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Chapter 5: VIRUS

18 | Lacap, Dixie Mae N. Lacap

02. .15

Introduction
Poliomyelitis one of the earliest evidences
- Egyptian priest
- From hieroglyph
Rabies also led to the discovery of the virus when it could not be explained by the presence of bacteria.
1940s Electron Microscopy- start of study (chemical nature of virus)
- infectivity mainly studied in animal models

General Structure of Virus


Virus
-

diverse in size and structure


Poliovirus smallest virus (28 nm)
Mimivirus largest virus (750 nm)
Capsid aka coat
protein core w/c holds the viral nucleic acid
possibly surrounded by lipidic envelope
Viral Classification- based on morphology, structure, physical and chemical properties

Viral Nucleic Acid


Viral Genome- composed of either DNA or RNA
- Maybe:
double or single stranded
linear (Poliovirus) or circular (Hepa B virus)
segmented (Influenza- 8 segments) or one molecule(Poliovirus)
*Virus that contains Plus-sense ss RNA can have their genome translated directly by the host ribosome
Retroviridae- ex. HIV
- requires a specific virus-encoded enzyme (reverse transcriptase) to convert its ss RNA to ss DNA,
to be able to replicate within the host cell.
- This enzyme is a primary target of antiviral drugs
Mulitplicity Reactivation- in poliovirus
- Random damage to viral capsid and nucleic acid following treatment with Hypochlorite (a biocide
intensively used for surface disinfection) resulted in complementary reconstruction of an infectious
particle by hybridization of gene pool of inactive virus.

Viral Capsid

protect the viral nucleic acid from detrimental physical and chemical conditions.

- for attachment to host cell


Capsomere subunit that makes up a viral capsid
- Gives the shape of the capsid
- Provide the virus with resistance to chemical and physical agents
- 3 different architectural styles:
Icosahedral - have capsomeres in the form of Pentons & Hexons
- Adenovirus- 240 hexons & 12 pentons

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- Poiliovirus- 20 hexons & 12 pentons


Helical symmetries- influenza and mumps virus
- Subunits symmetrically packed in helical array, like coils of wound rope
under electron microscopy
- Core is hallow, viral nucleic acid is embedded into the ridges on the inside
of each subunit
Complex -mammalian virus; several percutaneous structure envelopes the viral genome
core
Bacteriophages- aka bacteria virus
- Shows a complex structure consisting of:
Capsid head
Tail
Tail fibres

Viral Envelope-

a lipidic envelope that surrounds the viral capsid

originated from the host cell


added during replication or after excision of viral progeny from the host cell
may come from Cell Nuclear Membrane (Herpes simplex virus) or the Cytoplasmic Membrane
(influenza virus)
Enveloped Viruses are the most susceptible to chemical and physical conditions and do not survive
well on their own, although they can persist longer in inorganic soil (blood, exudates, feces)

Viral Receptors-

glycoproteins protruding from the viral capsid or embedded in the envelope

important for viral infectivity


recognizes the host cell receptor site conveying the viral specificity

Virus-host cell interaction


*Viruses are True intracellular parasites that grow within living cells and use their energy and synthetic
machinery to produce viral components
Virus progeny/Virions- new virus from the replication of one virus within the host cell
- can be released and infect adjacent cells
Major viral groups based on host specificity:
1. Viruses of bacteria & blue-green algae
2. Plant viruses
3. Animal(insects included) viruses
Rabies & Infulenza can cause diseases in both animals and humans
- these are exceptions because virus rarely cross species barriers due to their extreme specificity
Virus-Host cell interaction:
1. Multiplication of virus and destruction of the host cell upon release of viral progeny
2. Multiplication of the virus and release of the virions w/o the immediate destruction of the host cell
3. Survival of the virus in a latent stage w/o noticeable changes in a dramatically altered or transformed
state
4. Incorporation of the viral nucleic acid to the host cell genome w/o noticeable changes in the infected
cell

Chapter 5: VIRUS

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Asymptomatic Viral Infections- virus replicates w/in the host but doesnt produce symptoms of a disease

HIV-

enveloped virus with a cone-shaped nucleoplasmid


-

H IV co re
p e ne tra te s
ce ll cy to p la sm

2 copies of positive-sense ss RNA & enzyme reverse transcriptase


the viral genome encodes for the ff. genes:
env, envelope proteins
gag, capsid proteins
pol, enzymes involves in viral multiplication
tat, enzymes regulating host metabolism
70 glycoprotein spikes (gp120) projects from the envelope & interacts specifically with the CD4
protein receptor on the T-lymphocyte
M em b ra n e
fu sio n

U nc o a tin g ,
re le a se o f 2
RNA &
re v e rse
tran scrip tas
e in to
cy top la sm

Re v e rse
tra nscrip ta se
co p ie s R N A
itno ss D N A

ss D N A
du plica te d to
fo rm ds D N A
(cop y of th e
o rigin a l v ira l R N A
ge n o m e )

D N A m o ve s
in to h o st ce ll
n ucle u s

DNA
in te g ra te d a s
P R O V IR U S
in to a h ost
c ell
ch ro m o so m e

the provirus may lie dormant or can produce viral mRNA (polycistronic) & proteins to resume the
multiplication cycle producing virions

HAART Highly Activated Antiretroviral Therapy


-antiviral txt, combining a protease inhibitor & 2 reverse transcriptase inhibitor
-reduce the number of HIV particles (

10

10

viral particles produced continuously per day) & slow the

progression of the disease by restoring & maintaining the number of T4 helper lymphocytes.

Tumour Virus
-

virus infected cells change dramatically, acquiring the characteristic of tumour cells exhibiting
uncontrolled growth.
Acquisition of viral genes by the host must be followed by other events as environmental or dietary
exposures to chemical carcinogen for cancer to occur

Multiplication of human viruses


Objective of replication cycle: to ensure the multiplication of the virus with the formation of the identical
viral progeny
Multiplication time of Human Virus is 4- more than 40 hours; Bacterial Virus takes only 20 minutes.
Six distinct phases of the replication cycle:
1. Attachement to the host cell
Initial contact mainly dependent on
Brownian motion

Reversible phase during w/c electrostatic


repulsion is reduced

Irreversible changes in virus-receptorhost-receptor configuration that initiated


viral penetration through the cell
membrane

-Virus-cell Recognition Event- similar to any protein-protein interaction in that it occurs


through a stereospecific network of hydrogen bonds and lipophilic association
2. Penetration of the viral particle

Chapter 5: VIRUS

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Direct Injection- only the naked DNA will enter the host cell
Fusion- viral envelope will bind with host cell membrane, liberating the viral capsid w/in the
cell cytoplasm
- Endocytosis- the whole virus will be engulfed by the host cell, forming a Cystolic Vacuole.
3. Uncoating of the viral particle
- Viral nucleic acid needs to be released from the capsid
- Penetration by Endocytosis: acidification of cystolic vacuole induces a conformational change in
the capsid & the release of viral nucelocapsid into the cytoplasm
- Some like Reovirus only needs a partial uncoatingfor the expression of the viral genome
4. Replication of viral nucleic acids & translation of the genome
- the host cell synthesis is taken over by the virus
transcription of viral genes into viral
mRNA

translation of the viral genome into


proteins

replication of the viral genome

the viral genome is replicated


some viruses can encode for genes for the products of w/c regulate the synthetic process
according to the needs of the virus
- host cell mRNA encodes directly for functional proteins, whereas viral mRNA is polycistronic,
w/c means several distinct proteins are encoded w/in a single piece of mRNA. Thus, virus needs
a virus-specific protease to cut the correct place
5. Maturation/Assembly of virions
- Replicated viral genome and some viral proteins become packaged w/in the capsid
- Most non-enveloped viruses accumulate in the cytoplasm or nucleus and are only released
during cell lyses.
6. Release of the virions into the surrounding environment
- Mature virions are released from the host cell
- Some viruses produce a lytic enzyme or protease to lyse the host enabling the release of
infectious particles
- Enveloped viruses are released by budding process
- Ultimately cell will die following damage to its metabolism and house keeping function during
viral replication

Cultivation of human viruses


The study & ID of viruses depends on our ability to propagate them
Cultivation of many viruses enabling a better understanding of their replication properties, more rapid
diagnosis and the easier production of vaccine.
Cell Culture
Sources of cell samples: Humans, primates and rodents

Cell culture according to history


a. Primary- diploid cell line
- Derived directly from an intact tissue (human embryo, kidney or monkey kidney)
b. Secondary- diploid cell line
- Derived from primary culture, usually those arising from embryonic tissue
- Limited number of subcultures can be performed w/ these cells generally up to a mmaximum
of about 50 before the cells degenerate
c. Continuous Cell Line- usually derived from malignant tissue
- has the capacity to multiply in vitro indefinitely

Chapter 5: VIRUS

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In principle, cell culture for propagating viruses replies on the growth of cells in a Semiconfluent
Monolayer attached to a surface
To subculture, the cells are separated from the monolayer w/ the use of Trypsin to form a suspension of

single cells. Growth temp of

37 C

Bovine Albumin- serum to supplement a culture media


The established cell monolayer will support viral replication from w/c viruses can be harvested
Cytopathic Effect- characteristic morphological change in the infected cells, & usually indicates cell
death.
- Cell shrinkage, ballooning, detachment of cells from the surface of the flask

The Chick Embryo


-

9-11 days old fertile chicken eggs

Used as a convenient cell system to grow human pathogenic viruses

Control of Virus
Antiviral Chemotherapy- most are pro drugs that need to be activated within the cell (by Kinase & Other
cellular
enzymes)
- Role: slow or halt disease progression. Not cure.
- target Viral enzymes: Protease; Polymerases; Reverse Transcriptase
- Protease- particularly important for uncoating process preventing the release of viral
nucleocapsids
- Neuraminidase Inhibitors- prevents the release of mature virions

HIV
Antiretrovirals- antiviral drugs
- Aim to reduce HIV plasma levels for as much and as long as possible
- Side effects:
Immune Recinstitution syndrome
Cough
Headache
Lipodystrophy syndrome- includes:
Insomnia
fat redistribution insulin resistance;
Dizziness
hyperglycaemia; dyslipidaemia
Fatigue
Liver damage
Blood disorder
Osteonecrosis
Myalgia
GI Disturbance
Arthralgia
Anorexia
Rash
Pancreatitis
Urticaria
Dyspnoea
fever
-

Protease Inhibitors- metabolized by Cytochrome P-450 & therefore have significant potential for
drug

Interaction
Non-nucleoside Reverse Transcriptase Inhibitor- interact with a number of drugs metabolized in
the liver
Side effects:
Rash

Chapter 5: VIRUS

18 | Lacap, Dixie Mae N. Lacap

Psychiatric disturbance
CNS disturbance
Fatal hepatitis

02. .15

Herpesvirus Infection

Herpesviridae- a family of viruses that include:

Herpes simplex virus


Chickenpox (Varicella)
Shingles (Herpes zoster)
Cytomegalovirus

Mild herpes simples- txt with topical antiviral drug

Herpetic gingivostomatitis- txt with change of diet and analgesics

Systemic antivirals- given within 24 hrs onset of rashes, for adults infected w/
chickenpox
- Given within 27hrs onset of rashes, for adults infected w/ shingles
Side effects of Herpes Antivirals:
Nausea
Fatigue
Vomiting
Rash
Stomach pain
Increase in serum
Headache
Increase in urine uric acid

Influenza

Oseltamivir- Antiviral that was recommended for txt of influenza by National Institute & Clinical
Excellence (NICE)
- extensively used for prevention and control of the swine flu outbreak in UK 2009
- LIMITATIONS: Drug needs to be taken w/in a few hours of the onset of symptoms

Severe side effects for children and adolescents

Respiratory Syncytial Virus-

responsible for severe bronchiolitis notably in infants

Txt: Palivizumab (monoclonal antibody) & Ribavirin (antiviral drug), both associated with
side effects

Vaccination-

most successful measure against viral infections

Contains antigens that elicit a specific and active immunity against an infecting agent
Can induce the innate and adaptive (cellular, humoral) parts of the immune system

Small pox vaccine- by Jenner


- Inoculation of cowpox well before the germ theory of diseases were postulated

Most common methods of viral vaccine preparation:


1. Use of Live attenuated viruses- will cause a strong immune response w/o causing the disease
- used in prep. for MMR vaccine and Poliomyelitis vaccine
2. Use of inactivated viruses- Hepatitis A virus & Influenza vaccine
3. Use of viral components

Hepatitis B virus vaccine- recombinant vaccine where the viral DNA encoding for a virus surface
antigen (HBsAg) is

expressed in yeast (Saccharomyces cerevisiae) or mammalian cells (Chinese hamster


ovary cells)

Chapter 5: VIRUS

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02. .15

Human Papillomavirus (HPV)- contains virus-like particles & recombinant capsid protein expressed
in yeast or using a

baculovirus as an expression system.

Adjuvants in vaccines:
Aluminum Salts- antigens are absorbed to the aluminum salts
Monophosphoryl lipid A- increase or modulate host immunes response to the antigen

Human normal immunoglobulin (HNIG)- prepared from a pool of donated human plasma and
contains immunoglobulin G (igG) and antibodies against viruses like:
Hepa A
Rubella
Measles
Varicella
Mumps

Intramuscular normal immunoglobulin- used to protect against hepa A in


immunocompromised patients
- can also be used for pregnant ladies against rubella virus
Disease-specific immunoglobulins- prepared from a pool of plasma obtained from a human
donor who have high-levels of the specific antibody required
Disease-specific Hepatitis B immunoglobulins- treating infants born from mothers infected
with the virus
Disease-specific Varicella Zoster immunoglobulins- treatment for individuals who are at a
high risk such as:
a. neonates whose mothers developed chickenpox
b. for those exposed to the virus while requiring intensive care or proloned special care
c. for immunocompromised individuals

Viricidal effects of chemicals & physical agents on viruses

Viruses are generally transmitted via surfaces & therefore the use of viricidal disinfectants on hard
surfaces & viricidal antiseptics on skin is important
Susceptibility to viricidal agents:
a. Small non-enveloped viruses are more resistant than large enveloped viruses
b. Lipid-rich envelope being damaged easily by chemical & physical agents, large enveloped
virus
Biocidal activity depends upon a number of factors, such as:
a. Concentration
b. Contact time
c. Presence of soiling
d. Formulation
Target site for biocide:
a. Envelope
b. Glycoprotein
c. Capsid
d. Viral nucleic acid
Highly reactive biocide that damages the capsid:
a. Aldehyde ex. Gulataldehyd
b. Oxidizing agents- ex. Peracetic acid, Hydrogen peroxide
H 2 O2 & Chlorine dioxide have been shown to penetrate within the capsid and
damage viral nucleic acid
Lesser reactive biocide that can only damage the capsid to a certain extent:
a. Quaternary ammonium compounds (QACs)
b. Biguanides- ex. Chlorhexidine
Viruses with a HELICAL CAPSID STRUCTURE are more susceptible since the destruction/alteration of
capsid is more likely to cause damage to the viral nucleic acid w/c is closely associated to this type of
structures
More viruses are susceptible to exposure to temperatures above 60 for 30 minutes
- used for inactivation of viral contaminants (HIV) in blood products
Viruses survive well at low temperatures and they can be routinely stored at -40 to -70

Control of Viruses in Pharmaceutical Products

Factors that affect contamination of pharmaceutical products:


a. origin of product component
b. history of donor
c. amount of material used

d. manufacturing process
e. capacity to remove/destroy and contaminate
A risk assessment/stringent control are applied to samples containing a component from human or
animal origin
For preparation of viruses, the inactivation process must ensure that it does not affect antigenicity
while killing the virus and other potential contaminants such as mycoplasmas
Prep of Influenza Vaccine, the inactivation process must cause minimum alteration of the
HAEMAGGLUTININ & NEURAMINIDASE antigens

Viruses and Gene Therapy

certain viruses are used as vectors for the delivery of genes to targeted cells
Viruses used for Gene Transfer Medicinal Products:
a. (AAV) Adenoviruses
b. poxviruses
c. retroviruses
d. lentiviruses
e. adeno-associated viruses
f. herpesviruses
Viral vectors- for human use are freeze-dried/liquid prep of recombinant viruses, genetically modified
to transfer

genetic material to human somatic cells in vivo or ex vivo


Stringent control:
a. Complete sequence of the genome of the viral vector
b. Verification of the genomic integrity of the vector
c. Determination of the concentration of the infectious vector
d. Residual host cell protein and DNA
e. Residual reagent including antibiotics
Retroviridae-derived vector- genetic modifications aims to ensure that the recombinant
retroviruses are

rendered replication-incompetent.
Adeno-associated virus vectors- deficient adenovirus in which certain genes necessary for viral
replication have been replaced

Viruses as Antimicrobial

Bacteriophages- viruses that infect only bacteria


- 20-200 nm
- all bacterial species can be infected by a phage
Phages- tadpole-shaped consisting of a head that contains the viral genome & a tail which recognizes the
host receptor, attach and subsequently serve as a nucleic acid injection device
Lytic Cycle- phage replication resulting to the lysis of the host cell
Lysogenic Cycle- phage replication resulting in the viral nucleic acid being integrated into the host
genome
- Prophage- the viral nucleic acid that has integrated the hos genome
- Lysogenic- the host cell that contains the viral nucleic acid
Virulent phage- aka Lytic Phage
- Infection of these result in the replication of the phage within the susceptible bacteria and
the release of infectious phage progeny from the host cell following lysis
- Each lysis will for plaques (holes) in the sample
Transduction- when prophage takes the adjacent bacterial gene that becomes incorporated in the virion
and
transmits it to a new susceptible host cell
- Responsible for the gene transfer between bacteria

Epidemiological Uses and Diagnosis

Phage typing- method that differentiates distinct strains of the same bacterial species on the basis of
their susceptibility to phages

Prions- can be recovered from the brains of infected individuals as rod-like structures which are
oligomers of a 30kDa glycoprotein

Devoid of nucleic acid


Extremely resistant to heating and ultraviolet irradiation
Fail to produce an immune response in the host
Autocatalytic replication

end

praise the lord

hallelujah

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