Bio-chemistry diploma

Faculty of veterinary medicine

Al Sadat city University

Microbiology
(Virology)

Prof. Mohammed abo Elkhair

Professor of virology
Faculty of veterinary medicine, Microbiology department
Al Sadat city University

Files build by and most of images produced by:

Mohammed Abd El Moneem El Sayed

Head of department

‫طلعت حرب راجع‬

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Lec. 1 Introduction to Virology

Virus: is a Latin word means poison.
Viruses: are submicroscopic (light microscope), obligated and intra cellular parasites and they are entirely
dependent on the host cell metabolic machinery for replication.
Discovery:
1. Russian scientist observed that some of plants is infected with Mosaic.
2. He takes some of infected plant leaf and chewed them and put them in non-infected plant and he
observed that the healthy plant become infected.
3. He filtrated the chawed leafs of infected plant by filter paper (to get rid of Bactria as he was
expected that Bactria is causing that infection) and injected the filtrate to another healthy plant but
he observed that the healthy plant become infected.
4. He named that filtrate as poisonous liquid.
5. He made a serial dilution to that liquid and inject it to some health plants and all healthy was
infected.
6. After all more and more research started at that poisonous liquid.
Virus is a unique class of microorganism that characterized by:
1. Extremely small about (20-300 nm) can’t seen under the ordinary microscope.
2. Obligatory dependent in living cell for replication.
3. Contains only one type of nucleic acid (DNA or RNA) and only nucleic acid can make infection.
4. Has no functional organelles as mitochondria of nucleus of ribosomes so need host as Bactria to
replicate.
5. Can’t live on non-living media (eg. Agar) as Bactria.
6. Non-sensitivity to antibiotics
• some organisms as Rickettsia and chlamydia (Bactria) are sometimes mentioned as viruses because
some of the are obligatory intracellular parasites and <300 nm (with few exceptions) and can’t live in
agar (non-living media) and non-sensitive to antibiotics.
Origen of viruses: (three theories)
1. Thay may have originated and evolved in parallel with primordial life forms.
2. They may have arisen from cellular nucleic acid which acquired the ability to replicate at the expense
of the host cell.
3. They may arise from free-living organism which gradually lost genetic information until they
become totally dependent on the biosynthesis pathway of their host cells (regressive theory of the
origin of viruses).

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Viruses structure:

1. Capsid: a protein coat composed of morphological units called capsomer each capsomer composed
of protomers (structure unit)
each protomer composed of a lot of protein subunits (single folded poly-peptide chain) and together
form the chemical building block of larger assembly unit eg. VP1, Vp2, VP3 and VP4 (VP= viruses
protein)
Capsomers are self-assemble in a defined manner giving the capsid a characteristic symmetry and
two kinds of symmetry have been recognized as icosahedral or helical symmetry and other viruses
that don’t fall in a form are referred as complex eg. Poxviruses.
a. Icosahedral symmetry: A number of solid shapes can be constructed from repeated subunits.
I. Tetragon (4 triangle faces)
II. Cube (6 Squair faces)
III. Octagon (8 triangle faces)
IV. Dodecahedron (12 pentagonal faces)
V. Icosahedrons (20 triangle faces, 12 vertices “corner”, 30 edges, 2 or 3 axis and fivefold
rotational symmetry)
Viruses with icosahedral capsids:
1. All known animal DNA viruses (except poxviruses)
2. All animal viruses with double strand RNA genomes as (picornaviruses, caliciviruses,
flaviviruses, togaviruses and retro viruses).

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b. Helical symmetry: Self assembles as a cylindrical structure which the protein structural units are
arranged as a helix
The genomic RNA forms a spiral shape in within the helical nucleocapsid.
Capsomer consist of a single polypeptide molecule.
Viruses with helical capsid:
1. All animal enveloped viruses.
2. All viruses with single strand RNA with negative sense eg. Rhabdo-viridae expect
(picornviruses, caliciviruses, flaviviruses, togaviruses and retro viruses).

c. Complex capsids: Not helical or icosahedral symmetry

Viruses with complex symmetry:
1. Poxviruses.
2. Bacteriophage

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2. Viruses envelope (not in all viruses).
Viruses forming the envelope after it replicate in the cells.
All animal viruses with helical and icosahedral capsid is enveloped viruses.
Envelop composed of:
a. lipid bilayer: that derived from host cellular membrane.
Viruses have tow type of lipids:
1. Phospholipid (60-60%):
2. Cholesterol
They can acquire the envelop lipid during replication in the host cells
by budding with an organelle that are living beside it (forming it from one of two sites):
a. internal parts cell membranes as Golgi apparatus, rough endoplasmic reticulum or nuclear
membrane (Viruses envelop that bud and replicate in that organ contain only phospholipid).
b. From the plasma membrane (Viruses envelop that bud and replicate in plasma membrane
contain phospholipid and cholesterol) eg. Influenz viruses.
b. Glycoproteins: viral coded protein (Viral proteins “viral ligands”): forming the viral epitopes
(antigen determine site) that are important in:
i. Determination of viruses type.
ii. Immune response (immune system definition site): and main factor vaccine from it.
• Epitopes is composed mainly of carbohydrate.
• Viral glycans are synthesized by cellular glycosyl transferase so glycans of viruses are
similar to glycoproteins of host cell membrane.
Envelop function and role in viruses replication:
a. Receptor binding with cells to enter by peplomers eg. HA in orthmyxo-viridae.
b. Membrane fusion: with host cell eg. F protein in paramyxo-viride.
c. secondery un coting: getting rid of the envelop for short time for transcriptase activation.
d. Receptor destruction eg. Neuraminidase in Orthomyxoviridae
Enveloped viruses feature (IMPORTANT IN EXAM)
1. Enveloped viruses are easy to disinfection than non-enveloped viruses.
2. Enveloped viruses are more sensitive to environmental condition than non-enveloped viruses.
3. Enveloped viruses are quickly in replication in host cells than non-enveloped viruses.
• The more viruses are non-enveloped and small in size the more is highly resistance to
environmental condition.
• Some viruses that contain envelop have a layer after the envelop could Matrix protein that
helping to give the viruses it’s shape.
• In viruses that haven’t Envelop the capsid functional instead.
V.I.Q: Mention the function of capsid in non-enveloped viruses?

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3. DNA or RNA: the genomic martial:
Viruses has a single type of nucleic acid DNA or RNA.
Nucleic acid is bult from small units called nucleotides, each nucleoid is consisted of:
a. Pentose sugar (Ribose for RNA and Deoxyribose for DNA) (Carbohydrate in viruses).
b. Link between sugar molecules (Phosphoric acid)
c. Nitrogen bases (Chemical composition):
I. Adenine, Thiamine, Cytosine and guanin for
DNA.
II. Adenine, Uracil, Cytosine and guanin for DNA.
All viruses genomes are haploid (N) except retroviruses are
diploid (2N).
All RNA-contain viruses are single strand except retro-
viridae and birna-viridae are double strand RNA.
• Single strand or double strand is descriptive to the statue
of the genomic material but haploid or diploid mention
the number of gene copes (N) or (2N)
• RNA-contain viruses are smaller that DNA viruses
about (20-30 K. base)
Single strand RNA are two types:
1. Positive sense genome: RNA that the host ribosomes can translate its codons to amino acids
directly (originally mRNA).
2. Negative sense genome: RNA that the host ribosomes and translate it before it replicated (by
transcriptase enzyme found in negative sense viruses) first then the result in replication is
translated by the ribosome.
• Negative sense RNA is a single strand of RNA with 3→5 direction.
Positive sense RNA is a single strand RNA with 5→3 direction.
mRNA is a single strand RNA with 5→3 direction
So, +ve sense RNA is mRNA directly without templet.
So, -ve sense RNA must give +ve sense first to give mRNA.
• Polymerize dependent RNA (enzyme that give mRNA from the genome in the hos) can’t read
the -ve sense RNA so -ve first Duplicate to give +ve and +ve sense is itself is mRNA that will
transcript to give +ve sense mRNA and get the right protein.
• Positive sense RNA genome can be infectious itself.
• RNA viruses can be founded in multipartite (Segments) eg. Orthomyxoviridae

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Stability of viral infectivity:

1. Temperature: low heat inactivates viruses (viruses preserved in -70 and -196 CO)
2. pH: viruses is inactivated at pH 5-6 and each viruses have a specific pH stability range (can differs
viruses family by pH).
3. Humidity: Increase in humidity is combined with high temperature to promotes virus inactivation.
Dryness promotes preservation for viruses.
4. Ultraviolet: Completely damaging viral genome in 230 nm and damaging protein at 260 nm
5. Organic solvent: can completely inactive the envelop.
6. Oxidative agent: as quaternary ammonium chloride and sodium hypochlorite used as excellent
disinfectants with most viruses.
7. Protein denaturation agent: good disinfectants but not work well with large amount of proteinaceous
martial as formaldehyde, glutaraldehyde or alcohol

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Lec. 2 Viral taxonomy

Early virus classification was based on virus living site eg. Viruses causing diarrhea is mentioned as
enteric virus

Now viral classified by four classifications:

Taxonomy importance:
1. Viral bio control
2. Diagnosis of viruses in lab.
1. Hieratical classification: classify the viruses according to capsid symmetry and viruses structure.
System criteria:
a. Viral genome (dsDNA, dsDNA, ssRNA +, ssRNA -, dsRNA).
b. Size and symmetry of capsid (LOOK ABOVE).
c. Presence of envelop or not.

Some important identification

ssDNA Single strand DNA ssRNA Single strand RNA
dsDNA Douple strand DNA dsRNA Douple strand RNA
ssRNA (+) Single strand positive sense RNA ssRNA (-) Single strand negative sense RNA
Hepadna-viridae: is a double strand DNA genome and contain reverse transcriptase (RT) enzyme.
Retroviridae: is single strand negative sense RNA genome and contain reverse transcriptase (RT) enzyme.

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2. Baltimore classification: (By David Baltimore) based on viral replication and mRNA Synthes (7 groups).
System criteria:
a. Based on complete or partial (Genome based on reverse transcriptase enzyme) genome type of the
viral genome
b. Classification according to mRNA production

Group I dsDNA viruses:

a. invade host
b. Free it’s genomic DNA
c. Virus DNA will enter nuclease and will use
host transcriptase enzyme to get its own
mRNA directly.

Group II ssDNA viruses:

a. invade host
b. Free it’s genomic DNA
c. Virus DNA will enter nuclease and
duplicated its genomic ssDNA to make
dsDNA by replication process (using host
polymerize enzyme).
d. Then use host transcriptase enzyme to get
its own mRNA.

Group III dsRNA viruses: As Group I

Group IV ssRNA positive sense viruses:

a. invade host
b. Free it’s genomic RNA
c. Virus +ve sense RNA will replicate its
genome to give -ve sense and another +ve RNA to duplicate and preserve its genome first.
I. +ve RNA will preserved in the virus (keep the original)
II. -ve RNA used as templet that will transcript to give mRNA process (using host polymerize).
• That group is a special case for +ve and -ve sense RNA to forming mRNA.

Group V ssRNA negative sense viruses: As Group I

• Negative sense RNA Can give mRNA directly because it with 3→5 direction.

Group VI ssRNA positive sense viruses with reverse transcriptase:

a. invade host and free it’s genomic RNA
b. RNA is giving DNA first using reverse transcriptase (RT) from the virus to form DNA from RNA.
c. The Reverse transcriptase incorporate its new DNA (made by RT) with host chromosomes in
nuclease to make host DNA and virus DNA as one DNA (That method is called latency and
retroviruses do that method to escape from immune system)
d. Virus DNA start to transcript mRNA when immune is dropped.
Group VII dsDNA with reverse transcriptase viruses:
a. invade host
b. Free it’s genomic DNA
c. Forming complete its partial DNA to duple strand DNA
d. Virus new complete DNA will enter nuclease and will use host transcriptase enzyme to get its own
mRNA directly.

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3. Phylogenetic taxonomy or genotyping classification: Based
on the sequence of viral genome.
System criteria:
a. Based on complete or partial sequencing of viral genome
b. Comparison of the nucleotide sequencing using computer
software (sequencer).
c. Classify the viral strains into different linage.

4. Universal system of viruses taxonomy: The most widely used.

The international committee on taxonomy of viruses (I.C.T.V) established in 1973.
Taxonomy pyramid:
a. Order: Ended with virales eg. Mononegavirales (single strand RNA negative sense unisegment order)
b. Family: Ended with viridae
c. Subfamily: Ended with virinae
d. Genius: Ended with virus
e. Species: some important identification related with species (Ever identification below is a viral
mutation that makes differences between the same species) (VERY IMPORTANT):
Strains: Variation (mutations) of one species they are resembles type viruses in major properties and
different in minor properties as vector specificity or symptoms.
Pathotype: The variation in virulence of virus strains. Eg. Newcastle disease virus.
Species classified according to its pathotype to:
i. Lentogenic: Less in virulence
ii. Mesogenic: Moderate in virulence
iii. Velogenic: Highly virulence
• Virulence determined according to the viruses capacity to generate a new virus
(Replication and transcription) in short time.
Biotypes: Variation of cytopathogenicity of the virus strain eg. Bovine viral disease (have two types
cytopathogenicity and non- cytopathogenicity type).
Serotype: Variation in characteristic set of antigens of virus (variation of antibody developed against
viruses “Difference between strains”) eg. Toung fever (One serotype).
• Each serotype has a specific chemical kit.
Body release two types of antibodies to the same serotype:
i. Neutralized
ii. Non-neutralized.
Serogroup: a group of viruses that cross react with each other antigenically (antibody that react to
specific virus antigen can react also with other virus antigen from the same strain).
Genotype: Variation in genetic make-up of virus species (variation in genotype but not affect the
antigens if affect it called serotypes of species not genotype) (can affect something else as
way of reproduction).
Quasi-species: a population of genetic variation within individual isolates.
Variant: Two species with the same genome but everyone has its own symptoms and signs.
• Order, family, subfamily and genus first letter are capitalized and written in italic or underline
and species is written in small letters and also italic or underline.

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Lec. 3 Viral replication

Viral Replication
When a virus infects a cell (only living cells), nucleic acid must be uncoated and gain access to metabolic
machinery of cell to be asymptotic and infect the individuals (host) and generate new viruses.

How to study the viral replication?

By One-step growth curve is an experimental procedure for studying the viral replication.
• You should infect all cells at the same time with the same virus at this method to be able to see
(study) the viral replication

Replication and growth steps (generally):

1. Attachment of the virus.
2. Penetration of virus to the host cell.
3. Uncoating the capsid or envelop to leave naked genome in the host cytoplasm.
4. Transcription of viral nucleic acid.
5. Translate the viral nucleic acid to Protein (protein Synthes).
6. Replication of nucleic acid.
7. New individuals are released from the host cell by budding or lysis.

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Virus-host life cycle is characterized:
1. Attachment: by binding with specific receptor to enter the cell (Specificity between receptor of host
cell and the viral attachment).
• One specific receptor on the host cell surface to the virus if virus wasn’t found it, it will not
symptom to the host (due to no replication occurs due to can’t enter the cell).

2. Penetration: with entry of nucleic acid into cell and virus can det in by two methods:
a. Fusion: Fusion occur between envelop and plasma membrane in enveloped viruses.
• Some viruses need and acidic pH to fuse with plasma membrane as influenz and acidic pH
not found in cell surface so it uses the second method.

b. Receptor mediated endocytosis: Via cell receptor (Invagination).

• In this method non-enveloped viruses can destroy the cell endosomal membrane to access
the nucleus.
• Non-enveloped viruses can only enter the cell with only Endocytosis method but
enveloped viruses with both methods.
• Some nonenveloped viruses can pass the plasma membrane without any of the two
methods.
• Bacteriophage needn’t to enter the cell but invade its own DNA down in the cell.

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3. Uncoating: in that step virus remove its outer capsid or envelop and shown as genome only in host
cell cytoplasm.
You can see viruses under electron microscope in attachment or penetration phase, but you can’t
with the beginning of uncoating phase, so it known as (Eclipse phase)
Eclipse phase: that defined as phase that start with virus taking off the capsid or envelop to start new
infection to the host and end with new viruses are made.
• Viruses start the last phase (transcription phase) after it ends uncoating phase.
• Reoviruses: can start the transcription phase without ending the uncoating phase (harry up the
process).
• Pox viruses: uncoated the capsid in tow steps:
a. First: it used the host cell enzymes.
b. Second: it uses its own enzyme.
• Picornaviruses: make some conformal changes in capsid protein when attach to the host cell
receptor and those confirmatory changes (attachment) triggers uncoating (poring the capsid to
make the nucleic acid leave the viruses and enter the host cell when it attaches with receptor and
that known as confirmatory changes).

4. Replication and transcription:

Host cell provides the energy & machinery for the synthesis of viral proteins & nucleic acids.
Viral genome must be able to produce mRNA host cell protein-synthesizing machinery may be able
to synthesize viral proteins
Replication: Method used to duplicate the genomic material.
Transcription: method used to main factor mRNA from the nucleic acid.
Translation: Method used to convert the mRNA to protein.
First step before virus start to replicate is control the whole cell genome and prevent her from
translate any of its proteins.
• All DNA viruses replicate in nucleus except pox and asfa virus replicates in cytoplasm.
• All RNA viruses replicate in the cytoplasm except retroviruses, influenza and hepatitis D which
require intranuclear step.
• Translation method done in the cytoplasm.
• Transcription makes both structural and non-structural protein.
• Please, review the seven groups in Baltimore classification to understand mRNA correctly.

5. Assembly:
the stage after replication and transcription in which all
structure component of the viruses made by host cell is
come together to make a new virus.
In non-enveloped icosahedral viruses:
a. Protein (from translation) associated to for the
capsomer.
b. Capsomer are self-assemble to form procapsid.
c. Viral nucleic acid (from replication) is lastly entering
the procapsid and forming new individual.
• In some enveloped viruses: last step is linked
with viral release by budding.

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6. Maturation:
The stage in which new viruses become infectious.

7. Release:
a. Cell lysis in naked virus: Host cell damaging with release eg. Picornaviruses.

b. Budding released in enveloped viruses: To from the envelop from the host cell organs (REVIEW)
that method is the opposite of endocytosis.
• Envelop from cell membrane eg. Retroviruses.

c. Exocytosis
If the virus gained envelop from the intra organs as endoplasmic reticulum it released by
exocytosis.
But if virus gained the viruses from the cell membrane it released by budding.
• Envelop from nuclear membrane eg. Herpesviruses.
• Envelop from endoplasmic reticulum or Golgi complex eg. Flaviviruses.

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Lec. 4 Viral pathogenicity and shedding

Viral pathogenicity: Process by which virus produce disease in the host (generating disease).
Steps in general:
1. Routs of entry
2. Host specificity and tissue tropism (Attachment).
3. Spread within the host (infection of major target organs and tissue).
4. Shedding from the host.

1. Routs of entry:
A. By skin: Human host cell is covered by skin in the whole-body surface and skin is not loved to the
virus because it’s dry and high in salinity so, virus can’t enter the body without help to invade the
outer cutaneous layer (dead layer).
Help as Inclousion by needle or injury’s (skin breaches).

a. Skin breaks entry:

i. Epidermal skin: that have a few blood vessel and lymphatics.
viruses can’t replicate if the skin breaks wasn’t go farther than epidermis.
except Papilloma viruses that can replicate if arrived to the epidermal layer.
ii. Dermal layer: contain rich blood supply, lymphatics and nerve nourishing.
If breaks reached the dermis and viruses rich dermis the virus will be able to spread and induce
generalized infection or stay in dermis and make a localize infection.
Virus that can produce generalized and localized infection as it wants.
eg. Pox viruses.

b. Transmission of virus by Arthropods through skin (external and ectoparasite):

Arthropods that have sucking mouse part can deliver virus to the blood stream directly.
i. Mechanical transition by carrying the viruses in their mouthpart (without viral replication on the
arthropods) like a contaminated needle.
ii. Biological transition: the virus rich the Arthropoda while insects is tacking a blood meal from
infected hosts.
eg. Equine infectious anemia.
Then virus will enter the Arthropoda gut and then spread to salivary gland of the insect.
Finally if contaminated Arthropoda penetrate (pit) any other healthy human will infect him.
eg. West Nile fever and yellow fever.

c. Entry by animal pit

eg. Dogs, cates, wolves, foxes …. etc.
The animal bit deliver virus through skin (outer dead skin) to the subcutaneous or muscle through
their contaminated saliva
eg. Rabies.

d. Iatrogenic:
Virus can be introduced directly to the body by penetration during veterinary practice by needle, by
transfusion or surgical interference.
eg. HIV, papilloma virus or HCV and HBV

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B. Entry through respiratory tract: the most common portal for virus.
Respiratory tract mucosa is covered by lining epithelial cells with support viral replication.

Respiratory system has defense mechanisms:

i. Goblet cells: releases mucosa to cover the respiratory inner wall.
ii. Coordinated beating of ciliated epithelial cells: Some cilia that can move out the virus by cilia
movement to the pharynx that will go out the body again by cough.
iii. Activity of local antibody and macrophages: that engulf forging particles.

How the infection occurs:

If infected person coughed give out some droplets.
If health person is standing in short distance from the infected person will get infected by these
droplets.
• If the droplets size is 10 um or more: it trapped on nasal mucosa over the turbinate bone.
• If the droplets size is 5-10 um: may reach trachea and bronchioles and trapped in mucosa
blanket.
• If the droplets size is 5 um or less: inhaled directly to the lung and some may rich alveoli
where that may be ingested by macrophage (the less infectious)

Virus in respiratory system is divided to two categories:

i. Localized: infect the lung or trachea only.
ii. Genralized: leave the lung and enter the blood stream.

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C. Entry through Gastrointestinal tract:
Viruses enter the body through oral-fecal rout when you eat contaminated food with virus.
Esophagus hard to be infected with viruses.

Gastrointestinal defense mechanisms:

i. Acid pH in stomach and alkalis in intestine.
ii. Proteases by gastric and pancreatic cells (trypsin, pancreatin and elastase) affect capsid and
envelop.
iii. Mucus layer secreted by gastric and intestinal cells (physical barrier between viruses and
intestinal epithelial cells).
iv. Bile salts: also help in soluble the virus (kill it).
v. Peristaltic movement of the gut (limits the virus ability to attach to intestinal epithelial cells).
vi. Secretory antibodies (IgA) and non-specific (macrophages) inhibitor of viral infection
• Some viruses as Routa-viruses and Entero-viruses are resistance to the acidic PH that causing
some problems to gastric.
• Corona virus not acid resistance but in young ages enter the body with milk feeding and
secretes a wall around itself to buffer the pH.

As respiratory; gastrointestinal viruses are divided into two main categories:

i. Localized: infect the gastric only eg. Rota and corona viruses.
ii. Genralized: leave the gastric and enter the blood stream eg. Adeno and Entero-viruses.

D. Entry through urino-genital tract:

Localized infection: eg. IBRV and Papilloma viruses.
Generalized (Systemic infection): eg. HIV and Hepatitis B.

E. Entry through Conjunctiva

It is rare rout (opposite of respiratory) for virus entry.

Conjunctiva defense mechanisms:

It is protected by Tears that flash the eyes and wash out any virus and contain lysozymes that
inactivate viruses.

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2. Attachment (Host specificity and tissue spread):
Viruses have a specific receptor in the body if it not found it will leave the body without any problems
but if found it will attach to it this attachment will activate a specific sequence on the viral genome called
(Enhancer) that will define the virus found its specific receptor in the host and attach it in that time
enhancer will start to work to wake up the genome and activate it to start replication and transcription.

Tropism: The capacity of virus selectively to infect cells in particular organs

This depends on the presence on the cell surface receptors specific for the viruses

3. Spread in the body:

A. Local infection:
Some viruses (only) produce tissue injury at or near the body surface of entry (In epithelial surface).
• To infect another cell in different place in body but infect the neighboring cells by viral genome
replication to increases in number then infect the neighboring cells.
• Cells have two surfaces Apical and basal virus leave the cell from the apical surface to make
localized infection.

B. Generalized (Systematic) infections:

Others invade the subepithelial tissue and spread to specific tissues or organs via the lymphatic,
Hematogenous or nervous pathways.
• Can infect large number in cells in different places in the body in a short time through circulating
with the blood or lymph.
• if virus left the cell from the Basel surcease the probability to make generalized infection will
increased.

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Generalized infection have several ways to viral spread:

a. Sub-epithelial invasion and lymphatic spread

When a virus pass epithelium and basement membrane to sub-epithelial tissues; it may:
i. Engulfed by the tissue macrophage
ii. Enter the blood directly through an injured blood capillary
iii. Be carried by the lymphatic to a regional lymph node.

In the lymph node, the viruses may:

i. Be engulfed: inactivated and presented to the adjacent lymphocytes to initiate specific immune
response
ii. Be engulfed but replicated in macrophages, eg. canine
iii. Invade and replicate in the lymphocyte that can disseminate viruses throughout the body.

b. Blood spread: virus existence and spread through blood is called viremia.
Two phases of viremia are usually recorded:

Primary viremia Secondary viremia

Occurs after virus
Occurs after propagation in
Occurrence entry and primary
the target organs.
replication.
The virus is carried
It establishes infection in the
Pathways to the target organ or
body
tissue
Virus concentration
Viral conc. Virus concentration is high
is low
It stays for short
It stays for long period of
Residence period of time.1-2
time
days (transient).
Clinical Usually silent Usually active
picture (no symptoms) (symptoms is activated).

• If the vaccine contains an active virus (not killed or inactive virus) it may recombined with
the existing virus in the host to generate a new type of strain or pathotype.

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During viruses circulation in blood it may be:
i. Free in plasma (plasma viraemia): easily cleared from the body by Antigen precenting cells,
phagocytic, killer cells and complement ……. etc.
eg. Parvoviruses and flaviviruses.
ii. Associated with blood cells (cell associated viraemia): Protected from the body defense, so
viraemia may persist for months or years.
Some viruses either replicate in or just carried
by a single or multiple types of blood cells.
• Monocyte-associated eg. canine distemper
and bluetongue
• Lymphocyte associated eg. Marek’s disease
virus and HIV
• Platelets eg. Mouse leukemia virus
• Erythrocyte (rare) eg. Rift valley fever
• Neutrophils: (very rare) due its very short life
span and powerful of its antimicrobial
activity

Virus interaction with macrophages (VERY IMPORTANT):

i. Macrophages fail to phagocyte phagocytose virions (prolonged high viraemia).
ii. Macrophages phagocytose virions and destruct it efficiently
(rapid virus clearance from the blood).
iii. Macrophages phagocytose virions and transmit them to the hepatocytes where they replicate
eg. RFV (sever hepatitis and high viraemia)
iv. Macrophages phagocytose virions where it can replicate with or without transmission to
hepatocytes (high viraemia).

Virus interaction with the vascular endothelium

i. Direct passage from fenestrated endothelium.
ii. Carried by lymphocyte or monocyte cells (viruses circulation associated with blood cells).
iii. Movement through the endothelial cells by a process of: (REVIEW REPLICATION)
• Endocytosis
• Transcytosis
• Exocytosis
iv. Infection and replication in the endothelial cells and lysis it (REVIEW REPLICATION).

With microphage With Endothelium

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c. Nervous system spread
The process by which certain viruses are transmitted within nerve fibers (axon) to the central nerves
system (CNS)
eg. Rabies, borna and herpes viruses.
Viruses passes through nerves system are two types:
i. Obligatory neuro tropic: Is a Nuro passage with no viraemia at all.
eg. rabies.
ii. Neurotropic: Only nerve passage in one stage in adults and viremogenic in young
eg. Herpesvirus
During viral passage in nerve have the following characters:
i. Move in a centripetal (from body surface to CNS) movement.
ii. Move in a centrifugal (from the centers to periphery) movement.
iii. Cross the cell-to-cell junctions
iv. Usually pass from synaptic junctions eg. rabies and pseudorabies
v. Infect the Schwann cells of nerve sheet eg. herpesviruses or not eg. Rabies
• Virus movement along nerves is very slow.
• Nervous route is less common than viraemia

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4. Virus shedding
Virus release: Virus leaving cell to infect another neighboring cell or to enter blood stream to spread.
Viral shedding: Virus leaving the host.
• Virus shedding is important for maintenance of the infection in populations.
• The amount of virus shed is important for the efficiency of transmission.

In localized viral infections: The same body-opening is involved in entry and exit.
eg. respiratory and gastrointestinal infections.
In generalized infections: a great variety of shedding sites is recognized.
A. Skin
It transmits viruses that cause localized skin lesions by direct contact
eg. poxviruses and papilloma viruses.
• Only few of viruses with generalized infections making skin lesions; shed from the skin.
B. Respiratory tract
Respiratory viruses are shed in the nasal and oral secretions.
eg. mucus and saliva and are expelled during coughing and sneezing in form of:
a. Droplets (REVIEW RESPIRATORY VIRUSES).
b. Aerosols: Small droplets that remain suspended in the air.
C. Oropharynx and intestinal tract
Few viruses are shed from the oral cavity through infected salivary gland.
eg. Rabies (Exception) (No neurotropic virus).
Other viruses are present in the saliva because of continuous mixing with the mouth lesions.
eg. FMDV
D. Urinary tract
Some viruses replicate in the tubular epithelial cells of the kidney are shed in urine
that called (Viruria).
eg. RPV, FMDV, infectious canine hepatitis and arena virus in rodents (Reservoir host).
E. Genital tract
Most of generalized infection are shed from the genital tract (Infect semen and vaginal secretion).
eg. HIV and Hepatitis B virus
F. Milk
Viruses are rare to shed from mammary gland
eg: caprine arthritis encephalitis virus (in goats).
G. Blood and tissues
Blood and tissues from slaughtered animals considered transmission.
Blood is a potential source of virus transmission through:
a. Arthropods bites.
b. Blood transfusion.
c. Vertical transmission: Source of virus transmission to avian egg or mammalian fetus from moms.
H. Infection with no shedding
Many sites of viral replication are “dead ends” since they are not connected with a body surface for
shedding.
eg. Brain
• Maintenance of these virus infections occurs through consumption of infected tissues by a
carnivore or an omnivore.

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Lec. 5&6 Anti-viral chemotherapy

BEFORE START LECTURE REVISION LEC. 3 FIRST
Chemotherapy of Viral Diseases
Some-identifications:
Antibiotic: is a chemical agent that has a lethal antibacterial effect and effectless on viruses.
Anti-viral: An agent that kills a virus or that suppresses its ability to replicate and, hence, inhibits its
capability to multiply (has an anti-viral effect and effectless on bacteria and most of them also
effect on the host living cells).
• There are not many antiviral drugs.
• Most anti-viral drugs agents that interfere with virus replication are toxic to the cell.
Antibody: is a body immune response to an infection or invasion of a foreign body (better than vaccine).
Vaccine: is used to prevent the viral or bacterial infection from beginning (is a dead or week virus or
bacteria to enhance body to from antibody against) (injected to the host to protect him from
expectable viral or bacterial infection that are pandemic in the region)
eg. Malaria vaccine in south Africa before infection.

Several steps in the virus replication cycle represent potential targets

for selective antiviral drug attack.
The best anti-viral drugs that are used; drugs that target viral enzymes and host enzymes needed for viral.
Enzymes that attacked with some anti-viral drug.
1. Viral enzymes
a. DNA-dependent DNA polymerase in DNA viruses.
b. RNA-dependent RNA polymerase in RNA viruses.
c. RNA-dependent DNA polymerase in Retro viruses.
2. Host enzymes needed for viruses to complete replication:
a. Protases enzyme: That needed by some viruses to cleavage its newly formed protein after
translation process in ribosome.
eg. Retroviruses
b. Integrase: Needed for viruses that need to insert their genome to the host chromosomes to
replicate their genome.
eg. Retroviruses
c. Neuraminidase enzyme: That used by some viruses to be released from infected of lysed cells.

The table below sets out the most vulnerable steps and provides examples of antiviral drugs that display
activity.
• Some of them have already been licensed for use in humans.
Target Prototype drug
(step that we focus on to stop or prevent) (chemical we use to stop or prevent viruses from doing specific process)
Attachment of virion to cell receptor Receptor analogs
Uncoating Rimantadine
Primary transcription from viral genome Transcriptase inhibitors
Reverse transcriptase Zidovudine – AZT
Regulation of transcription Lentivirus tat inhibitors
Proccing of RNA transcription Ribavirin
Translation of viral RNA to protein Interferons
Post-translation cleavage of protein Protease inhibitors
Replication of viral DNA genome Acyclo-guanosine (Acyclovir)
Replication of viral RNA genome Replicase inhibitors.

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Drugs that targeting the ATTACHMENT of virion to cell receptor:
A logical approach to the development of new antiviral drugs is to isolate or synthesize substances that
might be predicted to serve as inhibitors of a known virus-encoded enzyme such as a transcriptase, replicase,
or protease.
• There is no good drug that inhibits receptor binding.

Drugs that inhibit attachment:

Example: Using soluble CD4 protein to prevent HIV attachment to cell.

Overview
HIV have glycoprotein called glycoprotein 120 (Gp-120) that bind to T helper lymphocyte cell by CD-4
receptor and HIV also have a co-receptor that bind with chemokine receptor 4 or 5 called CXCR4.

Mechanism of action:
1. Soluble CD-4 injected into blood that similar to CD-4 TH-Cell receptor that would bind HIV
glycoprotein and stop it binding to the T- cell receptor.
• The soluble protein is rapidly broken down and cleared from the circulation.
• CD4-Ig2 (PRO 542 prototype drug) is a tetrameric form of soluble CD-4 antigen genetically
fused that is more stable form (not broken down).
2. AMD3100 is a co-receptor ligand that also injected in soluble form to bund with CXCR4 to
deactivate it.

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Drugs that targeting the FUSION
Drugs that inhibit penetration (fusion) of viruses: eg. enfuvirtide

Overview: HIV uses Viral glycoprotein 41 (Gp-41) to make its conformational changes and fuse with cell.

Mechanism of action:
a. Manufacturing glycoprotein (called enfuvirtide) from the main HIV glycoprotein 41.
b. Inject the manufactured glycoprotein to the infected host.
c. The manufactured glycoprotein will attach the main virus glycoprotein and Previn HIV from making
its confirmatory changes that by the way blocking fusion.

Other example with the same mechanism:

Using synthesized RFI-641 glycoprotein for Respiratory synthetical virus (RSV).

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Drugs that targeting the UNCOATING:
Uncoating of the virus: the loss of the lipid envelope of membrane-containing viruses or the loss of
nucleocapsid proteins in non-enveloped viruses and only nucleic acid remining.

Overview:
Uncoating often occurs in acidic pH (REVIEW UNCOATING)
• Some viruses do not need an acidic environment for fusion and fuse with plasma membrane.

Drugs that inhibit uncoating (Lysosomotrophic drugs):

a. Amantadine
b. Rimantadine

Mechanism of action of Amantadine and Rimantadine (Lysosomotrophic drug):

a. It’s known to act on a viral protein (On M2 ion channel)
• M2 (trans membrane protein) = Matrix protein two is a channel that allow ion transport to enter
the virus to fell acidity pH and uncoating will start.
b. Bloching the ion channels of viruses which is necessary for the acidification of the enveloped virus.
c. Which by the way blocking the uncoating.
eg. Effect of M2 channel on picorna-viruses.
d. These drugs act on maturation of influenza HA glycoprotein M2 protein.
e. Drug can interrupt the shedding of RNA from the virus as the example upper.
f. These drugs are good for oral prophylaxis against influenza A (but not influenza B or C) because M2
protein found only in Influenzas A.
• They are a good alternative to the vaccine in immunocompromised patients and the elderly.

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Drugs that targeting the NUCLEIC ACID SYNTHESIS:
A. DNA Synthesis Inhibitors
The best anti-viral drugs because its selectivity.
eg. Acyclovir (Nucleoside analog to guanine base) chain terminator drug.
• Nucleoside analog: is manufactured nucleotide that similar to body original nucleotide but not
active.

Nucleoside: Is a nucleotide with di-phosphorus group (not active)

Nucleotide: is active form (tri-phosphorus) is a monomer used in DNA building.

Selectivity of drug (Only affect virus or infected cell only) is due to:
a. The virus uses its own enzyme to activate the drug eg. Thymidine kinase in herpes simplex
Thymidine kinase enzyme: Is the enzyme which add phosphate group and to the nucleotide to
complete forming the nucleotide (convert it from Di-phospho- nucleotide
as in active form to Tri-phospho- nucleotide) that sharing in building the
nucleic acids.
Enzyme has two forms:
Host form: Effect only thymidine nucleotide.
Virus form: Effect thymidine and other nucleotide.
Acyclovir contain Acyclo guanin (not complete Phosphorylated
guanine) to cross the plasma membrane
• Phosphorylated form (activated form) can’t cross the plasma
membrane.
Thymidine host kinase can’t interact with guanosine because its
specificity to thymidine, but the viral thymidine kinase enzyme interacts
with it to transform it to active form and then acyclovir starts its action
to the virus.

b. The viral polymerases may be much more sensitive to the drug than the corresponding host enzymes.
Mechanism of action
a. Acyclovir is a nucleoside analog for guanin
and act as in active form.
b. After ingestion of acyclovir, it wouldn’t
start action without virus containing
Thymidine kinase enzyme presence as
herpes simplex.
c. Virus Thymidine kinase add phosphate
group to guanine analog and transform it to
its active form (tri-phospho-guanine).
d. Acyclovir activated.
e. Acyclovir is a nucleoside that while
building the complimentary DNA strand by
DNA host polymerase if the original strand
has C nucleotide the DNA-polymerase will
complement it with G nucleotide, but acyclovir injected with 10 times action more than cell host
DNA polymerase and cell free nucleotide.
f. The cell DNA-Polymerase will use the nucleoside analog (Acyclovir) instead of G nucleotide.
g. After the G nucleoside analog is added viral replication will stop and chain termination occurs.
h. Chain termination means cutting to the viral DNA that will make it useless and effective less.

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B. RNA Synthesis Inhibitors
eg (1). Ribavirin
Overview:
This drug has been used in the treatment of human respiratory syncytial virus and hepatitis C virus
infections.
mRNA contain forepart called 5’ part that cap normally contains methyl guanosine and the caping of the
mRNA is important in leading the mRNA to the ribosomes.

Mechanism of action:
It may act as a guanosine analog and inhibit 5' cap formation on mRNA (guanosine).
• Ribavirin is known to inhibit the production of infectious polio virus and this virus does not have a
methyl guanosine cap.
So there must be alternative mechanisms for ribavirin action.
It is likely that this drug introduces multiple mutations into viral RNA rendering it incapable of a new
round of cell infection.

Therapy trials against HIV.

This prototype drug (ribavirin) inhibits the caping process.
So, the mRNA won't proceed the translation process with the ribosomes.
Thus, translation process is terminated, and viral protein wouldn’t be synthesized.

eg (2): Zanamivir (Relenza):

Zanamivir (Relenza): used as inner respiratory aerosols.

Overview
Influenza requires some enzymes as neuraminidase enzyme to:
a. Escaping from the cell.
b. Penetrate the mucus.

Mechanism of action:
Is a neuraminidase inhibitor that activated against Influenzas A and B

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Interferon:
In 1957, Isaacs and Lindemann reported that cells of the chorio-allanntoic membrane of embryonated hen’s
eggs infected with influenza virus release into the medium a nonviral protein (interferon).
Its name drifted from its action that interfere with viral nucleic acid replication.
Interferon is synthesized normally by body and classified as chemokine.
Interferon is now in-vetro synthesized and used as viral drug therapy.

There are three distinct types of interferon that each utilize to different cellular response:
Alpha Beta Gamma
leukocytes, many other Fibroblasts and T lymphocytes and NK cells
source
cells Epithelial cells
Inducing agent
Virus infection
for release
Number of At least 22 in humans.
1
subtypes Fewer in animals
Glycosylation No (most subtypes) Yes Yes
Functional form Monomer Dimer Tetramer
Principal activity Antiviral Antiviral Immuno-modulation
Enhances MHC (major histocomplement)
Mechanism of
Inhibits protein synthesis antigens, activates cytotoxic cells,
action
macrophages and NK (Nature killer) cells
• These proteins (IFN) are key elements of antiviral resistance and are central to both innate and
adaptive immune responses to viral infections.

Mechanism of Synthes release: (VERY IMPORTANT)

1. After the viral attachment to the host cell, it is
inducing some changes to the cell as presence of
duple strand RNA (viral nucleic acid during
replication).
2. Some receptors in the cell as (Toll like
receptors)
a. TLR3 sense the double strand RNA in the
cell (During replication of viral RNA).
b. TLR9: sense the un methylated DNA
(Viral genome of DNA viruses).
c. TLR7: sense the single strand RNA
(in +ve and -ve sense RNA genome).
3. Receptor sensation of any of above start to
enhance some genes in the cell genome as
interferon stimulating genes.
4. After the gene is activated, the interferon started
to be manufactured.
5. Interferon starts to inhibit the viral protein
Synthes (Translation).
• If the infected cell virus made the translation process Interferon can’t do anything anymore
because the virus nucleic acid had been translated.
6. The interferon also released out from the infected cell to the intercellular space and attach with some
specific receptor in neighboring healthy not infected cell.
7. After the interferon attachment to the healthy neighboring cells make that cell as (Anti-viral state)
that can’t be infected any more (as bacterial coting itself in hard environmental conditions).

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Mechanism of interferon to stop protein translation:
A. Oligoadenylate pathway:
1. After the interferon entry to the healthy cell, it activates 2’-5’ oligoadenylate enzyme.
2. That enzyme activates another enzyme called RNAase that responsible for degradation of RNA.
3. If the virus even enters the cell and make the transcription process and made its own mRNA the
mRNA will automatically broke down by RNAase enzyme.
• The cell will then apoptosis.

B. Protein kinase pathway:

1. Interferon also activates another enzyme called PKR kinas (Protein-Serine-Threonine Kinases).
2. That PKR enzyme is activated in the presence of Viral DNA.
3. After PKR activation its phosphorylation another enzyme called eIF (Elongation initiation
factor).
• eIF: enzyme that help on protein translation.
4. The phosphorylation of eIF will inactivate it and by the way will stop protein formation that will
inhibit and block viral protein synthesis.

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Lec. 7 Viral Oncogenesis

Oncogenic viruses:
A. RNA viruses:
a. Retroviridae: Leukemia (B-cell lymphomas), sarcomas and Carcinoma.
b. Flaviviridae: Hepatocellular carcinoma (hepatitis C virus) in man.

B. DNA viruses:
a. Hepadenaviridae: hepatocellular carcinoma (Hepatic B viruses)
b. Papillomaviruses: Papillomas
c. Poliomaviruses: various solid tumors.
d. Adenoviridae: various solid tumors
e. Herpesviridae: lymphomas, carcinomas and sarcomas
f. Poxviridae: Myxomas and fibromas

Characteristics and behavior of transformed cells = Cancer cell = Oncogenes = Tumorigeneses.

a. Immortal (can grow indefinitely).
b. Presence of integrated viral DNA (pro-virus).
c. Reduced requirement for serum growth factors (that enhance cell to cell control and division).
• Capacity for growth upon nutrient deprivation
d. High saturation densities (staked to each other to form a mass).
e. Loss of contact inhibition that normal cell in a sheet from but in cancer tissue (can grow forming
three-dimensional colonies).
f. Anchorage independence (can grow in soft agar and in cell suspension without anchor point).
g. Altered morphology (appear rounded and retractile with no uniform that result in presence a lot of
different type of antigens).
h. Virus-specific tumor associated antigens (some at the cell surface transplantation antigens, others
intracellular-T antigens).
i. Chromosomal abnormalities.
j. Tumorigenicity: capacity to produce tumors when inoculated in isologues animals.

How do viruses transform cells to cancer cell?

This occurs by subverting the normal cell control mechanisms.
But, to understand how the cancer cell is produced, first you must have a background in cell cycle

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There are two important classes of cellular genes which control cell replication:
1. Genes which promote cell replication these are called: Oncogenes, Protooncogenes, C-oncogenes
(cellular) or V- oncogenes (virus).
• Viruses, especially Retroviruses can affect the activity of these genes to amplify their action.
• Some retroviruses carry their own version of these genes, called v-oncogenes.
C-Oncogenes are involved in regulation of cell growth, division and differentiation.
Thay act as:
a. Growth factors (Effect on Growth factor receptors).
b. Intracellular signal transducers (deliver massages).
c. Nuclear transcription factors.
Retrovirus oncogenes and cell cycle control protein:
a. Oncogenes are not essential for virus replication
b. They are acquired over time by the viruses from the cells they infected
so, V-Oncogenes are derivative from normal C-Oncogene (through transduction process).
c. Retroviruses have Long terminal release gene (LTRs) in their genome which include strong
promoters and enhancers that will control the C-Oncogenes of host cell.

2. Genes which suppress cell replication: these are called Tumor suppressor genes.
Many viruses, especially DNA viruses prevent their proper functioning.
Tumor suppressor genes
They play an essential regulatory role in normal cell division and involved in the negative regulation
of cell cycle and hold the cell at the G1 phase.
eg (1). p53 protein:
Tetrameric protein that binds specifically to promoter elements specifically trans-activate gene
expression and suppress it to stop cell division and if fails it may trigger programmed cell death
(apoptosis).
eg (2). RB1 (Retina blastoma)
It acts as a brake on the advancement of cells from G0/G1 into S phase.
The role of tumor production by tumor suppressor genes
a. Mutations in tumor suppressor genes causing the produced proteins lose their DNA binding
capacity that will enter the cell to unstoppable cell division.
b. Binding of other viral oncoproteins to these tumor suppressor proteins causing their sequestering
or degradation host cell suppressor genes.

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A. RNA viruses:
Retroviruses produce tumors by one of three ways:
1. Cis-activating retroviruses: (insertional mutagenesis) (not carry v-oncogenes but activate it).
a. While Retrovirus entry in the host cell as mentioned previously it insert its own genome to the
host chromosomes.
b. There is a probability that insertion of the viral genome to the host chromosomes being neighbor
to host cell oncogenes (C-Oncogenes).
• Cancer making by Retrovirus or not is a probability due to the location of viral gene insertion.
c. If the viral genome inserted beside to C-oncogenes that will enhance viral LTRs to work.
d. After the LTRs is start working it produce V-Oncogenes to work and control the C-oncogenes
completely.
e. The cell only divided when the Retrovirus DNA is replicated, and retrovirus replicate harry and
rapidly.
f. That will enter the cell to Rapidly uncontrolled cell division (cancer) state.

2. Transducing retroviruses (carry its own oncogenes).

a. They carry their own v-oncogenes incorporated in their genome
eg. gag, pol, env and onc.
b. The viruses are rapidly replicate, and their own v-oncogene will replicate and amplified with it.
c. The V-oncogenes will affect the cell and entering it to cell rapidly uncontrolled division state.

3. Trans-activating retroviruses (Haven no v-oncogenes and wasn’t inserted beside to c-oncogene)

a. The virus has gene that encodes to generate regulatory proteins.
b. This protein either increase transcription or interfere with transcriptional control of specific
cellular genes.
c. This mechanism enhances at the end of oncogenesis.

B. DNA tumor viruses

Infected cells with DNA viruses may resist infection or become transformed into cancer cells.
Transformation of normal cell to cancer cell is associated with integration of viral genome (or portions) into
the host cell DNA and its stable passage to daughter cells.

V-oncogenes in DNA tumor viruses were recognized as:

a. T antigens of SV40 in polyoma viruses.
b. E1A and E1B of adenoviruses.
c. E6, E7 of papilloma viruses.

The v-oncogenes of DNA tumor viruses encode oncoproteins which play an important role in virus life cycle
as initiation of DNA replication and transcriptional regulation of viral genes.
• Unlike oncogenes of RNA tumor, the DNA tumor virus oncogenes have no cellular counterparts.

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