MEDICAL VIROLOGY

Assist Prof. Dr. Baydaa Hameed

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 VIRUSES CAN BE USEFUL

• VACCINE DEVELOPMENT
• GENE THERAPY
• TOOLS TO INVESTIGATE HOST
CELLS

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WHAT ARE VIRUSES?

“A PIECE OF BAD NEWS

WRAPPED UP IN A PROTEIN”

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 WHAT ARE VIRUSES?

• NUCLEIC ACID GENOME:

– DNA OR RNA
• PROTEIN COAT
– PROTECTION, ENTRY
• LIPID ENVELOPE IN SOME VIRUSES
• SMALL
– (20-400nm)
• OBLIGATE INTRACELLULAR PARASITES

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 What are viruses?

• Small obligate intracellular parasites

• Virion
– Complete virus particle : nucleic acid + protein coat, which may be surrounded
by an envelope
– It is the form in which the virus moves between cells or hosts

• Viral Genome
– EITHER RNA or DNA genome surrounded by a protective virus-coded protein
coat (Capsid)

• Propagation depends on specialized host cells supplying the machinery for

replication, metabolism and biosynthesis
• The DNA or RNA genome may be :
– ss – single stranded or
– ds – double stranded

• Genomes may be either:

– (+) sense: Positive-sense viral RNA is identical to viral mRNA

and thus can be immediately translated into protein by the host
cell.

OR
– (-) sense: Negative-sense viral RNA is complementary to mRNA
and thus must be converted to positive-sense RNA by an
RNA polymerase before translation.

• Retroviruses?
 Definitions

• Bacteriophage
– Virus that infects prokaryotic (bacterial)
cells.
• Nucleocapsid:
– viral nucleic acid + the protein coat that
encloses it.
– Represents the packaged form of the viral
genome.
Koneman et al. Color Atlas and Textbook of Microbiology 5th Ed. 1997
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  Growth on Division Contain Contain Contain Sensitive
artificial by binary DNA and protein muramic to
media fission RNA synthesis acid antibiotics
machinery

             
Bacteria often yes yes yes often yes

             
Viruses never no Either no* no no
DNA or
RNA

* The arenavirus family appears to ‘accidentally’ package ribosomes, but these appear to
play no role in protein synthesis.
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 CONSEQUENCES

• HEAVILY PARASITIC ON HOST CELL

• NO BROAD RANGE ANTIBIOTICS

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 HOST RANGE

• MAY BE WIDE OR NARROW

• MAY BE INSECT/ANIMAL,
INSECT/PLANT
• DO NOT CROSS EUCARYOTE /
PROCARYOTE BOUNDARY

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FACTORS AFFECTING HOST RANGE
- CELL SURFACE RECEPTORS

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 FACTORS AFFECTING
HOST RANGE

• CELL SURFACE RECEPTORS

• AVAILABILITY OF REPLICATION
MACHINERY
• ABILITY TO GET OUT OF CELL AND
SPREAD
• HOST ANTI-VIRAL RESPONSE
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VIRAL STRUCTURE – SOME
TERMINOLOGY
• virus particle = virion
• protein which coats the genome =
capsid
• capsid usually symmetrical
• capsid + genome = nucleocapsid
• may have an envelope

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 Viral Structure - Overview

Nucleic acid
Nucleocapsid
Capsid

Envelope protein
Viral envelope**
Membrane protein
Spike protein

Fig 1. Schematic overview of the structure of animal viruses

** does not exist in all viruses

 Viral Structure

• Varies in size, shape and symmetry

• VIP for classification
• 3 types of capsid symmetry:
– Cubic (icosahedral)
• Has 20 faces, each an equilateral triangle. Eg. adenovirus
– Helical
• Protein binds around DNA/RNA in a helical fashion eg.
Coronavirus
– Complex
• Is neither cubic nor helical eg. poxvirus
http://micro.magnet.fsu.edu/cells/virus.html
 ICOSAHEDRAL SYMMETRY

• 20 faces
• 12 vertices

http://www.tulane.edu/~dmsander/WWW/Video/Video.html 18
ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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ICOSAHEDRAL SYMMETRY

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Adenovirus

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Adenovirus

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ICOSAHEDRAL SYMMETRY

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 SYMMETRY OF
NUCLEOCAPSID

• ICOSAHEDRAL

• HELICAL

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TOBACCO MOSAIC VIRUS

adapted from:
Klug and Caspar Adv. Virus Res. 7:225

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 Helical symmetry

• Length controlled by nucleic acid

• Helix may be stiff or flexible

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 COMPLEX SYMMETRY

surface view cross section

White, DO and Fenner, FJ.

Medical Virology, 4th Ed. 1994 POXVIRUS FAMILY 34
 ENVELOPE

• OBTAINED BY BUDDING THROUGH

A CELLULAR MEMBRANE (except
poxviruses)
• POSSIBILITY OF EXITING CELL
WITHOUT KILLING IT
• CONTAINS AT LEAST ONE VIRALLY
CODED PROTEIN

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 ENVELOPE

• OBTAINED BY BUDDING THROUGH A

CELLULAR MEMBRANE (except poxviruses)
• POSSIBILITY OF EXITING CELL WITHOUT
KILLING IT
• CONTAINS AT LEAST ONE VIRALLY
CODED PROTEIN
– ATTACHMENT PROTEIN
• LOSS OF ENVELOPE RESULTS IN LOSS
OF INFECTIVITY

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 5 BASIC TYPES OF VIRAL STRUCTURE

icosahedral nucleocapsid nucleocapsid

lipid bilayer

ICOSAHEDRAL ENVELOPED ICOSAHEDRAL

helical nucleocapsid

COMPLEX

nucleocapsid

lipid bilayer

glycoprotein spikes
= peplomers

HELICAL ENVELOPED HELICAL

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Adapted from Schaechter et al., Mechanisms of Microbial Disease
 Viral Structure

Figure 1 An array of viruses. (a) The helical virus of rabies. (b) The segmented
helical virus of influenza. (c) A bacteriophage with an icosahedral head and helical
tail. (d) An enveloped icosahedral herpes simplex virus. (e) The unenveloped polio
virus. (f) The icosahedral HIV with spikes on its envelope.
 UNCONVENTIONAL
AGENTS
• VIROIDS
– RNA only
– Small genome
– Do not code for protein
– So far, only known viroids are in plants

• hepatitis delta virus (agent)

- some viroid, some virus features
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 UNCONVENTIONAL
AGENTS

• PRIONS

– protein only?

– do not contain any nucleic acid?

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 Viroids & Prions
• Viroids
– ss RNA genome and the smallest known pathogens.
– Affects plants

• Prions
– Infectious particles that are entirely protein.
– No nucleic acid
– Highly heat resistant
– Animal disease that affects nervous tissue
– Affects nervous tissue and results in
• Bovine spongiform encepahltits (BSE) “mad cow disease”,
• scrapie in sheep
• kuru & Creutzfeld-Jakob Disease (CJD) in humans
 CLASSIFICATION

• NUCLEIC ACID
• CAPSID
• PRESENCE OF ENVELOPE

• REPLICATION STRATEGY

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 CLASSIFICATION
NUCLEIC ACID
• RNA or DNA
• segmented or non-segmented
• linear or circular
• single-stranded or double-stranded
• if single-stranded RNA
– is genome mRNA (+) sense or
complementary to mRNA (-) sense

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 CLASSIFICATION
CAPSID
• symmetry
– icosahedral, helical, complex

• number of capsomers if icosahedral

• enveloped or non-enveloped

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 CLASSIFICATION

• REPLICATION STRATEGY

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 DNA VIRUSES

DOUBLE STRANDED SINGLE STRANDED COMPLEX

NON-ENVELOPED ENVELOPED

ENVELOPED NON-ENVELOPED PARVOVIRIDAE POXVIRIDAE

HERPESVIRIDAE
HEPADNAVIRIDAE
CIRCULAR LINEAR

PAPILLOMAVIRIDAE ADENOVIRIDAE All families shown are

POLYOMAVIRIDAE icosahedral except for
(formerly grouped together as the poxviruses
PAPOVAVIRIDAE)

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Modified from Volk et al., Essentials of Medical Microbiology, 4th Ed. 1991
 RNA VIRUSES

SINGLE STRANDED SINGLE STRANDED DOUBLE STRANDED

positive sense negative sense

ENVELOPED NONENVELOPED ENVELOPED NONENVELOPED

ICOSAHEDRAL HELICAL ICOSAHEDRAL HELICAL ICOSAHEDRAL

FLAVIVIRIDAE CORONAVIRIDAE PICORNAVIRIDAE ORTHOMYXOVIRIDAE REOVIRIDAE

TOGAVIRIDAE CALICIVIRIDAE PARAMYXOVIRIDAE
RETROVIRIDAE ASTROVIRIDAE RHABDOVIRIDAE
FILOVIRIDAE
BUNYAVIRIDAE
ARENAVIRIDAE

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Modified from Volk et al., Essentials of Medical Microbiology, 4th Ed. 1991
 CYTOPATHIC EFFECT

• ANY DETECTABLE CHANGES IN THE

HOST CELL
– MORPHOLOGICAL CHANGES

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 CYTOPATHIC EFFECT

• ANY DETECTABLE CHANGES IN THE

HOST CELL
– MORPHOLOGICAL CHANGES
– DEATH
– APOPTOSIS
– INDEFINITE GROWTH

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 tissue culture cells

epithelial epithelioid fibroblastic

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slides from CDC
 epithelial cells - adenovirus

uninfected early infection late infection

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slides from CDC
 epithelial cells - respiratory syncytial virus

uninfected respiratory syncytial virus

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slides from CDC
 fibroblastic cells - herpes simplex virus

uninfected early infection late infection

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slides from CDC
 fibroblastic cells - poliovirus

uninfected early infection late infection

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slides from CDC
PLAQUE ASSAY

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PLAQUE ASSAY

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PLAQUE ASSAY

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Diluted 10 fold Diluted 100 fold Diluted 1000 fold
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PLAQUE FORMING UNIT

P.F.U.
pfu

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 SOME POINTS TO
REMEMBER
• INFECTIVITY
– NOT EVERY RELEASED PARTICLE
IS INFECTIOUS

• ASSAYS
– detect every particle (e.g. electron
microscope)
– detect infectious particles only (e.g. plaque
assay) 64
Virus particle = virion White, DO and Fenner, FJ.
Medical Virology, 4th Ed. 1994

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Laboratory Diagnosis of Viral
Infection
 Difficulties

• Can not be seen under light microscope

• Can not be cultivated easily
• Do not grow on culture media
• Treatment was not available
 Microscopy

• Electron Microscope
• Light microscope – Inclusion bodies
• Fluorescent Microscope -Fluorescent
antibody technique
 Demonstration of Viral
Antigens
• Precipitation on gel eg HBsAg
• Immunofluorescence
• Counter Immuno Electro
Phoresis (CIEP)
• Enzyme Linkes Immuno
Sorbant Assay (ELISA)
 Isolation of Virus

• Laboratory animals
• Fertilized Hen’s Egg
– Chorioallantoic membrane
– Allantoic cavity
– Amniotic cavity
– Yolk sac
• Organ/Tissue/Cell Culture
• Growth identified by serological method like
neutralization.
 Serological Reactions

• Rising titre of antibody in paired sample of

sera
– First sample – At the earliest
– Second sample – After 2 weeks
• Single sample IgM type of antibody
detection
• Techniques – Neutralization, ELISA, CFT,
Haemagglutination Inhibition (HAI)Test
 Specimens

• According to the disease

– Respiratory – Throat swab
– CNS – CSF
– Eyes- Conjunctival scrapings
– Liver – Blood
– PUO – Blood
– Skin - Scrapings
 Viral Hemagglutination
• Hemagglutination
– Originally seen with the Influenza virus by Hirst in
1941.
– A convenient method of detection & assay of
Influenza virus.
– Due to the presence of Hemagglutinin spikes on the
surface.

• Reversal of hemagglutination – Elution

– Due to the presence of Neuraminidase enzyme,
Receptor Destroying Enzyme (RDE)
– Destruction of receptor – reversal of
hemagglutination – release of virus from the red cell
surface
– Found only in Myxoviuses.
 Virus Culture

Embryonated Egg Chorioallantioc membrane (CAM)

Allantoic cavity
Amniotic cavity
Yolk Sac

Cell Lines/ Primary

Tissue cultures Diploid/ Secondary
Continuous

Animal inoculation Suckling mice

 Embryonated Hen’s Egg
• Chorioallantoic membrane (CAM) – visible lesions
called pocks. Each infectious virus particle forms one
pock. e.g. Variola, Vaccinia virus

• Allantoic cavity – Influenza virus (vaccine production) &

paramyxoviruses

• Amniotic cavity – primary isolation of Influenza virus

• Yolk sac – Chlmyadia, Rickettsiae & some viruses

Embryonated Hen’s Egg
 Cell Culture
• Routinely used for growing viruses
• Classified into 3 types:
– Primary cell culture – normal cells freshly taken from body &
cultured, limited growth
1. Rhesus monkey kidney
2. Chick embryo fibroblast
3. Human amnion cell culture

– Diploid cell strains – cells of single type (fibroblast cells) that can
be subcultivated for limited number of times, mostly 50
1. WI-38: human embryonic lung cell
2. HL-8: Rhesus embryo cell

– Continuous cell lines – malignant cells, indefinite subcultivtion

1. HeLa: Human Ca of cervix cell line
2. HEP-2: Human epithelioma of larynx
3. Vero: Vervet monkey kidney
4. McCoy, Detroit-6, BHK-21, Kb
 Cell Culture

• Tissues Individual cells

trypsin & mechanical shaking

• Cells are washed, counted & suspended in a

growth medium.

• Growth medium – Minimum Essential Medium

(MEM): essential aminoacids, vitamins, salts,
glucose & bicarbonate in 5% CO2 with 5%
fetal calf or calf serum, antibiotics & phenol
red indicator
Cell Culture Bottles / Tubes
 Detection of virus growth in cell
cultures
1. Cytopathic effects (CPE) – morphological
changes in cultured cells, seen under
microscope, characteristic CPE for different
groups of viruses

2. Metabolic Inhibition – no acid production in

presence of virus

3. Hemadsorption – influenza & parainfluenza

viruses, by adding guinea pig erythrocytes to
the culture
 Detection of virus growth in cell
cultures
4. Interference – growth of a non cytopathogenic
virus can be tested by inoculating a known
cytopathogenic virus: growth of first virus will
inhibit the infection by second

5. Transformation – oncogenic viruses induce

transformation & loss of contact inhibition –
microtumors

6. Immunofluorescence – test for viral Ag in cells

from viral infected cultures.