VIROLOGY

Dr. MUHAMMAD IRFAN SIDDIQUE

Assistant Professor (Pharmaceutics)
IPS, UVAS
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a) Animal Sources

b) Airborne

c) Contact Infections
d) Food born

e) Fomites

f) Human carrier
Virology

• Definition
• History
• Brief introduction
• Characteristics of virus
• Structure of virus
• Symmetry of virus
• Classification of virus
 Introduction to Virology
• Obligate intracellular parasite containing genetic
material surrounded by protein
• Virus particles can only be observed by an
electron microscope

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 Introduction to Virology
• Recognizing the shape, size, and structure of different
viruses is critical to the study of disease
– Inner core of nucleic acid surrounded by protein coat
known as an envelope
– Sizes range from 20 – 250 nm

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General history
• In 1892 Dimitri Ivanowsky find virus (“a
poison”) during filtration.
• In 1915, Fredrick Twort discovered virus that
infect bacteria called “bacteriophage(to eat)”.
• In 1935 TMV was crystallized suggesting virus
might be non-living.
• By 1941, virologists beginning to visualize
viruses.
• Polio virus in 1954.
 Introduction (Terminologies)
• An infectious Virus outside host = Virion
• Surrounded protein coat = Capsid
• Subunit of capsid = capsomeres (protein)
• Capsid + Genome = Neucleocapsid
• Envelope =
Protein + Lipid bilayer
Introduction
 Deliver DNA or RNA into the host cell so that
the genome can be expressed (transcribed and
translated) by the host cell.

 Enveloped viruses
 Unique to each type
 ssRNA, dsRNA
 dsDNA, ssDNA
 Characteristics of Viruses
• Intracellular parasites of bacteria, protozoa,
fungi, algae, plants and animals.

• Ultramicroscopic size, ranging from 20 nm

(polio-virus) up to 450 nm (diameter).

• Non-cellular in nature; structure is very

compact.
• Live or dead?
• Inactive outside the host cell and active only
inside host cells.
Characteristics of Viruses
• Surface impart high specificity.
e.g. HIV specificity, Rabies virus

• Specificity called tissue tropism (tissue

attraction).

• Multiply by taking control.

• Lack enzymes for most metabolic processes.

• Lack machinery for synthesizing proteins.

 The size of viruses

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 Viruses are Ultramicroscopic

Koneman et al. Color Atlas and Textbook of Microbiology 5th Ed. 1997

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Size Range
• So minute (0.2 m) that an electron microscope
require.

• More than 2,000 bacterial viruses could fit into an

average bacterial cell, and more than 50 million
polioviruses could be accommodated by an average
human cell.
Viral Morphology
Genome

• Unbroken, but in some instances (as in

influenza viruses) it exists in segments.

• Usually folded

• Condensed in “icosahedral viruses” and

coiled in helical fashion in “helical viruses”.
 Viral Morphology
Capsid

 Protective covering for the genome because the

construction of its amino acids resists temperature, pH
and other environmental fluctuations.

 In some viruses, capsid proteins are organized into

enzymes to assist cell penetration during replication.

Capsomers
 whose organization yields the symmetry.
 Viral Structure - Overview

Nucleic acid
Nucleocapsid
Capsid
Envelope
protein Viral
Membrane envelope**
protein
Fig 1. Schematic overview of the structure of animal viruses
** does not exist in all viruses

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 Viral Morphology
Nucleocapsid:

 Capsid + genome “genocapsid”

 to maintain the structure

Envelope:

 A flexible membrane known as envelope.

 Composed of lipids and proteins
• similar to host cell membrane.
 Chicken pox (Varisella Flu (Influenza virus)
zoster virus (VZV)

Rabies virus

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 How are viruses named?
• Based on:
- the disease they cause
poliovirus, rabies virus
- the type of disease
murine leukemia virus
- geographic locations
Sendai virus, Coxsackie virus
- their discovers
Epstein-Barr virus
- how they were originally thought to be contracted
dengue virus (“evil spirit”), influenza virus (the “influence” of bad air)
- combinations of the above
Rous Sarcoma virus

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 How are viruses named?

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1. Name after disease
e.g. Measles virus
smallpox virus

2. Name after the places of disease

exposure
e.g. Newcastle disease virus,
Ebola virus
Nomenclature
3. Host and sign of disease:
Tobacco Mosaic Virus, Cauliflower mosaic virus
Viral Symmetry
1. Helical Viruses:
• Elongated like spring or corkscrew. e.g.
Tobacco Mosaic Virus (TMV) and Rabies
virus.
Viral Symmetry
2. Icosahedral viruses:
Icos= 20, hedral= sides

• e.g. Herpes simplex virus

• Poliovirus
Viral Symmetry
3. Complex viruses:
• Combination of helical and icosahedral
symmetry. E.g. Bacteriophage, Smallpox virus.
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 3rd lecture
• Classification of virus
• Replication of viruses
• Cultivation of viruses
Classification
1. Viruses classify based on structural and
chemical composition.
2. International Committee on Taxonomy of
Viruses (ICTV).
 Classification
Classification of Human Viral Diseases by Tissue Affected

Group Tissues Affected Important Diseases

Pneumotropic Respiratory system Influenza, respiratory syncytial disease, adenovirus diseases,

rhinovirus infection.

Dermotropic Skin and subcutaneous Chickenpox, herpes simplex, measles, mumps, smallpox,
tissues molluscum, contagiosum, rubella.

Viscerotropic Blood and visceral organs Yellow fever, dengue fever, infectious mononucleosis,
cytomegatovirus disease, viral fevers, Marburg disease, viral
gastroenteritis, hepatitis A, hepatitis B, AIDS

Neurotropic Central nervous system Rabies, lymphocytic choriomeningitis, polio, slow virus disease,
arboviral encepthalitis.
Classification
• Baltimore classify virus based on genome type
and mode of replication and transcription.
 DNA VIRUSES

DOUBLE STRANDED SINGLE STRANDED COMPLEX

NON-ENVELOPED ENVELOPED

ENVELOPED NON-ENVELOPED
PARVOVIRIDAE POXVIRIDAE

HERPESVIRIDAE
CIRCULAR LINEAR

PAPILLOMAVIRIDAE ADENOVIRIDAE
POLYOMAVIRIDAE All families shown are
(formerly grouped together as the icosahedral except for
PAPOVAVIRIDAE) poxviruses

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

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 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

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

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• Some are positive in that they have a "sense"
strand of RNA (coded information about how
to build proteins) as their genetic material.
And other RNA viruses tare negative in that
they have an "antisense" strand (the paired
opposite of the coded information). Positive-
strand or sense-strand RNA viruses are
therefore as opposed to negative-strand or
antisense-strand RNA viruses.
 Latest Classification of Human Viruses
Sr# Characteristic Viral Family Viral Genus Dimension Special Features
Features (With of virion
representative Diameter
species)and in nm
Unclassified
Members*
1 Single-stranded Parvoviridae Dependovirus 18-25 Depend on coinfection with
DNA, adenoviruses ; invariably cause
nonenveloped faetal death, and gastroenteritis.
2 Double-stranded Adenoviridae Mastadenovirus 70-90 Medium-sized viruses that cause
DNA, (adenovirus) various respiratory infections in
nonenveloped humans ; a few of them even
produce neoplasms (tumours) in
animals.
3 Double-stranded Poxviridae Orthopoxvirus 200-350 Very large, complex, brick shaped
DNA, enveloped (vaccinia and viruses that cause diseases e.g.
smallpox viruses) smallpox (variola), molluscum
Molluscipoxvirus contagiosum (wartlike skin
lesion), cowpox and vaccinia.
Vaccinia virus provides specific
immunity to smallpox.
4 Single-stranded Picornviridae Enterovirus 28-30 Upto 70 human entero-viruses
RNA, Rhinovirus are known, including the polio-,
nonenveloped (Common cold coxsackie-, and echoviruses; more
+ strand virus), Hepatitis A than 100 rhinoviruses exist and
virus prove to be the most common
cause of colds.
5 Single-stranded Togaviridae Alphavirus, 60-70 Essentially include several viruses
RNA, enveloped Rubivirus, (rubella transmitted by arthropods
+ strand virus) (Alphavirus); diseases include
Eastern Equine Encephalitis (EEE),
Rubella virus is transmitted by the
respiratory route.

6 one strand of Rhabdoviridae Vesiculovirus 70-180 Bullet-shaped viruses having a

RNA (vesicular spiked envelope ; invariably cause
stomatitis virus), rabies and several animal diseases.
Lyssavirus (rabies
virus)

7 multiple strands Orthomyxovirid Influenzavirus 80-200 Envelope spikes can agglutinate

of RNA ae (Influenza viruses red blood cells (RBCs).
A and B),
Influenza C virus.

8 Produce DNA Retroviridae Oncoviruses 100-120 Includes all RNA neoplasm viruses
Lentivirus (HIV) and double-stranded RNA viruses.
The oncoviruses invariably cause
leukemia and neoplasms in
animals, and the lentivirus HIV
causes AIDS.

9 Double-stranded Reoviridae Reovirus Colorado 60-80 Involved in mild respiratory

RNA tick fever virus infections ; an unclassified species
nonenveloped causes Colorado tick
fever.
 BACTERIOPHAGES

Example : The spindle tuber disease of potatoes is a glaring example

of a specific disease invariably caused by the viriods.

A good number of bacteriophages infecting various microorganisms

have now been duly isolated, characterized, and recognized. The
following table records the various bacteriophages, host(s), particle
dimensions (viz., head and tail in nm), structure, and composition
adequately.
 Multiplication of Bacteriophages
• Basic mechanism of viral multiplication is
similar for all viruses. Bacteriophages can
multiply by two alternative mechanisms:

• The lytic cycle (death of the host cell)

• The lysogenic cycle (host cell remains alive)

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• One of the best studied processes of replica-
tion is that carried out by bacteriophages of the
T-even group (T for “type”). Bacteriophages
T2, T4, and T6 are in this group.
• Large, complex, naked DNA virions with the
characteristic head and tail of bacteriophages.
• Contain tail fibers, which function similar to
spikes on animal viruses and identify what
bacterial species the phage will be able to
infect.
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 Why Host is Needed
• Nucleic acid in a phage contains only a few of
the many genes needed for viral synthesis and
replication.
• It contains, for example, genes for synthesizing
viral structural components, such as capsid
proteins.
• It lacks the genes for many other key enzymes,
such as those used during nucleic acid
synthesis.
• Therefore, it depend on the host cell.
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 Replication
• Most remarkable event in nature.

• A virion invades a living cells.

• Use metabolism of the cell, and produces copies

of itself, often destroying the cell.

• The virion cannot replicate independently, but

within the cell, the replication takes place with
high efficiency.
Processes of replication
1. Attachment/ Adsorption
2. Penetration
3. Biosynthesis
4. Maturation
5. Release
1.Attachment
Replication of Bacteriophages
The replication of bacteriophages is a five step process.

1.Attachment:

• Contact/ adsorb.
• No long-distance chemical attraction.
• Phage’s tail must match with a complementary
receptor site on the cell wall.
• The actual attachment is a weak chemical union.
 2. Penetration
• The tail of the phage releases the enzyme ,lysozyme to
dissolve a portion of the bacterial cell wall.

•Then the tail sheath contacts, and the tail core drives
through the cell wall.

•As the tip of the core reaches the cell membrane below,
the DNA passes through the tail core and on through the
cell membrane into the bacterial cytoplasm.

•For most bacteriophages, the capsid remains outside.

2. Penetration
3. Biosynthesis
 3. Biosynthesis
• Disruption of the host chromosome.
• Synthesize multiple copies of itself.
•Messenger RNA molecules transcribed from phase
DNA appear in the cytoplasm.
•Bacterial ribosomes, aminoacids, and enzymes are
all enlisted for the biosynthesis.
•Genetic code can be used over and over.

•For a number of minutes, called the eclipse period,

no new viral capsids are present.
 4. Maturation
•Replicated bacteriophage DNAs and the capsids
re assembled into complete virions.

•The enzymes encoded by viral genes guide the

assembly in step-by-step fashion.

•Phage heads and tails are assembled from protein

subunits; the heads are packaged with DNA; and
in the third, the tails are attached to the heads.
 5.Release
•For bacteriophage, this is also called the lysis stage
because the cell lyses, or breaks open.

•The time that passes from phage attachment to the

release of new viruses is commonly referred to as the
burst time.
•For bacteriophages, the burst time averages from 20-40
minutes.
•At the conclusion of the process, 50-200 new phages
emerge form the host cell.
•This number is commonly called the burst size.
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 Animal Virus Replication

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• Like bacteriophages, animal viruses also lead
often brief but eventful “lives” as they
produce more viruses as a result of infection.

• Such a productive infection retains the five

replication stages described for the
bacteriophages.

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 Attachment
• Animal viruses infect host cells by binding to
receptors on the host cell’s plasma
membrane.

• This binding is facilitated by the spikes

distributed over the surface of the capsid
(e.g., adenovirus) or envelope (e.g., HIV).

• The spikes are what determine the host range

for a virus. 65
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 Penetration
• Some viruses, such as HIV and the adenoviruses,
require a second receptor, called a co-receptor, for viral
penetration into the cytoplasm.

• Viral entry also differs from that in phages, where only

the DNA entered the bacterial cell cytoplasm.

• Animal viruses often are taken into the cytoplasm as

intact nucleocapsids.

• For viruses like HIV, the viral envelope fuses with the
plasma membrane and releases the nucleocapsid into
the cytoplasm
68
 An enveloped virus, such as HIV, contacts the plasma
membrane and the spikes interact with receptor sites on the
membrane surface.

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 Endocytosis
• For other animal viruses, such as the adeno-
viruses and influenza virus, the virion is taken
into the cell by endocytosis.

• At the attachment site, the cell enfolds the

virion within a vacuole and brings it into the
cytoplasm.

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 A Specific Interaction Between Spikes And Receptor Sites On The
Plasma Membrane. However, For This Naked Adenovirus
Undergoes Endocytosis

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 PENETRATION

herpesviruses, paramyxoviruses, HIV

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 Uncoating
• Once in the cell, the vacuole membrane breaks
down, releasing the nucleocapsid or the
genome into the cytoplasm.

• In both examples, the capsid disassembles

from the genome in a process called uncoating.

• The genome is transported to the site where

transcription or replication will occur.
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 Biosynthesis and Maturation
• One way we split the virus families was based on whether
they have DNA or RNA as their genetic information.

• The DNA of a DNA virus supplies the genetic codes for

enzymes that synthesize viral parts from available building
blocks.

• Although the poxviruses replicate entirely in the host cell

cytoplasm, most of the DNA viruses employ a division of
labor:

• DNA genomes are synthesized in the host cell nucleus, and

capsid proteins are produced in the cytoplasm

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• The proteins are then trans-ported to the
nucleus and join with the nucleic acid
molecules for maturation.

• Adenoviruses and herpesviruses follow this

pattern.

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 RNA Viruses
• RNA viruses follow a slightly different pattern.
• Because the +ssRNA viruses act as a messenger
RNA, following uncoating, the RNA immediately
begins supplying the codes for protein synthesis
as genome replication occurs.
• Other ssRNA viruses, such as the influenza virus,
use their RNA as a template to synthesize a
complementary (+) strand of RNA.
• An RNA-dependent RNA polymerase is present in
the virus to synthesize the (+) strand.
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• The synthesized +ssRNA then is used as a
messenger RNA molecule for protein synthesis as
well as the template to form the ssRNA genome.
• The final steps of maturation may include the
acquisition of an envelope.
• In this step, envelope proteins (spikes) are
synthesized and, depending on the virus,
incorporated into a nuclear or cytoplasmic
membrane, or the plasma membrane.
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 Release
In the final stage, enveloped viruses either:
• (1) Push through the plasma membrane, forcing a
portion of the membrane ahead of and around the
virion, resulting in an envelope;
• (2) As with the herpesvirus, a membrane-enclosed
virus fuses with the plasma membrane, releasing the
virion. This process, called budding, need not
necessarily kill the cell during release.
• The same cannot be said for naked viruses. They
leave the cell when the cell membrane ruptures, a
process that generally results in cell death.
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 Latent Infection
• Unlike most RNA viruses that go through a productive
infection, many of the DNA viruses and the retroviruses can
establish a latent infection, characterized by repression of
most viral genes.
• Thus the virus lies “dormant.” For example, some
herpesviruses, such as herpes simplex virus-1 (HSV-1), can
generate a productive or latent infection.
• In an infected sensory neuron, HSV-1 under-goes latency
as the viral dsDNA enters the neuron’s cell nucleus and
circularizes.
• No viral particles are produced for months or years until
some stress event reactivates the viral dsDNA and a new
productive infection will be initiated

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 Transmission of Viruses
• Respiratory transmission
– Influenza A virus
• Faecal-oral transmission
– Enterovirus
• Blood-borne transmission
– Hepatitis B virus
• Sexual Transmission
– HIV
• Animal or insect vectors
– Rabies virus

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 Viruses enter the body of the host
in a variety of ways, for example...

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 The commonest forms of
transmission are via...

INHALED DROPLETS
in sneezing of coughing
for example the COMMON COLD
or INFLUENZA VIRUSES.

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 or by...

drinking water or
eating raw food, for example,
HEPATITIS A and POLIOVIRUS.

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 also...

vertical transmission -
from mother to baby for example
HIV, HEPATITIS B and RUBELLA...

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 also...

bites of vector arthropods such as

mosquitoes for example YELLOW FEVER,
RIFT VALLEY FEVER and DENGUE.

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 get well after a period of sickness
to be immune for the rest of their lives.
Examples are MEASLES INFECTION,
RUBELLA or German measles,
MUMPS and many others...

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Cultivation Of Viruses
1. Plaque method (Infect to cell culture)
• Animal and plants viruses may be grown in
cell culture.
– Continuous cell lines may be maintained
indefinitely.

Figure 13.8
• Animal cell separated from tissue with
enzymes and suspended in nutrient, growth
factor, pH buffer and salt.
• Primary cell culture monolayer
separated.
• Isolation of single cell type cell line.
• Viruses infect and grow like clear zone with
cloudy lawn Plaque.
 Cultivation Of Viruses
2. Chicken Embryos
• Fertile hen’s egg incubated for 5 to 12 days can be inoculated by
removing a small piece of the shell aseptically and introducing the
virus containing material through the opening.
• The shell opening is then closed with paraffin wax, and the egg is
incubated at 36°C for the length of time required for the growth of
virus.
• The embryo may be inoculated on the chorioallantoic membrane,
where same viruses, e.g. vaccinia, will grow and produce local
lesions.
• The yolk sac of the embryo also can be used to grow viruses.
HARVEST OF ALLANTOIC FLUID
Applications

The embryo technique has been used for production of

virus for vaccines against
• small pox
• yellow fever
• influenza and
other diseases and for immunologic tests…..etc.
Cultivation of Bacteriophages In The Laboratory

It is practically possible to grow the bacteriophages

in two different manners, namely:
• In suspensions of organisms in liquid media.
• In bacterial cultures on solid media.

Advantages of using Solid Media:

In actual practice, the use of solid media makes it
feasible and possible the plaque method for the easy
detection and rapid counting of the viruses.
Methodology (Plaque Method)
The various steps that are involved in the ‘plaque
method’ are as enumerated under :

1. Sample of bacteriophage is duly mixed with the

host bacteria and molten agar.

2. The resulting agar is then poured carefully into a

Petri-plate adequately containing a hardened layer
of the agar growth medium.
3. The mixture of virus-bacteria gets solidified into a
thin top-layer that invariably comprises of a Layer
of organisms nearly one-cell thick.

4. This specific step allows each virus to infect a

bacterium, multiplies subsequently, and helps to
release several hundred altogether new viruses.

5. Nevertheless, these newly generated viruses in turn

duly infect other organisms that are present in the
immediate close vicinity; and hence, more new
crop of viruses are produced ultimately.
5. All the organisms present in the area surrounding the
original virus are destroyed finally. In this way, a good
number of ‘clearings’ or plaques are produced, which may
be seen against a “lawn” of bacterial growth upon the
surface of the agar ; whereas, the plaques are observed to
form uninfected microorganisms elsewhere in the Petri dish
(or Petri plate) undergoing rapid multiplication and giving
rise to a turbid background finally.

Note: Each plaque corresponds theoretically to a single

virus in the initial suspension. Hence, the concentrations of
viral suspensions measured by the actual number of
plaques are invariably expressed in terms of plaque-
forming units (pfu).
 Inactivation Of Viruses
Inactivated by many of the physical and chemical agents routinely
used for other organisms.

Few methods for inactivating the viruses are described as below:

• Formaldehyde reacts with free amino groups on adenine,
guanine and cytosine molecules to modify the viral genome and
prevent replication.

• Heat alters the structure of viral proteins and nucleic acids,

causing them to unfold and denature.

• Ultraviolet light inactivates viruses by stimulating adjacent

thymine or cytosine bases on DNA molecules to bind together
and form pairs called dimers.
Inactivation Of Viruses
• The dimmers twist the molecule out of shape, and
distorted viral genome cannot replicate.

• X-rays cause breaks in the sugar phosphate backbone

of the nucleic acid.

• Lipid solvents such as ether, chloroform and

detergents, all of which dissolve the lipid in the
envelope of viruses and inactivate them.

• Heavy metal compounds such as mercury and silver

derivatives inactivate viruses.
 References

1. Lippincot. Microbiology by Lipponcott. William & Willkin, USA,

2001.

2. Alcamo. Introduction to Microbiology. John Bartlett Publishers,

6th Ed., 2003.

3. Pelczar, Microbiology, McGraw-Hill Inc, 1996.