glu, trp
and thr)
vitamins and co-enzyme
Group B vitamins (choline, folic acid, nicotinic acid,
pyridoxal, panthotenic acid, riboflavin, inositol)
PREVENTION OF DISEASES WITH THE USE OF
VACCINES
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Factors for basic survival of cells
o osmotic pressure 1.6 atm at 38'C NaCl
o pH 7.2-7.4
o glucose
o inorganic ions
o gases
Gene encoding for a specific protein Ag from a
pathogenic microorganism is isolated and incorporated
into the genome of nonpathogenic bacteria, yeast, or
other cells.
The GM cells are cultured in large quantities to produce
the desired Ag.
Influenza A/B
o Varigrip
o Inflexal
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MODULE 1 - 3: INTRODUCTION TO VIROLOGY – PREVENTION OF DISEASES
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Rabies
o
o
o
Hepa B
o
o
o
o
Hepa A
o
Mericux
Lyssavac
Verorab
Hepaccine
Hevac B
Engerix B
Hb vax
Havrix
Table No. 2 Polio vaccines (Salk vs. Sabin)
Salk
Sabin
inactivated
Live, attenuated
humoral immunity
humoral/cellular
IgG
IgG, Th, Tc cells
Tonsils-> mucosa-> lamina
prevents paralytic polio
propia > disease is prevented
reversion (1 in a million)
no reversion
(this means that 1 person in a
million who’ll received the vax can
develop the polio bc it’s alive)
Intramuscular
OPV
SUBUNIT VACCINES
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Consists of one or more purified components of a
pathogen that can stimulate an immune response.
The forms routinely used:
o Toxoids
▪
Like tetanus toxoid and diphtheria toxoid
(toxins that have been chemically inactivated)
o Capsular polysaccharides
o Purified proteins
o Recombinant protein antigens
POLYSACCHARIDE VACCINES
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Capsule which covers the bacterial outer membrane.
If antibodies to the capsular polysaccharides are present,
they can facilitate clearance of the bacteria by
opsonization or complement-mediated lysis.
PURIFIED PROTEIN VACCINES
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Composed of proteins from a pathogen.
Ex = B. pertussis vaccine is composed of two to five
proteins from B. pertussis.
RECOMBINANT PROTEIN ANTIGENS
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Recombinant DNA technology has made it possible to
develop more highly purified vaccines.
DOROTEO, MEGARA C. | 3CMT-4
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